+

WO1999032639A1 - Tyrosine-kinase recepteur, ar-1, regulateur de l'angiogenese - Google Patents

Tyrosine-kinase recepteur, ar-1, regulateur de l'angiogenese Download PDF

Info

Publication number
WO1999032639A1
WO1999032639A1 PCT/US1998/026800 US9826800W WO9932639A1 WO 1999032639 A1 WO1999032639 A1 WO 1999032639A1 US 9826800 W US9826800 W US 9826800W WO 9932639 A1 WO9932639 A1 WO 9932639A1
Authority
WO
WIPO (PCT)
Prior art keywords
human
antibody
sequence
tie
cell
Prior art date
Application number
PCT/US1998/026800
Other languages
English (en)
Inventor
David M. Valenzuela
Original Assignee
Regeneron Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regeneron Pharmaceuticals, Inc. filed Critical Regeneron Pharmaceuticals, Inc.
Priority to IL13683098A priority Critical patent/IL136830A0/xx
Priority to EP98964745A priority patent/EP1038006A1/fr
Priority to AU20003/99A priority patent/AU2000399A/en
Priority to CA002313804A priority patent/CA2313804A1/fr
Publication of WO1999032639A1 publication Critical patent/WO1999032639A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to the field of genetic engineering and more particularly to genes for receptor tyrosine kinases and their cognate ligands, their insertion into recombinant DNA vectors, and the production of the encoded proteins in recipient strains of microorganisms and recipient eukaryotic cells. More specifically, the present invention is directed to a novel human factor which is believed to be a regulator of angiogenesis and is therefore designated
  • the invention further provides nucleic acid sequences encoding human AR-1 , and methods for the production of the nucleic acids and the gene products.
  • the novel AR-1 is believed to be a regulator of angiogenesis and thus the factor, as well as nucleic acids encoding it, may be useful in the diagnosis and treatment of certain diseases such as neoplastic diseases involving tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis and inflammatory diseases.
  • the cellular behavior responsible for the development, maintenance, and repair of differentiated cells and tissues is regulated, in large part, by intercellular signals conveyed via growth factors and similar ligands and their receptors.
  • the receptors are located on the cell surface of responding cells and they bind peptides or polypeptides known as growth factors as well as other hormone-like ligands. The results of this interaction are rapid biochemical changes in the responding cells, as well as a rapid and a long-term readjustment of cellular gene expression.
  • Several receptors associated with various cell surfaces may bind specific growth factors.
  • tyrosine kinases The phosphorylation of tyrosine residues in proteins by tyrosine kinases is one of the key modes by which signals are transduced across the plasma membrane.
  • Several currently known protein tyrosine kinase genes encode transmembrane receptors for polypeptide growth factors and hormones such as epidermal growth factor (EGF), insulin, insulin- like growth factor-l (IGF-I), platelet derived growth factors (PDGF-A and -B), and fibroblast growth factors (FGFs).
  • EGF epidermal growth factor
  • IGF-I insulin- like growth factor-l
  • PDGF-A and -B platelet derived growth factors
  • FGFs fibroblast growth factors
  • growth factors exert their action by binding to the extracellular portion of their cognate receptors, which leads to activation of the intrinsic tyrosine kinase present on the cytoplasmic portion of the receptor.
  • Growth factor receptors of endothelial cells are of particular interest due to the possible involvement of growth factors in several important physiological and pathological processes, such as vasculogenesis, angiogenesis, atherosclerosis, and inflammatory diseases. (Folkman, et al. Science, 235: 442-447 (1987)).
  • the receptors of several hematopoietic growth factors are tyrosine kinases; these include c-fms, which is the colony stimulating factor 1 receptor, Sherr, et al., Cell, 41 : 665-676 (1985), and c-kit, a primitive hematopoietic growth factor receptor reported in Huang, et al., Cell, 63: 225-33 (1990).
  • the receptor tyrosine kinases have been divided into evolutionary subfamilies based on the characteristic structure of their ectodomains. (Ullrich, et al. Cell, 61 : 243-54 (1990)). Such subfamilies include, EGF receptor-like kinase (subclass I) and insulin receptor-like kinase (subclass II), each of which contains repeated homologous cysteine- rich sequences in their extracellular domains. A single cysteine-rich region is also found in the extracellular domains of the eph-like kinases. Hirai, et al., Science, 238: 1717-1720 (1987); Lindberg, et al. Mol. Cell.
  • PDGF receptors as well as c-fms and c-kit receptor tyrosine kinases may be grouped into subclass III; while the FGF receptors form subclass IV. Typical for the members of both of these subclasses are extracellular folding units stabilized by intrachain disulfide bonds. These so-called immunoglobulin (Ig)-like folds are found in the proteins of the immunoglobulin superfamily which contains a wide variety of other cell surface receptors having either cell-bound or soluble ligands. Williams, et al., Ann. Rev. Immunol., 6: 381 -405
  • Receptor tyrosine kinases differ in their specificity and affinity.
  • receptor tyrosine kinases are glycoproteins which consist of (1 ) an extracellular domain capable of binding the specific growth factor(s); (2) a transmembrane domain which usually is an alpha- helical portion of the protein; (3) a juxtamembrane domain where the receptor may be regulated by, e.g., protein phosphorylation; (4) a tyrosine kinase domain which is the enzymatic component of the receptor; and (5) a carboxyterminal tail which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.
  • tie mRNA is present in all human fetal and mouse embryonic tissues. Upon inspection, tie message has been localized to the cardiac and vascular endothelial cells. Specifically, tie mRNA has been localized to the endothelia of blood vessels and endocardium of 9.5 to 18.5 day old mouse embryos. Enhanced tie expression was shown during neovascularization associated with developing ovarian follicles and granulation tissue in skin wounds. Korhonen, et al. Blood 80: 2548-2555 (1992). Thus the TIEs have been suggested to play a role in angiogenesis, which is important for developing treatments for solid tumors and several other angiogenesis- dependent diseases such as diabetic retinopathy, psoriasis, atherosclerosis and arthritis.
  • tie_-1 is the rat homolog of human tie . . Maisonpierre, et al., Oncogene 8: 1631 -1637 (1993).
  • the other gene, tie-2 may be the rat homolog of the murine tek . gene which, like tie, has been reported to be expressed in the mouse exclusively in endothelial cells and their presumptive progenitors. Dumont, et al. Oncogene 8: 1293-1301 (1993).
  • the human homolog of tie-2 is described in Ziegler, U.S. Patent No. 5,447,860 which issued on September 5, 1995 (wherein it is referred to as "ork”), which is incorporated in its entirety herein.
  • TIE vascular endothelia
  • TIE plays a role in the development and maintenance of the vascular system. This could include roles in endothelial cell determination, proliferation, differentiation and cell migration and patterning into vascular elements.
  • Analyses of mouse embryos deficient in TIE-2 illustrate its importance in angiogenesis, particularly for vascular network formation in endothelial cells. Sato, T.N., et al., Nature 376:70-74 (1995). In the mature vascular system, the TIEs could function in endothelial cell survival, maintenance and response to pathogenic influences.
  • the TIE receptors are also expressed in primitive hematopoietic stem cells, B cells and a subset of megakaryocytic cells, thus suggesting the role of ligands which bind these receptors in early hematopoiesis, in the differentiation and/or proliferation of B cells, and in the megakaryocytic differentiation pathway.
  • B cells hematopoietic stem cells
  • megakaryocytic cells hematopoietic stem cells
  • the TIE receptors are also expressed in primitive hematopoietic stem cells, B cells and a subset of megakaryocytic cells, thus suggesting the role of ligands which bind these receptors in early hematopoiesis, in the differentiation and/or proliferation of B cells, and in the megakaryocytic differentiation pathway.
  • TIE-2 ligand-1 TIE-2 ligand-1
  • Ang1 angiopoietin-1
  • Ang2 angiopoietin-1
  • TIE- 2 ligand-2 TIE- 2 ligand-2
  • Ang2 angiopoietin-2
  • the absence of Ang1 causes severe vascular abnormalities in the developing mouse embryo.
  • Ang1 and Ang2 provide for naturally occurring positive and negative regulators of angiogenesis. Positive or negative regulation of TIE2 is likely to result in different outcomes depending on the combination of simultaneously acting angiogenic signals.
  • TIE-2 ligand-1 also referred to as angiopoietin-1 (Ang1 ); TIE-2 ligand-2 (TL2) or angiopoietin-2 (Ang2); Tie ligand-3 (TL3) and Tie ligand-4 (TL4).
  • TIE-2 ligand-1 also referred to as angiopoietin-1 (Ang1 )
  • TIE-2 ligand-2 TIE-2 ligand-2 (TL2) or angiopoietin-2 (Ang2)
  • Tie ligand-3 TL3
  • Tie ligand-4 Tie ligand-4
  • the present invention provides for a composition comprising human AR- 1 substantially free of other proteins.
  • the invention also provides for an isolated nucleic acid molecule encoding human AR-1
  • the isolated nucleic acid may be DNA, cDNA or RNA.
  • the invention also provides for a vector comprising an isolated nucleic acid molecule encoding human AR-1.
  • the invention further provides for a host-vector system for the production in a suitable host cell of a polypeptide having the biological activity of human AR-1 .
  • the suitable host cell may be bacterial, yeast, insect or mammalian.
  • the invention also provides for a method of producing a polypeptide having the biological activity of human AR-1 which comprises growing cells of the host-vector system under conditions permitting production of the polypeptide and recovering the polypeptide so produced.
  • the invention herein described of an isolated nucleic acid molecule encoding human AR-1 further provides for the development of the ligand, a fragment or derivative thereof, or another molecule which is a receptor agonist or antagonist, as a therapeutic for the treatment of patients suffering from disorders involving cells, tissues or organs which express the human AR-1 receptor.
  • the present invention also provides for an antibody which specifically binds such a therapeutic molecule.
  • the antibody may be monoclonal or polyclonal.
  • the invention also provides for a method of using such a monoclonal or polyclonal antibody to measure the amount of the therapeutic molecule in a sample taken from a patient for purposes of monitoring the course of therapy.
  • the present invention also provides for an antibody which specifically binds human AR-1.
  • the antibody may be monoclonal or polyclonal.
  • compositions comprising an antibody which specifically binds human AR-1 and a vehicle.
  • the invention further provides for compositions comprising human AR- 1 in a vehicle.
  • the invention also provides for a method of regulating angiogenesis in a patient by administering an effective amount of a composition comprising human AR-1 in a vehicle.
  • human AR-1 may be conjugated to a cytotoxic agent and a composition prepared therefrom.
  • Biologically active AR-1 may be used to promote the growth, survival, migration, and/or differentiation and/or stabilization or destabilization of cells expressing its receptor.
  • Biologically active AR-1 may be used for the in vitro maintenance of AR-1 receptor expressing cells in culture.
  • AR-1 may be used to support cells which are engineered to express its receptor.
  • the AR-1 and its receptor may be used in assay systems to identify agonists or antagonists of the receptor.
  • FIGURE 1 A-1 B Nucleotide and deduced amino acid (single letter code) sequences of TIE ligand-3. The coding sequence starts at position 47. The fibrinogen-like domain starts at position 929.
  • FIGURE 2 Comparison of Amino Acid Sequences of TIE Ligand Family Members.
  • the underlined regions indicate conserved regions of homology among the family members.
  • FIGURE 3A-3C Nucleotide and deduced amino acid (single letter code) sequences of TIE ligand-4. Arrow indicates nucleotide position 569.
  • FIGURE 4A-4B Nucleotide and deduced amino acid (triple letter code) sequences of Human AR-1.
  • TIE-2 ligand 1 As described in greater detail below, applicants have isolated and identified a novel factor related to the TIE-2 ligands that bind the TIE- 2 receptor.
  • the novel factor is referred to herein as human AR-1 .
  • the TIE ligand family members are referred to herein as TIE-2 ligand 1
  • TIE-1 also known as angiopoietin-1 (Ang1 ); TIE-2 ligand 2 (TL2) also known as angiopoietin-2 (Ang2); TIE ligand-3; and TIE ligand-4.
  • Ang1 angiopoietin-1
  • TIE-2 ligand 2 also known as angiopoietin-2 (Ang2)
  • TIE ligand-3 also known as angiopoietin-2
  • TIE ligand-4 TIE ligand-4.
  • the novel factor is expected to have a function and utility similar to that of the known TIE-2 ligands.
  • the present invention comprises novel nucleic acids and their deduced amino acid sequences, as well as functionally equivalent variants thereof comprising naturally occurring allelic variations, as well as proteins or peptides comprising substitutions, deletions or insertional mutants of the described sequences, which retain biological activity.
  • variants include those in which amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid(s) of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the class of nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • proteins or fragments or derivatives thereof which exhibit the same or similar biological activity as the human AR-1 described herein, and derivatives which are differentially modified during or after translation, e.g.. by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand.
  • Functionally equivalent molecules also include molecules that contain modifications, including N-terminal modifications, which result from expression in a particular recombinant host, such as, for example, N-terminal methylation which occurs in certain bacterial (e.g. E . coli) expression systems.
  • Functional equivalents also include mutants in which amino acid substitutions are made for cysteine molecules to improve stability of the molecules and to prevent unwanted crosslinking.
  • the present invention also encompasses the nucleotide sequence that encodes the protein described herein as human AR-1 , as well as host cells, including yeast, bacteria, viruses, and mammalian cells, which are genetically engineered to produce the protein, by e.g. transfection, transduction, infection, electroporation, or microinjection of nucleic acid encoding the human AR-1 described herein in a suitable expression vector.
  • the present invention also encompasses introduction of the nucleic acid encoding human AR-1 through gene therapy techniques such as is described, for example, in Finkel and Epstein FASEB J. 9:843-851 (1995); Guzman, et al. PNAS (USA) 91 :10732-10736 (1994).
  • the present invention encompasses DNA and RNA sequences that hybridize to a human AR-1 encoding sequence, under stringent conditions.
  • the invention also provides for nucleic acid hybridization probes and replication/amplification primers having a human AR-1 DNA specific sequence and sufficient to effect specific hybridization with human
  • AR-1 Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0.18 M NaCI, 0.01 M NaP0 4 , pH7.7, 0.001 M EDTA) buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2 x SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5 x SSPE buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2x SSPE buffer at 42°C.
  • Human AR-1 homologs can also be distinguished from other polypeptides using alignment algorithms, such as BLASTX (Altschul, et al. (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215: 403-410).
  • a nucleic acid molecule contemplated by the invention includes one having a sequence deduced from an amino acid sequence of a human AR-1 prepared as described herein, as well as a molecule having a sequence of nucleic acids that hybridizes to such a nucleic acid sequence, and also a nucleic acid sequence which is degenerate of the above sequences as a result of the genetic code, but which encodes a human AR-1 and which has an amino acid sequence and other primary, secondary and tertiary characteristics that are sufficiently duplicative of the human AR-1 described herein so as to confer on the molecule the same biological activity as the human AR-1 described herein.
  • the present invention encompasses an isolated and purified nucleic acid molecule comprising a nucleotide sequence encoding a human AR-1 , wherein the nucleotide sequence is selected from the group consisting of: (a) the nucleotide sequence comprising the coding region of human AR-1 as set forth in Figure 4A-4B;
  • the present invention further provides for an isolated human AR-1 encoded by an isolated nucleic acid molecule of the invention.
  • the invention also provides for a vector which comprises an isolated nucleic acid molecule comprising a nucleic acid sequence encoding human AR-1.
  • any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding human AR-1 using appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinations (genetic recombination). Expression of a nucleic acid sequence encoding human AR-1 or peptide fragments thereof may be regulated by a second nucleic acid sequence which is operably linked to the human AR-1 encoding sequence such that the human AR-1 protein or peptide is expressed in a host transformed with the recombinant DNA molecule.
  • expression of human AR-1 described herein may be controlled by any promoter/enhancer element known in the art.
  • Promoters which may be used to control expression of the ligand include, but are not limited to the long terminal repeat as described in Squinto et al., (Cell 65: 1 -20 (1991 )); the SV40 early promoter region (Bernoist and Chambon, Nature
  • the adenovirus promoter the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731 (1978)), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A.
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals; elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 38: 639- 646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58): alpha 1 -antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 31 5:338-340: Kollias et al., 1986, Cell 46_:89-94); myelin basic protein gene control region which is active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48 . :703-712); myosin light chain- 2 gene control region which is active in skeletal muscle (Shani, 1985, Nature 314:283-286). and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
  • the invention further encompasses the production of antisense compounds which are capable of specifically hybridizing with a sequence of RNA encoding TIE ligand-3 or TIE ligand-4 to modulate its expression.
  • Ecker U.S. Patent No. 5,166,195, issued November 24, 1992.
  • expression vectors capable of being replicated in a bacterial or eukaryotic host comprising a nucleic acid encoding human AR-1 as described herein, are used to transfect a host and thereby direct expression of such nucleic acid to produce human AR-1 , which may then be recovered in a biologically active form.
  • a biologically active form includes a form capable of causing a biological response such as a differentiated function or influencing the phenotype of a cell expressing the receptor for human
  • Expression vectors containing the gene inserts can be identified by four general approaches: (a) DNA-DNA hybridization, (b) presence or absence of "marker” gene functions, (c) expression of inserted sequences and (d)
  • the presence of a foreign gene inserted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to an inserted human AR-1 encoding gene.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector.
  • recombinants containing the insert can be identified by the absence of the marker gene function.
  • recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of a human AR-1 gene product, for example, by binding of the human AR-1 to its receptor or a portion thereof which may be tagged with, for example, a detectable antibody or portion thereof or by binding to antibodies produced against the human AR-1 protein or a portion thereof.
  • Cells of the present invention may transiently or, preferably, constitutively and permanently express human AR-1 as described herein.
  • DNA nucleotide primers can be prepared corresponding to a human AR-1 specific DNA sequence. These primers could then be used to PCR a human AR-1 gene fragment. (PCR Protocols: A Guide To Methods and Applications, Edited by Michael A. Innis et al., Academic Press (1990)).
  • the recombinant human AR-1 may be purified by any technique which allows for the subsequent formation of a stable, biologically active protein.
  • the ligand is secreted into the culture medium from which it is recovered.
  • the human AR-1 may be recovered from cells either as soluble proteins or as inclusion bodies, from which it may be extracted quantitatively by 8M guanidinium hydrochloride and dialysis in accordance with well known methodology.
  • affinity chromatography conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration may be used.
  • a recombinant AR-1 encoding gene may be used to inactivate or "knock out" the endogenous gene by homologous recombination, and thereby create an AR-1 deficient cell, tissue, or animal.
  • the recombinant AR-1 encoding gene may be engineered to contain an insertional mutation, for example the neo gene, which would inactivate the native AR-1 encoding gene.
  • Such a construct under the control of a suitable promoter, may be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, transduction, or injection. Cells containing the construct may then be selected by G418 resistance.
  • Cells which lack an intact AR-1 gene may then be identified, e.g. by Southern blotting, PCR detection, Northern blotting or assay of expression. Cells lacking an intact AR-1 encoding gene may then be fused to early embryo cells to generate transgenic animals deficient in such ligand. Such an animal may be used to define specific in vivo processes, normally dependent upon the factor.
  • the present invention also provides for antibodies to human AR-1 described herein which are useful for detection of the factor in, for example, diagnostic applications.
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497). as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp. 77- 96) and the like are within the scope of the present invention.
  • the monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g.. Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16).
  • Chimeric antibody molecules may be prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81 :6851 , Takeda et al., 1985, Nature 314:452).
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum a molecular clone of an antibody to a selected human AR-1 epitope can be prepared by known techniques. Recombinant DNA methodology (see e.g..
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • Antibody molecules may be purified by known techniques, e.g., immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.
  • the present invention further encompasses an immunoassay for measuring the amount of human AR-1 in a biological sample by a) contacting the biological sample with at least one antibody which specifically binds human AR-1 so that the antibody forms a complex with any human AR-1 present in the sample; and b) measuring the amount of the complex and thereby measuring the amount of the human AR-1 in the biological sample.
  • the present invention also provides for the utilization of human AR-1 to support the survival and/or growth and/or migration and/or differentiation of human AR-1 receptor expressing cells.
  • the invention further provides for both a method of identifying antibodies or other molecules capable of neutralizing the factor or blocking the binding ability of the factor, as well as the molecules identified by the method.
  • the method may be performed via an assay which is conceptually similar to an
  • human AR-1 antibody may be bound to a solid support, such as a plastic multiwell plate.
  • a known amount of human AR-1 which has been Myc-tagged may then be introduced to the well and any tagged human AR-1 which binds the antibody may then be identified by means of a reporter antibody directed against the Myc-tag.
  • This assay system may then be used to screen test samples for molecules which are capable of i) binding to the tagged factor or ii) binding to the antibody and thereby blocking binding to the antibody by the tagged factor.
  • a test sample containing a putative molecule of interest together with a known amount of tagged factor may be introduced to the well and the amount of tagged factor which binds to the antibody may be measured. By comparing the amount of bound tagged factor in the test sample to the amount in the control, samples containing molecules which are capable of blocking factor binding to the antibody may be identified.
  • the molecules of interest thus identified may be isolated using methods well known to one of skill in the art.
  • a blocker of factor binding is found, one of skill in the art would know to perform secondary assays to determine whether the blocker is binding to the antibody or to the factor, as well as assays to determine if the blocker molecule can neutralize the biological activity of the facor. For example, by using a binding assay which employs BIAcore biosensor technology (or the equivalent), in which either human AR-1 antibody or human AR-1 is covalently attached to a solid support (e.g. carboxymethyl dextran on a gold surface), one of skill in the art would be able to determine if the blocker molecule is binding specifically to the factor or to the antibody.
  • BIAcore biosensor technology or the equivalent
  • factorbodies which comprise the AR-1 coupled to the Fc domain of IgG ("fFc's"). These factorbodies may be used as targeting agents, in diagnostics or in therapeutic applications, such as targeting agents for tumors and/or associated vasculature as indicated.
  • the invention herein further provides for the development of the AR-1 , a fragment or derivative thereof as a therapeutic for the treatment of patients suffering from disorders involving cells, tissues or organs which express the AR-1 receptor.
  • Such molecules may be used in a method of treatment of the human or animal body, or in a method of diagnosis.
  • AR-1 has been identified as related to the TIE family of ligands, applicants expect that the AR-1 may be useful for the induction or prevention of vascularization in diseases or disorders where such function is indicated.
  • diseases or disorders would include wound healing, ischaemia and diabetes or for preventing or attenuating, for example, tumor growth.
  • the AR-1 may be tested in animal models and used therapeutically as described for other agents, such as vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • AR-1 may be used alone or in combination with one or more additional pharmaceutically active compounds such as, for example, VEGF or basic fibroblast growth factor (bFGF), as well as cytokines, neurotrophins, etc.
  • additional pharmaceutically active compounds such as, for example, VEGF or basic fibroblast growth factor (bFGF), as well as cytokines, neurotrophins, etc.
  • Antagonists of the AR-1 such as antibodies or receptorbodies, would be useful to prevent or attenuate its biological activity. These agents may be used alone or in combination with other compositions.
  • AR-1 may be useful for the delivery of toxins to a receptor bearing cell.
  • AR-1 receptor is associated with a disease state
  • AR-1 may be useful as diagnostic reagents for detecting the disease by, for example, tissue staining or whole body imaging.
  • diagnostic reagents include radioisotopes, flurochromes, dyes, enzymes and biotin.
  • diagnostics or targeting agents may be prepared as described in Alitalo, et al. WO 95/26364 published October 5, 1995 and Burrows, F. and P. Thorpe, PNAS (USA) 90:8996-9000 (1993) which is hereby incorporated by reference in its entirety.
  • the human AR-1 of the present invention may be used alone, or in combination with another pharmaceutically active agent such as, for example, ctyokines, neurotrophins, interleukins, etc.
  • the human AR-1 may be used in conjunction with any of a number of the above referenced factors which are known to induce stem cell or other hematopoietic precursor proliferation, or factors acting on later cells in the hematopoietic pathway, including, but not limited to, hemopoietic maturation factor, thrombopoietin, stem cell factor, erythropoietin, G-CSF, GM-CSF, etc.
  • the present invention also provides for compositions comprising the human AR-1 described herein, peptide fragments thereof, or derivatives in a vehicle.
  • the human AR-1 , peptide fragments, or derivatives may be administered systemically or locally. Any appropriate mode of administration known in the art may be used, including, but not limited to, intravenous, intrathecal, intraarterial, intranasal, oral, subcutaneous, intraperitoneal, or by local injection or surgical implant. Sustained release formulations are also provided for.
  • the present invention also provides for an antibody which specifically binds such a therapeutic molecule.
  • the antibody may be monoclonal or polyclonal.
  • the invention also provides for a method of using such a monoclonal or polyclonal antibody to measure the amount of the therapeutic molecule in a sample taken from a patient for purposes of monitoring the course of therapy.
  • the invention further provides for a composition comprising a human AR-1 and a cytotoxic agent conjugated thereto.
  • the cytotoxic agent may be a radioisotope or toxin.
  • the invention also provides for an antibody which specifically binds a human AR-1 .
  • the antibody may be monoclonal or polyclonal.
  • the invention further provides for a method of purifying human AR-1 comprising: a) coupling at least one human AR-1 binding substrate to a solid matrix; b) incubating the substrate of a) with a cell lysate so that the substrate forms a complex with any human AR-1 in the cell lysate ; c) washing the solid matrix; and d) eluting the human AR-1 from the coupled substrate.
  • the substrate may be any substance that specifically binds the human AR-1 .
  • the substrate is selected from the group consisting of anti-human AR-1 antibody, human AR-1 receptor and human AR-1 receptorbody.
  • the invention also provides for a composition comprising human AR-1 in a vehicle, as well as a method of regulating angiogenesis in a patient comprising administering to the patient an effective amount of the therapeutic composition.
  • the present invention provides for a method for identifying a cell which expresses human AR-1 receptor which comprises contacting a cell with a detectably labeled human AR-1 or factorbody, under conditions permitting binding of the detectably labeled factor to the human AR-1 receptor and determining whether the detectably labeled factor is bound to the human AR-1 receptor, thereby identifying the cell as one which expresses human AR-1 receptor.
  • the present invention also provides for a composition comprising a human AR-1 and a cytotoxic agent conjugated thereto.
  • the cytotoxic agent may be a radioisotope or toxin.
  • the invention also provides a method of detecting expression of human AR-1 by a cell which comprises obtaining mRNA from the cell, contacting the mRNA so obtained with a labeled nucleic acid molecule encoding human AR-1 , under hybridizing conditions, determining the presence of mRNA hybridized to the labeled molecule, and thereby detecting the expression of the human AR-1 in the cell.
  • the invention further provides a method of detecting expression of human AR-1 in tissue sections which comprises contacting the tissue sections with a labeled nucleic acid molecule encoding a human AR-1 , under hybridizing conditions, determining the presence of mRNA hybridized to the labelled molecule, and thereby detecting the expression of human AR-1 in tissue sections.
  • TIE ligand-3 (TL3) was cloned from a mouse BAC genomic library (Research Genetics) by hybridizing library duplicates, with either mouse TL1 or mouse TL2 probes corresponding to the entire coding sequence of those genes. Each copy of the library was hybridized using phosphate buffer at 55°C overnight. After hybridization, the filters were washed using 2xSSC, 0.1 % SDS at 60°C, followed by exposure of X ray film to the filters. Strong hybridization signals were identified corresponding to mouse TL1 and mouse TL2. In addition, signals were identified which weakly hybridized to both mouse TL1 and mouse TL2.
  • DNA corresponding to these clones was purified, then digested with restriction enzymes, and two fragments which hybridized to the original probes were subcloned into a bacterial plasmid and sequenced.
  • the sequence of the fragments contained two exons with homology to both mouse TL1 and mouse TL2.
  • Primers specific for these sequences were used as PCR primers to identify tissues containing transcripts corresponding to TL3.
  • a PCR band corresponding to TL3 was identified in a mouse uterus cDNA library in lambda gt-11 . (Clontech Laboratories, Inc., Palo Alto, CA).
  • Plaques were plated at a density of 1 .25 x 10 6 /20x20 cm plate and replica filters taken following standard procedures (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • Duplicate filters were screened at "normal" stringency (2 x SSC, 65°C) with a 200 bp PCR radioactive probe made to the mouse TL3 sequence.
  • Hybridization was at 65°C in a solution containing 0.5 mg/ml salmon sperm DNA. Filters were washed in 2 x SSC at 65°C and exposed for 6 hours to X- ray film. Two positive clones that hybridized in duplicate were picked.
  • PCR reactions were performed using expression libraries derived from the mouse cell lines C2C12ras and MG87.
  • the specific primer US2 was used in conjunction with vector-specific oligos to allow amplification in either orientation.
  • PCR was in a total volume of 100ml using 35 cycles of 94 ° C, 1 min; 42 ° C or 48 ° C for 1 min; 72 ° C, 1 min.
  • the secondary PCR reaction included the second specific primer, US1 , which is contained within the primary PCR product, in conjunction with the same vector oligos. The secondary reactions were for 30 cycles, using the same temperatures and times as previous. PCR products were gel isolated and submitted for sequence analysis.
  • the mouse TL3 sequence may then be used to obtain a human clone containing the coding sequence of its human counterpart by hybridizing either a human genomic or cDNA library with a probe corresponding to mouse TL3 as has been described previously, for example, in Example 8 in International Publication No. WO 96/31598 published 10 October 1 996.
  • TIE ligand-4 was cloned from a human BAC genomic library (BAC HUMAN (II), Genome Systems Inc.) by hybridizing library duplicates, with either a human TL1 radioactive probe corresponding to the entire fibrinogen coding sequence of TL1 (nucleotides 1 153 to 1806) or a mouse TL3 radioactive probe corresponding to a segment of 186 nucleotides from the fibrinogen region of mouse TL3 (nucleotides 1307 to 1492 of Figure 1 A-1 B). Each probe was labeled by PCR using exact oligonucleotides and standard PCR conditions, except that dCTP was replaced by P 32 dCTP.
  • PCR mixture was then passed through a gel filtration column to separate the probe from free P 32 dCTP.
  • Each copy of the library was hybridized using phosphate buffer, and radiactive probe at 55°C overnight using standard hybridization conditions. After hybridization, the filters were washed using 2xSSC, 0.1 % SDS at 55°C, followed by exposure of X ray film. Strong hybridization signals were observed corresponding to human TL1 . In addition, signals were identified which weakly hybridized to both human TL1 and mouse TL3. DNA corresponding to these clones was purified using standard procedures, then digested with restriction enzymes, and one fragment which hybridized to the original probes was subcloned into a bacterial plasmid and sequenced.
  • the sequence of the fragments contained one exon with homology to both human TL1 and mouse TL3 and other members of the TIE ligand family. Primers specific for these sequences may be used as PCR primers to identify tissues containing transcripts corresponding to TL4.
  • the complete sequence of human TL4 may be obtained by sequencing the full BAC clone contained in the deposited bacterial cells. Exons may be identified by homology to known members of the TIE-ligand family such as TL1 , TL2 and TL3. The full coding sequence of TL4 may then be determined by splicing together the exons from the TL4 genomic clone which, in turn, may be used to produce the TL4 protein. Alternatively, the exons may be used as probes to obtain a full length cDNA clone, which may then be used to produce the TL4 protein.
  • Exons may also be identified from the BAC clone sequence by homology to protein domains such as fibrinogen domains, coiled coil domains, or protein signals such as signal peptide sequences. Missing exons from the BAC clone may be obtained by identification of contiguous BAC clones, for example, by using the ends of the deposited BAC clone as probes to screen a human genomic library such as the one used herein, by using the exon sequence contained in the BAC clone to screen a cDNA library, or by performing either 5' or 3' RACE procedure using oligonucleotide primers based on the TL4 exon sequences.
  • novel TIE ligand-4 sequence may be used in a rational search for additional members of the TIE ligand family using an approach that takes advantage of the existence of conserved segments of strong homology between the known family members. For example, an alignment of the amino acid sequences of the TIE ligands shows several regions of conserved sequence (see underlined regions of Figure 2). Degenerate oligonucleotides essentially based on these boxes in combination with either previously known or novel TIE ligand homology segments may be used to identify new TIE ligands.
  • TL1 , TL2 and TL3 may be used in designing degenerate oligonucleotide primers with which to prime PCR reactions using cDNAs.
  • cDNA templates may be generated by reverse transcription of tissue RNAs using oligo d(T) or other appropriate primers. Aliquots of the PCR reactions may then be subjected to electrophoresis on an agarose gel. Resulting amplified DNA fragments may be cloned by insertion into plasmids, sequenced and the DNA sequences compared with those of all known TIE ligands.
  • Size-selected amplified DNA fragments from these PCR reactions may be cloned into plasmids, introduced into E. coli by electroporation, and transformants plated on selective agar. Bacterial colonies from PCR transformation may be analyzed by sequencing of plasmid DNAs that are purified by standard plasmid procedures.
  • Cloned fragments containing a segment of a novel TIE ligand may be used as hybridization probes to obtain full length cDNA clones from a cDNA library.
  • the human TL4 genomic sequence may be used to obtain a human cDNA clone containing the complete coding sequence of human TL4 by hybridizing a human cDNA library with a probe corresponding to human TL4 as has been described previously.
  • Both 5' and 3' coding sequence from the genomic human TL-4 clone encoding human TIE ligand-4 was obtained by restriction enzyme digestion, Southern blotting and hybridization of the hTL-4 clone to coding sequences from mouse TL3, followed by subcloning and sequencing the hybridizing fragments. Coding sequences corresponding to the N-terminal and C-terminal amino acids of hTL4 were used to design PCR primers (shown below), which in turn were used for PCR amplification of TL4 from human ovary cDNA.
  • a PCR band was identified as corresponding to human TL4 by DNA sequencing using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). The PCR band was then subcloned into vector pCR-script and several plasmid clones were analyzed by sequencing. The complete human TL4 coding sequence was then compiled and is shown in Figure 3A-3C.
  • the nucleotide at position 569 is changed from A to G, resulting in an amino acid change from Q to R.
  • PCR primers used as described above were designed as follows: hTL4atg 5'-gcatgctatctcgagccaccATGCTCTCCCAGCTAGCCATGCTGCAG-3'
  • Human AR-1 was cloned from a human BAC genomic library (Human Genome Sciences, Inc., Cat. No. FBAC 4435, Release II) by hybridizing library duplicates with a mouse TL3 probe.
  • the probe was labeled by PCR using exact oligonucleotides and standard PCR conditions, except that dCTP was replaced by P 32 dCTP.
  • the PCR mixture was then passed through a gel filtration column to separate the probe from free P 32 dCTP.
  • the library was hybridized using phosphate buffer at 55°C overnight. After hybridization, the filters were washed using 2xSSC, 0.1 % SDS at 60°C, followed by exposure of X-ray film to the filters. Strong hybridization signals were identified corresponding to mouse TL3.
  • Genomic DNA corresponding to these clones was purified, digested with restriction enzymes, and subcloned into a bacterial plasmid and sequenced.
  • the sequence of the fragment contained a portion of the 3' end of the human AR-1 gene.
  • the 3' sequence was used to design oligonucleotide primers for use in a standard RACE procedure.
  • the RACE product was sequenced and found to contain the upstream sequence of the gene including the 5' untranslated sequence.
  • the 5' sequence was used to design oligonucleotide primers that were used in conjunction with Adaptor primer 1 and Adaptor primer 2 (Clontech Laboratories, Inc., Catalog # 7413-1 ) to amplify Human Skeletal Muscle Marathon-ReadyTM cDNA (Clontech Laboratories, Inc., Catalog # 7413- 1 ).
  • the amplification product was cloned into the pMT21 vector and sequenced.
  • the nucleotide and deduced amino acid sequence of human AR-1 is set forth in Figure 4A-4B.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Vascular Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Cette invention se rapporte à une molécule d'acide nucléique isolée codant le régulateur d'angiogenèse AR-1 humain. Cette invention se rapporte également à une protéine d'AR-1 isolée, pour ainsi dire exempte d'autres protéines, à un système hôte-vecteur servant à la production de l'AR-1 humain et à un procédé de production de l'AR-1 humain, ainsi qu'à un anticorps qui se fixe spécifiquement à l'AR-1 humain et à une composition contenant l'AR-1 humain.
PCT/US1998/026800 1997-12-19 1998-12-17 Tyrosine-kinase recepteur, ar-1, regulateur de l'angiogenese WO1999032639A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
IL13683098A IL136830A0 (en) 1997-12-19 1998-12-17 Receptor tyrosine kinase, ar-1
EP98964745A EP1038006A1 (fr) 1997-12-19 1998-12-17 Tyrosine-kinase recepteur, ar-1, regulateur de l'angiogenese
AU20003/99A AU2000399A (en) 1997-12-19 1998-12-17 Receptor tyrosine kinase, ar-1, is a regulator of angiogenesis
CA002313804A CA2313804A1 (fr) 1997-12-19 1998-12-17 Tyrosine-kinase recepteur, ar-1, regulateur de l'angiogenese

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US99430997A 1997-12-19 1997-12-19
US08/994,309 1997-12-19

Publications (1)

Publication Number Publication Date
WO1999032639A1 true WO1999032639A1 (fr) 1999-07-01

Family

ID=25540529

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/026800 WO1999032639A1 (fr) 1997-12-19 1998-12-17 Tyrosine-kinase recepteur, ar-1, regulateur de l'angiogenese

Country Status (5)

Country Link
EP (1) EP1038006A1 (fr)
AU (1) AU2000399A (fr)
CA (1) CA2313804A1 (fr)
IL (1) IL136830A0 (fr)
WO (1) WO1999032639A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029085A3 (fr) * 1999-10-21 2002-01-31 Royal Netherlands Academy Of A Cdt6 capable d'inhiber l'angiogenese et la croissance tumorale et d'induire ou d'augmenter la formation de structures contenant du collagene v
WO2011051276A1 (fr) * 2009-10-26 2011-05-05 Externautics S.P.A. Marqueurs de tumeurs du rectum et du côlon, et leurs méthodes d'utilisation
US8921058B2 (en) 2009-10-26 2014-12-30 Externautics Spa Prostate tumor markers and methods of use thereof
US10288617B2 (en) 2009-10-26 2019-05-14 Externautics Spa Ovary tumor markers and methods of use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011269A2 (fr) * 1994-10-07 1996-04-18 Regeneron Pharmaceuticals, Inc. Ligands tie-2, peocedes de production et utilisations de ces ligands
WO1999015653A2 (fr) * 1997-09-19 1999-04-01 Genentech, Inc. Homologues du ligand 'tie'

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011269A2 (fr) * 1994-10-07 1996-04-18 Regeneron Pharmaceuticals, Inc. Ligands tie-2, peocedes de production et utilisations de ces ligands
WO1999015653A2 (fr) * 1997-09-19 1999-04-01 Genentech, Inc. Homologues du ligand 'tie'

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PEEK R. ET AL.: "Molecular cloning of a new angiopoietinlike factor from the human cornea.", INVESTIGATIVE OPHTALMOLOGY AND VISUAL SCIENCE, vol. 39, no. 10, September 1998 (1998-09-01), pages 1782 - 1788, XP002103341 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029085A3 (fr) * 1999-10-21 2002-01-31 Royal Netherlands Academy Of A Cdt6 capable d'inhiber l'angiogenese et la croissance tumorale et d'induire ou d'augmenter la formation de structures contenant du collagene v
WO2011051276A1 (fr) * 2009-10-26 2011-05-05 Externautics S.P.A. Marqueurs de tumeurs du rectum et du côlon, et leurs méthodes d'utilisation
US8921058B2 (en) 2009-10-26 2014-12-30 Externautics Spa Prostate tumor markers and methods of use thereof
US10288617B2 (en) 2009-10-26 2019-05-14 Externautics Spa Ovary tumor markers and methods of use thereof

Also Published As

Publication number Publication date
AU2000399A (en) 1999-07-12
CA2313804A1 (fr) 1999-07-01
IL136830A0 (en) 2001-06-14
EP1038006A1 (fr) 2000-09-27

Similar Documents

Publication Publication Date Title
US6433143B1 (en) Tie-2 ligands
US5650490A (en) Tie-2 ligand 2
US6627415B1 (en) TIE-2 ligands, methods of making and uses thereof
US5879672A (en) Tie-2 ligand 1
US6441137B1 (en) Expressed ligand-vascular intercellular signalling molecule
WO1996011269A9 (fr) Ligands tie-2, peocedes de production et utilisations de ces ligands
EP0939809B1 (fr) Ligand de recepteurs de tie-2 (ligand-4 tie) et ses utilisation
US5851797A (en) Tie ligand-3, methods of making and uses thereof
US7063840B2 (en) TIE-2 ligands, methods of making and uses thereof
US20030166858A1 (en) TIE-2 ligands, methods of making and uses thereof
WO1999032639A1 (fr) Tyrosine-kinase recepteur, ar-1, regulateur de l'angiogenese
WO2000052167A9 (fr) Proteine associee a l'angiopoietine et acides nucleiques correspondants
US20050100906A1 (en) TIE-2 ligands, methods of making and uses thereof
US6846914B2 (en) Tie-2 ligand-3
AU741134B2 (en) Tie-2 receptor ligands (tie ligand-3; tie ligand-4) and their uses

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 136830

Country of ref document: IL

AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWE Wipo information: entry into national phase

Ref document number: 20003/99

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2313804

Country of ref document: CA

Ref country code: CA

Ref document number: 2313804

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1998964745

Country of ref document: EP

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 525557

Kind code of ref document: A

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: KR

WWP Wipo information: published in national office

Ref document number: 1998964745

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1998964745

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载