WO2000005369A2 - Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese - Google Patents

Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese Download PDF

Info

Publication number
WO2000005369A2
WO2000005369A2 PCT/US1999/012839 US9912839W WO0005369A2 WO 2000005369 A2 WO2000005369 A2 WO 2000005369A2 US 9912839 W US9912839 W US 9912839W WO 0005369 A2 WO0005369 A2 WO 0005369A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleotide
polynucleotide
protein
amino acid
sequence
Prior art date
Application number
PCT/US1999/012839
Other languages
English (en)
Other versions
WO2000005369A3 (fr
Inventor
Richard A. Shimkets
Original Assignee
Curagen Corporation
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 Curagen Corporation filed Critical Curagen Corporation
Priority to AU50818/99A priority Critical patent/AU5081899A/en
Priority to JP2000561315A priority patent/JP2002522019A/ja
Priority to CA002335326A priority patent/CA2335326A1/fr
Priority to EP99935312A priority patent/EP1098974A2/fr
Publication of WO2000005369A2 publication Critical patent/WO2000005369A2/fr
Publication of WO2000005369A3 publication Critical patent/WO2000005369A3/fr

Links

Classifications

    • 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
    • C07K14/50—Fibroblast growth factors [FGF]
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • 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

Definitions

  • the present invention provides novel amino acid sequences of potent angiogenesis/anti- angiogenesis secreted proteins and polynucleotide sequences encoding such proteins, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins, and derivatives, fragments and analogs thereof.
  • angiogenesis i.e., generation of new blood vessels into a tissue or organ, only in restricted situations.
  • endothelial cells react to stimulation with finely tuned signaling responses.
  • the "endothelium” is a thin layer of flat epithelial cells that lines serous cavities, lymph vessels, and blood vessels.
  • neovascularization is controlled by a balance of stimulatory and inhibitory angiogenic factors.
  • ischemic heart disease ischemic peripheral vascular disease
  • tumor growth and metastasis reproduction, embryogenesis, wound healing, bone repair, rheumatoid arthritis, diabetic retinopathy and other diseases (for review, see e.g. Battegay, 1995).
  • Endothelial cells and pericytes surrounded by a basement membrane, form capillary blood vessels.
  • Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes.
  • the endothelial cells which line the lumen of blood vessels, then protrude through the basement membrane.
  • Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane.
  • the migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate.
  • the endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
  • Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells and supports the pathological damage seen in these conditions.
  • the diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases.
  • angiogenesis regulators control the fate of vascular wall cells. They remain either in a state of vascular homeostasis, or they proceed to neovascularization, e.g., tumor growth and the switch to an angiogenic tumor pheno type correlates with increased secretion of angiogenic molecules such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and others.
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • tumors also acquire a more angiogenic phenotype because inhibitors of angiogenesis are down-regulated during turnorigenesis (e.g. thrombospondin)(Dameron et al., 1994, Science 265:1582-1584).
  • Angiogenic and antiangiogenic (or angiostatic) molecules control the formation of new vessels via different mechanisms.
  • Antiangiogenic molecules, or angiogenesis inhibitors e.g. angiostatin, angiopoeitin-1 (Angl 1), rat microvascular endothelial differentiation gene (MEDG), somatostatin, thrombospondin. platelet factor 4
  • angiogenesis inhibitors can repress angiogenesis, and therefore, maintain vascular homeostasis (see, e.g. for review Bicknell, 1994, Ann. Oncol. 5 (suppl) 4:45- 50).
  • Angiogenic molecules are capable of inducing the formation of new vessels and include, for example, but not for limitation, fibroblast growth factor (FGF), angiopoeitin 2 (Ang-2), erythroipoietin, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and others (for review, see e.g. Folkman & Shing, 1992, J. Biol. Chem. 267: 10931-10934). FGF elicit its effects mainly via direct action on relevant endothelial cells via its endothelial receptor (e.g. Folkman & Shing, 1992. J. Biol. Chem. 267:10931-10934). FGF lacks a signal sequence for secretion.
  • FGF fibroblast growth factor
  • Ang-2 angiopoeitin 2
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • FGF elicit its effects mainly via direct action on
  • Angiogenesis has been implicated in ischemic heart and ischemic vascular disease. In myocardial infarction new vessels penetrate the necrotic area and the surrounding schemic tissue.
  • Neovascularizations together with inflammatory cells, remove cellular debris and play a role in tissue repair and remodeling that results in myocardial scar formation.
  • FGF-induced mycoardial infarction and neovascularization show that angiogenesis contributes to the preservation of ischemic tissue and myocardial pump function in myocardial necrosis. This . suggests a therapeutic use of angiogenic factors in clinical situations.
  • angiogenesis has been implicated in various cancers.
  • Angiogenesis is an essential component of the metastatic pathway (see, e.g. Zetter, 1998, Ann. Rev. Med. 49:407- 427). These blood vessels provide the principal pathway by which rumor cells exit the primary tumor site and enter the circulation.
  • Tumor angiogenesis is regulated by the production of angiogenic stimulators including members of the FGF and VEGF families (see, e.g. Fernig & Gallaher, 1994, Prog. Growth Factor Res. 5:353-377). Tumors may also activate angiogenic inhibitors such as angiostatin (U.S.
  • Angiopoietin-1 is an angiogenic factor that signals through the endothelial cell- specific Tie2 receptor tyrosine kinase.
  • Ang-1 is essential for normal endothelial developmental processes in the mouse (Davis et al., 1996, Cell 87: ).
  • Ang-1 induces the formation of capillary sprouts (Koblizek et al., 1998, Curr. Biol. 8:529-532).
  • the protein is expressed only on endothelial cells and early hemopoietic cells (e.g., see Suri et al., 1996, Cell 87:1171-1180).
  • Angiopoietin-2 (Ang-2) is a naturally occurring antagonist for Angl and Tie2 and can disrupt blood vessel formation in the mouse embryos (see, eg. Maisonpierre et al., 1997, Science 277:55-). Ang-2 is expressed only at sites of vascular remodeling. In animal models some angiogenesis-dependent diseases can be controlled via induction or inhibition of new vessel formation. Treatment of diseases by modulation of angiogenesis are currently tested in clinical trials. Thus the manipulation of new vessel formation in angiogenesis- dependent conditions such as wound healing, inflammatory diseases, ischemic heart and peripheral vascular disease, myocardial infarction, diabetic retinopathy, and cancer is likely to create new therapeutic options.
  • angiogenesis is believed to play a significant role in the metastasis of a cancer and in the ischemic heart and ischemic vascular disease. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. If this angiogenic activity could be stimulated or induced, ischemic tissues in the heart and brain and mycocardial necrosis could be prevented. In the disease state, stimulation or induction of angiogenesis could avert the damage. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases.
  • compositions and methods of this invention are useful in inhibiting the activity of endogenous growth factors in premetastatic tumors and preventing the formation of the capillaries in the tumors thereby inhibiting the growth of the tumors.
  • the composition, and antibodies specific to the composition should also be able to modulate the formation of capillaries in other angiogenic processes, such as wound healing and reproduction.
  • the composition and method for inhibiting angiogenesis should preferably be non-toxic and produce few side effects.
  • compositions and methods of this invention are useful in stimulating the growth of blood vessels, especially in mycocardial infarction and other heart diseases or brain infarcts (strokes).
  • the composition should be able to overcome the necrotic effects of ischemic tissue and thereby prevent the effects of heart diseases or strokes.
  • the composition and method for stimulating angiogenesis should preferably be non-toxic and produce few side effects.
  • angiopoeitin-3 novel molecules, referred to herein as "angiopoeitin-3
  • Ang-3 human microvascular endothelial differentiation gene 1 (hMEDGl)” and “heart specific growth factor-8b (FGF-8b)” polypeptides, as well as nucleic acid sequences encoding those molecules.
  • the novel nucleic acid sequences of this invention is operatively linked to one or more expression control sequences.
  • the invention also provides a host cell, including bacterial, plant, yeast, insect and mammalian cells, that produce the novel polypeptides, whether the cell is transformed with the nucleic acid sequences encoding those proteins, or whether the cell is transformed with regulatory sequences to activate or enhance production of these proteins from an endogenous nucleic acid sequence encoding same.
  • Processes are also provided for producing a protein, which comprise growing a culture of host cells producing such proteins (as described above) in a suitable culture medium, and purifying the protein from the culture.
  • the protein produced according to such methods is also provided by the present invention.
  • the protein comprises an angiopoeitin-3, hMEDGl and FGF-8b amino acid sequence or fragments thereof, the protein being substantially free from other mammalian proteins.
  • Such compositions may further comprise a pharmaceutically acceptable carrier.
  • Compositions comprising an antibody which specifically reacts with such protein are also contemplated by the present invention.
  • Methods are also contemplated for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
  • compositions and methods of this invention are useful in anti-cancer and heart disease therapies. Diagnostic, prognostic and screening kits are also contemplated. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts the nucleotide sequence and deduced amino acid sequence of angiopoeitin-3 (Ang-3). The signal sequence is underlined.
  • FIG. 2 depicts the nucleotide sequence and deduced amino acid sequence of hMEDGl.
  • the signal sequence is underlined.
  • FIG. 3 depicts the nucleotide sequence and deduced amino acid sequence of heart specific growth factor 8b (FGF-8b).
  • Panel A shows the preliminarily determined sequence, with the signal sequence underlined.
  • Panel B shows the confirmed sequence.
  • direct angiogenic/antiangiogenic molecules means a molecule that elicits an effect on angiogenesis/antiangiogenesis in vivo upon exogenous administration or overexpression, that has an effect on relevant endothelial cells in vitro that is compatible with angiogenesis/antiangiogenesis. and the role of the molecule has been established in a process or disease.
  • This invention further provides for novel angiopoeitin-3, hMEDGl, or FGF-8b proteins and genes encoding those proteins, as well as contemplating derivatives, homologs, active fragments and analogs, from various species, particularly vertebrates, and more particularly mammals.
  • the foregoing proteins and genes are of human origin. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is also contemplated in the present invention.
  • the fragment, derivative or analog is functionally active, i.e.. capable of exhibiting one or more functional activity associated with wild type Ang3, hMEDGl . or FGF-8b protein.
  • Such functional activities include, but are not limited to, the stimulation or inhibition of angiogenesis and related disorders and the treatment of heart disease and related disorders.
  • Such functional activities include further, but are not limited to, antigenicity [ability to bind (or compete with Ang-3, hMEDGl, or FGF-8b for binding) to an anti-Ang3, anti-hMEDGl. or anti-FGF-8b antibody, respectively], immunogenicity (ability to generate an antibody that binds to anti-IP-Ang3, anti-IP-hMEDGT, or anti-IP-heart specific growth factor, respectively), etc.
  • RNA messenger RNA
  • Clontech e.g., Fetal brain, heart, kidney, fetal liver, liver, lung, skeletal muscle, pancreas and placenta
  • Oligotex TM cDNA synthesis kit QIAGEN, Inc.; Chatsworth, CA
  • E. coli DNA polymerase and 3.5 units of E. coli RNase H (all supplied by BRL; Grand Island, NY), second-strand synthesis was performed at 16°C for 2 hours. Five units of T 4 DNA polymerase was then added, and incubation was continued for an additional 5 minutes at 16°C. The reaction was then treated with 5 units of arctic shrimp alkaline phosphatase (U.S. Biochemicals; Cleveland OH) at 37°C for 30 minutes, and the cDNA was purified by standard phenol/chloroform (50:50 v/v) extraction. The yield of cDNA was estimated using fiuorometry with the Picogreen TM Labeling System (Molecular Probes; Eugene, OR).
  • the double-stranded cDNA was digested with various restriction enzymes, ligated to linkers compatible with the over-hanging termini generated by the restriction digestion.
  • the restriction fragments were amplified utilizing 30 cycles of polymerase chain reaction (PCR) by the addition of the following reagents: 2 ⁇ l 10 mM dNTP; 5 ⁇ l 10X TB buffer (500 mM Tris, 160 mM (NH4) : SO 4 ,; 20 mM MgC , pH 9.15); 0.25 ⁇ l Klentaq (Clontech Advantage) : PFU (Stratagene; La Jolla CA) in a 16: 1 v/v ratio; 32.75 ⁇ l ddH2O.
  • PCR polymerase chain reaction
  • TA TM cloning vector Invitrogen. Individual clones were subjected to dye-primer, double-stranded DNA sequencing utilizing PCR products which were derived from amplification using vector-specific primers, which flanked the insertion, site as templates. Sequencing was performed using a standard chemistry methdology on ABI Model 377 sequencers (Molecular Dynamics). 2. ISOLATED PROTEINS AND POLYNUCLEOTIDES, AND
  • Ang-3 The sequence of a polynucleotide encoding an Ang-3 protein of the present invention is set forth in Figure 1, with the coding region extending from nucleotides 352 to 1824. This polynucleotide has been named "Angiopoeitin-3 (Ang-3)".
  • Ang-3 The amino acid sequence of the protein encoded by Ang-3 is also shown in Figure 1.
  • Ang-3 was isolated from a human heart library, cloned into a vector and sequenced by methods known in the art.
  • the nucleic acid sequence in Figure 1 encodes a novel protein, Ang-3.
  • Sequence homology algorithms e.g., BLASTN/BLASTX or FASTA searches, revealed no exact sequence matches.
  • a BLASTX search revealed 59 % homology between Ang-3 (in the approximate region of nucleotides 1067 and 1833 of Figure 1), and human angiopoietin proteins (including human Angl [GenBank Accession Number U83508] and human Ang-2 [GenBank Accession Number AF004327].
  • a BLASTP search also revealed 63 % homology between Ang-3 (in the approximate region of amino acids 269 to 491 of Figure 1), and human angiopoietin proteins (including human angiopoetin-like protein (CDT6 gene) [GenBank Accession Number Y16132]). Furthermore, a BLASTP search revealed 51 % homology between Ang-3 (in the approximate region of amino acids 8 to 491 of Figure 1), and mouse angiopoietin- 1 protein [GenBank Accession Number U83509]).
  • Ang-3 in the approximate region of amino acids 277 to 491 of Figure 1
  • fibrinogen-like proteins including fibrinogen-like protein 1 precursor [TREMBL Accession Number Q08830]), fibrinogen-related protein HFREP-1 precursor [PER. Accession Number JN0596] and fibrinogen-like protein [TREMBLAccession Number Q143114]).
  • Ang-3 has a secretion signal peptide.
  • Ang-1 and Ang-2 are 60 % identical.
  • Ang-3 is homologous in the carboxy-terminal fibrinogen-like domain to Ang-1 and Ang-2 (Maisonpierre et al., 1997, Science 277:55-60). Based upon these homologies, Ang-3 and these homologous proteins are expected to share at least some activities.
  • the present invention provides an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence shown m Figure 1 from nucleotide 352 to nucleotide 1824, or nucleotide 415 to nucleotide 1824;
  • a polynucleotide comprising a fragment of the nucleotide sequence shown in Figure 1 encoding a protein having biological activity
  • a polynucleotide encoding a protein comprising the amino acid sequence shown in Figure 1, from nucleotide 352 to nucleotide 1824 (i.e., including the signal sequence) or from nucleotide 415 to nucleotide 1824 (the mature peptide), herein called angiopoetin-3;
  • hMEDGl The sequence of a polynucleotide encoding another protein of the present invention is set forth in Figure 2, with the coding region extending from nucleotides 190 to 858.
  • This polynucleotide has been named "Microvascular Endothelial Differentiation Gene” (hMEDGl).
  • the amino acid sequence of the protein encoded by the endothelial differentiation gene (hMEDGl) is set forth in Figure 2.
  • HMEDGl was isolated from a human heart library, cloned into a vector and sequenced by methods known in the art.
  • the nucleic acid sequence in Figure 2 encodes a novel protein, hMEDGl .
  • Sequence homology algorithms e.g., BLASTN/BLASTX or FASTA searches revealed no exact sequence matches.
  • a BLASTX search revealed 84 % homology between the hMEDGl protein (in the approximate region of nucleotides 56-1013 of Figure 2 and rat microvascular endothelial differentiation gene 1 (GenBank Accession Number X98993).
  • BLASTP search revealed 94 % homology between the hMEDGl protein (in the approximate region of amino acids 51-223 of Figure 2) and rat microvascular endothelial differentiation gene 1 (SPTREMBL-Acc. No. P97554).
  • hMEDGl contains 50 amino acids that were not reported in the rat MEDGl protein. These 50 amino acids comprise a signal sequence; therefore hMEDGl does encode a secreted factor. Based upon these homologies, hMEDGl and these homologous proteins are expected to share at least some activities.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence shown in Figure 2 from nucleotide 51 to nucleotide 858, or from nucleotide 259 to nucleotide 858; (b) a polynucleotide comprising a fragment of the nucleotide sequence shown in Figure 2 encoding a protein having biological activity;
  • the sequence of a polynucleotide encoding another protein of the present invention is set forth in Figure 3, with the coding region extending from nucleotides 39 to 618 (Panel A) and 39 to 659 (Panel B).
  • This polynucleotide has been identified as "human heart specific fibroblast growth factor 8b (FGF-8b)"
  • FGF-8b human heart specific fibroblast growth factor 8b
  • the amino acid sequence of the protein encoded by FGF-8b is set forth in Figure 3.
  • the human FGF-8b was isolated from a human heart library using a trap which selects for nucleotides encoding secreted proteins; therefore, FGF-8b encodes a secreted factor.
  • the nucleic acid sequence in Figure 3 encodes a novel protein, FGF-8b.
  • Panel A shows a preliminarily determined sequence.
  • Panel B shows a confirmed sequence.
  • Sequence homology algorithms e.g., BLASTN/BLASTX or FASTA searches revealed no exact sequence matches.
  • a BLASTP search revealed about 80 % homology between the FGF-8b (particularly in the approximate region of amino acids 1-181 of Figure 3) and various human growth factors including fibroblast growth factor 8, androgen-induced growth factor, keratinocyte growth factor, heparin-binding growth factor- 1, and beta-endothelial cell growth factor (including without limitation those assigned accession numbers G2660747,
  • the present invention provides a composition . comprising an isolated polynucleotide selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • Nucleic acids encoding Ang3, hMEDGl, or FGF-8b can be obtained by any method known in the art. e.g, by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence and/or by cloning from a cDNA or genomic library using an oligonucleotide specific for the gene sequence, e.g., as described infra.
  • Homologs e.g., of nucleic acids encoding Ang3, hMEDGl, or FGF-8b of species other than human, or other related sequences, e.g., paralogs, can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning, e.g., as described infra, for Ang3, hMEDGl, or FGF-8b nucleotide sequences.
  • polynucleotides hybridizing to the polynucleotides of the present invention under stringent conditions and highly stringent conditions are also part of the present invention.
  • Preferred high stringency conditions are hybridization in 4X SSC, 5X
  • the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
  • an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991)
  • Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).
  • operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
  • Mammalian host cells include, for example, monkey COS cells.
  • yeast eukaryotes
  • prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis. Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
  • Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.
  • Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al, Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference.
  • Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.
  • fragments of the protein may be fused through "linker" sequences to the Fc portion of an immuno globulin.
  • a bivalent form of the protein such a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulin isotypes may also be used to generate such fusions.
  • a protein-IgM fusion would generate a decavalent form of the protein of the invention.
  • the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin- toyopearl R TM. or Cibacrom blue 3GA Sepharose RTM; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will facilitate purification.
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New York.
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from Kodak (New Haven, Conn.).
  • RP-HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
  • the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
  • the protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art.
  • the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
  • the proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.
  • modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
  • Modifications of interest in the protein sequences may include the replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Mutagenic techniques for such replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584, incorporated by reference).
  • polynucleotides of the present invention and the proteins encoded thereby are expected to exhibit one or more of the uses or biological activities identified below.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
  • the biological activity of the proteins of this invention can be assayed by any suitable method known in the art.
  • the angiogenic/antiangiogenic potential can be characterized in angiogenesis assays in vivo such as the chick chorionic allantoic membrane (CAM) assay or different cornea micropocket assays (Klagsbrun & Folkman, 1990, In: Sporn & Roberts (eds). Peptide growth factors and their receptors II, pp. 549-574).
  • An a in vivo angiogenesis assay is described in, eg., U.S. Pat. No. 5,382,514, incorporated by reference), and a mouse model of hindlimb ischemia was described by Couffinhal et al., 1998, Am.
  • Assays for angiogenic/antiangiogenic activity include methods for inhibition of angiogenesis (see, for example, but not limited to, U.S. Pat. No. 5733876, 5639725, 5712291, 5698586, 5753230, 5733876, 5766591.5434185, 5721226,56293405593990, 5629327, 5744492, 5646136, 5610166, 5574026, 5567693, 5563130, each herein fully incorporated by reference).
  • a protein of the present invention may exhibit angiogenic (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity.
  • Many protein factors discovered to date including all known angiopoeitins and growth factors, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of angiogenic activity.
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10,B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DAI, 123, Tl 165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • Methods of diagnosis, prognosis, and screening for diseases and disorders associated with aberrant levels of an Ang3, hMEDGl, or heart specific growth factor are contemplated.
  • the invention also contemplates methods of treating or preventing diseases or disorders associated with aberrant levels of an Ang3, hMEDGl, or heart specific growth factor, or aberrant levels of activity of one or more of the components of the complex, comprising administration of the Ang3, hMEDGl, or heart specific growth factor.
  • Methods of assaying an Ang3, hMEDGl, or FGF-8b, for activity as therapeutics or diagnostics as well as methods of screening modulators (i.e., inhibitors, agonists and antagonists) are also contemplated.
  • a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubihzers, and other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment.
  • protein of the present invention may be included in formulations of the particular Ang3, hMEDGl, or FGF-8b to minimize side effects of the Ang3, hMEDGl, or FGF-8b agent.
  • protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection.
  • a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection
  • protein of the present invention will be in the form of a pyrogen-free, parenterally-acceptable aqueous solution.
  • the preparation of such parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a meaningful patient benefit i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated.
  • Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing Ang3, hMEDGl, or heart specific growth factor.
  • protein of the present invention may be administered either simultaneously with the Ang3, hMEDGl, or FGF-8b, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with Ang3, hMEDGl, or FGF-8b.
  • the amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
  • Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
  • the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
  • Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.
  • neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
  • Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Vascular Medicine (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Oncology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Cette invention concerne de nouvelles protéines angiogéniques/anti-angiogéniques sécrétées et les séquences d'acides aminés codant pour ces protéines.
PCT/US1999/012839 1998-07-20 1999-07-20 Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese WO2000005369A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU50818/99A AU5081899A (en) 1998-07-20 1999-07-20 Nucleotide sequences and amino acid sequences of secreted proteins involved in angiogenesis
JP2000561315A JP2002522019A (ja) 1998-07-20 1999-07-20 新脈管形成に関与する分泌タンパク質のヌクレオチド配列およびアミノ酸配列
CA002335326A CA2335326A1 (fr) 1998-07-20 1999-07-20 Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese
EP99935312A EP1098974A2 (fr) 1998-07-20 1999-07-20 Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US9339798P 1998-07-20 1998-07-20
US60/093,397 1998-07-20
US15068498A 1998-09-10 1998-09-10
US09/150,684 1998-09-10

Publications (2)

Publication Number Publication Date
WO2000005369A2 true WO2000005369A2 (fr) 2000-02-03
WO2000005369A3 WO2000005369A3 (fr) 2000-06-15

Family

ID=26787485

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/012839 WO2000005369A2 (fr) 1998-07-20 1999-07-20 Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese

Country Status (5)

Country Link
EP (1) EP1098974A2 (fr)
JP (1) JP2002522019A (fr)
AU (1) AU5081899A (fr)
CA (1) CA2335326A1 (fr)
WO (1) WO2000005369A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003078648A2 (fr) * 2002-03-11 2003-09-25 Curagen Corporation Sequences de nucleotides et sequences d'acides amines de proteines secretees impliquees dans l'angiogenese
US8153586B2 (en) 1996-10-16 2012-04-10 Zymogenetics, Inc. FGF homologs compositions and uses thereof
US8507430B2 (en) 2002-10-07 2013-08-13 Zymogenetics, Inc. Methods for administering FGF18

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012084561A (ja) * 2010-10-06 2012-04-26 Fujitsu Semiconductor Ltd 半導体装置およびその製造方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2106550A (en) 1980-09-15 1983-04-13 Nuovo Pignone Spa Weft transfer devices in shuttleless looms
EP0123005B1 (fr) 1983-04-18 1986-09-17 Maschinenfabrik Sulzer-Rüti Ag Pince pour l'insertion du fil de trame dans un métier à tisser à pince

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5989866A (en) * 1996-10-16 1999-11-23 Zymogenetics, Inc. FGF homologs
US6057435A (en) * 1997-09-19 2000-05-02 Genentech, Inc. Tie ligand homologues
WO1999040193A1 (fr) * 1998-02-04 1999-08-12 Zymogenetics, Inc. Zapo3 homologue d'angiopoietine, adn le codant et sa methode de production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2106550A (en) 1980-09-15 1983-04-13 Nuovo Pignone Spa Weft transfer devices in shuttleless looms
EP0123005B1 (fr) 1983-04-18 1986-09-17 Maschinenfabrik Sulzer-Rüti Ag Pince pour l'insertion du fil de trame dans un métier à tisser à pince

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8153586B2 (en) 1996-10-16 2012-04-10 Zymogenetics, Inc. FGF homologs compositions and uses thereof
US8349590B2 (en) 1996-10-16 2013-01-08 Zymogenetics, Inc. FGF homologs compositions and uses thereof
WO2003078648A2 (fr) * 2002-03-11 2003-09-25 Curagen Corporation Sequences de nucleotides et sequences d'acides amines de proteines secretees impliquees dans l'angiogenese
WO2003078648A3 (fr) * 2002-03-11 2004-11-25 Curagen Corp Sequences de nucleotides et sequences d'acides amines de proteines secretees impliquees dans l'angiogenese
US8507430B2 (en) 2002-10-07 2013-08-13 Zymogenetics, Inc. Methods for administering FGF18

Also Published As

Publication number Publication date
JP2002522019A (ja) 2002-07-23
CA2335326A1 (fr) 2000-02-03
EP1098974A2 (fr) 2001-05-16
WO2000005369A3 (fr) 2000-06-15
AU5081899A (en) 2000-02-14

Similar Documents

Publication Publication Date Title
US5607918A (en) Vascular endothelial growth factor-B and DNA coding therefor
US6689580B1 (en) Growth factor
KR100728405B1 (ko) Ｎｏｇｏ 유전자의 뉴클레오티드와 단백질 서열 및 이들에기초한 방법
EP0750628B1 (fr) Facteur-10 de croissance du fibroblaste
JP3492999B2 (ja) 脂肪細胞特異的タンパク質の同族体
JPH10510718A (ja) 血管内皮増殖因子−ｂ
JP2002523035A (ja) ４９個のヒト分泌タンパク質
JP2003159083A (ja) 新規な神経栄養因子
JP2002509432A (ja) 哺乳類サイトカイン様因子７
JP2002532083A (ja) ４７個のヒト分泌タンパク質
JP2002506625A (ja) サイトカインレセプター共通γ鎖様
JP2002530062A (ja) １２個のヒト分泌タンパク質
JP2002540763A (ja) ３３個のヒト分泌タンパク質
JP2002505871A (ja) ３１個のヒト分泌タンパク質
WO1999032515A2 (fr) Homologue de l'angiopoietine, adn le codant et procede de production dudit homologue
EP0962530A2 (fr) Séquence du gène codant pour la protéine scarface 1 apparentée a l'angiopoiétine
EP1098974A2 (fr) Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese
US6537554B1 (en) Nucleotide sequences and amino acid sequences of secreted proteins involved in angiogenesis
US20030104573A1 (en) Nucleotide sequences and amino acid sequences of secreted proteins involved in angiogenesis
JPH11507504A (ja) 線維芽細胞増殖因子１３
JP2000515726A (ja) 分泌蛋白をコードする成人ｐｂｍｃ由来のポリヌクレオチド
JP2002530058A (ja) ３１個のヒト分泌タンパク質
KR20010043090A (ko) 신규인 폴리펩티드, 그 폴리펩티드를 코드화하는 ｃＤＮＡ및 그 용도
EP0962466A1 (fr) Scarface 3, gène liée aux angiopoietins
JP2002504361A (ja) ３６個のヒト分泌タンパク質

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE 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 US US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL 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)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE 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 US US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL 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

ENP Entry into the national phase in:

Ref document number: 2335326

Country of ref document: CA

ENP Entry into the national phase in:

Ref country code: JP

Ref document number: 2000 561315

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1999935312

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 50818/99

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 09744184

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1999935312

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1999935312

Country of ref document: EP