WO1999040193A1 - Zapo3 homologue d'angiopoietine, adn le codant et sa methode de production - Google Patents

Zapo3 homologue d'angiopoietine, adn le codant et sa methode de production Download PDF

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WO1999040193A1
WO1999040193A1 PCT/US1999/002303 US9902303W WO9940193A1 WO 1999040193 A1 WO1999040193 A1 WO 1999040193A1 US 9902303 W US9902303 W US 9902303W WO 9940193 A1 WO9940193 A1 WO 9940193A1
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polypeptide
zapo3
seq
residues
sequence
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PCT/US1999/002303
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English (en)
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James L. Holloway
Kimberly E. Shoemaker
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Zymogenetics, Inc.
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Priority to AU24936/99A priority Critical patent/AU2493699A/en
Publication of WO1999040193A1 publication Critical patent/WO1999040193A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • polypeptide growth factors In multicellular animals, cell growth, differentiation, and migration are controlled by polypeptide growth factors. These growth factors play a role in both normal development and pathogenesis, including the development of solid tumors.
  • Polypeptide growth factors influence cellular events by binding to cell-surface receptors, many of which are tyrosine kinases. Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes such as cell division, protease production, and cell migration.
  • Angiogenesis the sprouting of capillaries from existing blood vessels, is one such growth factor-dependent developmental process.
  • angiogenesis vascular endothelial cells re-enter the cell cycle, degrade underlying basement membrane, and migrate to form new capillary sprouts.
  • Angiogenesis occurs during embryonic development, as well as in the adult organism during pregnancy, the female reproductive cycle, and wound healing. In addition, angiogenesis occurs during a variety of pathological conditions, including diabetic retinopathy, macular degeneration, atherosclerosis, psoriasis, rheumatoid arthritis, and solid tumor growth. For review, see Breier et al., Thrombosis and Haemostasis 78:678-683, 1997.
  • vascular endothelial growth factors VEGFs
  • angiopoietins vascular endothelial growth factors
  • the VEGFs act through at least three cell surface receptors, designated Flt-1 , Flk-1 , and Flt-4. The expression of these receptors is limited to certain cell types and/or 2 developmental stages, thereby defining the functions of the ligands. Data obtained from receptor- and growth factor-deficient mice indicate that the VEGFs are essential for vascular development in the embryo.
  • Angiopoietin-1 acting through the Tie-2 receptor (also known as Tek), is believed to regulate a later stage of vascular development (reviewed by Hanahan, Science 277:48-50, 1997), directing the maturation and stabilization of blood vessels through its action on endothelial cells and the surrounding matrix or mesenchyme.
  • the recently discovered angiopoietin-2 (Ang-2) is an antagonist of Tie-2-mediated activity. Ang-2 causes a loosening of vessel structure and loss of contact between endothelial cells and the matrix, making the endothelial cells more accessible to VEGF. This destabilization is an initial step in angiogenesis, and both VEGF and Ang-2 are up-regulated at sites of ongoing angiogenesis.
  • Ang-2 is also highly expressed during vascular regression in non-productive ovarian follicles. Individual factors may exhibit different effects depending on such variables as developmental stage, the the presence or absence of other factor(s), and target cell type.
  • VEGF acting through the receptor Flk-1 , stimulates the proliferation of endothelial cells. Acting through the receptor Flt-1 , VEGF promotes endothelial cell-cell interactions and the formation of capillary tubes.
  • Ang-2 in the absence of VEGF or other proliferative signals, may induce apoptosis in nonproductive ovarian follicles, but Ang-2 also is believed to make endothelial cells more responsive to VEGF and thereby promote cell proliferation and vascular development.
  • angiopoietins may be regulators of hematopoiesis. Endothelial cells and hematopoietic stem cells are believed to be derived from a common precursor cell, and Tie receptors are expressed on both cell types. Tie receptors are expressed in several leukemia cell lines with predominantly megakaryoblastic markers (Batard et al., Blood 8Z:2212-2220, 1996; Kukk et al., Brit. J. Haematol. 98:195-203, 1997).
  • Platelet-derived growth factor for example, has been disclosed for the treatment of periodontal disease (U.S. Patent No. 5,124,316) and gastrointestinal ulcers (U.S. Patent No. 5,234,908). Inhibition of PDGF receptor activity has been shown to reduce intimal hyperplasia in injured baboon arteries (Giese et al., Restenosis Summit VIII, Poster Session #23, 1996; U.S. Patent No. 5,620,687). The hematopoietic cytokine erythropoietin has been developed for the treatment of anemias (e.g., EP 613,683).
  • anemias e.g., EP 613,683
  • thrombopoietin has been shown to stimulate the production of platelets in vivo (Kaushansky et al., Nature 369:568-571 , 1994).
  • Vascular endothelial growth factors have been shown to promote the growth of blood vessels in ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and have been proposed for use as wound-healing agents, for treatment of periodontal disease, for promoting endothelialization in vascular graft surgery, and for promoting collateral circulation following myocardial infarction (WIPO Publication No. WO 95/24473; U.S. Patent No. 5,219,739).
  • VEGFs are also useful for promoting the growth of vascular endothelial cells in culture.
  • a soluble VEGF receptor (soluble flt-1) has been found to block binding of VEGF to cell-surface receptors and to inhibit the growth of vascular tissue in vitro (Biotechnology News !6(17):5-6, 1996).
  • Experimental evidence suggests that inhibition of angiogenesis may be used to block tumor development (Biotechnology News, Nov. 13, 1997) and that angiogenesis is an early indicator of cervical cancer (Br. J. Cancer 76:1410- 1415, 1997).
  • an isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical in amino acid sequence to residues 22 to 491 of SEQ ID NO:2.
  • the polypeptide is at least 95% identical in amino acid sequence to residues 22 to 491 of SEQ ID NO:2.
  • the polypeptide is from 440 to 495 amino acids in length.
  • the polypeptide comprises residues 26-491 of SEQ ID NO:2.
  • Polypeptides comprising residues 26-491 of SEQ ID NO:2 include polypeptides of from 440 to 490 amino acids in length.
  • a polypeptide disclosed above is covalently linked to a moiety selected from the group consisting of affinity tags, toxins, radionuclides, enzymes, and fluorophores.
  • the affinity tag is selected from the group consisting of polyhistidine, protein A, glutathione S transferase, substance P, and an immunoglobulin heavy chain constant region.
  • a proteolytic cleavage site is included between the polypeptide and the affinity tag.
  • an isolated multimeric protein comprising a first polypeptide chain that is at least 90% identical in amino acid sequence to residues 22-491 of SEQ ID NO:2, and a second polypeptide chain, wherein the protein is angiogenic.
  • first and second polypeptide chains are the same.
  • first and second polypeptide chains are different. 5
  • a protein produced by a method comprising the steps of (a) cultu ng a cell containing a DNA construct comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as disclosed above; and a transcription terminator; and (b) isolating the protein encoded by the DNA segment and produced by the cell.
  • the DNA construct further comprises a secretory signal sequence operably linked to the DNA segment.
  • the secretory signal sequence may encode, for example, residues 1 to 21 of SEQ ID NO:2.
  • an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as disclosed above; and a transcription terminator.
  • the DNA construct further comprises a secretory signal sequence operably linked to the DNA segment.
  • the secretory signal sequence may encode, for example, residues 1 to 21 of SEQ ID NO:2.
  • a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses the DNA segment and produces a polypeptide encoded by the DNA segment.
  • an antibody that specifically binds to a polypeptide as disclosed above.
  • an isolated polynucleotide encoding a polypeptide as disclosed above.
  • the isolated polynucleotide is from 1410 nucleotides to about 4.0 kb in length or from 1410 nucleotides to about 2.8 kb in length.
  • the polynucleotide is DNA.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31 , 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus.
  • allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference 7 sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • Angiogenic denotes the ability of a compound to stimulate the formation of new blood vessels from existing vessels, acting alone or in concert with one or more additional compounds. Angiogenic activity is measurable as endothelial cell activation, stimulation of protease secretion by endothelial cells, endothelial cell migration, capillary sprout formation, and endothelial cell proliferation. Angiogenesis can also be measured using any of several in vivo assays as disclosed herein.
  • a "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide).
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules 8 of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
  • an "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • Operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • a "polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt”), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single- stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs”.
  • the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide 9 molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
  • polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • a “secretory signal sequence” is a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • the present invention provides novel growth factor polypeptides and proteins.
  • This novel growth factor termed "zapo3” exhibits significant amino acid sequence homology to the previously described angiopoietin-2 (Maisonpierre et al., Science 277:55-60, 1997).
  • SEQ ID NO:2 a representative human zapo3 sequence, is approximately 31 % identical to the sequence of angiopoietin-2 when the two sequences are aligned to produce a 470 amino acid overlap (residues 49-491 of SEQ ID NO:2), accounting for gaps as disclosed below.
  • the zapo3 sequence is approximately 31% identical to angiopoietin-1 when the two sequences are aligned to produce a 473 amino acid overlap.
  • angiopoietins 1 and 2 and zapo3 are characterized by an amino-terminal coiled coil domain and a carboxyl- terminal fibrinogen-like domain. Based on an analysis of the amino acid sequence for zapo3 shown in SEQ ID NO:2, residues 63-253 are also expected to form a coiled coil.
  • Coiled coils are bundles of ⁇ -helices (generally 2-4 helices) wound into a superhelix.
  • the sequence shows a heptad repeat in the chemical nature of sidechains. This structure is characterized by a "knobs- into-holes" packing of amino acid sidechains in the core of the bundle. See, Lupas, TIBS 21:375-382, 1996.
  • Residues 279-490 of SEQ ID NO:2 show marked homology (approximately 40% sequence identity) to residues 631-864 of human fibrinogen alpha chain (SEQ ID NO:3).
  • This domain of zapo3 contains conserved cysteine residues at positions 280, 309, 432, and 445 of SEQ ID NO:2.
  • zapo3 and angiopoietin-2 are approximately 40% identical. Analysis of the zapo3 cDNA also predicts that residues 1 through 21 of SEQ ID NO:2 are a secretory peptide.
  • the present invention is not limited to the expression of the sequence shown in SEQ ID NO:1.
  • a number of truncated zapo3 polynucleotides and polypeptides are provided by the present invention. These polypeptides can be produced by expressing polynucleotides encoding them in a variety of host cells. In many cases, the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a zapo3 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide.
  • expressing the complete zapo3 sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved.
  • a variety of zapo3 polypeptides can thus be produced. Differential processing of individual chains may result in heterogeneity of expressed polypeptides and the production of heterodimeric zapo3 proteins.
  • zapo3 polypeptides can be produced by other known methods, such as solid phase synthesis, methods for which are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce
  • the present invention thus includes both heteromultimers and homomultimers comprising zapo3 polypeptide subunits.
  • Heteromultimers may be formed by association of distinct zapo3 polypeptides or by association of one or more zapo3 polypeptides with, for example, an angiopoietin-1 or -2 polypeptide.
  • multimer includes dimers and higher order multimers. Multimerization can occur either in vivo or in vitro.
  • Zapo3 proteins of the present invention are characterized by their angiogenic or hematopoietic activity.
  • angiogenesis and vasculogenesis are complex processes involving cell differentiation, proliferation, and migration, as well as reorganization of tissues comprising a plurality of cell types.
  • in vivo assays are preferred for detection and analysis of zapo3 activity, although certain in vitro models, such as the three-dimensional collagen gel matrix model of Pepper et al. (Biochem. Biophys. Res. Comm. 189:824-831 , 1992), are sufficiently complex to assay histological effects.
  • Assays can be performed using exogenously produced proteins, or may be carried out in vivo or in vitro using cells expressing the polypeptide(s) of interest. Assays for angiogenic activity are disclosed below.
  • hzapo3 FACS analysis of BHK cells expressing recombinant human zapo3 (hzapo3) revealed that cell surface-associated hzapo3 was released within one hour of treatment with 100 ng/mL LPS, suggesting inflammatory- regulated membrane shedding of zapo3 protein.
  • Many cytokines, receptors, and other proteins are initially synthesized as membrane-associated proteins that are subsequently released from the cell by proteolysis.
  • Membrane shedding is primarily regulated by members 13 of the family of metalloproteases, including MMP-2, MMP-9, TACE, or disintegrin (Gallea-Robache et al., ibid; Peschon et al., ibid.; Black et al., Nature 385:729, 1997; Crowe et al., J. Exp. Med. 181, 1995).
  • metalloproteases are activated by inflammatory signals such as TNF ⁇ and LPS.
  • membrane bound cytokines, chemokines, and growth factors may not be simply to provide a ready supply of soluble signaling molecules, but also to provide cell-attached signaling molecules that mediate cell-cell adhesion and intracrine signaling.
  • Membrane- anchored TGF ⁇ for example, can interacted with EGF receptors on hematopoietic progenitor cells, stimulating adhesion and proliferation of those cells (Anklesaria et al., Proc. Natl. Acad. Sci. USA 87:3289, 1990).
  • membrane-anchored EGF is important in controlling mammary epithelial cell organization (Wiley et al., J. Cell Biol. 143:1317, 1998).
  • Northern and western blot analysis indicates that zapo3 is produced in adrenal glands. Combined with the membrane shedding results, a similar role is suggested for zapo3 in adrenal.
  • Adrenals are endocrine organs and depend on close association between vascular bends and endocrine cells for the delivery of molecules to the blood stream.
  • Cell-cell adhesion molecules are intregal for this association.
  • Adrenal cell membrane-anchored zapo3 could act as a cell adhesion molecule through interaction with angiopoietin-like receptors on vascular cells in capillary beds. This is not whithout precedent; direct cell-cell adhesion between adrenal cells and adrenal medullary endothelial cells has been described (Mizrachi, et al., Cell Tissue Res. 256:365, 1989). These data taken together suggest a possible role for zapo3 in modulation of endocrine-vascular cell association.
  • SEQ ID NO:4 is a degenerate DNA sequence that encompasses all DNAs that encode the zapo3 polypeptide of SEQ ID NO: 2.
  • SEQ ID NO:4 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T.
  • zapo3 polypeptide- encoding polynucleotides comprising nucleotide 64 to nucleotide 1473 of SEQ ID NO: 4 and their RNA equivalents are contemplated by the present invention, as are polynucleotides encoding the other polypeptides listed in Table 1 and polynucloeotides encompassing nucleotides 1-1473 of SEQ ID NO:4.
  • Table 2 sets forth the one-letter codes used within SEQ ID NO:4 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter. “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NO:4, encompassing al possible codons for a given amino acid, are set forth in Table 3, below.
  • Met M ATG ATG lie 1 ATA ATC ATT ATH
  • degenerate codon representative of all possible codons encoding each amino acid.
  • WSN can, in some circumstances, encode arginine
  • MGN can, in some circumstances, encode serine
  • some polynucleotides encompassed by the degenerate sequence encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be readily tested for functionality as described herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO.1 , or a sequence complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for preparing DNA and RNA are well known in the art.
  • Complementary DNA (cDNA) clones are prepared from RNA that is isolated from a tissue or cell that produces large amounts of zapo3 RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201 , 1980), and include heart, placenta, skeletal muscle, kidney, pancreas, spleen, testis, ovary, small intestine, colon, stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, and bone marrow, with thyroid preferred.
  • Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCI gradient
  • RNA is prepared 18 from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972).
  • cDNA is prepared from poly(A) + RNA using known methods.
  • genomic DNA can be isolated.
  • Methods for identifying and isolating cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
  • Polynucleotides encoding zapo3 polypeptides are identified and isolated by, for example, hybridization or PCR.
  • Expression libraries can be probed with antibodies to zapo3, receptor fragments, or other specific binding partners.
  • sequences disclosed in SEQ ID NOS:1 , 2, and 4 represent a single allele of human zapo3. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs).
  • zapo3 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • Species orthologs of human zapo3 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zapo3 as disclosed above. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a zapo3-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the 19 representative human zapo3 sequence disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zapo3 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • the polynucleotides of the present invention can also be prepared by automated synthesis. Automated synthesis of polynucleotides is within the level of ordinary skill in the art, and suitable equipment and reagents are available from commercial suppliers. See, in general, Glick and Pasternak, Molecular Biotechnology. Principles & Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994; Itakura et al., Ann. Rev. Biochem. 53: 323-56, 1984; and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.
  • polypeptides can be produced by engineering amino acid changes into the representative human polypeptide sequence shown in SEQ ID NO:2 or an allelic variant or ortholog thereof. It is preferred that these engineered variant polypeptides are at least 80% identical to the polypeptide of SEQ ID NO:2. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
  • Engineered variant zapo3 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 5) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag as disclosed above.
  • the present invention thus includes polypeptides of from 440-495 amino acid residues in length; polypeptides comprising affinity tags may be longer. Affinity tags can be used in combination.
  • Polypeptides comprising one or more affinity tags can further comprise a proteolytic cleavage site between the zapo3 polypeptide and the affinity tag(s). Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • Aromatic phenylalanine tryptophan tyrosine
  • the proteins of the present invention can also comprise non- naturally occuring amino acid residues.
  • Non-naturally occuring amino acids include, without limitation, fr * at7s-3-methylproline, 2,4-methanoproline, c/s-4- hydroxyproline, frat7s-4-hydroxyproline, ⁇ /-methylglycine, a//o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, terMeucine, norvaline, 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylalanine.
  • Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins, including in vitro 24
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occuring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) (Koide et al., Biochem. 33:7470- 7476, 1994).
  • Naturally occuring amino acid residues can be converted to non-naturally occuring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395- 403, 1993).
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502. 1991). Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989).
  • phage display e.g., Lowman et al., Biochem. 30:10832-10837, 1991 ; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204
  • region-directed mutagenesis e.g., region-directed mutagenesis
  • Variants of the disclosed zapo3 DNA and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391 , 1994 and Stemmer, Proc. Natl. Acad. Sci. 25
  • variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations.
  • This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process.
  • Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes. Amino acid sequence changes are made in zapo3 polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • coiled coil structure in the amino-terminal region of zapo3 and the sequence homology to fibrinogen in the carboxyl-terminal region, particularly the conserved cysteine residues. Changes in the coiled-coil region can be analyzed using analytical software available on the World Wide Web at http://ulrec3.unil.ch/software/COILS_form.html and http://ostrich.lcs.mit.edu/cgi-bin/score.
  • Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zapo3 variant polypeptides.
  • the chick chorioallantoic assay disclosed below can be carried out on large numbers of samples.
  • Assays that can be scaled up for high throughput include mitogenesis assays, which can be run in a 96-well format.
  • Mutagenized DNA molecules that encode active zapo3 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. 26
  • any zapo3 polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 2 and 3, above.
  • the zapo3 polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • a DNA sequence encoding a zapo3 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such 27
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of zapo3 (e.g., encoding residues 1-21 of SEQ ID NO:2), or may be derived from another secreted protein (e.g., t-PA; see, U.S. Patent No. 5,641 ,655) or synthesized de novo.
  • the secretory signal sequence is operably linked to the zapo3 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly sythesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
  • zapo3 polypeptides via a host cell secretory pathway are expected to result in the production of multimeric proteins.
  • multimers include both homomultimers and heteromultimers, the latter including proteins comprising only zapo3 polypeptides and proteins including zapo3 and heterologous polypeptides.
  • a heteromultimer comprising a zapo3 polypeptide and an angiopoietin-1 polypeptide can be produced by co-expression of the two polypeptides in a host cell.
  • a cDNA sequence encoding angiopoietin-1 is disclosed by Davis et al., Cell 87:1161-1169, 1996.
  • angiopoietin-2 cDNA is disclosed by Maisonpierre et al., Science 277:55-60, 1997. If a mixture of proteins results from expression, individual species are isolated by conventional methods. Monomers, dimers, and higher order multimers are separated by, for example, size exclusion chromatography. Heteromultimers can be separated from homomultimers by immunoaffinity chromatography using antibodies specific for individual dimers or by sequential immunoaffinity 28
  • Multimers may also be assembled in vitro upon incubation of component polypeptides under suitable conditions, which will generally include physiological pH and salt concentration. Recovery and assembly of proteins expressed in bacterial cells is disclosed below.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993).
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573;
  • adenovirus vectors can be employed. See, for example, Gamier et al., Cytotechnol. 15:145-55, 1994. 29
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • hygromycin resistance multi-drug resistance
  • puromycin acetyltransferase can also be used.
  • eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV).
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • Recombinant baculovirus can also be produced through the use of a transposon-based system described by Luckow et al. (J. Virol. 67:4566-4579, 1993).
  • This system which utilizes transfer vectors, is commercially available in kit form (Bac-to-BacTM kit; Life Technologies, Rockville, MD). See also, Hill— Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311 ; Kawasaki et al., U.S. Patent No. 4,931 ,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931 ,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311 ; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S.
  • Patent No. 4,977,092) and alcohol dehydrogenase genes See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661 ,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465. 1986 and Cregg, U.S. Patent No. 4,882,279. 31
  • Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533. The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in U.S. Patents No. 5,716,808 and No. 5,736,383, and WIPO Publications WO 97/17450 and WO97/17451.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
  • the polypeptide When expressing a zapo3 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the pehplasmic space by a bacterial secretion sequence.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the pehplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the pehplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as 32
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
  • proteins of the present invention it is preferred to purify the proteins of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • a purified protein is substantially free of other proteins, particularly other proteins of animal origin.
  • Zapo3 proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles & Methods. Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid.
  • coturnix japonica embryos can also be used (Drake et al., Proc. Natl. Acad. Sci. USA 92:7657-7661 , 1995). Briefly, a solution containing the protein is injected into the interstitial space between the endoderm and the splanchnic mesoderm of early-stage embryos using a micropipette and micromanipulator system. After injection, embryos are placed ventral side down on a nutrient agar medium and incubated for 7 hours at 37°C in a humidified CO 2 /air mixture (10%/90%). Vascular development is assessed by microscopy of fixed, whole-mounted embryos and sections.
  • Efficacy of zapo3 polypeptides in promoting wound healing can be assayed in animal models, including the linear skin incision model (Mustoe et al., Science 237:1333, 1987; Mustoe et al., J. Clin. Invest. 87:694, 1991); the rabbit ear ischemic wound model (Ahn et al., Ann. Plast. Surg. 24:17, 1990); and partial-thickness skin wound models using pigs or guinea pigs (LeGrand et al., Growth Factors 8:307, 1993).
  • Impaired wound healing models are also known in the art (e.g., Cromack et al., Surgery 113:36, 1993; Pierce et al., Proc. Natl. Acad. Sci. USA 86:2229, 1989; Greenhalgh et al., Amer. J. Pathol. 136:1235, 1990).
  • Subcutaneous implants can be used to assess compounds acting in the early stages of wound healing (Broadley et al., Lab. Invest. 61:571 , 1985; Sprugel et al., Amer. J. Pathol. 129: 601 , 1987).
  • Stimulation of coronary collateral growth can be measured in known animal models, including a rabbit model of peripheral limb ischemia and hind limb ischemia and a pig model of chronic myocardial ischemia (Ferrara et al., Endocrine Reviews 18:4-25, 1997). Zapo3 proteins are assayed in the presence and absence of VEGF and basic FGF to test for combinatorial effects. These models can be modified by the use of adenovirus or naked DNA for gene delivery as disclosed in more detail below, resulting in local expression of the test protein(s).
  • Angiogenic factors are also expected to find use in the reduction or prevention of restenosis following invasive procedures such as balloon 34
  • VEGF vascular endothelial growth factor
  • Angiogenic activity can also be tested in a rodent model of corneal neovascularization as disclosed by Muthukkaruppan and Auerbach, Science 205:1416-1418. 1979, wherein a test substance is inserted into a pocket in the cornea of an inbred mouse.
  • proteins are combined with a solid or semi-solid, biocompatible carrier, such as a polymer pellet.
  • Angiogenesis is followed microscopically. Vascular growth into the corneal stroma can be detected in about 10 days.
  • Angiogenic activity can also be tested in the hampster cheek pouch assay (H ⁇ ckel et al., Arch. Surg. 128:423-429, 1993).
  • a test substance is injected subcutaneiously into the cheek pouch, and after five days the pouch is examined under low magnification to determine the extent of neovascularization.
  • Tissue sections can also be examined histologically.
  • Induction of vascular permeability is measured in assays designed to detect leakage of protein from the vasculature of a test animal (e.g., mouse or guinea pig) after administration of a test compound (Miles and Miles, J. Physiol. 118:228-257, 1952; Feng et al., J. Exp. Med. 183:1981- 1986, 1996).
  • test animal e.g., mouse or guinea pig
  • a variety of in vitro assays can also be used to measure activity of zapo3 proteins.
  • a tridimensional collagen gel matrix model can be used to test the effects of zapo3 proteins on the formation of tube-like structures by microvascular endothelial cells. See, Pepper et al. Biochem. Biophys. Res. Comm. 189:824-831 , 1992 and Ferrara et al., Ann. NY Acad. Sci. 732:246-256, 1995.
  • Assays are carried out in the presence and absence of VEGF to assess possible combinatorial effects.
  • Matrigel models (Grant et al., "Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, 35
  • Mitogenic activity of zapo3 proteins can be measured using known assays, including 3 H-thymidine incorporation assays (as disclosed by, e.g., Raines and Ross, Methods Enzymol. 109:749-773, 1985), dye incorporation assays (Mosman, J. Immunol. Meth. 65: 55-63, 1983; Raz et al., Acta Trop. 68:139-147, 1997) or cell counts.
  • a preferred mitogenesis assay measures the incorporation of [ 3 H]-thymidine into pe -endothelial cells (including vascular smooth muscle cells, pericytes, and myocardiocytes), fibroblasts, or endothelial cells.
  • human dermal fibroblasts are plated at a density of approximately 8,000 cells/well in 24-well culture plates and grown for approximately 72 hours in a suitable culture medium, such as DMEM containing 10% fetal calf serum.
  • a suitable culture medium such as DMEM containing 10% fetal calf serum.
  • the cells are allowed to become quiescent, then exposed to a test solution.
  • [ 3 H]-thymidine is added and incubation is continued to allow growing cells to incorporate the label.
  • the cells are then harvested, and incorporation of label is determined according to standard procedures. See also, Gospodarowicz et al., J. Cell. Biol. 70:395-405, 1976; Ewton and Florini, Endocrinol. 106:577-583. 1980; and Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 86:7311 -7315, 1989.
  • Chemotaxis assays are carried out as generally described by Seppa et al. (J. Cell Biol. 92:584-588, 1982). Test solutions are placed in Boyden chambers and covered with 8 ⁇ m pore size polycarbonate filters coated with gelatin. 0.8 ml of a cell suspension ( ⁇ 3 x 10 5 cells/ml; peri- endothelial or mesodermal cells preferred) is added to the top compartment of 36
  • Hematopoietic activity of zapo3 proteins can be assayed on various hematopoietic cells in culture.
  • Preferred assays include primary bone marrow colony assays and later stage lineage-restricted colony assays, which are known in the art (e.g., Holly et al., WIPO Publication WO 95/21920).
  • Marrow cells plated on a suitable semi-solid medium e.g., 50% methylcellulose containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotic mix
  • Known hematopoietic factors are used as controls. Mitogenic activity of zapo3 proteins on hematopoietic cell lines can be measured as disclosed above.
  • Proteins of the present invention can be assayed in vivo through the use of viral delivery systems.
  • viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see Becker et al., Meth. Cell Biol. 43:161-189, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997).
  • adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. 37
  • adenovirus By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential E1 gene is deleted from the viral vector, and the virus will not replicate unless the E1 gene is provided by the host cell (e.g., the human 293 cell line).
  • the host cell e.g., the human 293 cell line.
  • the host's tissue e.g., liver
  • the host's tissue will express and process (and, if a signal sequence is present, secrete) the heterologous protein.
  • Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
  • Transgenic mice engineered to express a zapo3 gene, and mice that exhibit a complete absence of zapo3 gene function, referred to as "knockout mice” (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-742, 1993). These mice can be employed to study the zapo3 gene and the protein encoded thereby in an in vivo system. Transgenic mice are particularly useful for investigating the role of zapo3 proteins in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or 38
  • Zapo3 proteins may be used therapeutically to stimulate the revascularization of tissue.
  • Specific applications include, without limitation: the treatment of full-thickness skin wounds, including venous stasis ulcers and other chronic, non-healing wounds, particularly in cases of compromised wound healing due to diabetes mellitus, connective tissue disease, smoking, burns, and other exacerbating conditions; fracture repair; skin grafting; within reconstructive surgery to promote neovascularization and increase skin flap survival; to establish vascular networks in transplanted cells and tissues, such as transplanted islets of Langerhans; to treat female reproductive tract disorders, including acute or chronic placental insufficiency (an important factor causing perinatal morbidity and mortality) and prolonged bleeeding; to promote the growth of tissue damaged by periodontal disease; to promote endothelialization of vascular grafts and stents; in the treatment of acute and chronic lesions of the gastrointestinal tract, including duodenal ulcers, which are characterized by a defic
  • the proteins are also useful additives in tissue adhesives for promoting revascularization of the healing tissue.
  • Zapo3 proteins can be administered alone or in combination with other vasculogenic or angiogenic agents, including VEGF.
  • VEGF vasculogenic or angiogenic agents
  • basic and acidic FGFs and VEGF have been found to play a role in the development of collateral circulation, and the combined use of zapo3 with one or more of these factors may be advantageous.
  • VEGF has also been implicated in the survival of transplanted 39
  • islet cells (Gorden et al. Transplantation 63:436-443, 1997; Pepper, Arteriosclerosis, Throm. and Vascular Biol. 17:605-619, 1997).
  • Basic FGF has been shown to induce angiogenesis and accelerate healing of ulcers in experimental animals (reviewed by Folkman, Nature Medicine 1:27-31 , 1995).
  • the two compounds can be administered simultaneously or sequentially as appropriate for the specific condition being treated.
  • zapo3 proteins are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • pharmaceutical formulations will include a zapo3 protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
  • a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
  • Zapo3 will generally be used in a concentration of about 10 to 100 ⁇ g/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 ⁇ g/ml may be used.
  • the protein will be applied in the range of 0.1-10 ⁇ g/cm 2 of wound area, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
  • the therapeutic formulations will generally be administered over the period required for neovascularization, typically from one to several months and, in treatment of chronic conditions, for a year or more. Dosing is daily or intermittently over the period of treatment. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed. In general, a 40
  • therapeutically effective amount of zapo3 is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant reduction in time required for wound closure, a significant reduction in wound area, a significant improvement in vascularization, a significant reduction in morbidity, or a significantly increased histological score.
  • Zapo3 proteins are useful as research reagents, such as in the expansion of hematopoietic cells (including stem cells) and endothelial cells. Zapo3 proteins are added to tissue culture media for these cell types.
  • Zapo3 can also be used to identify inhibitors of its activity.
  • Test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of zapo3.
  • samples can be tested for inhibition of zapo3 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zapo3- dependent cellular responses.
  • zapo3-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zapo3- stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zapo3-activated serum response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase.
  • SRE zapo3-activated serum response element
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zapo3 on the target cells as evidenced by a decrease in zapo3 stimulation of reporter gene expression.
  • Assays of this type will detect compounds that directly block zapo3 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding.
  • compounds or other samples can be tested for direct blocking of zapo3 binding to receptor using zapo3 tagged with a detectable label (e.g., 125 l, biotin, horseradish peroxidase, FITC, and the like).
  • a detectable label e.g., 125 l, biotin, horseradish peroxidase, FITC, and the like.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • Zapo3 proteins and polypeptide fragments thereof can also be used to prepare antibodies that specifically bind to zapo3 polypeptides.
  • Polypeptides for use as immunogens include mature, full-length zapo3 polypeptides and fragments thereof. Multimeric proteins can also be used.
  • Preferred peptide immunogens include residues 43-48, 422-427, 421-426, 93- 98, and 120-125 of SEQ ID NO:2.
  • the term "antibodies” includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies.
  • Non-human antibodies can be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • One skilled in the art can generate humanized antibodies with specific and different constant domains (i.e., different Ig subclasses) to facilitate or inhibit various immune functions associated with particular antibody constant domains.
  • Antibodies are defined to be specifically binding if they bind to a zapo3 polypeptide with an affinity at least 10-fold greater than the binding affinity to control (non-zapo3) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) of 10 6 M “1 or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “1 or greater, and most preferably 10 9 M "1 or greater.
  • K a binding affinity
  • the affinity of a monoclonal antibody can be readily determined by 42
  • polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.
  • the immunogenicity of a zapo3 polypeptide may be increased through the use of an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zapo3 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid tetanus toxoid
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zapo3 polypeptide or protein, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zapo3 polypeptide).
  • Human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
  • Antibodies to zapo3 can be used for affinity purification of zapo3 proteins; within diagnostic assays for determining circulating levels of zapo3 proteins; for detecting or quantitating soluble zapo3 protein as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block protein activity in vitro and in vivo.
  • Antibodies to zapo3 may also be used for tagging cells that express zapo3; in analytical methods employing fluorescence-activated cell sorting (FACS); for screening expression libraries; and for generating anti-idiotypic antibodies.
  • FACS fluorescence-activated cell sorting
  • Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targetting of those compounds to cells expressing receptors for zapo3.
  • Antibodies of the present invention can, for example, be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • DNA probes and anti-zapo3 antibodies can be used to detect sites of angiogenesis. Because angiogenesis in adult animals is limited to wound healing and the female reproductive cycle, it is a very specific indicator of pathological processes. Angiogenesis is indicative of, for example, developing solid tumors, retinopathies, and arthritis.
  • the probe or antibody is labeled with a moiety that produces a detectable signal, such as a radionuclide, enzyme, contrast agent, or fluorophore, although labeled second antibodies or other labeled secondary agents can be employed.
  • the probe or antibody can be administered to the patient and detected in vivo by 44
  • Inhibitors of zapo3 activity include anti- zapo3 antibodies and soluble zapo3 receptors, as well as other peptidic and non-peptidic agents (including bozymes). Such antagonists can be used to block the vasculogenic/angiogenic effects of zapo3. Inhibition of angiogenesis should be well tolerated in adult patients because angiogenesis is required in the adult only for wound healing and reproduction.
  • antagonists of zapo3 activity in cancer therapy.
  • Angiogenesis inhibitors are also expected to be useful in adjunct therapy after surgery to prevent the growth of residual cancer cells.
  • Inhibitors can also be used in combination with other cancer therapeutic agents.
  • Inhibitors of zapo3 may also prove useful in the treatment of ocular neovascularization, including diabetic retinopathy and age-related macular degeneration. Experimental evidence suggests that these conditions result from the expression of angiogenic factors induced by hypoxia in the retina.
  • Zapo3 antagonists are also of interest in the treatment of inflammatory disorders, such as rheumatoid arthritis and psoriasis.
  • VEGF plays an important role in the formation of pannus, an extensively vascularized tissue that invades and destroys cartilage.
  • Psoriatic lesions are hypervascular and overexpress the angiogenic polypeptide IL-8.
  • Zapo3 antagonists may also prove useful in the treatment of infantile hemangiomas, which exhibit overexpression of VEGF and bFGF during the proliferative phase. 45
  • zapo3 inhibitors include small molecule inhibitors and angiogenically or mitogenically inactive receptor-binding fragments of zapo3 polypeptides.
  • Inhibitors are formulated for pharmaceutical use as generally disclosed above, taking into account the precise chemical and physical nature of the inhibitor and the condition to be treated. The relevant determinations are within the level of ordinary skill in the formulation art.
  • Other angiogenic and vasculogenic factors, including VEGF and bFGF, have been implicated in pathological neovascularization. In such instances it may be advantageous to combine a zapo3 inhibitor with one or more inhibitors of these other factors.
  • Polynucleotides encoding zapo3 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zapo3 activity.
  • Isner et al. The Lancet (ibid.) reported that VEGF gene therapy promoted blood vessel growth in an ischemic limb.
  • Additional applications of zapo3 gene therapy include stimulation of wound healing and repopulation of vascular grafts.
  • a gene encoding a zapo3 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus
  • AAV a defective herpes simplex virus 1 vector
  • HSV1 herpes simplex virus 1
  • HSV1 herpes simplex virus 1
  • attenuated adenovirus vector such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest.
  • a zapo3 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; WIPO Publication No.
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-8031 , 1988).
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages, including targeting of liposomes to specific cells. Directing transfection to particular cell types is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids can be chemically coupled to other molecules according to known procedures for the purpose of targeting. Peptides (e.g., hormones or neurotransmitters), proteins (including antibodies), and non-peptidic molecules can be chemically coupled to liposomes. It is also possible to remove target cells from the body, introduce a vector into the cells as a naked DNA plasmid, then re-implant the transformed cells into the body as disclosed above.
  • Antisense methodology can be used to inhibit zapo3 gene transcription, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a zapo3-encoding protein e.g., a polynucleotide as set forth in SEQ ID NO: 1 are designed to bind to zapo3- encoding mRNA and to inhibit translation of such mRNA.
  • Such antisense oligonucleotides are used to inhibit expression of zapo3 polypeptide-encoding genes in cell culture or in a patient. 47
  • Zapo3 proteins and anti-zapo3 antibodies can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • proteins or antibodies of the present invention may used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance).
  • zapo3 proteins or anti-zapo3 antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues, or organs that express the anti- complementary molecule.
  • Suitable detectable molecules can be directly or indirectly attached to the proteins or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like.
  • Suitable cytotoxic molecules can be directly or indirectly attached to the proteins or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic radionuclides, such as iodine-131 , rhenium-188 or ytt um-90. These can be either directly attached to the protein or antibody, or indirectly attached according to known methods, such as through a chelating moiety.
  • Proteins or antibodies can also be conjugated to cytotoxic drugs, such as adhamycin.
  • cytotoxic drugs such as adhamycin.
  • the detectable or cytotoxic molecule may be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the protein or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins may be used for targeted cell or tissue inhibition or ablation, such as in cancer therapy.
  • conjugates of a zapo3 polypeptide and a cytotoxin which can be 48
  • cytotoxin used to target the cytotoxin to a tumor or other tissue that is undergoing undesired angiogenesis or neovascularization.
  • zapo3-cytokine fusion proteins or antibody/fragment-cytokine fusion proteins may be used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers).
  • target tissues for example, blood and bone marrow cancers.
  • cytokines are toxic if administered systemically.
  • the described fusion proteins enable targeting of a cytokine to a desired site of action, such as a cell having binding sites for zapo3, thereby providing an elevated local concentration of cytokine.
  • Suitable cytokines for this purpose include, for example, interleukin-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Such fusion proteins may be used to cause cytokine-induced killing of tumors and other tissues undergoing angiogenesis or neovascularization.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • a bioactive conjugate is used to localize a radionuclide into a vessel that has been treated with angioplasty, endartarectomy, or stent emplacement.
  • a zapo3 protein or anti-zapo3 antibody is conjugated with a radionuclide, particularly with a beta-emitting or gamma-emitting radionuclide, and used to reduce restenosis in the treated area.
  • Localized delivery of the radionuclide is used to decrease tissue growth in the vessel, reduce revascularization and stent thrombosis, and increase luminal diameter.
  • bioactive proteins or antibody conjugates described herein can be delivered intravenously, intra-arterially or intraductally, or may be introduced locally at the intended site of action.
  • the present invention also provides reagents for diagnostic use.
  • a zapo3 gene, a probe comprising zapo3 DNA or RNA, or a subsequence thereof can be used to determine if the zapo3 gene is present on chromosome 1 of a human patient or if a mutation has occurred.
  • Detectable chromosomal aberrations at the zapo3 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; A.J. Marian, Chest 108:255-265. 1995).
  • RFLP restriction fragment length polymorphism
  • STR short tandem repeat
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-250, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels.
  • Commercially available radiation hybrid mapping panels that cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL), are available. These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest.
  • This technique allows one to establish directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
  • the precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining relationships between short sequences and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have. 50
  • EST expressed sequence tag
  • a human testis cDNA library was screened to obtain additional sequence for zapo3. PCR reactions were performed on DNA from a pooled human testis library representing one million clones and a pooled human testis library representing ten million clones. Using oligonucletide primers ZC15,851 (SEQ ID NO:5) and ZC15.858 (SEQ ID NO:6), an annealing temperature of 65°C, 35 cycles and a commercially available DNA polymerase/antibody mix (50X AdvantageTM KlenTaq Polymerase Mix, obtained from Clontech Laboratories, Inc., Palo Alto, CA), a -570 bp fragment was obtained in both reactions.
  • testis library representing one million clones was selected for further cloning.
  • PCR reactions were run on the testis library working plate, representing 80 pools of 12,000 colonies each.
  • oligonucleotide primers ZC15,851 SEQ ID NO:5
  • ZC15,858 SEQ ID NO:6
  • 12 positive pools with -570 bp fragments were obtained.
  • a 5' RACE reaction was performed on the positive pools to discern the longest product.
  • oligonucleotides ZC13,006 SEQ ID NO:7
  • ZC15,858 SEQ ID NO:6
  • an annealing temperature of 65°C 35 cycles and DNA polymerase/antibody mix (50X AdvantageTM KlenTaq Polymerase Mix, Clontech Laboratories, inc.)
  • pool D9 was found to have the longest product (-1.5 kb in size).
  • PCR reactions were then run on subpools of D9 (transfer plate 9 pools 5 E-H and 6 A-H).
  • oligonucleotide primers ZC15,851 SEQ ID NO:5
  • ZC15,858 SEQ ID 51
  • Lpif #107326 (also known as omega 52) was sequenced. The insert was found to be 1324 bp in size and appeared not to be full length. The 5' end was missing, but the 3' end had an apparent in-frame stop codon plus some 3' untranslated sequence. The 5' RACE sequence was found to overlap with the Lpif #107326 clone and contained a starting Met codon and upstream stop plus some 5' untranslated sequence. A full-length cDNA encoding zapo3 was then assembled.
  • the 5' race fragment was digested with EcoRI (GibcoBRL, Grand Island, NY) and BamHI (Boehringer Mannheim, Indianapolis, IN) in buffer B (Boehringer Mannheim, Indianapolis, IN), then purified using the QIAquickTM gel extraction kit.
  • Clone Lpif #107326 was digested with BamHI (Boehringer Mannheim) and Notl (New England Biolabs, Beverly, MA) in universal buffer (Stratagene, La Jolla, CA), and gel purified using the QIAquickTM gel extraction kit.
  • the mammalian expression vector pzp9 (comprising a mouse metallothionein-1 gene promoter) was linearized by digestion with EcoRI (GibcoBRL) and Notl (New England Biolabs) in buffer H (Boehringer Mannheim) and purified using the QIAquickTM gel extraction kit. The three fragments were then assembled in a three part ligation using T4 DNA ligase and T4 DNA ligase buffer (both from Life Technologies, Inc.). E. coli host cells (Electromax DH10BTM cells; obtained from Life Technologies, Inc., Gaithersburg, MD) were transformed with the ligation mixture. Clones were screened by colony PCR using oligonucleotide primers ZC15,858 (SEQ ID NO:6) and ZC13,006 (SEQ ID 52
  • Northern blot analysis was performed using a series of commercially available Northern blots (Multiple Tissue Northern Blots,
  • the DNA probe was generated using oligonucleotide primers ZC15,851 (SEQ ID NO:5) for the 5' end and ZC15,858 (SEQ ID NO:6) for the 3' end, and adrenal gland cDNA (Marathon-ReadyTM cDNA; Clontech Laboratories, Inc.) as template according to the supplier's instructions.
  • the DNA probe was gel purified using a QIAquickTM gel extraction kit.
  • the probe was radioactively labeled with 32 P using a commercially available labeling kit (MultiprimeTM DNA labeling system, Amersham Corp., Arlington Heights, IL) according to the manufacturer's specifications.
  • the probe was purified using a push column (NucTrap® column; Stratagene, La Jolla, CA; see U.S. Patent No. 5,336,412).
  • a commercially available hybridization solution (ExpressHybTM Hybridization Solution; Clontech Laboratories, Inc.) was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 65°C, and the blots were then washed four times in 2X SCC and 0.05% SDS at room temperature, followed by two washes in 0.1X SSC and 0.1 % SDS at 50°C. The blots were then exposed to film overnight at -80°C. Two transcript sizes were observed: a transcript of -4.0 kb was seen in heart, placenta, skeletal muscle, spleen, testis, ovary, small intestine, thyroid, and 53
  • a transcript of -2.8 kb was seen in heart, placenta, skeletal muscle, kidney, pancreas, spleen, testis, ovary, small intestine, colon, stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, and bone marrow. Signal intensity was highest in thyroid for both transcript sizes.
  • HUVEC human umbilical vein endothelial cells; Cascade Biologies, Inc., Portland, OR
  • HPAEC human pulmonary artery endothelial cells
  • Cascade Biologies, Inc. human aortic endothelial cells
  • the DNA probe was gel-purified using a QIAquickTM gel extraction kit (Qiagen).
  • the probe was radioactively labeled with 32 P using a commercially available labeling kit (MultiprimeTM DNA labeling system, Amersham Corp., Arlington Heights, IL) according to the manufacturer's specifications.
  • the probe was purified using a push column (NucTrap® column; Stratagene). Prehybridization and hybridization were carried out essentially as disclosed in Example 2. The blots were then washed four times in 2X SCC and 0.05% SDS at room temperature, followed by two washes in 0.1X SSC and 0.1 % SDS at 50°C.
  • a third wash was done at 56°C in 0.1X SSC and 0.1 % SDS, with a final wash at 61 °C in 0.1X SSC and 0.1 % SDS.
  • the blots were then exposed to film for three days at -80°C.
  • a transcript of ⁇ 2.4kb was seen in HISM, SK-5 and NHLF cells. Signal intensity was highest in HISM cells.
  • the zapo3 sequence was mapped to chromosome 1 using the commercially available GeneBridge 4 Radiation Hybrid Panel (Research).
  • This panel contains PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient).
  • a publicly available WWW server http://www- genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR” radiation hybrid map), which was constructed with the GeneBridge 4 Radiation Hybrid Panel. For mapping zapo3, 20- ⁇ l reaction mixtures were set up in a
  • PCRable 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a thermal cycler (RoboCycler® Gradient 96; Stratagene) and commercially available reagents (AdvantageTM KlenTaq Polymerase Kit, Clontech Laboratories, Inc.).
  • Each of the 95 PCR reactions contained 2 ⁇ l buffer (10X KlenTaq PCR reaction buffer, Clontech Laboratories, Inc.), 1.6 ⁇ l dNTPs mix 55
  • the PCR cycler conditions were as follows: an initial 5-minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 60°C, and 1.5 minute extension at 72°C; followed by a final extension of 7 minutes at 72°C.
  • the reaction products were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).
  • the antibody was affinity purified on a CNBR-Sepharose- peptide column built using 10mg peptide per gram of Sepharose. Purified antibody was dialyzed overnight against PBS.
  • Plasmid pC4His contains a Bam HI cloning site downstream of a mouse metalothionein (mMT-1) promoter and upstream of DNA encoding two glycine residues followed by 6 histidine residues and a stop codon. In-frame insertion into the Bam HI site results in a sequence encoding a protein with a carboxy terminal extension of two glycine residues and 6 histidine residues.
  • the plasmid also contains an ampicillin resistance gene for selection in E. coli and a dihydrofolate reductase gene for selection in mammalian cells.
  • Plasmid pC4His was cut with Bam HI and treated with calf intestine alkaline phosphatase (Boehringer Mannheim) according to the maufacturer's directions.
  • the linear plasmid DNA was purified by agarose gel electrophoresis.
  • the zapo3 cDNA was amplified in two seperate reactions using DNA polymerase mix (ExpandTM; Boehringer Mannheim, Indianapolis, IN) and the maufacturer's suggested conditions.
  • the cDNA template was amplified in 25 cycles, denaturing 94°C for 30 seconds, annealing 60°C for 30 seconds, and extending at 68°C for 45 seconds.
  • the DNA was cut with Bgl II and purified by agarose gel electrophoresis.
  • amplification was directed with the oligonucleotides zc20538 (SEQ ID NO:11) and zc20539 (SEQ ID NO:12).
  • the amplified DNA included the 57
  • BHK-570 cells were transfected with 10 ⁇ g pC4zAPO3 or 10 ⁇ g pC4zAPO3.His using a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1- propaniminium-trifluoroacetate and the neutral lipid dioleoyl phosphatidylethanolamine in membrane-filtered water (LipofectamineTM Reagent; Life Technologies, Gaithersburg, MD) and following the manufacturer's instructions. Stable transfectants were selected by 10 days culture in the presence of 5 ⁇ M methotrexate.
  • Fluorescence activated cell sorting analysis, using the rabbit poyclonal antibody Rb-antizAPO3.1 (D8449), revealed that hzapo3 was localized to the cell surface of the BHK cells transfected with either pC4zAPO3 or pC4zAPO3.His. Further analysis of BHK cells transfected with pC4zAPO3.His, using a monoclonal antibody directed against the His tag, revealed that the His epitope was also localized to the cell surface. 58
  • CM Conditioned media
  • CM from untreated cells or those treated with 250 ng/mL hFGF-2, 100 ng/mL hVegF, 100 mM PMA, or 1 ⁇ M forskolin contained no hzAPO3.
  • FACS analysis of BHK cells expressing hzapo3 revealed that cell surface-associated hzapo3 was released within one hour of treatment with 100 ng/mL LPS, suggesting inflammatory-regulated membrane shedding of zapo3 protein.
  • zapo3 Native human and mouse zapo3 were analyzed by analysis of tissues that in which the proteins were expressed. By northern blot analysis zapo3 was highly expressed in adrenals in both human and mouse.

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Abstract

L'invention concerne des polypeptides angiogéniques, leurs méthodes de production, des polynucléotides les codant et leurs méthodes d'utilisation. Les polypeptides comprennent une séquence de résidus d'acides aminés identique au moins à 90 % dans la séquence d'acides aminés aux résidus 22 à 491 de SEQ. ID NO 2. Les polypeptides s'utilisent pour l'étude et la régulation d'une angiogenèse et pour le développement d'inhibiteurs d'angiogenèse.
PCT/US1999/002303 1998-02-04 1999-02-03 Zapo3 homologue d'angiopoietine, adn le codant et sa methode de production WO1999040193A1 (fr)

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WO2000011164A1 (fr) * 1998-08-20 2000-03-02 Incyte Pharmaceuticals, Inc. Angiopoietine-3 humaine
WO2000005369A3 (fr) * 1998-07-20 2000-06-15 Curagen Corp Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese
US6537554B1 (en) 1998-09-10 2003-03-25 Curagen Corporation Nucleotide sequences and amino acid sequences of secreted proteins involved in angiogenesis
EP1308511A1 (fr) * 2001-10-31 2003-05-07 Mermaid Pharmaceuticals GmbH Utilisation des proteines 1 et 2 associes a l'angiopoietine et des acides nucleiques correspondants pour le traitement des troubles associes au sang et des defauts du systeme vasculaire
US6753321B2 (en) 2000-09-15 2004-06-22 Genvec, Inc. Method of modulating neovascularization
WO2005003782A1 (fr) * 2003-06-30 2005-01-13 Genova Ltd. Especes polypeptides secretees dont le taux diminue en cas de maladies cardiovasculaires

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000005369A3 (fr) * 1998-07-20 2000-06-15 Curagen Corp Sequences nucleotidiques et sequences d'acides amines de proteines secretees en rapport avec l'angiogenese
WO2000011164A1 (fr) * 1998-08-20 2000-03-02 Incyte Pharmaceuticals, Inc. Angiopoietine-3 humaine
US6537554B1 (en) 1998-09-10 2003-03-25 Curagen Corporation Nucleotide sequences and amino acid sequences of secreted proteins involved in angiogenesis
US6753321B2 (en) 2000-09-15 2004-06-22 Genvec, Inc. Method of modulating neovascularization
EP1308511A1 (fr) * 2001-10-31 2003-05-07 Mermaid Pharmaceuticals GmbH Utilisation des proteines 1 et 2 associes a l'angiopoietine et des acides nucleiques correspondants pour le traitement des troubles associes au sang et des defauts du systeme vasculaire
WO2005003782A1 (fr) * 2003-06-30 2005-01-13 Genova Ltd. Especes polypeptides secretees dont le taux diminue en cas de maladies cardiovasculaires

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