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WO1999032515A2 - Homologue de l'angiopoietine, adn le codant et procede de production dudit homologue - Google Patents

Homologue de l'angiopoietine, adn le codant et procede de production dudit homologue Download PDF

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Publication number
WO1999032515A2
WO1999032515A2 PCT/US1998/027055 US9827055W WO9932515A2 WO 1999032515 A2 WO1999032515 A2 WO 1999032515A2 US 9827055 W US9827055 W US 9827055W WO 9932515 A2 WO9932515 A2 WO 9932515A2
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Prior art keywords
polypeptide
zapol
sequence
amino acid
residues
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PCT/US1998/027055
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English (en)
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WO1999032515A3 (fr
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Scott R. Presnell
Darrell C. Conklin
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Zymogenetics, Inc.
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Priority to AU19315/99A priority Critical patent/AU1931599A/en
Publication of WO1999032515A2 publication Critical patent/WO1999032515A2/fr
Publication of WO1999032515A3 publication Critical patent/WO1999032515A3/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. These cells then differentiate, and mature vessels are formed. This process of growth and differentiation is regulated by a balance of pro-angiogenic and anti- angiogenic factors.
  • Angiogenesis occurs during embryonic development, as well as in the adult organism during pregnancy, the female reproductive cycle, and wound healing.
  • 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 act through at least three cell surface receptors, designated Flt-1 , Flk-1 , and Flt-4.
  • Flt-1 cell surface receptors
  • Flk-1 cell surface receptors
  • Flt-4 cell surface receptors
  • the expression of these receptors is limited to certain cell types and/or 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 Ang-1 ; see, Davis et al., Cell 87: 1161 -1169, 1996; and Davis et al., U.S. Patent No.
  • 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 87:2212-2220, 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).
  • 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 16(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). The hematopoietic cytokine erythropoietin has been developed for the treatment of 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).
  • growth factors and cytokines there is a need in the art for additional such molecules for use as both therapeutic agents and research tools and reagents.
  • an isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical in amino acid sequence to residues 31 to 406 of SEQ ID NO:2.
  • the polypeptide is at least 95% identical in amino acid sequence to residues 31 to 406 of SEQ ID NO:2.
  • the polypeptide is from 346 to 409 amino acids in length.
  • the polypeptide comprises residues x-406 of SEQ ID NO:2, wherein x is 23, 24, or 31.
  • the polypeptide comprises residues x-406 of SEQ ID NO:2, wherein x is 23, 24, or 31 , and is from 346 to 409 amino acids in length.
  • the polypeptide is covalently linked to a moiety selected from the group consisting of affinity tags, toxins, radionuclides, enzymes, and fluorophores.
  • affinity tags include polyhistidine, protein A, glutathione S transferase, substance P, and an immunoglobulin heavy chain constant region.
  • Polypeptides comprising an affinity tag may further comprise a proteolytic cleavage site to facilitate removal of the affinity tag.
  • an isolated multimeric protein comprising a first polypeptide chain as disclosed above, and a second polypeptide chain, wherein said protein is angiogenic.
  • the first and second polypeptide chains may be the same or different.
  • a protein produced by a method comprising the steps of (a) culturing 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.
  • 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 vector further comprises a secretory signal sequence operably linked to the DNA segment.
  • 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.
  • the proteins and antibodies of the present invention can be used as medicaments for the regulation of angiogenesis.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection 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.
  • 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.
  • the terms "ammo-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 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. Such 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 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.
  • 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.
  • 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 "zapol” exhibits significant amino acid sequence homology to the previously described angiopoietin-1 (Davis et al., Cell 87:1161-1169, 1996).
  • one of the polypeptides of the present invention is approximately 35% identical to Ang-1 when the sequences are aligned to produce a 295 amino acid residue overlap (residues 109-400 of SEQ ID NO:2), accounting for overlaps as disclosed below.
  • the polypeptide is approximately 33% identical to Ang-2 when the sequences are aligned to produce a 296 amino acid residue overlap (residues 108-399 of SEQ ID NO:2)
  • angiopoietin-1 , angiopoietin-2, and zapol are characterized by an amino-terminal coiled coil domain and a carboxyl-terminal fibrinogen-like domain.
  • residues 46-150 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.
  • Residues 183-366 of SEQ ID NO:2 show marked homology (approximately 40% sequence identity) to residues 627-825 of human fibrinogen alpha chain (SEQ ID NO:3).
  • This domain of zapol contains conserved cysteine residues at positions 188, 216, 341 , and 354 of SEQ ID NO:2.
  • conserved residues are also located at positions 224, 230, 237, 244, 247, 248, 254, 256, 257, 258, 300, 326, 332, 349, 350, 361, and 363 of SEQ ID NO:2.
  • SEQ ID NO:2 further indicates the presence of an amino-terminal secretory peptide of from 22 to 30 residues. Based on alignment with related proteins and prediction of proteolytic cleavage sites, the amino terminus of the mature protein is predicted to begin at residue 23 (Ala), 24 (Gin), or 31 (Lys). Those skilled in the art will recognize that domain boundaries are approximations based on sequence alignments, intron positions, and splice sites, and that the actual structure may vary somewhat from the prediction. For example, the coiled-coil domain of zapol may extend up to 20-25 residues beyond the boundaries specified above.
  • the present invention is not limited to the expression of the sequence shown in SEQ ID NO:1.
  • a number of truncated zapol 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 zapol polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide.
  • expressing the complete zapol 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.
  • zapol polypeptides can thus be produced.
  • zapol polypeptides can be produced by other known methods, such as solid phase synthesis, methods for which are well known in the art. Differential processing of individual chains may result in heterogeneity of expressed polypeptides and the production of heterodimeric zapol proteins.
  • the presence of a coiled coil domain in the angiopoietins suggests that these proteins are assembled into multimers.
  • the present invention thus includes both heteromultimers and homomultimers comprising zapol polypeptide subunits.
  • Heteromultimers may be formed by association of distinct zapol polypeptides or by association of one or more zapol polypeptides with, for example, an angiopoietin-1 or -2 polypeptide.
  • the term "multimer” includes dimers and higher order multimers.
  • Zapol 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 zapol 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.
  • SEQ ID NO:4 is a degenerate DNA sequence that encompasses all DNAs that encode the zapol polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:4 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T.
  • zapol polypeptide- encoding polynucleotides comprising nucleotide 1 to nucleotide 1218 of SEQ ID NO: 4 and their RNA equivalents are contemplated by the present invention.
  • Table 1 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. TABLE 1
  • degenerate codons used in SEQ ID NO:4, encompassing all possible codons for a given amino acid, are set forth in Table 2, below.
  • Met M ATG ATG lie 1 ATA ATC ATT ATH
  • Gap - One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine WSN
  • the degenerate codon for arginine AGR
  • the degenerate codon for arginine MGN
  • some polynucleotides encompassed by the degenerate sequence may 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.
  • RNA is isolated from a tissue or cell that produces large amounts of zapol RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201 , 1980), and include placenta, umbilical vein endothelial cells, lung, liver, thyroid, spinal cord, fetal brain, heart, and uterus. Total RNA can be prepared using guanidine HCI extraction followed by isolation by centrifugation in a CsCI gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972).
  • Complementary DNA cDNA is prepared from poly(A) + RNA using known methods.
  • genomic DNA can be isolated.
  • Polynucleotides encoding zapol polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding zapol can be obtained by conventional cloning procedures.
  • Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
  • Methods for preparing 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.
  • Expression libraries can be probed with antibodies to zapol , receptor fragments, or other specific binding partners.
  • sequences disclosed in SEQ ID NOS:1 , 2, and 4 represent a single allele of human zapol . 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").
  • zapol polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • Species orthologs of human zapol 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 zapol 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 zapol -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 representative human zapol 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 zapol polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • 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 species ortholog thereof. It is preferred that these engineered variant polypeptides are at least 90% identical to the polypeptide of SEQ ID NO:2. Such polypeptides will more preferably be 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 zapol 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 4) 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.
  • the present invention thus includes polypeptides of from 346 to 409 amino acid residues that comprise a sequence that is at least 90%, and preferably 95% or more identical to the corresponding region of SEQ ID NO:2.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zapol polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • the proteins of the present invention can also comprise non- naturally occuring amino acid residues.
  • Non-naturally occuring amino acids include, without limitation, frat7s-3-methylproline, 2,4-methanoproline, cis-4- hydroxyproline, frans-4-hydroxyproline, ⁇ /-methylglycine, a//o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tet ⁇ -leucine, norvaline, 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylalanine.
  • E. 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.
  • mutagenesis and screening 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).
  • Other methods that can be used include 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) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA T ⁇ 27, 1988).
  • Amino acid sequence changes are made in zapol polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • it is generally preferred to retain the coiled coil structure in the amino-terminal region of zapol 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.
  • variant genes of the disclosed zapol DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391 , 1994 and Stemmer, Proc. Natl. Acad. Sci. USA 9J . :10747-10751 , 1994. Briefly, 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.
  • Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zapol 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 zapol 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.
  • any zapol 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.
  • zapol 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.
  • 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 Graham et al., J. Gen. Virol. 36:59-72, 1977
  • Chinese hamster ovary e.g. CHO-K1 ; ATCC No. CCL 61
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA.
  • Other higher 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.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV).
  • baculovirus commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See, King and Possee, The Baculovirus Expression System: A Laboratory Guide. London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual. New York, Oxford University Press., 1994; and Richardson, Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Humana Press, Totowa, NJ, 1995.
  • Recombinant baculovirus can also be produced through the use of a transposon-based system described by Luckow et al. (J. Virol.
  • 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. 4,845,075. Transformation systems for other yeasts, including Hansenula polymorpha ,
  • Kluyveromyces fragilis , ⁇ stilago 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. 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.
  • a DNA sequence encoding a zapol 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 elements are described in the literature and are available through commercial suppliers.
  • 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 zapol , 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 zapol 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).
  • 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 growth factors or serum, as required.
  • 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.
  • polypeptides of the present invention it is preferred to purify the polypeptides 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 polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Zapol polypeptides 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.
  • Polypeptides comprising a polyhistidine affinity tag are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988.
  • zapol polypeptides on primordial endothelial cells in angiogenesis can be assayed in the chick chorioallantoic membrane angiogenesis assay (Leung, Science 246: 1306-1309, 1989; Ferrara, Ann. NY Acad. Sci. 752:246-256. 1995). Briefly, a small window is cut into the shell of an eight-day old fertilized egg, and a test substance is applied to the chorioallantoic membrane. After 72 hours, the membrane is examined for neovascularization. Embryo microinjection of early stage quail (Coturnix coturnix japonica) embryos can also be used (Drake et al., Proc. Natl. Acad. Sci.
  • a solution containing the polypeptide is injected into the interstitial space between the endoderm and the splanchnic mesoderm of early-stage embryos using a micropipette and micomanipulator 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 zapol 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.
  • 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). Zapol 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 angioplasty and stent placement.
  • VEGF has been shown to promote vessel re-endothelialization and to reduce intimal hyperplasia in animal models of restenosis (Asahara et al., Circulation 91 :2802-2809, 1995; Callow et al., Growth Factors 10:223-228, 1994); efficacy of zapol polypeptides can be tested in these and other known models.
  • 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; and in the hampster cheek pouch assay (H ⁇ ckel et al., Arch. Surg. 128:423-429, 1993), wherein a test substance is injected subcutaneously 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 zapol polypeptides.
  • a tridimensional collagen gel matrix model can be used to test the effects of zapol polypeptides 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, Birkhauser Veriag, 1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997) are used to determine effects of zapol polypeptides on tube formation by endothelial cells.
  • Matrigel a basement membrane extract enriched in laminin, is used to coat tissue culture plates and allowed to polymerize. The gel is then seeded with endothelial cells, and test samples are applied. Angiogenic activity is indicated by cell migration and reorganization resulting in the formation of tube-like structures. It is preferred to test zapol polypeptides in angiogenesis assays in the presence and absence of VEGF because of potential combinatorial effects of the two factors. It is also preferred to use VEGF as a control within in vivo assays.
  • Hematopoietic activity of zapol 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 zapol polypeptides on hematopoietic cell lines can be measured using 3 H-thymidine incorporation assays, dye incorporation assays, or cell counts (Raines and Ross, Methods Enzymol. 109:749-773. 1985; Foster et al., U.S. Patent No. 5,641 ,655; Mosman, J. Immunol. Meth. 65: 55-63, 1983; and Raz et al., Ada Trop. 68:139-147, 1997).
  • 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.
  • 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.
  • the adenovirus system can also be used for protein production in vitro.
  • the cells By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al., Cvtotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non-secreted proteins can also be effectively obtained.
  • Transgenic mice engineered to express a zapol gene, and mice that exhibit a complete absence of zapol gene function, referred to as "knockout mice” (Snouwaert et al., Science 257:1083, 1992), may also be generated (Lowell et al., Nature 366:740-742, 1993). These mice may be employed to study the zapol gene and the protein encoded thereby in an in vivo system. Transgenic mice are particularly useful for investigating the role of zapol polypeptides in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or underexpression of a specific factor. See also, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science 277:48-50, 1997.
  • Zapol polypeptides 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
  • the polypeptides are also useful additives in tissue adhesives for promoting revascularization of the healing tissue.
  • Zapol polypeptides 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 zapol with one or more of these factors may be advantageous.
  • VEGF has also been implicated in the survival of transplanted islet cells (Gorden et al. Transplantation 63:436-443, 1997; Pepper, Arteriosclerosis, Throm. and Vascular Biol. 17:605-619, 1997).
  • the zapol polypeptides are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • pharmaceutical formulations will include a zapol polypeptide 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.
  • Zapol will preferably 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 0J-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.
  • a therapeutically effective amount of zapol is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant reduction in time required by wound closure, a significant reduction in wound area, a significant improvement in vascularization, a significant reduction in morbidity, or a significantly increased histological score.
  • Zapol polypeptides are useful as research reagents, such as in the expansion of hematopoietic cells (including stem cells) and endothelial cells. Zapol polypeptides are added to tissue culture media for these cell types.
  • Zapol 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 zapol In addition to those assays disclosed above, samples can be tested for inhibition of zapol activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zapol - dependent cellular responses.
  • zapol -responsive cell lines can be transfected with a reporter gene construct that is responsive to a zapol - stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zapol -activated serum response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase.
  • SRE zapol -activated serum response element
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zapol on the target cells as evidenced by a decrease in zapol stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zapol binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of zapol binding to receptor using zapol 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.
  • Zapol polypeptides can also be used to prepare antibodies that specifically bind to zapol polypeptides.
  • 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 only 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.
  • 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.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zapol polypeptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zapol polypeptide).
  • Antibodies are defined to be specifically binding if they bind to a zapol polypeptide with an affinity at least 10-fold greater than the binding affinity to control (non-zapo1) polypeptide.
  • 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 one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51 660-672, 1949).
  • 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 zapol 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 zapol 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
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid for immunization.
  • Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to zapol polypeptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zapol polypeptide).
  • assays known to those skilled in the art can be utilized to detect antibodies that specifically bind to zapol polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include concurrent immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
  • ELISA enzyme-linked immunosorbent assays
  • Antibodies to zapol may be used for affinity purification of zapol polypeptides; within diagnostic assays for determining circulating levels of zapol polypeptides; for detecting or quantitating soluble zapol polypeptide 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 zapol may also be used for tagging cells that express zapol ; for affinity purification of zapol polypeptides; in analytical methods employing FACS; for screening expression libraries; and for generating anti-id iotypic antibodies.
  • DNA probes and anti-zapol 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 conventional scanning methodologies, or can be used to screed biopsy or other tissue samples in vitro.
  • Inhibitors of zapol activity include anti- zapol antibodies and soluble zapol receptors, as well as other peptidic and non-peptidic agents (including ribozymes). Such antagonists can be used to block the vasculogenic/angiogenic effects of zapol 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 zapol 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 zapol 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.
  • Zapol 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.
  • Zapol antagonists may also prove useful in the treatment of infantile hemangiomas, which exhibit overexpression of VEGF and bFGF during the proliferative phase.
  • zapol inhibitors include small molecule inhibitors and angiogenically or mitogenically inactive receptor-binding fragments of zapol polypeptides.
  • Inhibitors are formulated for pharmaceutic 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.
  • zapol inhibitor may be advantageous to combine a zapol inhibitor with one or more inhibitors of these other factors.
  • Polynucleotides encoding zapol polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zapol activity. For example, Isner et al., The Lancet (ibid.) reported that VEGF gene therapy promoted blood vessel growth in an ischemic limb. Additional applications of zapol gene therapy include stimulation of wound healing and repopulation of vascular grafts.
  • a gene encoding a zapol protein 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), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.
  • HSV1 herpes simplex virus
  • adenovirus vector such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-630, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol. 613096-3101, 1987; Samulski et al., J. Virol. 63:3822-3828, 1989). 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 zapol gene transcription, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a zapol -encoding protein e.g., a polynucleotide as set forth in SEQ ID NO: 1 are designed to bind to zapol- encoding mRNA and to inhibit translation of such mRNA.
  • Such antisense oligonucleotides are used to inhibit expression of zapol polypeptide-encoding genes in cell culture or in a patient.
  • Zapol polypeptides and anti-zapol 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.
  • polypeptides or antibodies of the present invention may be used to identify or treat tissues or organs that express a corresponding anti- complementary molecule (receptor or antigen, respectively, for instance).
  • zapol polypeptides or anti-zapol 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 polypeptide 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 polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131 , rhenium-188 or yttrium-90. These can be either directly attached to the polypeptide or antibody, or indirectly attached according to known methods, such as through a chelating moiety. Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin.
  • the detectable or cytotoxic molecule may be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide 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 zapol polypeptide and a cytotoxin which can be used to target the cytotoxin to a tumor or other tissue that is undergoing undesired angiogenesis or neovascularization.
  • zapol -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 zapol 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.
  • a zapol polypeptide or anti-zapol antibody can be conjugated with a radionuclide, particularly with a beta- emitting or gamma-emitting radionuclide, and used to reduce restenosis.
  • bioactive polypeptide 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 zapol gene, a probe comprising zapol DNA or RNA, or a subsequence thereof can be used to determine if the zapol gene is present on chromosome 19 of a human patient or if a mutation has occurred.
  • Detectable chromosomal aberrations at the zapol 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).
  • 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).
  • a partial zapol cDNA was radiolabeled using a commercially available labeling kit (MultiprimeTM DNA labeling system, Amersham Corp., Arlington Heights, IL). The labeled DNA was used to probe a series of
  • the zapol sequence was mapped to chromosome 19 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.
  • WWW server http://www- genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl
  • PCRable 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a RoboCycler® Gradient 96 thermal cycler (Stratagene) and commercially available reagents (AdvantageTM KlenTaq Polymerase Kit, Clontech Laboratories, Inc., Palo Alto, CA).
  • Each of the 95 PCR reactions contained 2 ⁇ l buffer (10X KlenTaq PCR reaction buffer, Clontech Laboratories, Inc.), 1.6 ⁇ l dNTPs mix (2.5 mM each, Perkin-Elmer, Foster City, CA), 1 ⁇ l sense primer, ZC 14,749 (SEQ ID NO:7), 1 ⁇ l antisense primer, ZC 14,745 (SEQ ID NO:8), 2 ⁇ l of a density increasing agent and tracking dye (RediLoad, Research Genetics, Inc., Huntsville, AL), 0.4 ⁇ l of a commercially available DNA polymerase/antibody mix (50X AdvantageTM KlenTaq Polymerase Mix, obtained from Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH 2 O to a total volume of 20 ⁇ l.
  • 2 ⁇ l buffer 10X KlenTaq PCR reaction buffer, Clontech Laboratories, Inc.
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • 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 64°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).
  • Proximal and distal framework markers were AFMA134XB9 and RP_S28_1 , respectively.
  • the use of surrounding markers positions zapol in the 19p13.3-p13.2 region on the integrated LDB chromosome 19 map (The Genetic Location Database, University of Southhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html/).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Biochemistry (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Vascular Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

On décrit des polypeptides angiogéniques, des procédés concernant leur production, des polynucléotides les codant et des procédés relatifs à leur utilisation. Les polypeptides comprennent une séquence d'acides aminés résiduaires identique au moins à 90% en acides aminés aux résidus 31 à 406 de la SEQ ID NO:2. Ces polypeptides sont utiles pour l'étude et la régulation de l'angiogenèse et pour la mise au point d'inhibiteurs de l'angiogenèse.
PCT/US1998/027055 1997-12-19 1998-12-17 Homologue de l'angiopoietine, adn le codant et procede de production dudit homologue WO1999032515A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU19315/99A AU1931599A (en) 1997-12-19 1998-12-17 Angiopoietin homolog, dna encoding it, and method of making it

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6826897 1997-12-19
US60/068,268 1997-12-19

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WO1999032515A2 true WO1999032515A2 (fr) 1999-07-01
WO1999032515A3 WO1999032515A3 (fr) 1999-09-10

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999067382A3 (fr) * 1998-06-24 2000-04-27 Compugen Ltd Sequences d'un facteur de croissance apparente a l'angiopoietine
WO2001002429A3 (fr) * 1999-07-02 2001-03-15 Bayer Ag Angiopoietine 6 et ses utilisations
KR20010070139A (ko) * 1999-10-14 2001-07-25 오철준, 윤혜진 사람 안지오포이에틴-2-443 단백질 및 그 암호화 유전자
WO2001047951A3 (fr) * 1999-12-23 2002-04-25 Duncan John Stewart Analogues de l'angiopoietine
WO2001077151A3 (fr) * 2000-04-07 2002-09-12 Millennium Pharm Inc Nouvelles molecules de proteines et d'acide nucleique fdrg et utilisation desdites molecules
EP1058737A4 (fr) * 1998-03-02 2003-01-02 Millennium Pharm Inc Nouvelles molecules fdrg de proteines et d'acide nucleique et utilisation desdites molecules
EP1484401A1 (fr) * 1999-07-20 2004-12-08 Curagen Corporation Protéines secrètées et polynicleotides correspondant
US7371384B2 (en) 2004-07-20 2008-05-13 Genentech, Inc. Compositions and methods of using angiopoietin-like 4 protein antibody
US7740846B2 (en) 2004-07-20 2010-06-22 Genentech, Inc. Inhibitors of angiopoietin-like 4 protein, combinations, and their use
US8084200B2 (en) 2002-11-15 2011-12-27 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
US8604185B2 (en) 2004-07-20 2013-12-10 Genentech, Inc. Inhibitors of angiopoietin-like 4 protein, combinations, and their use

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2240492A (en) * 1991-06-20 1993-01-25 United States Of America, As Represented By The Secretary, Department Of Health And Human Services, The Sequences characteristic of human gene transcription product
US5814464A (en) * 1994-10-07 1998-09-29 Regeneron Pharma Nucleic acids encoding TIE-2 ligand-2
US6030831A (en) * 1997-09-19 2000-02-29 Genetech, Inc. Tie ligand homologues

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1058737A4 (fr) * 1998-03-02 2003-01-02 Millennium Pharm Inc Nouvelles molecules fdrg de proteines et d'acide nucleique et utilisation desdites molecules
WO1999067382A3 (fr) * 1998-06-24 2000-04-27 Compugen Ltd Sequences d'un facteur de croissance apparente a l'angiopoietine
WO2001002429A3 (fr) * 1999-07-02 2001-03-15 Bayer Ag Angiopoietine 6 et ses utilisations
EP1484401A1 (fr) * 1999-07-20 2004-12-08 Curagen Corporation Protéines secrètées et polynicleotides correspondant
KR20010070139A (ko) * 1999-10-14 2001-07-25 오철준, 윤혜진 사람 안지오포이에틴-2-443 단백질 및 그 암호화 유전자
WO2001047951A3 (fr) * 1999-12-23 2002-04-25 Duncan John Stewart Analogues de l'angiopoietine
WO2001077151A3 (fr) * 2000-04-07 2002-09-12 Millennium Pharm Inc Nouvelles molecules de proteines et d'acide nucleique fdrg et utilisation desdites molecules
US8084200B2 (en) 2002-11-15 2011-12-27 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
US7371384B2 (en) 2004-07-20 2008-05-13 Genentech, Inc. Compositions and methods of using angiopoietin-like 4 protein antibody
US7740846B2 (en) 2004-07-20 2010-06-22 Genentech, Inc. Inhibitors of angiopoietin-like 4 protein, combinations, and their use
US8604185B2 (en) 2004-07-20 2013-12-10 Genentech, Inc. Inhibitors of angiopoietin-like 4 protein, combinations, and their use
US8633155B2 (en) 2004-07-20 2014-01-21 Genentech, Inc. Methods of using angiopoietin-like 4 protein to stimulate proliferation of pre-adipocytes

Also Published As

Publication number Publication date
AU1931599A (en) 1999-07-12
WO1999032515A3 (fr) 1999-09-10

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