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WO2000020595A1 - Homologues de l'interleukine-1 - Google Patents

Homologues de l'interleukine-1 Download PDF

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Publication number
WO2000020595A1
WO2000020595A1 PCT/US1999/023533 US9923533W WO0020595A1 WO 2000020595 A1 WO2000020595 A1 WO 2000020595A1 US 9923533 W US9923533 W US 9923533W WO 0020595 A1 WO0020595 A1 WO 0020595A1
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Prior art keywords
protein
zil1a3
seq
amino acid
cells
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PCT/US1999/023533
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English (en)
Inventor
Paul O. Sheppard
Robert R. West
Christopher H. Clegg
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Zymogenetics, Inc.
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Priority to AU62968/99A priority Critical patent/AU6296899A/en
Publication of WO2000020595A1 publication Critical patent/WO2000020595A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the interleukins are a family of cytokines that mediate immune and inflammatory responses
  • IL-1 lnterleukin-1
  • IL-1 ⁇ and IL-1 ⁇ are pro-inflammatory cytokines
  • IL-1 receptor antagonist (IL-1ra) is an antagonist of IL-1 ⁇ and IL-1 ⁇ activities.
  • IL-1ra is unusual in that it is the only known, naturally occuring cytokine receptor antagonist with no apparent agonist function. The ability of IL-1ra to bind, but not activate, the IL-1 receptor suggests that IL-1ra is a negative regulator of inflammation (Dripps et al., J. Biol. Chem. 266:10331-10336, 1991 ; Granowitz et al., Blood 79:2356-2363, 1992).
  • the interleukins mediate a variety of inflammatory pathologies.
  • 11-1 ⁇ and IL-1 ⁇ act locally on mononuclear phagocytes and the vascular endothelium to induce further IL-1 and IL-6 synthesis.
  • IL-1 does not act directly on leukocytes and neutrophils, but causes the mononuclear phagocytes and endothelial cells to activate leukocytes.
  • IL-1 When secreted in large quantities into the bloodstream, IL-1 has endocrine effects, including fever, synthesis of acute phase plasma proteins, and cachexia.
  • IL-1 receptor antagonist human peripheral blood mononuclear cells (PMNC) cultured on IgG-coated plates and from the human monocytic cell line U937 activated with phorbol ester (PMA) (Carter et al., Nature 344:633-638, 1990; Hannum et al., Nature 343:336-340, 1990; Eisenberg et al., Nature 343:341-346, 1990).
  • PMNC peripheral blood mononuclear cells
  • PMA phorbol ester
  • Serum from healthy people has a low level of circulating IL-1ra activity (200-400 pg/ml). Serum IL-1ra levels are dramatically increased in patients with acute or chronic inflammatory disease, certain cancers, infectious diseases, and septic shock (Fisher et al., Blood 79:2196-2200, 1992), major surgery for Hirshsprung's disease (O Nuallain et al., C//t7. Exp. Immunol. 93:218-222, 1993), liver disease (Sekiyama et al., Clin. Exp. Immunol.
  • IL-1ra has been investigated for use in treating several chronic inflammatory disorders including rheumatoid arthritis (Henderson et al., Cytokine 3:246-249, 1991), chronic myelogenous leukemia (CML) (Schiro et al., Blood 83:460-465, 1994), and inflammatory bowel disease (IBD) (Cominelli et al., Gastroenterolo ⁇ y 103:65-71 , 1992).
  • CML chronic myelogenous leukemia
  • IBD inflammatory bowel disease
  • IL-1ra normal skin expresses IL-1ra mainly in the differentiated stratum granulosum of the epidermis, whereas psoriatic skin expresses IL-1ra in basal midbasal layers (Hammerberg et al, d. Clin. Invest. 90:571-583, 1992). Changes in the IL- 1 ⁇ :IL1-ra ratio in different strata of the epidermis may affect keratinocyte proliferation and differentiation. Chronic inflammatory bowel disease may also involve an altered IL-1 :ll-1 ra ratio since it is markedly increased in Crohn's disease and ulcerative colitis (Cominelli et al., Cytokine 6:A171 , 1994).
  • IL-1ra may be useful in chronic and acute cerebral neuropathologies (Relton et al., Exp. Neurol. 138:206-213, 1996; Loddick et al., Biochem. Biophys. Res. Comm. 234:211-215, 1997), insulin dependent diabetes mellitus (Madrup-Poulsen et al., Cytokine 5:185- 191 , 1993), glomerulonephritis (Lan et al., Kidney Int. 47:1303-1309, 1995), and pancreatitis (Norman et al., Ann. Surg. 221 :625. 1995).
  • Increased IL-1 production has been reported in patients with various viral, bacterial, fungal, and parasitic infections; intravascular coagulation; high-dose IL-2 therapy; solid tumors; leukemias; Alzheimer's disease; HIV-1 infection; autoimmune disorders; trauma (surgery); hemodialysis; ischemic diseases (myocardial infarction); noninfectious hepatitis; asthma; UV radiation; closed head injury; pancreatitis; peridontitis; graft-versus-host disease; transplant rejection; and in healthy subjects after strenuous exercise (Dinarello, Blood 87:2095-2147, 1996).
  • IL-1ra has been shown to be well tolerated in clinical trials in humans (Campion et al., Arthritis and Rhematism 39: 1092- 1101 , 1996), and to be potentially efficacious in the treatment of septic shock (Fisher et al., dAMA 271:1836-1843, 1994), rheumatoid arthritis (Campion et al., Arthritis & Rheumatism 39:1092-1101 , 1996), and graft vs. host disease (GVHD) (Antin et al., Blood 84:1342-1348, 1994).
  • GVHD graft vs. host disease
  • the present invention provides novel interleukin-1 (IL-1) homologs, as well as materials and methods for making the IL-1 homologs, compositions comprising them, and methods for using them.
  • the invention thus provides an isolated protein comprising a sequence of amino acid residues as shown in SEQ ID NO:7.
  • the protein comprises a Lys residue at position 148 of SEQ ID NO:7 or an Asp residue at position 148 of SEQ ID NO:7.
  • the protein comprises the amino acid sequence of SEQ ID NO:8.
  • the protein is from 155 to 1200 amino acid residues in length.
  • the protein is 155 amino acid residues in length.
  • an isolated polypeptide of at least 15 amino acid residues comprising an epitope- bearing portion of a protein of SEQ ID NO:7.
  • an expression vector comprising the following operably linked elements: (a) a transcription promoter; (b) a DNA segment encoding a protein comprising a sequence of amino acid residues as shown in SEQ ID NO:7; and (c) a transcription terminator.
  • the expression vector further comprises a secretory signal sequence operably linked to the DNA segment.
  • the protein comprises a Lys residue or an Asp residue at position 148 of SEQ ID NO:7.
  • the protein comprises the amino acid sequence of SEQ ID NO:8.
  • the protein is from 155 to 1200 amino acid residues in length.
  • the protein is 155 amino acid residues in length.
  • a cultured cell comprising an expression vector as disclosed above.
  • the invention provides a method of making a protein, comprising culturing a cell as disclosed above under conditions wherein the DNA segment is expressed, and recovering the protein encoded by the DNA segment.
  • the invention provides an antibody that specifically binds to a protein as disclosed above.
  • a method of modulating an immune response in an animal comprising administering to the animal a composition comprising a protein as disclosed above in combination with a pharmaceutically acceptable vehicle.
  • the figure is a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2.
  • the profile is based on a sliding six- residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO d. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31 , 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • 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.
  • a "complement of a polynucleotide molecule” is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3" is complementary to 5' CCCGTGCAT 3'.
  • 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).
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78. 1985).
  • An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • isolated does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • a "motif is a series of amino acid positions in a protein sequence for which certain amino acid residues are required. A motif defines the set of possible residues at each such position.
  • 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.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ -globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • polynucleotide is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides (“nt”), or kilobases ("kb”). Where the context allows, the latter two terms may describe polynucleotides that are single- stranded or double-stranded.
  • double-stranded molecules When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
  • polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non- coding regions of genes.
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • the term "receptor” denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction.
  • Many cell-surface receptors are, in their active forms, multi-peptide structures in which the ligand-binding and signal transduction functions may reside in separate subunits. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are linked to receptor- ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids, and hydrolysis of phospholipids.
  • receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • secretory signal sequence denotes 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.
  • a “segment” is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes. For example, a polynucleotide or polypeptide having specified attributes.
  • 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.
  • the term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence.
  • the term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
  • the present invention provides a group of novel proteins, designated "zil1a3", that are members of the IL-1 family.
  • Analysis of the human zil1a3 sequence indicates that this protein, like other members of the family, contains a core structure of 12 ⁇ -strands wound into a ⁇ -barrel, with the ⁇ -strands separated from each other by loops.
  • loops between these ⁇ -strands are highly variable among the family members and are believed to be involved in receptor binding. These loops, which each contain at least three amino acid residues and may contain up to
  • 17 residues do not form ⁇ -strands or helices, but may (and often do) contain ⁇ -turns.
  • the twelve ⁇ -strands are formed by residues 8-13, 17-21 , 26-28, 41-47, 56-61 , 66-71 , 77-83, 99-105, 109-113, 119-123, 131-133, and 149-153.
  • These strands are characterized by a high content of hydrophobic amino acid residues (Leu, Val, Phe, and lie).
  • the loops include residues 14-16, 22-25, 29-40, 48-55, 62-65, 72-76, 84-98, 106- 108, 114-118, 124-130, and 134-148.
  • the zil1a3 proteins are characterized by the presence of conserved motifs at positions corresponding to (1) residues 111-115 of SEQ ID NO:2, (2) residues 117-121 of SEQ ID NO:2, (3) residues 130-134 of SEQ ID NO:2, and (4) residues 146-149 of SEQ ID NO:2. These motifs are shown in Table 1 using the standard single- letter codes for amino acid residues.
  • the higher-order structure of the IL-1 family of proteins can also be envisioned as three 4-stranded covalent monomers assembled into a 12- stranded structure (a "trefoil").
  • a "trefoil” When these monomers are superimposed on each other, residues 41 , 99, and 148 of SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:8 occupy like positions in their respective monomers.
  • the proteins of the present invention have pro-inflammatory (agonist) or anti-inflammatory (antagonist) activity, depending on the particular amino acid sequence.
  • zil1a3 proteins having a Lys residue corresponding to position 148 of SEQ ID NO:2 will have anti-inflammatory activity, while those having an Asp or Glu residue at this position will have pro-inflammatory activity.
  • Sequence variations at positions corresponding to residues 41 and 99 of SEQ ID NO:2 may also influence biological activity.
  • the present invention includes zil1a3 proteins having Lys or Glu at a position corresponding to residue 41 of SEQ ID NO:2 and Ala, lie, or Thr at a position corresponding to residue 99 of SEQ ID NO:2.
  • zil1a3 proteins act through IL-1 receptors. Pro- and anti-inflammatory activities can be assayed using standard assays of IL-1 activity known in the art.
  • Non-naturally occurring amino acids include, without limitation, trans-3- methylproline, 2,4-methanoproline, c/s-4-hydroxyproline, trans-4- hydroxyproline, ⁇ /-methylglycine, a//o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tetf-leucine, norvaline, 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylalanine.
  • 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 occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site- directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • the effects of amino acid sequence changes can be predicted by computer modeling using available software (e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA) or determined by alignment and analysis of crystal structures. See, Priestle et al., EMBO d. 7:339-343, 1988; Priestle et al., Proc. Natl. Acad. Sci. USA 86:9667-9671 , 1989; Finzel et al., d. Mol. Biol. 209:779-791 , 1989; Graves et al., Biochem. 29:2679-2684, 1990; Clore and Gronenbom, d. Mol. Biol. 221 :47-53.
  • available software e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, CA
  • Alignment of zil1a3 with other family members also provides guidance in selecting amino acid substitutions, particularly if information about the effects of amino acid substitutions in other family members is available. For example, alignment suggests that residue 148 (Asp) can be replaced with Lys, resulting in a change in activity from agonist to antagonist (Ju et al., Proc. Natl. Acad. Sci. USA 88:2658-2662, 1991 ; Oldfield et al., Protein Eng. 6:865-871 , 1993).
  • This variant of SEQ ID NO:2 (designated "hzil1a3-D148K”) is shown in SEQ ID NO:8.
  • Aromatic phenylalanine tryptophan tyrosine
  • the proteins of the present invention can further comprise amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag as disclosed above. Two or more affinity tags may be used in combination. Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zil1a3 polypeptide and the affinity tag. Preferred cleavage sites include thrombin cleavage sites and factor Xa cleavage sites. The present invention further provides a variety of other polypeptide fusions.
  • a zil1a3 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
  • lmmunoglobulin-zil1a3 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zil1a3 analogs.
  • a zil1a3 polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a multi-functional molecule.
  • zil1a3 polypeptide can be joined to another cytokine to enhance or otherwise modify its biological properties.
  • Auxiliary domains can be fused to zil1a3 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a zil1a3 polypeptide or protein can be targeted to a predetermined cell type by fusing a zil1a3 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • a zil1a3 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 996.
  • Polypeptide fusions of the present invention will generally contain not more than about 1 ,500 amino acid residues, preferably not more than about 1 ,200 residues, more preferably not more than about 1 ,000 residues, and will in many cases be considerably smaller.
  • a zil1a3 polypeptide of 155 residues can be fused to E. coli /?-galactosidase (1 ,021 residues; see Casadaban et al., d. Bacteriol.
  • residues 1-155 of SEQ ID NO:2 can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.
  • Essential amino acids in the proteins of the present invention can be identified according to procedures known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991).
  • site- directed mutagenesis or alanine-scanning mutagenesis
  • Single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity or other properties to identify amino acid residues that are critical to the activity of the molecule.
  • Variants of the disclosed zil1a3 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391 , 1994 and Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751, 1994. Briefly, variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zil1a3 variant proteins.
  • Mutagenized DNA molecules that encode active zil1a3 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.
  • Assays for IL-1 biological activity and receptor binding are known in the art.
  • Exemplary activity assays include mitogenesis assays in which IL-1 responsive cells (e.g., D10.N4.M cells) are incubated in the presence of IL-1 or a test zil1a3 protein for 72 hours at 37°C in a 5% CO 2 atmosphere.
  • IL-2 (and optionally IL-4) is added to the culture medium to enhance sensitivity and specificity of the assay. [ 3 H]thymidine is then added, and incubation is continued for six hours. The amount of label incorporated is indicative of agonist activity. See, Hopkins and Humphreys, J. Immunol. Methods 120:271-276, 1989; Greenfeder et al., J. Biol. Chem. 270:22460-22466, 1995. IL-1 stimulation of cell proliferation can also be measured using thymocytes cultured in IL-1 in combination with phytohemagglutinin.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide
  • Receptor binding can be measured by the competition binding method of Labriola-Tompkins et al., Proc. Natl. Acad. Sci. USA 88:11182-11186, 1991. Briefly, membranes pepared from EL-4 thymoma cells (Paganelli et al., J. Immunol. 138:2249-2253, 1987) are incubated in the presence of the test protein for 30 minutes at 37°C. Labeled IL-1 ⁇ or IL-1 ⁇ is then added, and the incubation is continued for 60 minutes. The assay is terminated by membrane filtration. The amount of bound label is determined by conventional means (e.g., ⁇ counter).
  • the ability of a ziha3 protein to compete with labeled IL-1 for binding to cultured human dermal fibroblasts is measured according to the method of Dower et al. (Nature 324:266-268, 1986). Briefly, cells are incubated in a round-bottomed, 96-well plate in a suitable culture medium (e.g., RPMI 1640 containing 1% BSA, 0.1% Na azide, and 20 mM HEPES pH 7.4) at 8°C on a rocker platform in the presence of labeled IL-1. Various concentrations of zil1a3 protein are added.
  • a suitable culture medium e.g., RPMI 1640 containing 1% BSA, 0.1% Na azide, and 20 mM HEPES pH 7.4
  • Receptor binding can also be measured using immobilized receptors or ligand-binding receptor fragments.
  • an immobilized IL-1 receptor can be exposed to labeled IL-1 and unlabeled test protein, whereby a reduction in IL-1 binding compared to a control is indicative of receptor-binding activity in the test protein.
  • a receptor or ligand-binding receptor fragment is immobilized on a biosensor (e.g., BIACoreTM, Pharmacia Biosensor, Piscataway, NJ) and binding is determined.
  • a biosensor e.g., BIACoreTM, Pharmacia Biosensor, Piscataway, NJ
  • IL-1 antagonists will exhibit receptor binding but will exhibit essentially no activity in IL-1 activity assays or will reduce the IL-1-mediated response when combined with IL-1.
  • a large excess of antagonist typically a 10- to 1000-fold molar excess may be necessary to neutralize IL-1 activity.
  • polypeptides can also include additional polypeptide segments as generally disclosed above.
  • the present invention further provides polynucleotide molecules, including DNA and RNA molecules, encoding zil1a3 proteins.
  • the polynucleotides of the present invention include the sense strand; the anti- sense strand; and the DNA as double-stranded, having both the sense and anti-sense strand annealed together by hydrogen bonds.
  • a representative DNA sequence encoding a human zil1a3 protein is set forth in SEQ ID NO:1.
  • DNA sequences encoding other zil1a3 proteins can be readily generated by those of ordinary skill in the art based on the genetic code.
  • Counterpart RNA sequences can be generated by substitution of U for T.
  • SEQ ID NO:9 is a degenerate DNA sequence that encompasses all DNAs that encode the zil1a3 polypeptide of SEQ ID NO:2.
  • SEQ ID NO: 10 is a degenerate DNA sequence that encompasses all DNAs that encode the zil1a3 polypeptide of SEQ ID NO:8.
  • the degenerate sequences of SEQ ID NO:9 and SEQ ID NO: 10 also provide all RNA sequences encoding SEQ ID NO:2 and SEQ ID NO:8, respectively, by substituting U for T.
  • zil1a3 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 465 of SEQ ID NO:9, nucleotide 1 to nucleotide 465 of SEQ ID NO: 10, and their respective RNA equivalents are contemplated by the present invention.
  • Table 3 sets forth the one-letter codes used within SEQ ID NOS:9 and 10 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter.
  • “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
  • WSN can, in some circumstances, encode arginine
  • MGN degenerate codon for arginine
  • AGY serine
  • some polynucleotides encompassed by a 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 shown in SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
  • codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequences disclosed in SEQ ID NOS:9 and 10 serve as templates for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferred codons can be tested and optimized for expression in various host cell species, and tested for functionality as disclosed herein.
  • zil1a3 polynucleotides provided by 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 zil1a3 RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201 , 1980), and include stomach and skin.
  • Total RNA can be prepared using guanidine-HCI extraction followed by isolation by centrifugation in a CsCI gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl.
  • cDNA Complementary DNA
  • genomic DNA can be isolated.
  • Polynucleotides encoding zil1a3 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • the polynucleotides of the present invention can also be synthesized using automated equipment ("gene machines") according to methods known in the art. See, for example, Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994; Itakura et al., Annu. Rev. Biochem. 53: 323-356, 1984; and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-637, 1990.
  • zil1a3 polynucleotide sequences disclosed herein can be used to isolate polynucleotides encoding other zil1a3 proteins.
  • Such other polynucleotides include alternatively spliced cDNAs (including cDNAs encoding secreted zil1a3 proteins) and counterpart polynucleotides from other species (orthologs).
  • orthologous polynucleotides can be used, inter alia, to prepare the respective orthologous proteins.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zil1a3 as disclosed herein.
  • Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a zil1a3-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • Hybridization will generally be done under low stringency conditions, wherein washing is carried out in 1 x SSC with an initial wash at 40°C and with subsequent washes at 5°C higher intervals until background is suitably reduced.
  • 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 zil1a3 sequence disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zil1a3 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • sequences disclosed in SEQ ID NO:1 and SEQ ID NO:2 represent a single allele of human ziha3, and that natural variation, including allelic variation and alternative splicing, is expected to occur. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1 , including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2.
  • cDNAs generated from alternatively spliced mRNAs that retain the inflammation modulating activity of zil1a3 are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • the sequence disclosed in SEQ ID NO:1 and SEQ ID NO:2 is believed to represent a cytoplasmically expressed form of the protein in view of the lack of a signal peptide sequence.
  • An additional, larger band is seen on northern blots, indicating the existence of such an alternate form.
  • sequence upstream of the initiation ATG may encode an alternatively spliced form of the protein or an alternative form that is translated from an upstream ATG.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • the proteins of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells.
  • Eukaryotic cells particularly cultured cells of multicellular organisms, are preferred.
  • a DNA sequence encoding a zil1a3 protein 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 a zil1a3 gene, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo.
  • the secretory signal sequence is operably linked to the zil1a3 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).
  • a zil1a3 protein is expressed cytoplasmically and is isolated after lysing the host cells.
  • Cultured mammalian cells are suitable 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 Cell Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO d.
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., d. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1 ; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus.
  • promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601 ,978) and the adenovirus major late promoter.
  • 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.”
  • An exemplary 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.
  • An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • Alternative markers that produce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, and placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., d. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV).
  • Recombinant baculovirus can also be produced through the use of a transposon-based system described by Luckow et al. (d. Virol. 67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (Bac-to-BacTM kit; Life Technologies, Rockville, MD).
  • the transfer vector (e.g., pFastBadTM; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, d. Gen. Virol. 71:971-976, 1990; Bonning et al., d. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and Rapoport, d. Biol. Chem. 270:1543-1549, 1995.
  • 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 zil1a3-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 zil1a3 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 (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveTM cells; Invitrogen, Carlsbad, CA).
  • host cells typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveTM cells; Invitrogen, Carlsbad, CA).
  • Spodoptera frugiperda e.g., Sf9 or Sf21 cells
  • Trichoplusia ni e.g., High FiveTM cells; Invitrogen, Carlsbad, CA.
  • 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.
  • MOI multiplicity of infection
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311 ; Kawasaki et al., U.S. Patent No. 4,931 ,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931 ,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311 ; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S.
  • 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. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Patents No. 5,716,808, 5,736,383, 5,854,039, and 5,888,768; and WIPO publications WO 99/14347 and WO 99/14320. Prokaryotic host cells, including strains of the bacteria
  • Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
  • the polypeptide When expressing a zil1a3 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the protein is recovered from the soluble fraction.
  • 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.
  • 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.
  • proteins of the present invention it is preferred to purify the proteins of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • a purified protein is substantially free of other polypeptides or proteins, particularly those of animal origin.
  • Expressed recombinant zil1a3 proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatographv: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice. Springer-Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988.
  • Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
  • Zil1a3 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. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides. Zil1a3 proteins of the present invention can be used to modulate inflammation and related processes.
  • zil1a3 e.g., SEQ ID NO:8
  • certain zil1a3 proteins may be used to treat or prevent chronic inflammatory diseases such as arthritis (including rheumatoid arthritis, osteoarthritis, and Lyme arthritis) and psoriasis; to reduce tissue damage after ischemia; and to treat septic shock, graft-versus-host disease, and leukemia.
  • arthritis including rheumatoid arthritis, osteoarthritis, and Lyme arthritis
  • psoriasis to reduce tissue damage after ischemia
  • septic shock graft-versus-host disease
  • leukemia leukemia
  • Antagonists are expected to have in vivo activity like of that of IL-1 receptor antagonist (IL-1 ra), which has shown beneficial effects in clinical trials directed to the treatment of rheumatoid arthritis (Campion et al., Arthritis & Rheumatism 39:1092-1101, 1996), graft-versus-host disease (Antin et al., Blood 84:1342-1348, 1994), septic shock (Fisher et al., JAMA 271 :1836-1843, 1994), and leukemia (Dinarello, Blood 87:2095-2147, 1996).
  • IL-1 ra IL-1 receptor antagonist
  • Agonists may promote wound healing in view of the effects of IL-1 on growth factor secretion and cell proliferation or be useful in the treatment of infections, in particular gastrointestinal infections.
  • Zil1a3 proteins can be tested in animal models of disease.
  • Animal models of psoriasis include the analysis of histological alterations in adult mouse tail epidermis (Hofbauer et al, Brit. J. Dermatol. 118:85-89, 1988; Bladon et al., Arch Dermatol. Res. 277:121-125, 1985).
  • anti- psoriatic activity is indicated by the induction of a granular layer and orthokeratosis in areas of scale between the hinges of the tail epidermis.
  • a topical ointment is applied daily for seven consecutive days, then the animal is sacrificed, and tail skin is examined histologically.
  • inflammation is induced in guinea pig epidermis by topically applying phorbol ester (phorbol-12-myristate-13- acetate; PMA), typically at ca. 2 g/ml in acetone, to one ear and vehicle to the contralateral ear.
  • PMA phorbol ester
  • Test compounds are applied concurrently with the PMA, or may be given orally. Histological analysis is performed at 96 hours after application of PMA.
  • This model duplicates many symptoms of human psoriasis, including edema, inflammatory cell diapedesis and infiltration, high LTB4 levels and epidermal proliferation. Cerebral ischemia can be studied in a rat model as disclosed by Relton et al.
  • Wound-healing models include the linear skin incision model of Mustoe et al. (Science 237:1333, 1987). In a typical procedure, a 6-cm incision is made in the dorsal pelt of an adult rat, then closed with wound clips. Test substances and controls (in solution, gel, or powder form) are applied before primary closure. It is preferred to limit administration to a single application, although additional applications can be made on succeeding days by careful injection at several sites under the incision. Wound breaking strength is evaluated between 3 and 21 days post wounding. In a second model, multiple, small, full-thickness excisions are made on the ear of a rabbit.
  • the cartilage in the ear splints the wound, removing the variable of wound contraction from the evaluation of closure.
  • Experimental treatments and controls are applied.
  • the geometry and anatomy of the wound site allow for reliable quantification of cell ingrowth and epithelial migration, as well as quantitative analysis of the biochemistry of the wounds (e.g., collagen content). See, Mustoe et al., J. Clin. Invest. 87:694, 1991.
  • the rabbit ear model can be modified to create an ischemic wound environment, which more closely resembles the clinical situation (Ahn et al., Ann. Plast. Surg. 24:17, 1990).
  • 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. Sci. USA 86:2229, 1989; Greenhalgh et al., Amer. J. Pathol. 136:1235, 1990).
  • 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).
  • 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.
  • Zil1a3-encoding polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.
  • test animals such as mice
  • viral vectors or naked DNA or transgenic animals can be produced.
  • viral delivery systems include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno- associated virus (AAV).
  • 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, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997.
  • the adenovirus system offers several advantages. Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential E1 gene is deleted from the viral vector, and the virus will not replicate unless the E1 gene is provided by the host cell (e.g., the human 293 cell line).
  • the host cell e.g., the human 293 cell line.
  • a zil1a3 gene can be introduced in a retroviral vector as described, for example, by Anderson et al., U.S. Patent No. 5,399,346; Mann et al., Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol.
  • the vector can be introduced by "lipofection" in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31 , 1988).
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages, including molecular targeting of liposomes to specific cells. Directing transfection to particular cell types is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • target cells are removed from the the animal, and the DNA is introduced as a naked DNA plasmid.
  • the transformed cells are then re-implanted into the body of the animal.
  • naked DNA vectors can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963- 7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.
  • Transgenic animals engineered to express a zil1a3 gene, and animals that exhibit a complete absence of zil1a3 gene function, referred to as "knockout mice” (Snouwaert et a!., Science 257:1083, 1992), can be generated (Lowell et al., Nature 366:740-42, 1993). See also, Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840, 1988; Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482, 1991 ; Whitelaw et al., Transgenic Res. V.
  • Polynucleotides used in generating transgenic animals that express a zil1a3 gene will preferably contain one or more introns; genomic sequences are thus preferred.
  • Antisense methodology can be used to inhibit zil1a3 gene transcription to examine the effects of such inhibition in vivo.
  • a polynucleotide as set froth in SEQ ID NO:1 are designed to bind to zil1a3-encoding mRNA and to inhibit translation of such mRNA.
  • the proteins of the present invention are formulated for local, including topical; or parenteral, including intravenous, subcutaneous, or intraperitoneal delivery according to conventional methods.
  • Intravenous administration will be by injection or infusion. In many instances it will be beneficial to administer the protein by infusion or multiple injections per day over a period of several days to several weeks, sometimes preceded by a bolus injection.
  • pharmaceutical formulations will include a zil1a3 protein in combination with 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.
  • inhibition of IL-1 activity requires a large molar excess of antagonist.
  • Doses of zil1a3 antagonist proteins will in general be quite large, particularly when treating life-threatening conditions. IL-1ra appears safe in high doses.
  • doses of zil1a3 antagonist proteins will range from as low as 10 mg per patient per day to as high as 100 mg or more per hour infused over a period of days.
  • Doses of IL-1ra found to be efficacious in clinical studies include 70 mg per patient per day in rheumatoid arthritis and up to 3,400 mg per patient per day in graft-versus-host disease. The exact dose will be 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 proteins may be administered for acute treatment, over one week or less, but will often be used in treatment of chronic conditions requiring administration over several weeks to several months or longer.
  • a therapeutically effective amount of a zil1a3 protein is an amount sufficient to produce a clinically significant improvement in one or more standard indicators appropriate to the treated condition. Therapeutic endpoints will be apparent to those skilled in the art.
  • Zil1a3 proteins can be used as standards in assays of IL-1 and IL-1 inhibitor.
  • Such assays can comprise any of a number of standard formats, include radioreceptor assays and ELISAs.
  • Zil1a3 protein standards can be prepared in labeled form using a radioisotope, enzyme, fluorophore, or other compound that produces a detectable signal.
  • the proteins can be packaged in kit form, such kits comprising one or more vials containing the zil1a3 protein and, optionally, a diluent, an antibody, a labeled binding protein, etc.
  • Assay kits can also be used in the research laboratory to detect IL-1 and IL-1 inhibitor activities produced by cultured cells or test animals.
  • Zil1a3 proteins are also useful as research reagents.
  • zil1a3 agonist proteins can be used as cell culture components to promote the growth of IL-1 responsive cells, including fibroblasts, smooth muscle cells, and mesangial cells.
  • Agonists can be combined with IL-3 and other cytokines to expand CD34 + peripheral blood cells, and with IL-3 and IL-6 to promote the proliferation of stem cells.
  • the invention further provides polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:7.
  • 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 8J_:3998-4002, 1984.
  • 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.
  • Antigenic, epitope-bearing zil1a3 polypeptides useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zil1a3 protein contain a sequence of at least six, preferably at least nine, more preferably from 15 to about 30 contiguous amino acid residues of a zil1a3 protein (e.g., SEQ ID NO:2 or SEQ ID NO:8). Polypeptides comprising a larger portion of a zil1a3 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. Sequences containing proline residues are preferred. Preferred such regions include residues 10-15, 38-43, 91-96, 92-97, and 124- 129 of SEQ ID NO:2. As noted above, it is generally preferred to use somewhat longer peptides as immunogens, such as a peptide comprising residues 91-104 SEQ ID NO:2.
  • antibodies includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies.
  • Non-human antibodies can be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • One skilled in the art can generate humanized antibodies with specific and different constant domains (i.e., different Ig subclasses) to facilitate or inhibit various immune functions associated with particular antibody constant domains.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to a zil1a3 protein, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zil1a3 polypeptide).
  • Antibodies are defined to be specifically binding if they bind to a zil1a3 protein with an affinity at least 10-fold greater than the binding affinity to control (non-zil1a3) polypeptide.
  • the antibodies exhibit a binding affinity (K a ) of 10 6 M “1 or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “1 or greater, and most preferably 10 9 M “1 or greater.
  • K a binding affinity
  • the affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51; 660-672, 1949). Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982).
  • 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 zil1a3 protein 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 zil1a3 protein 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.
  • polypeptide portion is "hapten-like"
  • such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • assays known to those skilled in the art can be used to detect antibodies that specifically bind to a zil1a3 protein. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include concurrent immunoelectrophoresis, radio- immunoassays, radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
  • ELISA enzyme-linked immunosorbent assays
  • Antibodies to zil1a3 may be used for affinity purification of zil1a3 proteins; within diagnostic assays for determining circulating levels of ziha3 proteins; for detecting or quantitating soluble zil1a3 protein as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; for screening expression libraries; and for other uses that will be evident to those skilled in the art. For certain applications, including in vitro and in vivo diagnostic uses, it is advantageous to employ labeled antibodies.
  • 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.
  • Zil1a3 polynucleotides can be used to determine the presence of mutations at or near the zil1a3 locus on human chromosome 2 (2q14).
  • Detectable chromosomal aberrations at the zil1a3 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.
  • 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.
  • 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 include genomic DNA, cDNA, and RNA.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise at least a portion of SEQ ID NO:1 , the complement of SEQ ID NO: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 Applications 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.
  • RFLP restriction fragment length polymorphism
  • STR short tandem repeat
  • 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
  • PCR assays 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 Applications 1:34-38, 1991 ). The invention is further illustrated by the following non-limiting examples.
  • SSCP single strand conformational polymorphism
  • Example 1 Recombinant human zil1a3 was produced in E. coli using a His 6 tag/maltose binding protein (MBP) double affinity fusion system as generally disclosed by Pryor and Leiting, Prot. Expr. Pur. 10:309-319, 1997. A thrombin cleavage site was placed at the junction between the affinity tag and zil1a3 sequences.
  • the fusion construct was assembled in the vector pTAP98, which comprised sequences for replication and selection in E. coli and yeast, the E. coli tac promoter, and a unique Smal site just downstream of the MBP- His 6 -thrombin site coding sequences.
  • the zil1a3 cDNA (SEQ ID NO:1) was amplified by PCR using primers each comprising 40 bp of sequence homologous to vector sequence and 25 bp of sequence that annealed to the cDNA.
  • the reaction was run using Pwo DNA polymerase (Boehringer Mannheim, Indianapolis, IN) for 30 cycles of 94°C, 30 seconds; 60°C, 60 seconds; and 72°C, 60 seconds.
  • One microgram of the resulting fragment was mixed with 100 ng of Smal-cut pTAP98, and the mixture was transformed into yeast to assemble the vector by homologous recombination (Oldenburg et al., Nucl. Acids. Res. 25:451-452, 1997). Ura + transformants were selected.
  • Plasmid DNA was prepared from yeast transformants and transformed into E. coli MC1061. Pooled plasmid DNA was then prepared from the MC1061 transformants by the miniprep method after scraping an entire plate. Plasmid DNA was analyzed by restriction digestion using Ncol and EcoRI.
  • E. coli strain BL21 was used for expression of zil1a3. Cells were transformed by electroporation and grown on minimal glucose plates containing casamino acids and ampicillin.
  • Protein expression was analyzed by gel electrophoresis. Cells were grown in liquid media containing ampicillin. After one hour at 37°C, IPTG was added to a final concentration of 1 mM, and the cells were grown for an additional 2-3 hours at 37°C. Cells were disrupted using glass beads, and extracts were prepared. Seven of nine isolates were found to produce large amounts of the fusion protein.
  • E. coli BL21 expressing the zil1a3-MBP-His 6 fusion protein are prepared essentially as disclosed in Example 3.
  • Cell pellets are resuspended in 100 ml of binding buffer (20 mM Tris, pH 7.58, 100 mM NaCI, 20 mM NaH 2 PO 4 , 0.4 mM 4-(2- Aminoethyl)-benzenesulfonyl fluoride hydrochloride [Pefabloc® SC; Boehringer-Mannheim], 2 ⁇ g/ml Leupeptin, 2 ⁇ g/ml Aprotinin).
  • the cells are lysed in a French press at 30,000 psi, and the lysate is centrifuged at 18,000 x g for 45 minutes at 4°C to clarify it. Protein concentration is estimated by gel electrophoresis with a BSA standard.
  • Recombinant zil1a3 fusion protein is purified from the lysate by affinity chromatography.
  • Immobilized cobalt resin (Talon® resin; Clontech Laboratories, Inc., Palo Alto, CA) is equilibrated in binding buffer.
  • One ml of packed resin per 50 mg protein is combined with the clarified supernatant in a tube, and the tube is capped and sealed, then placed on a rocker overnight at 4°C. The resin is then pelleted by centrifugation at 4°C and washed three times with binding buffer. Protein is eluted with binding buffer containing 0.2 M imidazole.
  • the resin and elution buffer are mixed for at least one hour at 4°C, the resin is pelleted, and the supernatant is removed. An aliquot is analyzed by gel electrophoresis, and concentration is estimated.
  • Amylose resin is equilibrated in amylose binding buffer (20 mM Tris-HCI, pH 7.0, 100 mM NaCI, 10 mM EDTA) and combined with the supernatant from the Talon resin at a ratio of 2 mg fusion protein per ml of resin. Binding and washing steps are carried out as disclosed above. Protein is eluted with amylose binding buffer containing 10 mM maltose using as small a volume as possible to minimize the need for subsequent concentration. The eluted protein is analyzed by gel electrophoresis and staining with Coomassie blue using a BSA standard, and by Western blotting using an anti-MBP antibody.
  • Zil1a3 was mapped to human chromosome 2 using the commercially available version of the Stanford G3 Radiation Hybrid Mapping
  • Each of the 85 PCR reaction mixtures consisted of 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 20,354 (SEQ ID NO: 11), 1 ⁇ l antisense primer, ZC 20,355 (SEQ ID NO:12), 2 ⁇ l of a density increasing agent and tracking dye (RediLoadTM, Research Genetics, Inc., Huntsville, AL), 0.4 ⁇ l of a commercially available DNA polymerase/antibody mix (50X AdvantageTM KlenTaq Polymerase Mix, obtained from Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and x ⁇ l ddH 2 O for a total volume of 20 ⁇ l.
  • 2 ⁇ l buffer 10X KlenTaq PCR
  • the mixtures were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 5-minute denaturation at 94°C; 35 cycles of 45 seconds denaturation at 94°C, 45 seconds annealing at 64°C, and 75 seconds extension at 72°C; followed by a final extension of 7 minutes at 72°C.
  • the reactions were separated by electrophoresis on a 2% agarose gel.
  • the 2q14 region also contains the genes for interleukin 1 alpha (IL-1 ⁇ ), interleukin 1 beta (IL-1 ⁇ ), and the interleukin 1 receptor antagonist (IL-1ra).
  • Example 6 Northern blot analysis was performed using human multiple tissue blots (MTN® I, MTN® II, and MTN® III) (Clontech Laboratories, Inc., Palo Alto, CA) and human fetal multiple tissue blots (Clontech Laboratories, Inc.).
  • MTN® I, MTN® II, and MTN® III human multiple tissue blots
  • human fetal multiple tissue blots Clontech Laboratories, Inc.
  • a 595-bp human probe was generated by amplification of an Xhol-Ncol cDNA fragement.
  • the 595-bp fragment was gel purified using a spin column containing a silica gel membrane (QIAquickTM Gel Extraction Kit; Qiagen, Inc., Valencia, CA).
  • the probe was radioactively labeled with 32 P using a commercially available kit (RediprimeTM II random-prime labeling system; Amersham Corp., Arlington Heights, IL) according to the manufacturer's specifications.
  • the probe was purified using a push column (NucTrap® column; Stratagene, La Jolla, CA).
  • a commercially available hybridization solution (ExpressHybTM Hybridization Solution; Clontech Laboratories, Inc.) was used for the hybridizing solution for the blots. Hybridization took place overnight at 65°C.
  • the blots were then washed 4 times in 2X SCC and 0.05% SDS at room temperature, followed by two washes in 0.1X SSC and 0.1% SDS at 50°C.
  • Two transcripts were detected at approximately 1.8kb and 2.8kb in placenta. Signal intensity was strong for placenta. A faint signal was seen at 2.8 kb in testis, thyroid, spinal cord, and trachea
  • Dot blots (Human RNA Master BlotsTM; Clontech Laboratories, Inc.) were analyzed essentially as disclosed above. A signal was seen in placenta.
  • Human northern blots (MTN® I, MTN® II, MTN® III, and human fetal multiple tissue blots; Clontech Laboratories, Inc.) were analyzed with a mouse zil1a3 probe.
  • a 350-bp mouse probe was generated from a mouse cDNA (SEQ ID NO:14) by PCR using oligonucleotide primers zc18,609 (SEQ ID NO:15) and zc18,610 (SEQ ID NO:16). The reaction was run at 94°C for 2 minutes; then 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 5 minutes.
  • the PCR product was gel purified using a spin column containing a silica gel membrane.
  • the probe was radioactively labeled with 32 P using a commercially available kit (RediprimeTM II random-prime labeling system; Amersham Corp.) according to the manufacturer's specifications.
  • the probe was purified using a push column. Hybridization and washing were carried out essentially as disclosed in Example 6, except the final wash was perfomed four times.
  • Two transcripts, of approximately 1.0 kb and 1.5 kb, were seen in heart, brain, lung, liver, skeletal muscle, kidney, pancreas, adrenal gland, spinal cord, peripheral blood leukocytes, colon, small intestine, testis, and prostate. Signal intensity was strongest in heart and liver.

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Abstract

L'invention concerne les homologues de l'interleukine-1, les matériaux et les techniques permettant de les produire, les compositions les renfermant ainsi que leur mode d'utilisation. Les homologues sont des protéines comprenant la séquence de résidus d'acides aminés indiquée par SEQ ID NO:7. Ces protéines ont une activité modulatrice de l'inflammation et sont utiles pour des applications thérapeutiques et de recherche.
PCT/US1999/023533 1998-10-08 1999-10-08 Homologues de l'interleukine-1 WO2000020595A1 (fr)

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Cited By (7)

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WO2001002571A2 (fr) * 1999-07-07 2001-01-11 Hyseq, Inc. Nouvel antagoniste du recepteur d'interleukin-1 et ses utilisations
WO2001005974A2 (fr) * 1999-07-16 2001-01-25 Interleukin Genetics, Inc. Gène il-1l1 et produits polypeptidiques
WO2003010291A2 (fr) * 2001-07-25 2003-02-06 Hyseq, Inc. Traitement de troubles concernant les cellules immunitaires et les cellules b
AU778759B2 (en) * 1998-12-23 2004-12-16 Genentech Inc. IL-1 related polypeptides
WO2007034465A2 (fr) * 2005-09-21 2007-03-29 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Compositions et methodes associees a la modulation d'une reaction a mediation immunitaire
US7285648B1 (en) 1998-01-09 2007-10-23 Immunex Corporation IL-1 delta DNA and polypeptides
JP2010162016A (ja) * 2002-09-25 2010-07-29 Genentech Inc 乾癬の治療のための新規組成物と方法

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WO1998047921A1 (fr) * 1997-04-21 1998-10-29 Schering Corporation Cytokines de mammifere; reactifs et procedes connexes
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SMITH D.E. ET AL: "Four new members expand the iL-1 superfamily", EMBL DATBASE ENTRY AAF25210, ACCESSION NUMBER AAF25210, 27 January 2000 (2000-01-27), XP002131487 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7285648B1 (en) 1998-01-09 2007-10-23 Immunex Corporation IL-1 delta DNA and polypeptides
AU778759B2 (en) * 1998-12-23 2004-12-16 Genentech Inc. IL-1 related polypeptides
US8628777B2 (en) 1998-12-23 2014-01-14 Genentech, Inc. Antibodies binding IL-1 related polypeptides
US7951916B2 (en) 1998-12-23 2011-05-31 Genentech, Inc. Il-1 related polypeptides
WO2001002571A3 (fr) * 1999-07-07 2001-05-31 Hyseq Inc Nouvel antagoniste du recepteur d'interleukin-1 et ses utilisations
WO2001002571A2 (fr) * 1999-07-07 2001-01-11 Hyseq, Inc. Nouvel antagoniste du recepteur d'interleukin-1 et ses utilisations
WO2001005974A3 (fr) * 1999-07-16 2001-05-10 Interleukin Genetics Inc Gène il-1l1 et produits polypeptidiques
WO2001005974A2 (fr) * 1999-07-16 2001-01-25 Interleukin Genetics, Inc. Gène il-1l1 et produits polypeptidiques
WO2003010291A3 (fr) * 2001-07-25 2004-02-19 Hyseq Inc Traitement de troubles concernant les cellules immunitaires et les cellules b
WO2003010291A2 (fr) * 2001-07-25 2003-02-06 Hyseq, Inc. Traitement de troubles concernant les cellules immunitaires et les cellules b
JP2010162016A (ja) * 2002-09-25 2010-07-29 Genentech Inc 乾癬の治療のための新規組成物と方法
WO2007034465A2 (fr) * 2005-09-21 2007-03-29 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Compositions et methodes associees a la modulation d'une reaction a mediation immunitaire
WO2007034465A3 (fr) * 2005-09-21 2007-11-29 Trinity College Dublin Compositions et methodes associees a la modulation d'une reaction a mediation immunitaire

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