US20030114410A1 - Pharmaceutical compositions and methods useful for modulating angiogenesis and inhibiting metastasis and tumor fibrosis - Google Patents

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

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US20030114410A1
US20030114410A1 US10/305,348 US30534802A US2003114410A1 US 20030114410 A1 US20030114410 A1 US 20030114410A1 US 30534802 A US30534802 A US 30534802A US 2003114410 A1 US2003114410 A1 US 2003114410A1
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gly
lysyl
ala
arg
oxidase
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Gera Neufeld
Gal Akiri
Zahava Vadasz
Stela Gengrinovitch
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Technion Research and Development Foundation Ltd
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Technion Research and Development Foundation Ltd
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Priority claimed from PCT/IL2001/000728 external-priority patent/WO2002011667A2/fr
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Priority to US10/305,348 priority Critical patent/US20030114410A1/en
Assigned to TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD. reassignment TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GENGRINOVITCH, STELA, VADASZ, ZAHAVA, AKIRI, GAL, NEUFELD, GERA
Publication of US20030114410A1 publication Critical patent/US20030114410A1/en
Priority to EP08020753.3A priority patent/EP2062919B1/fr
Priority to AU2003286391A priority patent/AU2003286391A1/en
Priority to PT08020753T priority patent/PT2062919E/pt
Priority to ES08020753.3T priority patent/ES2584847T3/es
Priority to US10/536,440 priority patent/US8163494B2/en
Priority to EP03777136A priority patent/EP1572100A4/fr
Priority to PCT/IL2003/001008 priority patent/WO2004047720A2/fr
Priority to EP08020752A priority patent/EP2062918A3/fr
Priority to DK08020753.3T priority patent/DK2062919T3/en
Priority to US12/571,167 priority patent/US8168180B2/en
Priority to HK09110803.7A priority patent/HK1131165A1/zh
Priority to US13/412,544 priority patent/US20120202206A1/en
Priority to US13/416,976 priority patent/US8815823B2/en
Priority to US14/311,110 priority patent/US20140302499A1/en
Priority to CY20161100760T priority patent/CY1117943T1/el
Priority to US15/881,339 priority patent/US20190040470A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03013—Protein-lysine 6-oxidase (1.4.3.13), i.e. lysyl-oxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to pharmaceutical compositions and methods useful for modulating angiogenesis and for inhibiting metastasis and fibrosis in a mammalian tissue.
  • vasculogenesis the transformation of pre-existing arterioles into small muscular arteries
  • angiogenesis the sprouting of existing blood vessels (which occurs both in the embryo and in the adult).
  • vascular endothelial growth factor VEGF
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • Platelet factor-4 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).
  • the present inventors 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.
  • LOR-1 lysyl oxidase protein
  • 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.
  • 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.
  • the molecule is a polynucleotide capable of down regulating expression of the at least one type of lysyl-oxidase.
  • polynucleotide is at least partially complementary with the polynucleotide set forth in SEQ ID NO: 1, 4, 5 or 7.
  • polypeptide is as set forth in SEQ ID NO: 2, 3, 6, 8 or 9.
  • a method of modulating angiogenesis in a mammalian tissue 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.
  • polypeptide is at least 75% homologous to the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.
  • step (a) is effected by binding assays and/or lysyl-oxidase activity assays.
  • 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.
  • a mammalian tissue 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
  • 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.
  • 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.
  • the molecule is a polynucleotide capable of downregulating expression of the at least one type of lysyl-oxidase.
  • step (a) is effected by binding assays and/or lysyl-oxidase activity assays.
  • 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 (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.
  • PEP Cells Pporcine aortic endothelial cells
  • 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 CentriconTM, and 10 ⁇ l 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. 5 a - b illustrate anti-factor-8 immunostaining of tumors generated by MCF-7 cells transfected with the expression vector alone (FIG. 5 a ) or with an expression vector containing the LOR-1 cDNA (FIG. 5 b ). 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. 5 b ) as compared to tumors generated by control cells which do not express LOR-1 (FIG. 5 a ).
  • 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. 9 a ) and LOR-2 (FIG. 9 b ) 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.
  • FIGS. 9 c - f illustrate the expression pattern of LOR-1 in normal human breast (FIG. 9 c ), in in-situ non-invasive breast carcinoma (FIG. 9 d ), in grade-1 invasive ductal carcinoma (FIG. 9 e ) and in grade-3 invasive ductal carcinoma (FIG. 9 f ).
  • 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. 9 c , open arrow); magnification ⁇ 100.
  • in-situ non-invasive breast carcinoma FIG.
  • 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. 9 d , empty arrow); magnification ⁇ 200.
  • LOR-1 high levels of LOR-1
  • the tumorigenic cells In grade-1 invasive breast carcinoma the tumorigenic cells have formed pseudo-ducts (FIG. 9 e , black arrows) but they do not express LOR-1 anymore. However, cells found in a nearby carcinoma in-situ express LOR-1 (FIG. 9 e , empty arrow); magnification ⁇ 200.
  • grade-3 invasive breast carcinoma the tumorigenic cells express large amounts of LOR-1 and the morphology is completely disorderly (FIG. 9f); magnification ⁇ 200.
  • 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.
  • 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.
  • 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).
  • 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. 11 a , 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. 11 b , arrows, magnification ⁇ 20).
  • 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. 11 d - 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. 11 d , arrows, magnification ⁇ 200).
  • Thick collagen bundles are seen between tumor cells generated from MCF-7 cells transfected with expression vector containing clone 12 (FIG. 11 e , 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. 11 f , magnification ⁇ 200).
  • 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. 11 g ) or expression vector containing clone 12 (FIG. 11 h ); magnification ⁇ 400.
  • 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. 12 a, c ) or vector expressing LOR-1 cDNA clone 12 (FIG. 12 b,d ); magnification ⁇ 200.
  • 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. 13 a, b ) or a vector expressing LOR-1 (FIGS. 13 c - 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. 13 e , red stain). Counterstain was with hematoxylin (light blue) in all sections.
  • 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.
  • PF4-LOR-1 binding assays Further support to the angiogenic activity of lysyl-oxidases is provided by the PF4-LOR-1 binding assays presented herein.
  • PF4 is an inhibitor of angiogenesis.
  • 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.
  • 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.
  • 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.
  • 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.
  • lysyl-oxidase 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”.
  • An antibody polyclonal, monoclonal or monospecific
  • an antibody portion e.g., Fab fragment
  • 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.
  • antibody inhibitors include inhibitors of angiogenesis which target angiogenic factors (Brooks et al., 1994; Brooks et al., 1998).
  • 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.
  • 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.
  • 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.
  • 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.
  • oligonucleotide binding affinity 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.
  • 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-).
  • 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.
  • RNA oligonucleotides may also be used for antisense inhibition as they form a stable RNA-RNA duplex with the target, suggesting efficient inhibition.
  • 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.
  • 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).
  • 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).
  • RNA interference an approach which utilizes small interfering dsRNA (siRNA) molecules that are homologous to the target mRNA and lead to its degradation (Carthew, 2001).
  • siRNA small interfering dsRNA
  • 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).
  • siRNAs small interfering RNA
  • 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).
  • 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • An example of a suitable expression vector is the pSUPERTM, 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.
  • 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.
  • 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.
  • bi-directional promoters are disclosed in U.S. pat. application No. 20020108142.
  • VEGF vascular endothelial growth factor
  • VPF potent blood vessel permeabilizing factor
  • 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.
  • 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.
  • Gene “knock-in” techniques can also be used to increase the expression levels of the lysyl oxidase.
  • affinity binding assays and/or activity assays 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.
  • 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.
  • 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.
  • administering refers to all modes of administration described hereinbelow with respect to the pharmaceutical compositions of the present invention.
  • 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.
  • the present invention also provide a method of identifying molecules capable of inhibiting metastasis and/or fibrosis.
  • 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.
  • lysyl-oxidase 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.
  • Screening for cross reactivity can be effected by lysyl-oxidase enzymatic activity assays, by binding assays and the like.
  • 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).
  • 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).
  • RBM reconstituted basement membrane
  • 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).
  • FCM fibroblast conditioned medium
  • 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.
  • 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).
  • determining phospholipase A-2 (PLA 2 ) activity, and the activity or abundance of factors that affect the activity of PLA 2 , such as uteroglobin protein (U.S. Pat. No.: 6,316,416) can serve as an indication of metastatic potential.
  • Determining pattern and level of expression of metastasis-associated markers can be effected by one of several methods known in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • 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.
  • 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.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • 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.
  • 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.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • compositions 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.
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • 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.
  • the present invention provides a method and pharmaceutical compositions useful modulating angiogenesis.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 35 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).
  • 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).
  • 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).
  • LOR-1 purification A PF4 affinity column was used to detect endothelial cell proteins which specifically interact with PF4.
  • 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.
  • LOR-1 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- ⁇ and inhibited by phorbol esters and retinoic acid (Saito et al., 1997).
  • LOR-1 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • LOR-1 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. 6 a and 6 c ) and antisense probes (FIGS. 6 b and 6 d ). Normal liver tissues expresses very low levels of LOR-1 (FIG. 6 b ). However, fibrotic liver tissues such as those observed in Wilson's disease, exhibit a strong increase in hepatocyte expression of LOR-1 (FIG. 6 d ).
  • 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.
  • Estrogen pellets (17 ⁇ -estradiol, 0.72 mg/pellet, 60-days release) were from Alternative 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).
  • 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).
  • FCS Fetal Calf Serum
  • 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 ⁇ g/ml G418, 150 ⁇ g/ml Hygromycin B and 1 ⁇ g/ml Tetracyclin.
  • LOR1 and LOR2 cDNAs Total RNA (4 ⁇ g) 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.
  • ROCHE Expand Long High Fidelity PCR system
  • LOR-1 SEQ ID NO: 1
  • SEQ ID NO: 4 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.
  • 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.
  • Hygro plasmid or pcDNA-LOR1 plasmid (10-20 ⁇ g) were stably transfected into MCF-7 cells using electroporation with a BioRad gene pulser (960 ⁇ F, 0.28 V). Stable transfectants were selected using 300 ⁇ g/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).
  • LOR1 cDNA SEQ ID NO: 1
  • pTET-Splice vector Clontech
  • Tet off system tetracycline
  • 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 ⁇ g/ml G418, 150 ⁇ g/ml hygromycin B and 1 ⁇ g/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.
  • C6 glioma cells were transfected and screened for LOR1 expression as described above.
  • Protein blot analysis Serum free conditioned media (40 ⁇ l) 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).
  • 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.
  • 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.
  • both LOR-1 expressing cells displayed extra protein bands of about 70 kDa (FIGS. 10 a, 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).
  • LOR-1 cDNA has been expressed in MCF-7 cells under the control of a Tetracycline inducible promoter (Shockett and Schatz, 1996).
  • 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. 10 c, d ).
  • 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. 11 b ) and only a few necrotic areas in tumors generated from parental (not shown) or MCF-7 cells transfected with expression vector alone (FIG. 11 a ).
  • 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. 11 c ).
  • 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. 11 d , arrows).
  • 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. 11 e ).
  • the fibrotic and necrotic areas in these tumors appeared choke full with collagen fibers (FIG. 11 f ).
  • the blood vessels within tumors that developed from vector-transfected MCF-7 cells remained almost unstained (FIG. 11 g , arrows)
  • the blood vessels in tumors derived from LOR-1 expressing MCF-7 cells were sheathed by a thick layer of collagen (FIG. 11 h , arrows).
  • VEGF Vascular endothelial growth factor
  • Integrin alpha(v)beta(3) antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157-1164.
  • Neuropilin-1 is expressed by endothelial and tumor cells as an isoform specific receptor for vascular endothelial growth factor. Cell 92, 735-745.
  • RNAi double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.
  • RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188-200.
  • RNA interference RNA interference

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US10/305,348 US20030114410A1 (en) 2000-08-08 2002-11-27 Pharmaceutical compositions and methods useful for modulating angiogenesis and inhibiting metastasis and tumor fibrosis
PCT/IL2003/001008 WO2004047720A2 (fr) 2002-11-27 2003-11-27 Compositions pharmaceutiques et procedes de modulation de l'angiogenese, d'enraiement de la metastase et de la fibrose tumorale et de determination de la malignite des tumeurs cancereuses du colon
EP08020752A EP2062918A3 (fr) 2002-11-27 2003-11-27 Compositions pharmaceutiques et procédés utiles pour moduler l'angiogénèse, inhiber la métastase et les fibroses de tumeur, et évaluer les tumeurs malignes parmi les tumeurs du cancer du colon
AU2003286391A AU2003286391A1 (en) 2002-11-27 2003-11-27 Methods of inhibiting angiogensis via modification of a lysyl oxidase
EP03777136A EP1572100A4 (fr) 2002-11-27 2003-11-27 Compositions pharmaceutiques et procedes de modulation de l'angiogenese, d'enraiement de la metastase et de la fibrose tumorale et de determination de la malignite des tumeurs cancereuses du colon
DK08020753.3T DK2062919T3 (en) 2002-11-27 2003-11-27 Pharmaceutical compositions and methods useful for the modulation of angiogenesis, inhibition of metastasis and tumor fibrosis, and evaluate malignancy of coloncancertumorer
PT08020753T PT2062919E (pt) 2002-11-27 2003-11-27 Composições farmacêuticas e métodos úteis para a modelação de angiogénese, inibição de metástase e fibrose tumoral, e avaliação da malignidade de tumores de cancro do cólon
ES08020753.3T ES2584847T3 (es) 2002-11-27 2003-11-27 Composiciones farmacéuticas y métodos útiles para modular la angiogénesis, inhibir la metástasis y la fibrosis tumoral, y evaluar la malignidad de tumores cancerígenos de colon
US10/536,440 US8163494B2 (en) 2002-11-27 2003-11-27 Method for assessing metastatic properties of breast cancer
EP08020753.3A EP2062919B1 (fr) 2002-11-27 2003-11-27 Compositions pharmaceutiques et procédés utiles pour moduler l'angiogénèse, inhiber la métastase et les fibroses de tumeur, et évaluer les tumeurs malignes parmi les tumeurs du cancer du colon
US12/571,167 US8168180B2 (en) 2002-11-27 2009-09-30 Methods and compositions for modulating angiogenesis
HK09110803.7A HK1131165A1 (zh) 2002-11-27 2009-11-19 幫助調控血管生成、抑制轉移和腫瘤纖維化以及評估結腸癌腫瘤的惡性程度的藥物組合物及方法
US13/412,544 US20120202206A1 (en) 2002-11-27 2012-03-05 Pharmaceutical compositions and methods useful for modulating angiogenesis, inhibiting metastasis and tumor fibrosis, and assessing the malignancy of colon cancer tumors
US13/416,976 US8815823B2 (en) 2002-11-27 2012-03-09 Pharmaceutical compositions and methods useful for modulating angiogenesis, inhibiting metastasis and tumor fibrosis, and assessing the malignancy of colon cancer tumors
US14/311,110 US20140302499A1 (en) 2002-11-27 2014-06-20 Pharmaceutical compositions and methods useful for modulating angiogenesis, inhibiting metastasis and tumor fibrosis, and assessing the malignancy of colon cancer tumors
CY20161100760T CY1117943T1 (el) 2002-11-27 2016-08-03 Φαρμακευτικες συνθεσεις και μεθοδοι χρησιμες για διαμορφωση της αγγειογενεσης, αναστολη μεταστασης και ινωσης νεοπλασματος, και αξιολογηση της κακοηθους καταστασης νεοπλασματων καρκινου παχεος εντερου
US15/881,339 US20190040470A1 (en) 2002-11-27 2018-01-26 Pharmaceutical compositions and methods useful for modulating angiogenesis, inhibiting metastasis and tumor fibrosis, and assessing the malignancy of colon cancer tumors

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