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WO1999055869A1 - Nouveaux facteurs et materiels de croissance polypeptidiques et leurs procedes de production - Google Patents

Nouveaux facteurs et materiels de croissance polypeptidiques et leurs procedes de production Download PDF

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WO1999055869A1
WO1999055869A1 PCT/US1999/008821 US9908821W WO9955869A1 WO 1999055869 A1 WO1999055869 A1 WO 1999055869A1 US 9908821 W US9908821 W US 9908821W WO 9955869 A1 WO9955869 A1 WO 9955869A1
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seq
residues
polypeptide
protein
zalpha5
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Darrell C. Conklin
Kenneth B. Lewis, Jr.
Debra G. Gilbertson
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Zymogenetics Inc
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Zymogenetics Inc
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Priority to AU36611/99A priority Critical patent/AU3661199A/en
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

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. Binding initiates a chain of signalling events within the cell, which ultimately results in phenotypic changes such as cell division, protease production, cell migration, expression of cell surface proteins, and production of additional growth factors.
  • 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 213:678-683, 1997.
  • Angiogenesis is regulated by the vascular endothelial growth factors
  • VEGFs vascular endothelial growth factor receptors
  • Flt-1 cell surface receptors
  • VEGFs are essential for vascular development in the embryo.
  • Angiopoietin-1 (Ang-1) acting through the Tie-2 receptor (also known as Tek) , is believed to regulate a later stage of vascular development
  • 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.
  • erythropoietin erythropoietin
  • thrombopoietin thrombopoietin
  • G-CSF G-CSF stimulate the production of erythrocytes, platelets, and neutrophils, respectively, from precursor cells in the bone marrow.
  • Development of mature cells from pluripotent progenitors may require the presence of a plurality of factors .
  • 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 82:2212-2220, 1996; Kukk et al . , Bri t . J.
  • 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 .
  • 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.
  • hematopoietic cytokine erythropoietin has been developed for the treatment of anemias (e.g., EP 613,683). More recently, thrombopoietin has been shown to stimulate the production of platelets in vivo (Kaushansky et al . , Na ture
  • growth factors are used in the laboratory to study developmental processes, and in laboratory and industry settings as components of cell culture media.
  • an isolated polypeptide of at least 15 amino acid residues comprising an epitope-bearing portion of a polypeptide of SEQ ID NO : 2 or SEQ ID NO: 15.
  • the polypeptide comprises at least 15 contiguous residues of SEQ ID NO: 2, such as residues 19- 42, 88-116, or 404-430.
  • an isolated polypeptide comprising a sequence of amino acids of the formula B x -L y -C z , wherein B is at least 70% identical to residues m to n of SEQ ID NO : 2 , wherein m is from 15 to 23 and n is from 199-207; L is at least 70% identical to residues o to p of SEQ ID NO: 2, wherein o is n+1 and p is from 234 to 242; C is at least 70% identical to residues q to r of SEQ ID NO : 2 , wherein q is from p+1 to 246 and r is from 408 to 460; and each of x, y, and z is individually 0 or 1 , subject to the limitations that at least one of x and z is 1 and if both x and z are 1, y is 1.
  • B is at least 80% identical to residues m to n of SEQ ID NO : 2
  • L is at least 80% identical to residues o to p of SEQ ID NO : 2
  • C is at least 80% identical to residues q to r of SEQ ID NO : 2.
  • x is 1 and B comprises residues 23-199 of SEQ ID NO : 2
  • z is 1 and C comprises residues 246-408 of SEQ ID NO : 2.
  • the polypeptide comprises residues XI -X2 of SEQ ID NO : 2 , wherein XI is selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 200, 201, 202, 203, 204, 205, 206, 207, 208, 225, 231, 232, 233, 235, 236, 237, 238, 239, 240, 241, 242, 243, and 246, and X2 is from 408 to 460; or XI is selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, and 23, and X2 is selected from the group consisting of 199, 200, 201, 202, 203, 204, 205, 206, 207, 224, 230, 231, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, and 408 through 460.
  • XI is selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 200, 201, 202, 203,
  • the polypeptide may comprise cysteine residues at positions corresponding to residues 246, 274, 394, and 408 of SEQ ID NO : 2.
  • the polypeptide is glycosylated.
  • the isolated polypeptide is covalently linked to a moiety selected from the group consisting of affinity tags, toxins, radionuclides , enzymes, and fluorophores .
  • Preferred affinity tags include polyhistidine, protein A, glutathione S transferase, substance P, and an immunoglobulin heavy chain constant region.
  • the isolated polypeptide further comprises a proteolytic cleavage site between the polypeptide and the affinity tag.
  • an isolated multimeric protein comprising a first polypeptide as disclosed above non-covalently associated with a second polypeptide, wherein the protein modulates cell proliferation, differentiation, migration, adhesion, or metabolism.
  • the first and second polypeptides are the same.
  • the first and second polypeptides are different .
  • an expression vector comprising the following operably linked elements: (a) a transcription promoter; (b) a DNA segment encoding a polypeptide as disclosed above; and (c) a transcription terminator.
  • the expression vector further comprises a secretory signal sequence operably linked to the DNA segment.
  • the polypeptide comprises a sequence of amino acid residues selected from the group consisting of residues 23-238 of SEQ ID NO : 2 and residues 17-238 of SEQ ID NO : 2.
  • the polypeptide comprises a sequence of amino acid residues selected from the group consisting of residues 23-460 of SEQ ID NO : 2 and residues 17-460 of SEQ ID NO:2.
  • a protein produced by a method comprising the steps of (a) culturing a cell containing an expression vector as disclosed above, and (b) isolating the protein encoded by the DNA segment and produced by the cell.
  • 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 .
  • a method of making a protein comprising the steps of (a) culturing a cell containing an expression vector as disclosed above, and (b) isolating the protein encoded by the DNA segment and produced by the cell .
  • antibodies that bind to the polypeptides and/or multimeric proteins disclosed above.
  • the antibodies specifically bind to a 7
  • Fig. 1 shows an alignment of representative human (SEQ ID NO:2) and mouse (SEQ ID NO:15) zalpha5 amino acid sequences. Amino acid residues are represented by the conventional single-letter codes.
  • Fig. 2 illustrates the vector pHB12-8.
  • 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 tag any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly-histidine tract, protein A (Nilsson et al . , EMBO J. 4 . : 1075, 1985; Nilsson et al . ,
  • 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 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 1
  • ATGCACGGG 3 ' is complementary to 5 ' CCCGTGCAT 3 ' .
  • corresponding to when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned.
  • 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) .
  • a "DNA segment” is a portion of a larger DNA molecule having specified attributes.
  • a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide .
  • 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 of the present invention are free of other genes 10
  • 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 vi tro, or prepared from a combination of natural and synthetic molecules.
  • a “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” .
  • the term “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 11
  • 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.
  • Molecular weights and lengths of polymers determined by imprecise analytical methods e.g., gel electrophoresis
  • the present invention provides novel growth factor polypeptides and proteins.
  • This novel growth factor termed "zalpha5", exhibits significant amino acid sequence homology to the previously described angiopoietin-1 (Davis et al . , Cell 12:1161-1169, 1996) and angiopoietin-2 (Maisonpierre et al . , Sci ence 277 : 55-60 ,
  • a representative polypeptide of the present invention is approximately 30% identical to Ang-1 throughout its length when the sequences are aligned to produce a 441 amino acid residue overlap (residues 17-455 of SEQ ID NO: 2), accounting for overlaps as disclosed below.
  • the polypeptide is approximately 29% identical to 12
  • Ang-2 when the sequences are aligned to produce a 406 amino acid residue overlap (residues 50-455 of SEQ ID NO: 2) .
  • zalpha5 is produced in cells of the liver. Using multiple tissue Northern blots, zAlpha5 transcript is detected primarily in liver. Immunohistochemistry (IHC) using a polyclonal antibody raised against full-length zalpha5 detects protein in normal human and monkey liver. These data suggest that one or more of the cell types that line the sinusoids (sinusoidal endothelial cells, Ito cells, or Kupffer cells) is the source of zalpha5. In normal liver, the space of Disse and Kupffer cells appear to stain strongly for zalpha5 protein. In si tu hybridization of mouse liver slices confirms the presence of transcript in the liver starting as early as embryonic day 13.5 as well as in adult liver.
  • IHC Immunohistochemistry
  • Neither cultured hepatocytic nor cultured sinusoidal endothelial cells contain zalpha5 transcript. While not wishing to be bound by theory, this result suggests that another non-parenchymal cell type in the liver produces zalpha ⁇ . When whole rat livers are perfused with collagenase, and the resulting cell mixture is fractionated, the Kupffer cell fraction contains noticeably more transcript than the other fractions. Experimental data further suggest that sinusoidal lining cells may also be a target for zalpha5. Alterations in the expression level and localization of zalpha5 are observed by IHC in liver cirrhosis, hepatitis, thrombosis and centrilobular necrosis.
  • Zalpha5 may thus be involved in organ-specific liver biology, such as through effects on the sinusoidal endothelial cells and/or hepatocytes . There is thought to be significant paracrine interaction between the cell types lining the liver sinusoids. Given that zalpha5 transcript is detected in adult liver rather than transiently during embryogenesis, it may function in vivo to maintain the liver sinusoidal 13
  • the proteins zalpha5, angiopoietin-1, angiopoietin-2 , fibrinogen-beta, and fibrinogen-gamma form an evolutionarily related family.
  • Their amino acid sequences include three distinct segments designated, from the amino terminus to the carboxyl terminus, A, B, and C.
  • Segment A comprises a secretory signal peptide.
  • Segment B is predicted to be an extended helical region which, in interaction with analogous extended helical regions in other chains, produces multimeric (e.g., dimeric, trimeric, or tetrameric) coiled coil structures.
  • Coiled coils are bundles of extended ⁇ -helices (generally 2-4 helices) wound into a superhelix.
  • segment B The sequence of a single component chain of a coiled coil shows a heptad repeat in the chemical nature of sidechains.
  • This "coiled coil” structure is characterized by a "knobs-into-holes" packing of amino acid sidechains in the core of the bundle. See, Lupas, TIBS 2JL:375-382, 1996.
  • segment B has some similarity to the primary structure of myosin, which forms a prototypical dimeric coiled coil.
  • Segment C as revealed by the crystal structure of the gamma chain of human fibrinogen (Spraggon et al .
  • Segment C is sometimes referred to as a "fibrinogen homology domain". This domain occurs in numerous other proteins, including tenascin-C (Erickson and Bourdon, Ann . Rev. Cell Biol . 5 : 71-92, 1989), restrictin, and ficolin.
  • segment A the secretory signal peptide
  • segment B the extended helical region
  • the core helices of segment B comprise residues Arg36-Leu50, Phe78-Ile92, 14
  • Segment C the fibrinogen homology domain, extends from residue 239 to the carboxyl terminus of the protein.
  • zalpha5 includes an interdomain region or linker (segment L) between segments B and C.
  • zalpha5 polypeptides and multimers thereof can be produced by selecting different expression systems, adding or deleting proteolytic cleavage sites through mutagenesis, synthesizing polypeptides in vi tro, or through the application of other methods known in the art.
  • 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 zalpha5 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide.
  • expressing the complete zalpha5 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.
  • Differential processing of individual chains may result in 15
  • heterogeneity of expressed polypeptides and the production of heterodimeric zalpha5 proteins are heterogeneity of expressed polypeptides and the production of heterodimeric zalpha5 proteins.
  • zalpha5 polypeptides will contain at least segment B residues 23-199 or segment C residues 246- 408 as shown in SEQ ID NO : 2.
  • These polypeptides can be represented by the formula B x -L y -C z , wherein B is at least 70% identical to residues m to n of SEQ ID NO: 2; L is at least 70% identical to residues o to p of SEQ ID NO : 2 ; and C is at least 70% identical to residues q to r of SEQ ID NO : 2.
  • m is from 15 to 23
  • n is from 199-207
  • o is from 234 to 242
  • q is from p+1 to 246
  • r is from 408 to 460
  • x, y, and z are each 0 (i.e., the indicated domain is absent) or 1 (i.e., the indicated domain is present), subject to the limitations that at least one of x and z is 1 and if both x and z are 1, y is 1.
  • Representative polypeptides (with reference to SEQ ID NO: 2) are shown below in Table 1.
  • Zalpha5 multimeric complexes may be heteromultimers, comprising other angiopoietin-like monomers (including dissimilar zalpha ⁇ polypeptides); or homomultimers , comprising two or more identical zalpha5 monomeric units.
  • Experimental data indicate that the protein is produced as a monomer or as a non-covalent multimer, depending upon the expression system. Any of the zalpha5 polypeptides disclosed above any form multimers.
  • Segment B in human fibrinogen gamma, beta, and alpha contains several cysteines which, with interchain 17
  • disulfide bonds form another "disulfide ring" that links the trimeric alpha/beta/gamma multimer to a second multimer, forming a hexameric complex.
  • cysteine residues are not present in zalpha5. While not wishing to be bound by theory, these sequence dissimilarities provide evidence that dimers or trimers of zalpha5 will not combine to form tetrameric or hexameric complexes .
  • segment C of each of human fibrinogen beta and gamma chains there are four cysteine residues that form intrachain disulfide bonds.
  • cysteines are also present, with similar spacing, in segment C of zalpha5.
  • disulfide bonds are predicted to occur in zalpha ⁇ between Cys246 and Cys274 of SEQ ID NO : 2 , and between Cys394 and Cys408 of SEQ ID NO: 2. This prediction is confirmed by analysis of recombinant zalpha5.
  • Zalpha5 has four potential N-linked glycosylation sites, at residues 23, 115, 296, and 357 of SEQ ID NO : 2.
  • Different lots of recombinant zalpha ⁇ showed different amounts of N-linked glycosylation, with two to three oligosaccharides per molecule on the average. The first site was found to be almost never occupied, the second site to be occupied about half the time, and the last two sites to be usually occupied. There were also several (range of 2-5) O-linked oligosaccharides per molecule .
  • Zalpha5 proteins of the present invention are characterized by their growth factor activity. These proteins modulate the proliferation, differentiation, migration, adhesion, or metabolism of responsive cell types. Biological activity of zalpha5 proteins is assayed using in vi tro or in vivo assays designed to detect cell proliferation, differentiation, migration or adhesion; or changes in cellular metabolism (e.g., production of other growth factors or other macromolecules) . Many suitable assays are known in the art, and representative assays are disclosed herein. Assays using cultured cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions.
  • in vivo assays will generally be employed to confirm and further characterize biological activity.
  • Certain in vi tro models such as the three-dimensional collagen gel matrix model of Pepper et al . ⁇ Biochem .
  • Assays can be performed using exogenously produced proteins, or may be carried out in vivo or in vi tro using cells expressing the polypeptide (s) of interest.
  • Assays can be conducted using zalpha5 proteins alone or in combination with other growth factors, such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor) . Representative assays are disclosed below.
  • 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 70% identical to the polypeptide of SEQ ID NO : 2. Such polypeptides will preferably be at least 75% identical, more preferably 80% identical, still more preferably at least 90% identical, and most preferably 95% or more identical to SEQ ID NO : 2. Preferred candidate amino acid substitutions within human zalpha5 are suggested by alignment of the human (SEQ ID NO: 2) and mouse (SEQ ID NO: 15) sequences as shown in Fig. 1, which sequences are approximately 76% identical overall.
  • the level of identity between amino acid sequences can be determined using the "FASTA" similarity search algorithm disclosed by Pearson and Lipman ⁇ Proc .
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman and Wunsch, J " . Mol . Biol .
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio 22
  • the ktup value can range between one to six, preferably from four to six.
  • the present invention includes polypeptides having one or more conservative amino acid changes as compared with the amino acid sequence of SEQ ID NO : 2.
  • the BLOSUM62 matrix (Table 2) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, ibid . ) .
  • the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention.
  • the term "conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least one 1 (e.g., 1, 2 or 3)
  • more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3) .
  • Engineered variant zalpha5 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 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 extension that facilitates purification (an affinity 23
  • affinity tags See, in general Ford et al . , Protein Expression and Purification 2 : 95-107, 1991, which is incorporated herein by reference.
  • Two or more affinity tags may be used in combination.
  • DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA) . It is preferred to limit amino acid deletions and substitutions to the globular region (when present) of a zalpha5 polypeptide, although conservative substitutions or deletions of one to a few residues can be made in the extended helical region.
  • Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zalpha ⁇ polypeptide and the affinity tag.
  • Preferred cleavage 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, trans-3-methylproline, 2 , 4-methanoproline, cis-4 -hydroxyproline, trans-4. -hydroxyproline, JV- methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine , hydroxyethylhomocysteine , nitroglutamine, homoglutamine, pipecolic acid, tert- leucine, norvaline, 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylalanine .
  • Proteins are purified by chromatography. See, for example, Robertson et al . , J. Am. Chem. Soc. 113 :2722 , 1991; Ell an et al . , Methods Enzymol . 202 :301, 1991; Chung et al . , Science 2_5_9: 806-809 , 1993; and Chung et al., Proc. Natl. Acad. Sci. USA . 90 . : 10145-10149 , 1993).
  • 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) .
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine
  • non-naturally occuring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 3_3: 7470-7476 , 1994. Naturally occuring amino acid residues can be converted to non-naturally occuring species by in vi tro chemical modification.
  • 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 . ,
  • Amino acid sequence changes are made in zalpha5 polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • 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 .
  • Variants of the disclosed zalpha5 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Na ture 370 : 389-391 , 1994 and Stemmer, Proc . Natl . Acad . Sci . USA 91:10747-
  • variant genes are generated by in vi tro 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 26
  • 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 zalpha5 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 zalpha ⁇ 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.
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zalpha5 polypeptides disclosed above.
  • a representative DNA sequence encoding the amino acid sequence of SEQ ID NO : 2 is shown in SEQ ID NO : 1.
  • a representative mouse zalpha ⁇ DNA sequence is shown in SEQ ID NO: 14, with the encoded protein shown in SEQ ID NO: 15.
  • SEQ ID NO : 4 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha5 polypeptide 27
  • SEQ ID NO: 4 also provides all RNA sequences encoding SEQ ID NO : 2 by substituting U for T.
  • zalpha5 polypeptide-encoding polynucleotides comprising nucleotide 70 to nucleotide 714 of SEQ ID NO : 4 , and their RNA equivalents are contemplated by the present invention, as are segments of SEQ ID NO : 4 encoding other polypeptides shown in Table 1.
  • Table 3 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) .
  • the code Y denotes either C or T
  • R denotes A or G
  • A being complementary to T
  • G being complementary to C.
  • degenerate codons used in SEQ ID NO: 4 encompassing all possible codons for a given amino acid, are set forth in Table 4, below.
  • degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR) , and the degenerate 3 0
  • codon for arginine can, in some circumstances, encode serine (AGY) .
  • 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.
  • One of ordinary skill in the art will also appreciate that different species can exhibit preferential codon usage. See, in general, Grantham et al . , Nuc . Acids Res . . 8:1893-912, 1980; Haas et al . Curr .
  • the degenerate codon sequence disclosed in SEQ ID NO : 4 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed 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. 31
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for preparing DNA and RNA are well known in the art.
  • RNA is isolated from a tissue or cell that produces large amounts of zalpha5 RNA. Liver is a preferred tissue. Zalpha5 transcripts have also been detected in liver tumor tissue.
  • Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al . , Biochemistry ⁇ :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).
  • cDNA Complementary DNA
  • poly (A) + RNA using known methods.
  • genomic DNA can be isolated.
  • Polynucleotides encoding zalpha5 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding zalpha5 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 zalpha5, receptor fragments, or other specific binding partners.
  • Zalpha5 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5 ' non-coding regions of a zalpha5 gene. In view of the tissue-specific expression observed for zalpha5 by Northern blotting, this gene region is expected to provide for liver- and kidney-specific expression. 32
  • Promoter elements from a zalpha ⁇ gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy.
  • Cloning of 5' flanking sequences also facilitates production of zalpha5 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zalpha5 gene in a cell is altered by introducing into the zalpha5 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a zalpha5 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zalpha5 locus, whereby the sequences within the construct become operably linked with the endogenous zalpha5 coding sequence.
  • an endogenous zalpha5 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • sequences disclosed in SEQ ID NOS : 1 and 2 represent a single allele of human zalpha5. 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" ) .
  • zalpha5 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • Orthologs of human zalpha5 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 zalpha5 as disclosed above.
  • a library is then prepared from mRNA of a 33
  • a zalpha5 -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human or mouse 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 or mouse zalpha ⁇ sequences 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 zalpha5 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • any zalpha5 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 3 and 4, above.
  • those of skill in the art can use standard software to devise zsig51 variants based upon the nucleotide and amino acid sequences described herein.
  • the present invention thus provides a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO:l, SEQ ID NO : 2 , SEQ ID NO : 4 , SEQ ID NO: 14, SEQ ID NO: 15, and portions thereof.
  • Suitable forms of computer-readable media include magnetic media and optically-readable media.
  • magnétique media examples include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT) , a disk cache, and a ZIP disk.
  • Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM) , CD-rewritable (RW) , and CD-recordable) , and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD- RAM, and DVD+RW) .
  • compact discs e.g., CD-read only memory (ROM) , CD-rewritable (RW) , and CD-recordable
  • DVD digital versatile/video discs
  • DVD digital versatile/video discs
  • the zalpha ⁇ polypeptides of the present invention including full-length polypeptides, biologically active fragments, and fusion polypeptides 34
  • 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. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al . , Molecular Cloning: A
  • a DNA sequence encoding a zalpha5 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 zalpha5, or may be derived from another secreted protein 35
  • the secretory signal sequence is operably linked to the zalpha5 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) .
  • Cultured mammalian cells are preferred hosts for use within the present invention.
  • 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
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No.
  • promoters from SV-40 or cytomegalovirus See, e.g., U.S. Patent No. 4,956,288.
  • suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited with the American Type Culture Collection, Manassas, VA USA under accession numbers 98669 and 98668, respectively.
  • 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
  • the adenovirus system (disclosed in more detail below) can also be used for protein production in vi tro . 37
  • adenovirus-infected non-293 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 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol . 15:145-55, 1994).
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell.
  • non-secreted proteins can also be effectively obtained.
  • Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells.
  • Agroba cterium 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 calif ornica nuclear polyhedrosis virus (AcNPV) according to methods known in the art.
  • recombinant baculovirus is produced through the use of a transposon- based system described by Luckow et al . (J. Virol .
  • the transfer vector (e.g., pFastBaclTM; Life Technologies) contains a Tn7 transposon to move the DNA encoding the 3 8
  • bacmid protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See,
  • transfer vectors can include an in- frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above.
  • a transfer vector containing a zalpha5- encoding sequence is transformed into E. coli host cells, and the cells are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells.
  • Recombinant virus that expresses zalpha5 protein is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art .
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda
  • Trichoplusia ni e.g., High
  • Serum- free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers. The cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally known in the art .
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces 39
  • 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.,
  • Transformation systems for other yeasts including Hansenula polymorpha , Schizosa ccharomyces pombe, Kl uyveromyces lactis , Kl uyveromyces fragilis , Ustilago maydis , Pichia pastoris , Pichia methanolica ,
  • Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen .
  • 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. 40
  • DNA molecules for use in transforming P . methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • a preferred method of transformation is electroporation as disclosed in U.S. Patent No. 5,854,039.
  • the promoter and terminator in the plasmid be that of a P . methanolica gene, such as a P . methanoli ca alcohol utilization gene ⁇ AUG1 or AUG2) .
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • Prokaryotic host cells including strains of the bacteria Escherichia coli , Ba cill us 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.). When 41
  • the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic 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 periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic 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 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.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors . It is preferred to purify the polypeptides and proteins of the present invention to >80% purity, more 42
  • a purified polypeptide or protein is substantially free of other polypeptides or proteins, particularly those of animal origin.
  • Expressed recombinant zalpha5 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,
  • Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al . , ibid .
  • the proteins of the present invention can be isolated by exploitation of their binding properties.
  • immobilized metal ion adsorption chromatography IMAC can be used to purify polyhistidine-tagged polypeptides. Briefly, a gel is first charged with divalent metal ions to form a chelate
  • Histidine-rich polypeptides will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • a zalpha ⁇ protein comprising a 43
  • polyhistidine affinity tag (typically about 6 histidine residues) can be purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al . , Bio/Technol . 6_ : 1321-1325, 1988.
  • Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol . , Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Academic Press, San Diego, 1990, 529-539) .
  • Fusion polypeptides comprising other affinity tags (e.g., maltose-binding protein, glu-glu tag, or an immunoglobulin domain) can be constructed and purified using the appropriate binding agent (e.g., an antibody), the selection of which will be evident to those skilled in the art .
  • affinity tags e.g., maltose-binding protein, glu-glu tag, or an immunoglobulin domain
  • Zalpha5 polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 15:2149, 1963; Stewart et al . , Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem .
  • zalpha5 proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non- pegylated; and may or may not include an initial methionine amino acid residue.
  • Target cells for use in zalpha5 activity assays include vascular cells (especially endothelial cells and smooth muscle cells) , hematopoietic (myeloid and lymphoid) cells, liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito 44
  • Endothelial cells and hematopoietic cells are derived from a common ancestral cell, the hemangioblast (Choi et al . , Development
  • Activity of zalpha ⁇ proteins can be measured in vi tro using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate animal model.
  • mitogenic activity 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 and Wahl et al . , Mol . Cell Biol . 1:5016-5025, 1988), dye incorporation assays (as disclosed by, for example, Mosman, J " . Immunol .
  • Preferred mitogenesis assays measure incorporation of 3 H-thymidine into (1) 20% confluent cultures to look for the ability of zalpha5 proteins to further stimulate proliferating cells, and
  • Differentiation can be assayed using suitable precursor cells that can be induced to differentiate into a more mature phenotype .
  • suitable precursor cells that can be induced to differentiate into a more mature phenotype .
  • mesenchymal stem cells can be used to measure the ability of zalpha ⁇ protein to stimulate differentiation into osteoblasts. Differentiation is indicated by the expression of osteocalcin, the ability of the cells to mineralize, and the expression of alkaline phosphatase, 45
  • Zalpha5 activity may also be detected using assays designed to measure zalpha ⁇ - induced production of one or more additional growth factors or other macromolecules .
  • Preferred such assays include those for determining the presence of hepatocyte growth factor (HGF) , epidermal growth factor (EGF) , transforming growth factor alpha (TGF ⁇ ) , interleukin-6 (IL-6) , VEGF, acidic fibroblast growth factor (aFGF) , angiogenin, and other macromolecules produced by the liver.
  • HGF hepatocyte growth factor
  • EGF epidermal growth factor
  • TGF ⁇ transforming growth factor alpha
  • IL-6 interleukin-6
  • VEGF acidic fibroblast growth factor
  • angiogenin angiogenin
  • Suitable assays include mitogenesis assays using target cells responsive to the macromolecule of interest, receptor-binding assays, competition binding assays, immunological assays (e.g., ELISA) , and other formats known in the art.
  • Metalloprotease secretion is measured from treated primary human dermal fibroblasts, synoviocytes and chondrocytes .
  • the relative levels of collagenase, gelatinase and stromalysin produced in response to culturing in the presence of a zalpha5 protein is measured using zymogram gels (Loita and Stetler- Stevenson, Cancer Biology 1:96-106, 1990).
  • Procollagen/collagen synthesis by dermal fibroblasts and chondrocytes in response to a test protein is measured using 3 H-proline incorporation into nascent secreted collagen.
  • 3 H-labeled collagen is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J " . Biol .
  • GAG Glycosaminoglycan secretion from dermal fibroblasts and chondrocytes is measured using a 1 , 9-dimethylmethylene blue dye binding assay (Farndale et al . , Biochim . Biophys . Acta 883 :173-
  • Collagen and GAG assays are also carried out in the presence of IL-l ⁇ or TGF- ⁇ to examine the ability of zalpha5 protein to modify the established responses to these cytokines.
  • Monocyte activation assays are carried out (1) to look for the ability of zalpha ⁇ proteins to further stimulate monocyte activation, and (2) to examine the ability of zalpha ⁇ proteins to modulate attachment - induced or endotoxin-induced monocyte activation
  • IL-l ⁇ and TNF ⁇ levels produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA) .
  • Monocyte/macrophage cells by virtue of CD14 (LPS receptor) , are extremely sensitive to endotoxin, and proteins with moderate levels of endotoxin-like activity will activate these cells.
  • Hematopoietic activity of zalpha5 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 zalpha5 polypeptides on hematopoietic cell lines can be measured as disclosed above.
  • a protein is considered to be chemotactic if it induces migration of cells from an area of low protein concentration to an area of high protein concentration.
  • the assay is performed using modified Boyden chambers with a polystryrene membrane separating the two chambers (Transwell; Corning Costar Corp.) .
  • the test sample diluted in medium containing 1% BSA, is added to the 47
  • microtiter plates are coated with the test protein, non-specific sites are blocked with BSA, and cells (such as smooth muscle cells, leukocytes, or endothelial cells) are plated at a density of approximately 10 4 - 10 s cells/well.
  • the wells are incubated at 37°C (typically for about 60 minutes) , then non-adherent cells are removed by gentle washing.
  • Adhered cells are quantitated by conventional methods
  • Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin.
  • Assays for angiogenic activity are also known in the art.
  • the effect of zalpha ⁇ proteins on primordial endothelial cells in angiogenesis can be assayed in the chick chorioallantoic membrane angiogenesis assay (Leung, Science 246 : 1306-1309 , 1989; 48
  • test substance is inserted into a pocket in the cornea of an inbred mouse; and the hampster cheek pouch assay (H ⁇ ckel et al . , Arch .
  • 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).
  • In vi tro assays for angiogenic activity include the tridimensional collagen gel matrix model (Pepper et al . Biochem .
  • 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 . , Circula tion 11:2802-2809, 1995; Callow et al . , Growth Factors JLO.: 223-228 , 1994); efficacy of zalpha5 polypeptides can be tested in these and other known models.
  • 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). Zalpha5 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) .
  • Efficacy of zalpha5 polypeptides in promoting wound healing can be assayed in animal models.
  • One such model is the linear skin incision model of Mustoe et al .
  • the rabbit ear model can be modified to create an ischemic wound environment, which more closely resembles the clinical situation (Ahn et al . , Ann . Plas t . Surg. 24 : 17 , 1990) .
  • healing of partial- thickness skin wounds in pigs or guinea pigs is evaluated (LeGrand et al . , Growth Factors 1:307, 1993).
  • Experimental treatments are applied daily on or under dressings. Seven days after wounding, granulation tissue thickness is determined. This model is preferred for dose-response studies, as it is more quantitative than other in vivo models of wound healing. A full thickness excision model can also be employed. Within this model, the epidermis and dermis are removed down to the panniculus carnosum in rodents or the subcutaneous fat in pigs. Experimental treatments are applied topically on or under a dressing, and can be applied daily if desired. The wound closes by a combination of contraction and cell ingrowth and proliferation. Measurable endpoints include time to wound closure, histologic score, and biochemical parameters of wound tissue. 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 .
  • Delay or prolongation of the wound healing process can be induced pharmacologically by treatment with steroids, irradiation of the wound site, or by concomitant disease states (e.g., diabetes) . Linear incisions or full-thickness excisions are most commonly used as the experimental wound. Endpoints are 51
  • Subcutaneous implants can be used to assess compounds acting in the early stages of wound healing (Broadley et al . , Lab .
  • Implants are prepared in a porous, relatively non-inflammatory container (e.g., polyethylene sponges or expanded polytetrafluoroethylene implants filled with bovine collagen) and placed subcutaneously in mice or rats.
  • the interior of the implant is empty of cells, producing a "wound space" that is well-defined and separable from the preexisting tissue. This arrangement allows the assessment of cell influx and cell type as well as the measurement of vasculogenesis/angiogenesis and extracellular matrix production.
  • Zalpha5 -encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.
  • viruses for this purpose include adenovirus, herpesvirus, retroviruses, 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 acids. For review, see Becker et al . , Meth . Cell Biol . 43 : 161-89,
  • 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 52
  • adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. 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 El gene is deleted from the viral vector, and the virus will not replicate unless the El 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 virus cannot replicate in the host cells.
  • 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 . 4_3: 353-365, 1994.
  • mice engineered to express a zalpha5 gene, and mice that exhibit a complete absence of zalpha ⁇ gene function, referred to as "knockout mice” 53
  • mice can be employed to study the zalpha5 gene and the protein encoded thereby in an in vivo system.
  • Transgenic mice are particularly useful for investigating the role of zalpha5 proteins 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,
  • promoters for transgenic expression include promoters from metallothionein and albumin genes.
  • Antisense methodology can be used to inhibit zalpha5 gene transcription to examine the effects of such inhibition in vivo .
  • Polynucleotides that are complementary to a segment of a zalpha5 -encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ID N0:1 are designed to bind to zalpha5 -encoding mRNA and to inhibit translation of such mRNA.
  • Such antisense oligonucleotides can also be used to inhibit expression of zalpha5 polypeptide-encoding genes in cell culture .
  • Zalpha5 polypeptides may be used therapeutically to stimulate tissue development or repair, or cellular differentiation or proliferation.
  • 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 54
  • 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 deficiency of microvessels ; to promote angiogenesis and prevent neuronal degeneration due to chronic cerebral ischemia; to accelerate the formation of collateral blood vessels in ischemic limbs; to promote vessel re-endothelialization and to reduce intimal hyperplasia following invasive procedures such as balloon angioplasty and stent placement; to promote vessel repair and development of collateral circulation following myocardial infarction so as to limit ischemic injury; and to stimulate hematopoiesis .
  • the polypeptides are also useful additives in tissue adhesives for promoting revascularization of the healing tissue.
  • Zalpha5 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 zalpha5 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 61:436-443, 1997; Pepper, Arterioscl erosis , Throm . and
  • zalpha5 proteins are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • pharmaceutical formulations will include a zalpha5 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.
  • Zalpha5 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 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.
  • a therapeutically effective amount of zalpha5 is an amount sufficient to produce a clinically significant 56
  • Proteins of the present invention are useful for modulating the proliferation, differentiation, migration, or metabolism of responsive cell types, which include both primary cells and cultured cell lines.
  • responsive cell types which include both primary cells and cultured cell lines.
  • hematopoietic cells including stem cells and mature myeloid and lymphoid cells
  • endothelial cells including stem cells and mature myeloid and lymphoid cells
  • smooth muscle cells including fibroblasts, and hepatocytes
  • Zalpha5 polypeptides are added to tissue culture media for these cell types at a concentration of about 10 pg/ml to about 100 ng/ml.
  • zalpha5 proteins can be advantageously combined with other growth factors in culture media.
  • zalpha5 proteins can also be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over- or under-production of zalpha5 protein or in the analysis of cell phenotype .
  • Zalpha ⁇ proteins can also be used to identify inhibitors of their activity.
  • Test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of zalpha5 protein.
  • samples can be tested for inhibition of zalpha ⁇ activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zalpha5-dependent cellular responses.
  • zalpha ⁇ -responsive cell lines can be transfected with a reporter gene construct that is responsive to a zalpha5-stimulated cellular pathway. Reporter gene constructs of this type are known in the 57
  • zalpha5-activated serum response element SRE
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zalpha5 on the target cells as evidenced by a decrease in zalpha5 stimulation of reporter gene expression.
  • Assays of this type will detect compounds that directly block zalpha5 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 zalpha5 binding to receptor using zalpha5 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like) .
  • a detectable label e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors .
  • the invention further provides polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO: 2.
  • An "epitope” is a region of a protein to which an antibody can bind. See, for example, Geysen et al . , Proc . Natl . Acad . Sci . USA 11:3998-4002,
  • Epitopes can be linear or conformational , the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein.
  • Linear epitopes are generally at least 6 amino acid residues in length.
  • Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al . , Science 219 : 660-666 , 1983. Antibodies that recognize short, linear epitopes are particularly useful in 58
  • Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zalpha5 protein.
  • Antigenic, epitope-bearing polypeptides contain a sequence of at least six, preferably at least nine, more preferably from 15 to about 30 contiguous amino acid residues of a zalpha5 protein (e.g., SEQ ID NO:2) .
  • Polypeptides comprising a larger portion of a zalpha ⁇ protein, i.e. from 30 to 50 residues up to the entire sequence are included.
  • amino acid sequence of the epitope- bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided.
  • Preferred such regions include residues 19-42, 88-108, 88-116, and 404- 430 of SEQ ID NO : 2.
  • antibodies includes polyclonal antibodies, monoclonal antibodies, antigen- binding fragments thereof such as F(ab')2 an( Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies.
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains
  • 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 59
  • Antibodies are defined to be specifically binding if they bind to a zalpha ⁇ polypeptide or protein with an affinity at least
  • 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 zalpha ⁇ 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 zalpha5 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 bovine serum albumin
  • tetanus toxoid tetanus toxoid
  • lymphocytes to zalpha5 polypeptides, and selection of antibody display libraries in phage or similar vectors
  • Human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in
  • 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 zalpha5 may be used for affinity purification of the protein, within diagnostic assays for determining circulating levels of the protein; for detecting or quantitating soluble zalpha5 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 vi tro and in vivo .
  • Antibodies to zalpha5 may also be used for tagging cells that express zalpha5 ,- for affinity purification of zalpha5 polypeptides and proteins; in analytical methods employing FACS; for screening expression libraries; and for generating anti- idiotypic antibodies.
  • Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, 61
  • Suitable direct tags or labels include radionuclides , enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates.
  • Antibodies of the present invention may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications (e . g . , inhibition of cell proliferation) . See, in general, Ramakrishnan et al., Cancer Res . 51:1324-1330, 1996.
  • Polypeptides and proteins of the present invention can be used to identify and isolate receptors.
  • Zalpha5 receptors may be involved in growth regulation in the liver, blood vessel formation, and other developmental processes.
  • zalpha5 proteins and polypeptides can be immobilized on a column, and membrane preparations run over the column (as generally disclosed in Immobilized Affinity Ligand Techniques, Hermanson et al . , eds., Academic Press, San Diego, CA, 1992, pp.195-202).
  • Proteins and polypeptides can also be radiolabeled ⁇ Methods Enzymol . , vol. 182, "Guide to
  • radiolabeled zalpha5 proteins and polypeptides can be used to clone the cognate receptor in binding 62
  • the present invention also provides reagents for use in diagnostic applications.
  • the zalpha5 gene, a probe comprising zalpha ⁇ DNA or RNA, or a subsequence thereof can be used to determine if the zalpha5 gene is present on chromosome 1 or if a mutation has occurred.
  • Detectable chromosomal aberrations at the zalpha5 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and rearrangements.
  • Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
  • PCR primers are at least 5 nucleotides in length, preferably 15 or more nt , more preferably 20-30 nt . Short polynucleotides can be used when a small region of the gene is targetted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more.
  • Probes will generally comprise a polynucleotide linked to a signal- generating moiety such as a radionucleotide .
  • these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (c) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention 63
  • RNA examples include genomic DNA, cDNA, and RNA.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID N0:1, the complement of SEQ ID N0:1, or an RNA equivalent thereof.
  • Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applica tions 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid . ; Ausubel et . al . , ibid . ; A.J. Marian, Chest
  • Ribonuclease protection assays comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion.
  • RNA-RNA hybrid reaction product
  • a patient genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient.
  • Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applica tions 1:34-38, 1991) .
  • SSCP single strand conformational polymorphism
  • polypeptides, nucleic acid and/or antibodies of the present invention may be used in diagnosis or treatment of disorders associated with cell loss or abnormal cell proliferation (including cancer), including impaired or excessive vasculogenesis or angiogenesis and liver disease.
  • cancers of the liver may be amenable to such diagnosis, treatment or prevention.
  • Elevated levels of zalpha5 polypeptides may be indicative of liver damage.
  • Labeled zalpha ⁇ polypeptides may be used for imaging tumors or other sites of abnormal cell proliferation.
  • 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 screen biopsy or other tissue samples in vi tro .
  • Inhibitors of zalpha5 activity include anti-zalpha5 antibodies and soluble zalpha5 receptors, as well as other peptidic and non- peptidic agents (including ribozymes) . Such antagonists can be used to block the effects of zalpha5 on cells or tissues. Of particular interest is the use of antagonists of zalpha5 activity in cancer therapy. As early detection methods improve it becomes possible to intervene at earlier times in tumor development, making it feasible to use inhibitors of growth factors to block cell proliferation, angiogenesis, and other events that lead to tumor development and metastasis. 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 zalpha ⁇ 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. 65
  • Zalpha5 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.
  • Zalpha5 antagonists may also prove useful in the treatment of infantile hemangiomas, which exhibit overexpression of VEGF and bFGF during the proliferative phase .
  • zalpha ⁇ inhibitors include small molecule inhibitors and angiogenically or mitogenically inactive receptor-binding fragments of zalpha5 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 zalpha ⁇ inhibitor with one or more inhibitors of these other factors .
  • Polynucleotides encoding zalpha5 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zalpha5 activity.
  • zalpha5 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 zalpha ⁇ gene therapy include stimulation of wound healing and repopulation of vascular grafts. Zalpha ⁇ polypeptides and anti-zalpha5 antibodies can be directly or indirectly conjugated to 66
  • 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) .
  • zalpha5 polypeptides or anti-zalpha5 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, saporin, and the like) , as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90.
  • Polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin.
  • 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 67
  • conjugates of a zalpha ⁇ 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.
  • Target cells i.e., those displaying the zsig51 receptor
  • bind the zsig51-toxin conjugate which is then internalized, killing the cell.
  • the effects of receptor-specific cell killing (target ablation) are revealed by changes in whole animal physiology or through histological examination.
  • ligand-dependent , receptor-directed cyotoxicity can be used to enhance understanding of the physiological significance of a protein ligand.
  • a preferred such toxin is saporin. Mammalian cells have no receptor for saporin, which is non-toxic when it remains extracellular .
  • zalpha5-cytokine fusion proteins or antibody/fragment-cytokine fusion proteins may be used for enhancing in vi tro 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 zalpha5, 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) .
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Such fusion proteins may be used to cause cytokine- induced killing of tumors and other tissues undergoing angiogenesis or neovascularization.
  • a zalpha5 polypeptide or anti-zalpha5 antibody can be conjugated with a radionuclide, particularly with a beta-emitting or gamma- emitting radionuclide, and used to reduce restenosis.
  • a radionuclide particularly with a beta-emitting or gamma- emitting radionuclide
  • iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered resulted in decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein.
  • 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.
  • a full length cDNA for zalpha ⁇ was obtained from a plasmid containing an insert corresponding to an expressed sequence tag (EST) from a fetal liver/spleen library (Hillier et al . , The WashU-Merck EST Project, 1995; EST319951; GenBank Accession No. R86161; obtained from Genome Systems, Inc., St. Louis, MO) .
  • the insert was sequenced and found to consist of 1534 bp with an open reading frame of 1383 bp (including a termination codon) .
  • Analysis of the encoded polypeptide showed significant sequence homology to angiopoietin-1 and angiopoietin-2.
  • a second EST (EST135867; GenBank Accession No. T73510; Hillier et al . , The WashU-Merck EST Project, 1985) was also analyzed and was found to be truncated at the 3' end.
  • tissue distribution was performed by the northern blotting technique using Human Multiple Tissue and Master Dot Blots from Clontech Laboratories, Inc. (Palo Alto, CA) .
  • a probe was obtained by restriction digest of the EST135867 clone with EcoRI and Xhol to remove the insert from the vector.
  • the reaction mixture was electrophoresed on a 2% agarose gel, and the 656 bp band corresponding to the original EST was excised and purified using commercially available gel purification reagents and protocol (QIAEX ® II Gel Extraction kit; Qiagen, Inc., Valencia, CA) .
  • the purified DNA was radioactively labeled with 32 P using a commercially available kit (RediprimeTM DNA labeling system; Amersham Corp., Arlington Heights, IL) .
  • the probe was purified using a commercially available 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., Palo Alto, CA) was used for prehybridization and hybridization. The final 70
  • hybridization solution contained 8 ml ExpressHybTM Hybridization Solution, 80 ⁇ l sheared salmon sperm DNA (10 mg/ml; obtained from 5 Prime-3 Prime, Boulder, CO), 48 ⁇ l human Cot-1 DNA (1 mg/ml, GibcoBRL, Gaithersburg, MD) and 45 ⁇ l labeled probe ( " 50 ng @ 305,000 CPM/ng) .
  • Hybridization took place overnight at 55°C. After hybridization the blots were washed in 2X SSC, 0.1% SDS at room temperature; then in 2X SSC, 0.1% SDS at 65°C; followed by a 0. IX SSC, 0.1% SDS wash at 65°C. The blots were exposed to film overnight.
  • liver Three transcript sizes were observed in liver at approximately 5.8 kb, 2.8 kb, and 1.7 kb, and a faint signal was detected in kidney at approximately 2 kb .
  • the dot blot showed signals in liver and fetal liver and a faint signal in kidney. While not wishing to be bound by theory, this expression pattern suggests a possible role for zalpha ⁇ in liver regeneration .
  • Example 2 Northern analysis was performed on Northern blots of human tumor RNA (Human Tumor Panel Blot I, Human Tumor Panel Blot II, Human Tumor Panel Blot IVb, and Human Tumor Panel Blot V from Invitrogen Corporation, Carlsbad, CA) .
  • a probe was obtained by PCR using primers ZC16,985 (SEQ ID NO: 5) and ZC17,250 (SEQ ID NO: 6) and EST319951 as template, yielding a DNA fragment corresponding to the coding sequence of zalpha5 with flanking restriction sites.
  • the PCR product was analyzed on a 1% agarose gel, and the band of interest was purified using a commercially available kit (QIAEX ® II gel purification reagents and protocol; Qiagen, Inc.) .
  • the purified DNA was labeled with 32 P using a commercially available kit (RediprimeTM DNA labeling system; Amersham Corp.) .
  • the probe was purified using a push column. Hybridization was carried out in 8 mis of hybridization 71
  • Example 3 Zalpha5 was mapped to human chromosome 1 using the commercially available GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) .
  • 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 world-wide web server http : //www-genome . wi .mit . edu/cgi- bin/contig/rhmapper .pi
  • WICGR Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome
  • Each of the 95 PCR reactions contained 2 ⁇ l buffer (10X KlenTaq PCR reaction buffer; Clontech Laboratories, Inc., Palo Alto, CA) , 1.6 ⁇ l dNTPs mix (2.5 mM each, Perkin-Elmer, Foster City, CA) , 1 ⁇ l sense primer ZC 17,332 (SEQ ID NO:7), 1 ⁇ l antisense primer ZC 17,336 (SEQ ID NO:8), 2 ⁇ l of a density increasing agent and tracking dye 72
  • 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 58°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 results showed that zalpha5 maps 2.74 cR_3000 from the framework marker D1S230 on the chromosome 1 WICGR radiation hybrid map.
  • Proximal and distal framework markers were D1S230 and WI-9515 (D1S2423), respectively.
  • ESTs 917237 and 744974 were obtained from the IMAGE Consortium.
  • EST917237 contained the 3' end of the mouse zalpha5 sequence.
  • EST744974 contained both 5' and 3' ends but contained an internal alternative splice. Splicing of the two clones was done by PCR.
  • a first reaction was carried out with primers ZC18,979 (SEQ ID NO: 10) and ZC18,967 (SEQ ID NO: 11) with EST744974 as a template.
  • the second reaction was carried out with primers ZC18,969 (SEQ ID NO:12) and ZC18,968 (SEQ ID NO:13) with EST917237 73
  • the products of the third reaction were separated by gel electrophoreses, and the 1.36 kb product was purified by adsorption to silica gel particles (QIAEX® II gel extraction kit; Qiagen, Valencia, CA) .
  • the fragment was subcloned into the vector pCR®2.1-T0P0 using a commercially available cloning kit (Invitrogen) . Positive clones were sequenced for PCR errors.
  • the full- length mouse zalpha5 sequence is shown in SEQ ID NO: 14.
  • Expression constructs were made for eukaryotic cell production of N- and C-terminally glu-glu tagged proteins.
  • a mammalian expression vector was constructed with the dihyrofolate reductase gene under control of the SV40 early promoter and SV40 polyadenylation site, and a cloning site to insert the gene of interest under control of the mouse MT-1 promoter and the hGH polyadenylation site.
  • the expression vector was designated pZP-9 and was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD on February 20, 1998 under Accession Number 98668. To facilitate purification of the protein of interest, the pZP-9 vector was modified by addition of the tPA leader sequence (U.S.
  • pZP9NEE terminally tagged vector
  • Another vector was similarly constructed with a C-terminal GluGlu tag (SEQ ID NO: 9) inserted just 5' to the hGH terminator and utilizes the native (or other fused) secretory signal sequence for secretion of the encoded polypeptide of interest; expression resulted in a C-terminally tagged protein.
  • the C-terminal GluGlu tagged vector was designated pZP9CEE.
  • zalpha5CEEpZP9 was created by ligation of a PCR-generated zalpha5 sequence into the vector pZP9CEE.
  • EST 319951 (Example 1) was used as a template in a PCR reaction using oligonucleotide primers ZC16,985 (SEQ ID N0:5) and ZC17,250 (SEQ ID NO:6).
  • PCR conditions were: one cycle at 94° for 1'30"; five cycles at 94° for 10", 32° for 20", 72° for 1'30”; 20 cycles at 94° for 10", 64° for 20", 72° for 1'30”; one cycle at 72° for 7'; followed by a 4° hold.
  • the reaction mixture was gel electrophoresed, and the resulting band was purified using silica gel particles (QIAEX® II gel extraction kit; Qiagen, Valencia, CA) .
  • the DNA was digested with EcoRI and BamHI following standard protocols.
  • the reaction mixture was gel electrophoresed, and the band was re-purified.
  • a portion of the purified DNA was ligated into pZP9CEE that had been previously digested with EcoRI and BamHI following standard protocols.
  • the ligated DNA was electroporated into E. coli host cells (Electromax DH10BTM cells; obtained from
  • Plasmid from a positive colony was purified using an anion-exchange kit (QIAGEN® Plasmid Maxi Kit, Qiagen Inc., Valencia, CA) according to the manufacturer's directions.
  • a second construct, zalpha5NEEpZP9 was created by ligation of a PCR-generated zalpha ⁇ sequence into the vector pZP9NEE.
  • EST 319951 was used as a template in a 75
  • PCR reaction using primers zcl6,987 and zcl7,548. PCR conditions were as disclosed above for zalpha5CEEpZP9.
  • the reaction mixture was gel electrophoresed, and the resulting band purified using silica gel particles.
  • the DNA was digested with BamHI and Xbal following standard protocols.
  • the reaction mixture was gel electrophoresed, and the band was re-purified.
  • a portion of the purified DNA was ligated into pZP9NEE that had been digested with BamHI and Xbal following standard protocols.
  • the ligated DNA was electroporated into E. coli host cells
  • Three expression vectors were prepared to express zalpha5 polypeptides in insect cells: palpha5 , designed to express a zalpha5 polypeptide with its native leader and no tag; zalpha5CEE pzbv4L, designed to express a zalpha5 polypeptide with a C-terminal Glu-Glu tag; and zalpha5NEE pzbv4L, designed to express a zalpha5 polypeptide with an N-terminal Glu-Glu tag.
  • baculovirus expression vector pFastBac; Life Technologies, Gaithersburg, MD
  • pFastBac baculovirus expression vector
  • the resulting vector was designated pZBV3L. 81.8 ng of a 1394-bp EcoRI/Xba I zalpha5 fragment and 95.6 ng of the EcoRI/Xba I-digested pZBV3L were ligated overnight.
  • the ligation mixture was diluted 3 -fold in TE (10 mM Tris-HCI, pH 7.5 and 1 mM EDTA), and 4 fmol of the diluted ligation mixture was transformed into competent E. coli cells (Library Efficiency DH5 ⁇ TM competent cells;
  • the ligated DNA was diluted in 980 ⁇ l SOC media and plated onto Luria Agar plates containing 50 ⁇ g/ml kanamycin, 7 ⁇ g/ml gentamicin, 10 ⁇ g/ml tetracycline, IPTG and halogenated indolyl- ⁇ -D- galactoside (bluo-gal; Life Technologies) .
  • the cells were incubated for 48 hours at 37°C. A color selection was used to identify those cells having virus that had incorporated into the plasmid (referred to as a "bacmid”) . Those colonies that were white in color were picked for analysis.
  • Bacmid DNA was isolated from positive colonies using a commercially available kit
  • Clones were screened for the correct insert by amplifying DNA using primers to the Basic Protein Promoter and to the SV40 terminator via PCR. Those having the correct insert were used to transfect Spodoptera frugiperda (Sf9) cells.
  • Vector zalpha5CEEpZP9 (Example 5) was digested with EcoRI and Xbal .
  • the reaction mixture was gel electrophoresed, and the band corresponding to zalpha5CEE was gel-purified as described in Example 5.
  • the purified DNA was ligated into pZBV4L (a modified pFastBacTM expression vector (Life Technologies) containing the late activating Basic Protein promoter) that had been digested with with EcoRI and Xbal .
  • pZBV4L a modified pFastBacTM expression vector (Life Technologies) containing the late activating Basic Protein promoter
  • E. coli host cells E. coli host cells
  • Positive ligations were identified by colony PCR using primers ZC7350 (SEQ ID N0:16) and ZC16,084 (SEQ ID N0:17).
  • PCR conditions were: one cycle at 94° for 1'30"; thirty cycles at 94° for 10", 52° for 20", 72° for 1'30"; one cycle at 72° for 1'30"; followed by a 4 ° hold.
  • Plasmid from a positive colony was purified using an anion-exchange kit as disclosed above.
  • One microliter of zalpha5CEEpZBV4L was used to independently transform 20 ⁇ l E. coli Max
  • zalpha5NEEpZBV4L An expression construct designated zalpha5NEEpZBV4L was constructed from zalpha5NEEpZP9 and pZBV4L essentially as disclosed above for zalpha5CEEpZBV4L. One microliter of zalpha5NEEpZBV4L was used to independently transform 20 ⁇ l of E. coli Max
  • Sf9 cells were seeded at 5 x 10 6 cells per 35- mm plate and allowed to attach for 1 hour at 27°C.
  • Five microliters of bacmid DNA was diluted with 100 ⁇ l serum- 78
  • Sf-900 II SFM Eight hundred microliters of Sf-900 II SFM was added to the lipid-DNA mixture.
  • the wash media was aspirated, and the DNA-lipid mixture was added to the cells.
  • the cells were incubated at 27°C for 4-5 hours.
  • the DNA-lipid mixture was aspirated, and 2 ml of Sf-900 II media was added to each plate.
  • the plates were incubated at 27°C, 90% humidity for 96 hours, after which the virus was harvested.
  • Sf9 cells were grown in 50 ml Sf-900 II SFM in a 125-ml shake flask to an approximate density of 0.41- 0.52 x 10 5 cells/ml. They were then infected with 100 ⁇ l of virus stock (disclosed above) and incubated at 27°C for 2-3 days, after which the virus was harvested.
  • the titer for palpha ⁇ was 2.9xl0 7 pfu/ml, for Zalpha5CEEpZBV4L it was 4xl0 6 pfu/ml, and for Zalpha5NEEpZBV4L it was 1.85xl0 7 pfu/ml.
  • zalpha5 cDNA is inserted into the expression vector pHB12-8 (see Fig. 2) .
  • Vector pHB12-8 was derived from P2999B4 (Palmiter et al . , Mol . Cell Biol . 13 :5266-5275,
  • the vector comprises a mouse metallothionein (MT- 1) promoter (ca. 750 bp) and human growth hormone (hGH) untranslated region and polyadenylation signal (ca. 650 79
  • the cDNA is inserted between the insulin II and hGH sequences.
  • Zalpha5 protein produced in a baculovirus or BHK cell system is purified by a combination of cation and anion exchange chromatography followed by size exclusion chromatography.
  • Conditioned cell culture medium is diluted to ⁇ 5mS with 20 mM MES, phosphate, or acetate/citrate buffer, pH 5.5-6.5.
  • the diluted medium is loaded onto a cation exchange column (Poros ® 20HS or 50HS (PerSeptive Biosystems, Framingham, MA) or SP Sepharose ® Fast Flow (Pharmacia Biotech, Piscataway, NJ) ) at a ratio of 100-200 volumes medium to resin.
  • the column is washed with 40-50 column volumes of the same buffer used to dilute the medium.
  • Protein is eluted with a 0-1M NaCl gradient over 40-50 column volumes.
  • the resulting eluate is diluted to ⁇ 5 mS with 20 mM Tris-HCI or HEPES buffer, pH 7.4-8.5.
  • the diluted eluate is loaded onta a Poros ® 20HQ (PerSeptive Biosystems) column at a ratio of ⁇ 50 volumes sample to resin.
  • the column is washed with 40-50 column volumes of the same buffer used to dilute the sample. Protein is eluted with a 0-1M NaCl gradient over 40-50 column volumes.
  • the eluate from the HQ column is concentrated using a centrifugal filter device (UltrafreeTM-15 ; Millipore, Bedford, MA) and loaded onto a Sephacryl ® S-200 or S-300 (Pharmacia Biotech) size exclusion column run in PBS.
  • a centrifugal filter device UltrafreeTM-15 ; Millipore, Bedford, MA
  • Sephacryl ® S-200 or S-300 Pharmacia Biotech
  • N-terminal glu-glu tagged zalpha ⁇ is produced in BHK cells according to conventional procedures.
  • the tagged protein is purified by affinity chromatography using an antibody to the glu-glu tag. Unless otherwise noted, all operations are carried out at 4°C. 80
  • BHK cell-conditioned medium is sequentially sterile filtered through a 4 inch, 0.2 mM capsule filter (OpticapTM; Millipore, Bedford, MA) and a 0.2 mM capsule filter (SupercapTM 50; Gelman, Ann Arbor, MI).
  • the material is then concentrated about 20x using a tangential flow concentrator (ProFlux® A30; Millipore) fitted with a 3000 kDa cutoff ultrafiltration membrane (S10Y3 membrane; Amicon, Bedford, MA) .
  • the concentrated material is again sterile-filtered with the Gelman filter as described above.
  • a mixture of protease inhibitors is added to the concentrated conditioned media to final concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.
  • a 100 ml bed volume of immobilized protein G (protein G-Sepharose®; Pharmacia Biotech) is washed 3 times with 100 ml of PBS containing 0.02% sodium azide using a 500 ml Nalgene 0.45 micron filter unit.
  • the gel is washed with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA; Sigma Chemical Co., St. Louis, MO), and an equal volume of anti-glu-glu antibody solution containing 900 mg of antibody is added. After an overnight incubation at 4°C, unbound antibody is removed by washing the resin with 5 volumes of 200 mM TEA as described above.
  • the resin is resuspended in 2 volumes of TEA, transferred to a suitable container, and dimethylpimilimidate-2HCl (Pierce, Rockford, IL) , dissolved in TEA, is added to a final concentration of 36 mg/ml of gel .
  • the gel is rocked at room temperature for 45 minutes, and the liquid is removed using the filter unit as described above. Nonspecific sites on the gel are then blocked by incubating for 10 minutes at room temperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA.
  • the gel is washed with 5 volumes of PBS containing 0.02% sodium azide and stored in this solution at 4°C.
  • a 25.0 ml sample of anti-GluGlu Sepharose is added to the concentrated culture medium for batch adsorption, and the mixture is gently agitated on a roller culture apparatus (Wheaton Science Products, Millville, NJ) for 18.0 hours at 4°C.
  • the mixture is poured into a 5.0 x 20.0 cm column (Econo-Column® ; Bio- Rad Laboratories, Hercules, CA) , and the gel is washed with 10 column volumes (cv) of phosphate buffered saline
  • IM glycine, pH 2.5, and the glycine wash is collected separately.
  • the pH of the glycine-eluted fraction is adjusted to 7.0 by the addition of a small volume of 10X PBS and stored at 4°C for future analysis if needed.
  • the peptide elution fraction is concentrated to 5.0 ml using a 15,000 molecular weight cutoff membrane concentrator (Millipore, Bedford, MA) according to the manufacturer's instructions.
  • the concentrated peptide elution is separated from free peptide by chromatography on a 1.5 x 50 cm Sephadex ® G-50 (Pharmacia Biotech) column equilibrated in PBS at a flow rate of 1.0 ml/min using a commercially available HPLC system (BioCadTM/SprintTM HPLC system; PerSeptive BioSystems, Framingham, MA) . Two-ml fractions are collected, and the 82
  • absorbance at 280 nM is monitored.
  • the first peak of material absorbing at 280 nM and eluting near the void volume of the column, which contains the purified zalpha ⁇ protein, is collected.
  • the pure material is concentrated as described above, analyzed by SDS-PAGE and Western blotting with anti-GluGlu antibodies, and samples are taken for amino acid analysis and N-terminal sequencing. The remainder of the sample is aliquoted and stored at - 80°C.
  • the molecular weight predicted for human zalpha ⁇ was 52 kDa.
  • the observed molecular weight of recombinant zalpha5 with an N-terminal Glu-Glu tag made in BHK cells was « 68kd.
  • Zalpha5 produced by a baculovirus expression system was found to have an amino terminus at residue 17
  • the baculovirus-produced protein included cleavage products of 36.5 kDa and 21.5 kDa, as well as full-length protein of approximately 60 kDa.
  • a second preparation of zalpha ⁇ protein from baculovirus was electrophoresed on a reducing 4-20% tris- glycine-SDS polyacrylamide gel, transferred to a PVDF membrane, and stained with coomassie blue.
  • the protein ran as a doublet of M r ⁇ 36 kDa. Sequencing indicated the presence of multiple species, the predominant species having an amino terminus at Thr225 and lesser species with amino termini at Leu231, Asn232, and Glu233.
  • Zalpha5 produced in BHK cells and by baculovirus expression ran as a multimer on size exclusion chromatography.
  • N-terminal glu-glu tagged zalpha5 was analyzed by sequencing, peptide mapping, carbohydrate analysis, and disulfide bond determination.
  • peptide mapping the protein was digested with several different enzymes to generate a peptide map.
  • the peptide mixtures were then analyzed by liquid chromatography/mass spectrometry using a C-18 column, a gradient of 1-81% B (90% acetonitrile, 0.1% trifluoroacetic acid), and an ion trap mass spectrometer. Peptides corresponding to most of the predicted amino acid sequence were identified. The C- terminal peptide was easily identified and showed no heterogeneity. The first N-linked glycosylation site (Asn-Ser-Ser, residues 23-25 of SEQ ID NO: 2) appeared to be unoccupied since the unmodified peptide was clearly present. Two sites of probable O-linked glycosylation were identified in peptides 190-205 of SEQ ID NO: 2
  • the first peptide was digested with V-8 protease in 50 mM sodium phosphate, pH 7.8.
  • This second digest mixture was spotted on MALDI targets (both non- reduced and reduced) using both alpha-cyano-4- hydroxycinnamic acid and sinapinic acid as matrices.
  • the data indicated the presence of a disulfide bond between the two cysteines (residues 394 and 408 of SEQ ID NO : 2 ) .
  • Material from the second fraction was spotted on MALDI targets (both non-reduced and reduced) using both alpha-cyano-4-hydroxycinnamic acid and sinapinic acid as matrices .
  • Anti-zalpha5 polyclonal antibodies were raised using the peptide immunogens shown below in Table 6. Peptides were prepared by conventional synthetic techniques. Sequence numbers in Table 6 refer to SEQ ID NO : 2. "Cys" indicates and additional C- or N-terminal cysteine residue.
  • Peptides huzalpha5-l and huzalpha5-2 were conjugated to maleimide-activated keyhole limpet hemocyanin through the terminal cysteine.
  • Antibodies were prepared in New Zealand white rabbits. Animals were given initial immunizations of 200 ⁇ g/animal with Freund ' s complete adjuvant, and three boosts, at 3 -week intervals, of 100 ⁇ g/animal with Freund ' s incomplete adjuvant. Animals were first bled after the second boost, then at 3 -week intervals thereafter. Antisera were affinity-purified on a CNBr- Sepharose-peptide column prepared with 10 mg peptide per gram of Sepharose. Purified antibodies were dialyzed overnight against PBS.
  • Immunocytochemical screening analyses were performed on normal and diseased human liver, monkey liver, normal human kidney, and normal human ileum using a polyclonal rabbit antibody to full-length zalpha5 protein as the primary antibody at dilutions of 1:5,000 and 1:10,000.
  • the detection system employed a commercially available biotinylated second antibody and staining kit (VECTASTAIN® ABC-AP kit; Vector 87
  • zalpha5 was strongly expressed in the space of Disse and within Kupffer cells of the liver, the convoluted tubules and collecting ducts of the kidney (staining concentrated along the brush border of the proximal tubule and along the luminal border or the distal tubules and collecting ducts) , and within nerves of the ileum and liver.
  • Vascular smooth muscle was negative.
  • the location of the signal in the liver was along the interface between the hepatocyte, sinusoidal lining cell, and Kupffer cell. Light microscopy did not reveal whether the sinusoidal lining cell was producing the protein independently of the signal seen in the Kupffer cell and along the hepatocyte cell membrane. Occasional staining was observed along the bile canaliculi between hepatocytes, suggesting that the protein is either membrane-bound by hepatocytes or produced by normal hepatocytes .
  • carcinomas In carcinomas, strong expression was seen in both hepatomas and adenocarcinomas . The level of positive staining was variable within the carcinomas and was seen along both the sinusoidal and biliary borders of the cell and within acinar spaces.
  • the positive signal was seen along the opposite border of the hepatocyte as compared to normal liver and was concentrated toward the bile canaliculi rather than the 88
  • a zalpha5- saporin conjugate was administered to cells, tissue slices, and animals.
  • Saporin was conjugated to zalpha5 using conventional methods.
  • the prostate epithelial cell line pz-HPV7 specifically bound 125 I-zalpha5 and was killed by saporin- zalpha5 with an LD50 of 10 nM, while saporin alone was at least 100-fold less toxic. Cell killing was abrogated by the addition of unconjugated zalpha ⁇ . Similar results were obtained with prostate tissue slices.
  • saporin-zalpha ⁇ showed no toxicity toward DU-145, a prostate cell line that does not bind 125j_ zalpha5. Saporin and saporin-zalpha ⁇ were injected at 4,
  • mice 40, and 400 ⁇ g/kg into the tail vein of mice at day 1, day 3, and day 8 for saporin and saporin-zAlpha5. On day 9 all animals were sacrificed, and samples were prepared for physioscreen.
  • Chemoattractant activity of zalpha5 was assayed using a matrigel assay.
  • the matrigel invasion assay assesses the ability of a cell to move through a basement membrane matrix coated on a PET membrane containing uniform 8 -micron pores.
  • the layer of matrigel derived from the Engelbreth-Holm-Swarm tumor as a soluble membrane extract, serves as a basement membrane for in vi tro studies. It occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. A cell must move through the matrigel, through 89
  • the matrigel invasion assay is obtained from
  • VEGF Human Umbilical Vein Endothelial Cells
  • Mvec Human Uterine Myometrial Microvascular Endothelial Cells
  • VEGF was purchased from RD Systems, Minneapolis, MN.
  • the invasion chambers are brought to room temperature in a laminar air- flow hood. Sterile technique is used throughout the assay.
  • the Matrigel is rehydrated with 250 ⁇ l of warm media for 2 hours.
  • Assay media for Huvec cells is EBM containing 1.0% fetal bovine serum and GA1000; Mvec media is EGM-2 containing 0.5 % fetal bovine serum, GA1000, and ascorbic acid (obtained from Clonetics) .
  • Cells are removed from tissue culture flasks using trypsinization reagents
  • ReagentPackTM reagents; Clonetics ReagentPackTM reagents; Clonetics
  • Positive control protein and zalpha5 are diluted in assay media such that when lO ⁇ l is added to the lower chamber of the kit, the VEGF concentration is 100 ng/ml and the zalpha ⁇ concentration varies.
  • Media 750 ⁇ l is added to the lower chamber of the assay kit followed by control or test protein.
  • the rehydration media placed into the top of the insert is removed, and 0.5 ml of cell suspension is added.
  • the insert is carefully placed into the well such that no air bubbles are trapped beneath the insert.
  • the plate is covered and placed into an incubator at 37° C, 5% C0 2 for 48 hours.
  • the media in the top of the insert Prior to staining the migrating cells, the media in the top of the insert is removed, and the top surface of the membrane is swabbed twice using a cotton 90
  • a commercially available staining kit (Diff-Quick, obtained from VWR Scientific Products, Salt Lake City, UT) is used to fix and stain the cells.
  • the inserts are placed in fixative for 2 minutes followed by stain 1 and then stain 2, 2 minutes each, followed by thorough rinsing in deionized water.
  • the inserts are numbered and allowed to air dry.
  • the membranes are removed using a #15 scalpel blade. The membrane is cut next to the plastic edge and the insert is rotated around the blade leaving only a small piece of the membrane attached and it is removed using forceps.
  • a drop of immersion oil is placed on a glass slide followed by the membrane placed bottom side down.
  • a drop of oil is placed on top followed by a glass coverslip.
  • the number of migrating cells is quantified using an image analysis program (Media Cybernetics, Optimus version 6.2).

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Abstract

L'invention concerne des facteurs de croissance polypeptidique, des procédés de production et d'utilisation desdits facteurs de croissance et des polynucléotides les codant. Ces polypeptides incluent les polypeptides zalpha5 de l'humain et de la souris indiqués dans les séquences SEQ ID NO.2 et SEQ ID NO.15, respectivement, ainsi que des homologues et des fragments desdits homologues. On décrit également des multimères desdits polypeptides. Ces polypeptides, protéines multimères et polynucléotides peuvent être utilisés dans l'étude de la régulation de la croissance cellulaire et tissulaire, en tant que constituants du milieu de culture cellulaire et en tant qu'agents diagnostiques.
PCT/US1999/008821 1998-04-27 1999-04-21 Nouveaux facteurs et materiels de croissance polypeptidiques et leurs procedes de production Ceased WO1999055869A1 (fr)

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WO2001042499A1 (fr) * 1999-12-09 2001-06-14 Sankyo Company, Limited Procede d'essai d'agent curatif ou preventif de l'hyperlipemie
WO2000053757A3 (fr) * 1999-03-08 2001-11-01 Genentech Inc Activation et inhibition de l'angiogenese et de la cardiovascularisation
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EP2087905A2 (fr) 2003-08-08 2009-08-12 Novo Nordisk A/S Interleukine-20 dans le traitement et le diagnostic des états associés à la neovascularisation
US9139633B2 (en) 2009-07-14 2015-09-22 Novartis Ag Mesenchymal stem cell differentiation
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EP1482041A1 (fr) * 1997-09-19 2004-12-01 Genentech Inc. Homologue NL6 du ligand du récepteur de kinase TIE
WO2000037638A3 (fr) * 1998-12-22 2000-11-09 Genentech Inc Methodes permettant d'inhiber la croissance de cellules neoplasiques
WO2000053757A3 (fr) * 1999-03-08 2001-11-01 Genentech Inc Activation et inhibition de l'angiogenese et de la cardiovascularisation
JP2003531811A (ja) * 1999-03-08 2003-10-28 ジェネンテック・インコーポレーテッド 血管形成及び心臓血管新生の促進又は阻害
JP2003505350A (ja) * 1999-07-20 2003-02-12 ジェネンテック・インコーポレーテッド 免疫関連疾患の治療のための組成物と方法
WO2001005972A1 (fr) * 1999-07-20 2001-01-25 Genentech, Inc. Compositions et methodes pour traiter des maladies d'ordre immunologique
WO2001042499A1 (fr) * 1999-12-09 2001-06-14 Sankyo Company, Limited Procede d'essai d'agent curatif ou preventif de l'hyperlipemie
EP1403367A4 (fr) * 2001-06-08 2005-08-17 Sankyo Co Procede de test de medicament destine a traiter ou a prevenir des maladies telles que l'hyperlipemie
US7267819B2 (en) * 2001-11-16 2007-09-11 Genentech, Inc. Composition comprising and method of using angiopoietin-like protein 3 Angptl3
US8541376B2 (en) 2001-11-16 2013-09-24 Genentech, Inc. Composition comprising and method of using angiopoietin-like protein 3 ANGPTL3
EP2087905A2 (fr) 2003-08-08 2009-08-12 Novo Nordisk A/S Interleukine-20 dans le traitement et le diagnostic des états associés à la neovascularisation
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US11389541B2 (en) 2018-10-03 2022-07-19 Novartis Ag Sustained delivery of angiopoetin-like 3 polypeptides

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