WO1997046686A2 - Tumor necrosis factor-related polypeptide - Google Patents

Tumor necrosis factor-related polypeptide Download PDF

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Publication number
WO1997046686A2
WO1997046686A2 PCT/US1997/009895 US9709895W WO9746686A2 WO 1997046686 A2 WO1997046686 A2 WO 1997046686A2 US 9709895 W US9709895 W US 9709895W WO 9746686 A2 WO9746686 A2 WO 9746686A2
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Prior art keywords
agp
seq
polypeptide
nucleic acid
ser
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PCT/US1997/009895
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French (fr)
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WO1997046686A3 (en
WO1997046686A9 (en
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Merrie J. Johnson
William S. Simonet
Dimitry M. Danilenko
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Amgen Inc.
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Priority to AU33810/97A priority Critical patent/AU3381097A/en
Priority to JP10500892A priority patent/JP2000515727A/en
Priority to EP97929846A priority patent/EP0918860A2/en
Publication of WO1997046686A2 publication Critical patent/WO1997046686A2/en
Publication of WO1997046686A9 publication Critical patent/WO1997046686A9/en
Publication of WO1997046686A3 publication Critical patent/WO1997046686A3/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the invention relates to AGP-1, a tumor necrosis factor-related polypeptide involved in inflammation, myelopoiesis and bone resorption.
  • Nucleic acid sequences, vectors and host cells for the expression of AGP-1 are disclosed.
  • pharmaceutical compositions comprising AGP-1, methods of identifying antagonists of AGP-1 and methods of treatment using AGP-1 or AGP-1 antagonists.
  • Tumor necrosis factor family is a growing group of cytokines which function as mediators of immune regulation, acute and chronic inflammatory responses, and programmed cell death.
  • Tumor necrosis factor (TNF ⁇ ) is the prototypical member of this family which also includes lymphotoxin (LT ⁇ , TNF ⁇ ) , lymphotoxin ⁇ (LT ⁇ ) , and ligands for CD27, CD30, CD40, OX40, 4-1BB, and Fas.
  • TNF ⁇ is a secreted protein
  • all the ligands in this family are type II membrane proteins.
  • the homologous carboxy-terminal domains are extracellular, and the shorter non-homologous amino-terminal regions are intracellular.
  • the membrane bound form of TNF ⁇ can be the target of proteolytic cleavage, generating a soluble form of TNF ⁇ which circulates in certain disease states.
  • TNF ⁇ may contribute to the morbidity and mortality associated with a variety of infectious diseases, including septic shock, autoimmune disorders and graft-versus-host disease.
  • TNF family of cytokines exert their biological effects through their interactions with a family of receptors which are generally characterized as Type I membrane proteins with cysteine-rich pseudorepeats in their extracellular domains.
  • T2 and A53R encode soluble, secreted receptors.
  • soluble forms of TNF ⁇ play an important role in the immune response
  • the interaction of membrane bound ligands and receptors of this family, particularly on T and B cells likely plays a major role in cell-cell cross-talk within the immune system.
  • signaling through FasL and its receptor is believed to play an important role in T-cell mediated cytotoxicity.
  • TNF ⁇ , LT ⁇ and FasL have all been demonstrated to induce apoptosis of certain cells under the correct conditions.
  • signalling through Fas has been demonstrated to induce apoptosis of numerous transformed cell lines and chronically activated T cell clones.
  • two mutations that accelerate autoimmune disease ( lpr and gld) resulting in lymphadenopathy and splenomegaly in mutant mice, are known to correspond to mutations within the genes encoding Fas and FasL, respectively.
  • TNF and TNF-related family members In view of the involvement of TNF and TNF-related family members in conditions associated with inflammation, infectious disease, immune system disorders and apoptotic cell death, it is desirable to identify additional related TNF family members.
  • a novel gene has been identified which encodes a polypeptide having significant homology to the TNF family member FasL.
  • the polypeptide has been termed AGP-1.
  • Transgenic mice expressing murine AGP-1 in the liver exhibit hepatic inflammation and necrosis, bile duct hyperplasia, as well as pathological findings supportive of direct or indirect systemic effects of the factor.
  • the nucleotide and amino acid sequence of AGP-1 was found to be identical to the sequence reported for TNF-related apoptosis-inducing ligand (TRAIL, see Wiley et al. Immunity 2, 673-682 (1995)) .
  • TRAIL was observed to induce apoptosis in a wide variety of transformed cell lines.
  • AGP-1 tumor necrosis factor family
  • AGP-1 is involved in myelopoiesis accompanied by an increase in neutrophils and lymphocytes, inflammation and necrosis of the liver, and bone resorption.
  • AGP-1 has also been identified.
  • the invention provides for nucleic acids encoding a polypeptide having at least one of the biological activities of AGP-1, vectors and host cells expressing the polypeptide, and method for producing recombinant AGP-1. Antibodies or fragments thereof which specifically bind AGP-1 are also provided. Methods of identifying antagonists of AGP-1 which reduce or eliminate at least one of the biological activities of AGP-1 are also encompassed by the invention.
  • Such antagonists include peptides, proteins, carbohydrates or small molecular weight organic molecules which bind to AGP-1 or to its receptor (s) and interfere with AGP-1 receptor activation.
  • AGP-1 may be used to treat hematopoeitic disorders characterized by a decrease in cell population of the bone marrow.
  • AGP-1 antagonists may be used to treat inflammatory conditions.
  • AGP-1 antagonists may also be used to treat bone disorders resulting from an increase in bone resorption.
  • Pharmaceutical compositions comprising AGP-1 and AGP-1 antagonists are also encompassed by the invention.
  • Figure 1 cDNA and amino acid sequence of murine AGP-1.
  • Figure 2 cDNA and amino acid sequence of human AGP-1.
  • FIG. 3 Hematoxylin and Eosin (H&E) stained sections of liver from non-transgenic mouse #12 (A) and HEAGP FI transgenic mouse #75-13 (B) .
  • Figure 4 Myeloperoxidase stained sections of HEAGP FI transgenic (B - mouse #75-13) and non-transgenic (A mouse #12) spleen.
  • AGP-1 refers to a polypeptide having an amino acid sequence of mammalian AGP-1 or a derivative thereof and having at least one of the biological activities of AGP-1.
  • AGP-1 is mouse or human AGP-1.
  • cDNA and amino acid sequences of mouse and human AGP-1 are shown in Figures 1 and 2, respectively.
  • the biological activities of AGP-1 include, but are not limited to, involvement in myelopoiesis, inflammation and necrosis, especially in the liver, and bone resorption.
  • nucleic acid represents cDNA, genomic DNA, wholly or partially synthetic DNA or RNA.
  • the nucleic acids of the invention are selected from the group consisting of: a) the nucleic acids as shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) ; b) nucleic acids which hybridize to the polypeptide coding regions of the nucleic acids shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) and remain hybridized to the nucleic acids under high stringency conditions; and c) nucleic acids which are degenerate to the nucleic acids of (a) or (b) .
  • Nucleic acid hybridizations typically involve a multi-step process comprising a first hybridization step to form nucleic acid duplexes from single strands followed by a second hybridization step carried out under more stringent conditions to selectively retain nucleic acid duplexes having a degree of homology which depends upon the stringency of hybridization during the second step.
  • the conditions of the first hybridization step are generally not crucial, provided they are not of higher stringency than the second hybridization step.
  • the second hybridization is carried out under conditions of high stringency, wherein "high stringency” conditions refers to conditions of temperature and salt which are about 12-20°C below the melting temperature (T m ) of a perfect hybrid of part or all of the complementary strands corresponding to SEQ. ID.
  • high stringency conditions refer to conditions of about 65°C and not more than about IM Na+. It is understood that salt concentration, temperature and/or length of incubation may be varied in either the first or second hybridization steps such that one obtains the hybridizing nucleic acid molecules according to the invention. Conditions for hybridization of nucleic acids and calculations of T m for nucleic acid hybrids are described in Sambrook et al. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, New York. (1989) .
  • nucleic acids of the invention may hybridize to part or all of the polypeptide coding regions of AGP-1 as shown in SEQ ID NO: 1 and SEQ ID NO: 3, and therefore may be truncations or extensions of the nucleic acids in SEQ ID NO: 1 and SEQ ID NO: 3. Truncated or extended nucleic acids are encompassed by the invention provided that they retain one or more of the biological properties of AGP-1, such as stimulating myelopoiesis, bone resorption or an inflammatory response.
  • the nucleic acid will encode a polypeptide of at least about 10 amino acids. In another embodiment, the nucleic acid will encode a polypeptide of at least about 20 amino acids.
  • the nucleic acid will encode polypeptides of at least about 50 amino acids.
  • the hybridizing nucleic acids may also include noncoding sequences located 5' and/or 3' to the AGP-1 coding regions. Noncoding sequences include regulatory regions involved in AGP-1 expression, such as promoters, enhancer regions, translational initiation sites, transcription termination sites and the like.
  • the nucleic acids of the invention encode mouse AGP-1 or human AGP-1.
  • Mouse AGP-1 is shown in Figure 1 and SEQ. ID. NO: 2
  • human AGP-1 is shown in Figure 2 and SEQ. ID. NO: 4.
  • Nucleic acids may encode a full-length form of AGP-1 which is a membrane-bound or soluble forms of AGP-1 lacking part or all of the transmembrane region.
  • the predicted transmembrane region for human AGP-1 includes residues 16-36 as shown in SEQ. ID. NO: 4. Deletions of part or all these residues would be expected to produce soluble forms of AGP-1.
  • the nucleic acids of the invention will be linked with DNA sequences so as to express biologically active AGP-1.
  • Sequences required for expression are known to those skilled in the art and include promoters and enhancer sequences for initiation of RNA synthesis, transcription termination sites, ribosome binding sites for the initiation of protein synthesis, and leader sequences for secretion.
  • Sequences directing expression and secretion of AGP-1 may be homologous, i.e., those sequences in the genome involved in AGP-1 expression and secretion, or may be heterologous.
  • a variety of plasmid vectors are available for expressing AGP-1 in host cells.
  • One example is plasmid pDSR ⁇ described in
  • PCT Application No. 90/14363 which may be used for expression in mammalian hosts.
  • AGP-1 coding regions may also be modified by substitution of preferred codons for optimal expression in a given host. Codon usage in bacterial, plant, insect and mammalian host systems is known and may be exploited by one skilled in the art to optimize mRNA translation.
  • vectors are available for the tissue-specific expression of AGP-1 in transgenic animals. Retroviral and adenovirus-based gene transfer vectors may also be used for the expression of AGP-1 in human cells for in vivo therapy (see PCT Application No. 86/00922) .
  • Procaryotic and eucaryotic host cells expressing AGP-1 are also provided by the invention.
  • Host cells include bacterial, yeast, plant, insect or mammalian cells.
  • AGP-1 may also be produced in transgenic animals such as mice or goats. Plasmids and vectors containing the nucleic acids of the invention are introduced into appropriate host cells using transfection or transformation techniques known to one skilled in the art.
  • Host cells may contain DNA sequences encoding the full-length AGP-1 gene as shown in Figure 1.
  • Host cells will also process AGP-1 encoded by the full-length gene to the mature form or produce the mature form without processing by expression of DNA sequences encoding same. Examples of mammalian host cells for AGP-1 expression include, but are not limited to COS, CHOd-, 293 and 3T3 cells.
  • the invention also provides AGP-1 as the product of procaryotic or eucaryotic expression of an exogenous DNA sequence, i.e., AGP-1 is recombinant AGP-1.
  • Exogenous DNA sequences include cDNA, genomic DNA and synthetic DNA sequences.
  • AGP-1 may be the product of bacterial, yeast, plant, insect or mammalian cells expression. AGP-1 produced in bacterial cells will have an N-terminal methionine residue.
  • the invention also provides for a process of producing AGP-1 comprising growing procaryotic or eucaryotic host cells transformed or transfected with nucleic acids encoding AGP-1 and isolating polypeptide expression products of the nucleic acids.
  • a derivative of AGP-1 refers to a polypeptide having an addition, deletion, insertion or substitution of one or more amino acids such that the resulting polypeptide has at least one of the biological activities of AGP-1.
  • the derivative may be naturally occurring, such as a polypeptide product of an allelic variant or a mRNA splice variant, or it may be constructed using techniques available to one skilled in the art for manipulating and synthesizing nucleic acids.
  • AGP-1 polypeptides may be full-length polypeptides or fragments thereof which, in preferred embodiments, are at least about ten amino acids, at least about 20 amino acids, or at least about 50 amino acids in length.
  • AGP-1 full-length polypeptides and fragments preferably have the amino acid sequence in Figure 1 or 2 or a portion thereof.
  • the polypeptides may or may not have an amino terminal methionine residue.
  • AGP-1 polypeptides which have undergone post-translational modifications (e.g., addition of N-linked or O-linked carbohydrate chains, processing of N-terminal or
  • AGP-1 C-terminal ends
  • attachment of chemical moieties to the amino acid backbone chemical modifications of N-linked or O-linked carbohydrate chains, and addition of an N-terminal methionine residue as a result of procaryotic host cell expression.
  • mouse and human AGP-1 are encoded as transmembrane proteins
  • soluble forms of AGP-1 are also envisioned. Such soluble forms may be readily constructed by removal of the transmembrane region of the polypeptide.
  • the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • AGP-1 chimeric proteins comprising part or all of an AGP-1 amino acid sequence fused to a heterologous amino acid sequence are also included.
  • the heterologous sequence may be any sequence which allows the resulting fusion protein to retain the activity of AGP-1.
  • the heterologous sequences include, for example, immunoglobulin fusions, such as an Fc region of IgG, which provide dimerization, or fusions to enzymes which provide a label for the polypeptide.
  • polypeptides of the invention are isolated and purified from tissues and cell lines which express AGP-1 and from transformed host cells expressing AGP-1, or purified from cell cultures containing the secreted protein. Isolated AGP-1 polypeptide is free from association with human proteins and other cell constituents.
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxy ethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • a method for the purification of AGP-1 from natural sources e.g. tissues and cell lines which normally express AGP-1) and from transfected host cells is also encompassed by the invention.
  • the purification process may employ one or more standard protein purification steps in an appropriate order to obtain purified protein.
  • the chromatography steps can include ion exchange, gel filtration, hydrophobic interaction, reverse phase, chromatofocusing, affinity chromatography employing an anti-AGP-1 antibody or biotin-streptavidin affinity complex and the like.
  • the invention also encompasses AGP-1 antagonists and the methods for obtaining them.
  • An antagonist will reduce or eliminate one or more of the biological activities of AGP-1.
  • an AGP-1 antagonist may act as an anti-inflammatory agent, or may act to inhibit bone resorption.
  • AGP-1 antagonists include substances which bind to AGP-1 or to AGP-1 receptors in a manner to prevent normal ligand-receptor interaction and substances which regulate the expression of AGP-1.
  • Substances which bind to AGP-1 or to AGP-1 receptors include proteins, peptides, carbohydrates and small molecular weight organic compounds.
  • protein inhibitors include anti-AGP-1 antibodies, anti-AGP-1 receptor antibodies and soluble forms of AGP-1 receptor comprising part or all of the extraceullular domain of the AGP-1 receptor.
  • Substances which regulate AGP-1 expression typically include nucleic acids which are complementary to nucleic acids encoding AGP-1 or AGP-1 receptors and which act as anti-sense regulators of expression.
  • Methods for indentifying compounds which interact with AGP-1 are also encompassed by the invention.
  • the method comprises incubating AGP-1 with a compound under conditions which permit binding of the compound to AGP-1 and measuring the extent of binding.
  • the compound may be substantially purified or present in a crude mixture.
  • Binding compounds may be proteins, peptides, carbohydrates or small mo_ecular weight organic compounds.
  • the compounds may be further characterized by their ability to enhance or reduce AGP-1 biological activity and therefore act as AGP-1 agonists or as AGP-1 antagonists.
  • the method is used to identify AGP-1 antagonists.
  • Antibodies specifically binding the AGP-1 polypeptides of the invention are also encompassed by the invention.
  • the antibodies may be produced by immunization with full-length membrane-bound AGP-1, soluble AGP-1, or a peptide fragment thereof, and the antibodies may be polyclonal or monoclonal.
  • the antibodies of the invention may be recombinant, such as chimeric antibodies wherein the murine constant regions on light and heavy chains are replaced by human sequences, or CDR-grafted antibodies wherein only the complementary determining regions are of murine origin.
  • Antibodies of the invention may also be human antibodies prepared, for example, by immunization of transgenic animals capable of producing human antibodies (see, for example, PCT Application No. W093/12227) .
  • the antibodies are useful for detecting AGP-1 in biological samples, thereby allowing the identification of cells or tissues which produce AGP-1.
  • antibodies which bind to AGP-1 and prevent receptor interaction may also be useful for blocking the effects of AGP-1.
  • the invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of the AGP-1 polypeptide of the invention together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
  • pharmaceutical compositions comprising a therapeutically effective amount of an AGP-1 antagonist.
  • therapeutically effective amount means an amount which provides a therapeutic effect for a specified condition and route of administration.
  • the composition may be in a liquid or lyophilized form and comprises a diluent (Tris, acetate or phosphate buffers) having various pH values and ionic strengths, solubilizer such as Tween or Polysorbate, carriers such as human serum albumin or gelatin, preservatives such as thimerosal or benzyl alcohol, and antioxidants such as ascrobic acid or sodium metabisulfite.
  • a diluent Tris, acetate or phosphate buffers
  • solubilizer such as Tween or Polysorbate
  • carriers such as human serum albumin or gelatin
  • preservatives such as thimerosal or benzyl alcohol
  • antioxidants such as ascrobic acid or sodium metabisulfite.
  • compositions comprising AGP-1 modified with water soluble polymers to increase solubility, stability, plasma half-life and bioavailability.
  • Compositions may also comprise incorporation of AGP-1 into liposomes, microemulsions, micelles
  • compositions of the invention may be administered by injection, either subcutaneous, intravenous or intramuscular, or by oral, nasal, pulmonary or rectal administration.
  • the route of administration eventually chosen will depend upon a number of factors and may be ascertained by one skilled in the art.
  • the invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of the nucleic acids of the invention together with a pharmaceutically acceptable adjuvant.
  • Nucleic acid compositions will be suitable for the delivery of part or all of the APG-1 coding region and/or flanking regions to cells and tissues as part of ⁇ . anti-sense therapy regimen.
  • AGP-1 may be used to treat hematopoietic disorders that are associated with a decrease in the population of cells in bone marrow.
  • AGP-1 may be used to treat conditions resulting in low white blood cell levels, particularly reduced levels of neutrophils and lymphocytes. Such conditions may result from disease, injury or exposure to certain environmental agents known to suppress bone marrow levels. It is understood that AGP-1 may be administered alone or in combination with other factors to treat hematopoietic disorders. In one embodiment, AGP-1 is used in conjunction with a therapeutically effective amount of a factor which stimulates hematopoiesis.
  • EPO erythropoietin
  • G-CSF granulocyte colony stimulating factor
  • MGDF megakaryocyte growth and differentiation factor
  • GM-CSF granulocyte-macrophage stimulating factor
  • SCF stem cell factor
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • AGP-1 Hepatic expression of AGP-1 in transgenic mice resulted in increased inflammation and necrosis, especially in the liver. This effect may be the result of a high local concentration of AGP-1 occurring in the liver during transgene expression.
  • antagonists of AGP-1 may be used as anti-inflammatory agents which are administered to patients susceptible to or suffering from an inflammatory condition. Inflammatory conditions include rhematoid arthritis, systemic lupus erythematosis, psoriasis, systemic and localized amyloidosis, Sjogerns syndrome, sclerodoma, dermatomyositis, glomerulonephritis, and inflammation arising from infections and parasitic diseass.
  • AGP-1 antagonists which reduce or eliminate inflammation may be administered alone or in combination with a therapeutically effective amount of an anti-inflam tory agent such as a corticosteroid, a non-steroidal anti-inflammatory agent (NSAID), or cyclosporin A.
  • an anti-inflam tory agent such as a corticosteroid, a non-steroidal anti-inflammatory agent (NSAID), or cyclosporin A.
  • AGP-1 antagonists may also reduce or eliminate necrosis associated with an inflammatory condition.
  • AGP-1 is also involved in stimulation of osteoclasts which promote bone resorption through mineralization of the bone matrix. Increase in bone resorption rates that exceed rates of bone formation can lead to various bone disorders including osteoporosis, osteomyelitis, hypercalcemia, osteopenia brought on by surgery or steroid administration, Paget's disease, osteonecrosis, bone loss due to rheumatoid arthritis, periodontal bone loss, and osteolytic metastasis. Antagonists of AGP-1 may be administered to patients suffering from disorders brought on by increased osteoclast activity and increased bone resorption.
  • AGP-1 antagonists may be administered alone or in combination with a therapeutically effective amount a bone growth promoting agent including bone morphogenic factors designated BMP-1 to BMP-12, transforming growth factor- ⁇ and TGF- ⁇ family members, interleukin-1 inhibitors, TNF ⁇ inhibitors, parathyroid hormone, E series prostaglandins, bisphosphonates and bone-enhancing minerals such as fluoride and calcium.
  • a bone growth promoting agent including bone morphogenic factors designated BMP-1 to BMP-12, transforming growth factor- ⁇ and TGF- ⁇ family members, interleukin-1 inhibitors, TNF ⁇ inhibitors, parathyroid hormone, E series prostaglandins, bisphosphonates and bone-enhancing minerals such as fluoride and calcium.
  • a cDNA library was constructed using mRNA isolated from 5, 6, and 7 day post-5FU treated bone marrow from C57/B6 female mice. Mice were treated with 150mg/kg 5-fluorouracil (5FU) , intraperitoneally, on each of three consecutive days. On day 5, 6, and 7 post-5FU treatment both femurs and tibias were harvested, and plugs flushed with PBS. Bones were crushed with mortar and pestle and combined with the bone marrow plugs.
  • the poly A+ mRNA was purified using Fast Track mRNA Kit (InVitrogen, San Diego, CA) using the manufacturer's recommended procedures.
  • a random primed cDNA library was prepared using the Superscript Plasmid System (Gibco BRL, Gaithersburg, MD) .
  • a random cDNA primer containing an internal Not I restriction site was used to initiate first strand synthesis and had the following double strand sequence:
  • the first strand cDNA synthesis reaction was assembled using l ⁇ g of the mRNA and 150 ng of the Not 1 random primer. After second strand synthesis, the reaction products were extracted with the phenol:chloroform:isoamyl alcohol mixture and ethanol precipitated. The double strand (ds) cDNA products were ligated to the following ds oligonucleotide adapter (Gibco BRL) :
  • the cDNA was digested to completion with Not 1, extracted with phenol:chloroform:isoamyl alcohol (25:24:1 ratio) and ethanol precipitated.
  • the resuspended cDNA was then size fractionated by gel filtration using the premade columns provided with the Superscript Plasmid System (Gibco BRL) as recommended by the manufacturer.
  • the fractions containing the largest cDNA products were ethanol precipitated and then directionally ligated into Not 1 and Sal 1 digested pMOB vector DNA (Strathmann et. al. Science 25Z, 802-808 (1991)) .
  • the ligated cDNA was introduced into electrocompetent XLl-Blue E. coli (Stratagene, LaJolla, CA) by electroporation.
  • thermocycler Perkin-Elmer 9600
  • the reactions were incubated in a thermocycler (Perkin-Elmer 9600) with the following cycle conditions: 94°C for 2 minutes; 94°C for 5 seconds, 50°C for
  • PCR reaction products were sequenced on an Applied Biosystems 373A automated DNA sequencer using T3 primer:
  • the resulting 5' nucleotide sequence obtained from randomly picked cDNA clones were translated and then compared to the existing database of known protein sequences using a modified version of the FASTA program (Pearson, et . al. Meth. Enzymol. 18_3_, 63-98 (1990)) .
  • Translated sequences were also analyzed for the presence of specific tumor necrosis factor superfamily motifs, using the sequence profile method of Gribskov, et . al . (Proc. Natl. Acad. Sci. USA £2, 4355-4359 (1987)) as modified by Luethy et al . (Protein Science 2/ 139-146 (1994) ) .
  • muAGP-ESTl was identified as a possible new member of the TNF family.
  • the muAGP-ESTl clone contained an 864 bp insert with an open reading frame of about 90 amino acids which was found to have significant homology to pig lymphotoxin- ⁇ precursor (TNF- ⁇ ) and rabbit tumor necrosis factor precursor (TNF- ⁇ )
  • an internal EST database was searched for overlapping clones and two other murine EST clones were identified.
  • One EST clone designated muAGP-EST2 from a murine irradiated small intestine library gave a sequence which overlapped the sequence obtained from the muAGP-ESTl clone.
  • the muAGP-EST2 clone was subsequently sequenced in its entirety.
  • the insert was 3048 bp and contained an open reading frame of 291 amino acids which was deduced to be the full-length AGP-1 sequence.
  • the nucleotide sequence and deduced amino acid of murine AGP-1 is shown in Figure 1.
  • a cDNA library was constructed using RNA from human bladder carcinoma cell line 5637 which had been stimulated with 20nM of PMA for about nine hours.
  • mRNA was isolated from a membrane bound polysomal fraction of RNA (Mechler Methods in Enzymology 152. 241-248 (1987)) .
  • the poly A+ mRNA fraction was isolated from the total RNA preparation by using the Fast Track mRNA Isolation Kit (InVitrogen) according to the manufacturer's recommended procedure.
  • a directional random primed cDNA library was prepared essentially as described for the 5-FU mouse bone marrow library above. The cDNA inserts were sequenced as described above for the mouse cDNA clones.
  • the resulting 5' nucleotide sequences obtained from randomly picked cDNA clones were translated and compared to the existing database of known protein sequences using a modified version of the FASTA program (Pearson et al . ibid) .
  • Translated sequences were also analysed for the presence of specific motifs found in the tumor necrosis factor superfamily using the sequence profile method of Gribskov et.al. ibid as modified by Luethy et.al. ibid.
  • huAGP-ESTl was identified as a possible new member of the TNF family.
  • huAGP-ESTl contained an 446 bp insert with an open reading frame of about 84 amino acids.
  • Translation of the huAGP-ESTl nucleotide sequence gave an amino acid sequence which was 77% identical to the deduced amino acid sequence of murine AGP-1 when compared using FASTA analysis. This high degree of sequence similarity identifies huAGP-ESTl as the human homolog of murine AGP-1.
  • huAGP-EST2 was from a human peripheral blood megakaryocyte cDNA library and had an insert of 1028 bp which overlapped the huAGP-ESTl clone.
  • the overlapping clones had an open reading frame of 281 amino acids.
  • the full-length human AGP-1 was obtained as a composite of the sequences from the huAGP-ESTl and huAGP-EST2 clones. The nucleotide sequence and deduced amino acid sequence of human AGP-1 is shown in Figure 2.
  • the TNF ⁇ -related clone muAGP-EST2 was used as template to PCR amplify the coding region for subcloning into an APOE-liver specific expression vector (Simonet et al. J. Clin. Invest. , 1310-1319 (1994), and PCT Application No. W094/11675) .
  • the oligonucleotides used for amplification were:
  • the conditions for PCR were: 94°C for 1 minute, followed by 25 cycles of 94 C for 20 sec, 63 C for 30 sec, and 74 C for 1 minute.
  • the PCR reactions contained 1 x PFU buffer, 50 uM dNTPs, 20 pmol of each oligo, 10 ng of DNA template and 2.5 units of PFU enzyme in a total volume of 50 ul.
  • the samples were purified over Qiagen PCR columns and digested overnight with Spel and NotI restriction enzymes. The digested products were extracted and precipitated and subcloned into the ApoE promoter expression vector.
  • Ligations were transformed into E.. coli strain DH5 ⁇ and colonies were inipreped for analysis of the insert. Two clones containing the desired size insert were grown in 100ml TB cultures and plasmid DNA was prepared. The two clones were sent to sequencing to verify the authenticity of the insert. One was selected for microinjection to generate transgenic mice. This transgene was designated HE-AGP.
  • the HE-AGP plasmid was purified through two rounds of CsCl .
  • the plasmid was digested with Xhol and Ase I, and the 3.4 kb transgene insert was purified on a 0.8% BRL ultrapure DNA agarose gel by electrophoresis onto NA 45 paper.
  • the purified fragment was diluted to 1 ug/ml in 5 mM Tris, pH 7.4, 0.2 mM EDTA.
  • Single-cell embryos from BDF1 x BDFl-bred mice were injected essentially as described (Brinster et al., 1985), except that injection needles were beveled and siliconized before use. Embryos were cultured overnight in a C ⁇ 2 incubator and 15 to 20 two-cell embryos were transferred to the oviducts of pseudopregnant CD1 female mice.
  • 105 offspring were obtained from implantation of microinjected embryos. Of the 105 offspring, 17 were identified as transgenic founders by screening for the HE-AGP transgene in DNA prepared from ear and tail biopsies. The PCR screening involved amplification of a 369 bp region of the human Apo E intron which was included in the expression vector. The oligos used for PCR amplification were:
  • the conditions for PCR were: 94 C for 2 minute, followed by 30 cycles of 94 C for 1 min, 63 C for 20 sec, and 72°C for 30 sec.
  • the PCR reactions contained 1 x Taq buffer, 100 uM each dNTPs, 20 pmol of each oligo, 1 ul of DNA template extract and 0.5 units of taq enzyme in a total volume of 50 ul.
  • mice were anesthetized with avertin and a lobe of liver was surgically removed.
  • Total cellular RNA was isolated from livers of all transgenic founders, and 5 negative control littermates as described (McDonald et al. (1987)) .
  • Northern blot analysis was performed on these samples to assess the level of transgene expression.
  • RNA from each animal liver was resolved by electrophoresis denaturing gels (Ogden et al. (1987)), then transferred to HYBOND-N nylon membrane (Amersham) , and probed with 32p CTP-labelled pBl.l insert DNA. Hybridization was performed overnight at 42°C in 50% Formamide, 5 x SSPE, 0.5% SDS, 5 x 686 PC17US97/09895
  • the Northern blot data indicate that 13 of the transgenic founders express detectable levels of the transgene mRNA (animal #'s 10, 42, 44, 45, 48, 50, 52, 53, 67, 69, 74, 75 and 76) .
  • the negative control mice expressed no transgene-related mRNA.
  • the highest expressing founders From the group that were necropsied were #'s 52, 69 and 76.
  • the highest expressing animals from the remaining group of founder's were #'s 42, 45, 67, and 75. Six of the founder's that were analyzed by hepatectomy were subsequently bred to generate FI offspring for further analysis.
  • mice which were founder transgenics for the murine AGP-1 molecule targeted to the liver via an apolipoprotein E promoter as well as four male non-transgenic littermate mice were necropsied for phenotypic analysis.
  • twelve BDF1 mice (nine females and three males) which were FI transgenics for the murine AGP molecule targeted to the liver via an apolipoprotein E promoter as well as four female non-transgenic littermate mice - 25 -
  • mice were necropsied for phenotypic analysis. In both studies, all mice were injected with BrdU one hour prior to harvest and sacrificed. Body and liver, spleen, kidney, stomach, and thymus weights were taken, blood was drawn for hematology and serum chemistries, and liver, spleen, lung, brain, heart, kidney, adrenal, stomach, small intestine, pancreas, cecum, colon, mesenteric lymph node, skin, mammary gland, trachea, esophagus, thyroid, parathyroid, salivary gland, urinary bladder, ovary or testis, uterus or seminal vesicle, bone, and bone marrow were examined were harvested for histologic analysis and BrdU labeling.
  • myeloperoxidase immunostaining sections were stained with rabbit polyclonal antisera directed at human myeloperoxidase (Dako, Carpinteria, CA) , followed by a biotinylated anti-rabbit/anti-mouse secondary cocktail (BioTek) and avidin-biotin complex (ABC) tertiary coupled to horseradish peroxidase.
  • the staining reaction was visualized with dia inobenzidine (DAB, Sigma) .
  • mice The livers from two transgenic founder mice(#s 69 and 76) and two FI transgenic mice (#s 75-13 and 75-18) were significantly increased in size and weight (8.42 ⁇ 1.26 SD % of body weight vs. 5.33 ⁇ 0.89 SD % of body weight in non-transgenic control mice) and were pale green-tan and more friable than normal. These four mice also had a significant increase in splenic weight (1.14 ⁇ 0.12 SD % of body weight vs. 0.41 ⁇ 0.09 SD % body weight in non-transgenic control mice. These results are summarized in Table 1.
  • the four transgenic mice with enlarged livers had marked and significant increases in total serum bilirubin and alkaline phosphatase levels, with moderate but significant increases in hepatic transaminase (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) levels.
  • the four transgenic mice had a mean total bilirubin level of 4.33 ⁇
  • H&E and BrdU stained sections of liver, spleen, lung, brain, heart, kidney, adrenal, stomach, small intestine, pancreas, cecum, colon, mesenteric lymph node, skin, mammary gland, trachea, esophagus, thyroid, parathyroid, salivary gland, urinary bladder, ovary or testis, uterus or seminal vesicle, bone, and bone marrow were examined from the 17 HE-AGP-1 transgenic mice and 8 non-transgenic control littermates.
  • Myeloperoxidase stained sections of spleen and bone marrow as well as tartrate resistant acid phosphatase (TRAP) stained sections of bone were also examined from all mice.
  • transgenic mice Major histologic changes in the transgenic mice included marked periportal inflammation and bile duct hyperplasia with scattered multifocal to coalescing areas of hepatocellular necrosis in transgenic mice #s 69 and 76 (founders) and #s 75-13 and 75-18 (Fls) ( Figure 3) . All four of these transgenic mice also had enlarged spleens primarily due to increased red pulp myelopoiesis and to a lesser extent, lymphoid hyperplasia ( Figure 4) . These four transgenic mice also appeared to have increased numbers of TRAP positive osteoclasts lining bony trabeculae in peripheral diaphyseal marrow compared to non-transgenic control mice ( Figure 5) .
  • Transgenic mice also exhibited increased intravascular neutrophils, and small atrophic/hypoplastic uteri (only founder transgenics #s 69 and 76) .
  • the two founder transgenic mice (#s 69 and 76) also exhibited moderate peritoneal mixed inflammatory cellular infiltration.
  • mice Four of the HE-AGP-1 transgenic mice (founder nos. 69 and 76 and FI nos. 75-13 and 75-18) had relatively severe phenotypic alterations, particularly in their livers with marked cholangiohepatitis, bile duct hyperplasia and hepatic necrosis. Accompanying these hepatic histologic abnormalities in these four transgenic mice was evidence of liver dysfunction with marked elevations in total serum bilirubin and alkaline phosphatase with moderate elevations in serum transaminases. In addition to hepatic findings, these four transgenic mice also exhibited increased myelopoiesis, with a less prominent increase in circulating platelets.
  • Founder mouse #69 had a circulating neutrophilia while all transgenic mice had a moderate increase in circulating lymphocytes. Evidence of peritoneal inflammation was also seen in the two founder transgenic mice with marked hepatic inflammation. Two of the other HEAGP founder transgenic mice, #'s 52 and 53, also had evidence of mild cholangiohepatitis, and a mild to moderate increase in myelopoiesis and neutrophilia, suggesting that these two mice were producing the transgenic AGP-1 protein at a lower level than founder mice #s 69 and 76 were.
  • At least four of the transgenic mice exhibited a marked increase in splenic myelopoiesis and moderate lymphoid hyperplasia as well as exhibiting an apparent increase in TRAP+ osteoclasts lining bony trabeculae in the bone marrow. All of these findings suggest that the AGP protein plays a role in inflammation, myelopoiesis, and bone resorption (osteoclasis) .
  • AGCATAAAAA TCACAGAATA TCTCACCTAC CAAATCAGAG TGGGTGTGCC CCTGTGTGTA 1580
  • TCATCAGTGG AACCTTGCCC AAAGAATGTA TGAAATCTCC AGGCAATGAA TGAGGGCAGC 1760 CCAAGAAAGA GGCCCGCAGA GCCATACCAC AGGGCTGCCC CACCCTGCTG GAGCTCAGAT 1820
  • GCT TGT TTC TTA AAA GAA GAT GAC AGT TAT TGG GAC CCC AAT GAC GAA 244 Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu 55 60 65 70

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Abstract

A novel member of the tumor necrosis factor (TNF) family was identified and observed to be involved in inflammation and necrosis, especially of the liver, myelopoiesis and bone resorption. The polypeptide is termed AGP-1. Nucleic acid sequences, vectors and host cells for the expression of AGP-1 are disclosed. Methods for identifying antagonists of AGP-1, pharmaceutical compositions comprising AGP-1 and methods of treatment using AGP-1 and AGP-1 antagonists are also disclosed.

Description

TUMOR NECROSIS FACTOR-RELATED POLYPEPTIDE
Field of the Invention The invention relates to AGP-1, a tumor necrosis factor-related polypeptide involved in inflammation, myelopoiesis and bone resorption. Nucleic acid sequences, vectors and host cells for the expression of AGP-1 are disclosed. Also encompassed are pharmaceutical compositions comprising AGP-1, methods of identifying antagonists of AGP-1 and methods of treatment using AGP-1 or AGP-1 antagonists.
Background of the Invention
The tumor necrosis factor family is a growing group of cytokines which function as mediators of immune regulation, acute and chronic inflammatory responses, and programmed cell death. Tumor necrosis factor (TNFα) is the prototypical member of this family which also includes lymphotoxin (LTα, TNFβ) , lymphotoxin β (LTβ) , and ligands for CD27, CD30, CD40, OX40, 4-1BB, and Fas.
Homology among these family members is confined to the carboxy-terminal 150 amino acid residues, with the highest degree of homology within the β-strand regions involved in subunit contacts which lead to oligomerization. With the exception of LTα, which is a secreted protein, all the ligands in this family are type II membrane proteins. The homologous carboxy-terminal domains are extracellular, and the shorter non-homologous amino-terminal regions are intracellular. The membrane bound form of TNFα can be the target of proteolytic cleavage, generating a soluble form of TNFα which circulates in certain disease states. As systemic delivery of TNFα induces toxic shock and widespread tissue necrosis, TNFα may contribute to the morbidity and mortality associated with a variety of infectious diseases, including septic shock, autoimmune disorders and graft-versus-host disease.
The TNF family of cytokines exert their biological effects through their interactions with a family of receptors which are generally characterized as Type I membrane proteins with cysteine-rich pseudorepeats in their extracellular domains. Of the twelve TNF receptor superfa ily members identified to date, only the two poxvirus genes, T2 and A53R, encode soluble, secreted receptors. Whereas soluble forms of TNFα play an important role in the immune response, the interaction of membrane bound ligands and receptors of this family, particularly on T and B cells, likely plays a major role in cell-cell cross-talk within the immune system. In this regard, signaling through FasL and its receptor is believed to play an important role in T-cell mediated cytotoxicity.
Perhaps the most intriguing activity associated with this family is their ability to induce programmed cell death through the apoptotic pathway, a phenomena which is crucial in many areas of vertebrae development, including T-cell development. Of the known TNF family members, TNFα, LTα and FasL have all been demonstrated to induce apoptosis of certain cells under the correct conditions. Although the apoptotic effects of TNFα and LTα appear to be limited to a minimal number of cell types, signalling through Fas has been demonstrated to induce apoptosis of numerous transformed cell lines and chronically activated T cell clones. Additionally, two mutations that accelerate autoimmune disease ( lpr and gld) , resulting in lymphadenopathy and splenomegaly in mutant mice, are known to correspond to mutations within the genes encoding Fas and FasL, respectively.
In view of the involvement of TNF and TNF-related family members in conditions associated with inflammation, infectious disease, immune system disorders and apoptotic cell death, it is desirable to identify additional related TNF family members.
It is an object of this invention to identify TNF-related molecules for the purpose of developing treatments for disorders related to TNF and TNF-related molecules.
A novel gene has been identified which encodes a polypeptide having significant homology to the TNF family member FasL. The polypeptide has been termed AGP-1. Transgenic mice expressing murine AGP-1 in the liver exhibit hepatic inflammation and necrosis, bile duct hyperplasia, as well as pathological findings supportive of direct or indirect systemic effects of the factor. The nucleotide and amino acid sequence of AGP-1 was found to be identical to the sequence reported for TNF-related apoptosis-inducing ligand (TRAIL, see Wiley et al. Immunity 2, 673-682 (1995)) . TRAIL was observed to induce apoptosis in a wide variety of transformed cell lines.
Summary of the Invention
A novel member of the tumor necrosis factor family, termed AGP-1, has been identified from a murine cDNA library and expressed in a transgenic mouse system.
AGP-1 is involved in myelopoiesis accompanied by an increase in neutrophils and lymphocytes, inflammation and necrosis of the liver, and bone resorption. Human
AGP-1 has also been identified. The invention provides for nucleic acids encoding a polypeptide having at least one of the biological activities of AGP-1, vectors and host cells expressing the polypeptide, and method for producing recombinant AGP-1. Antibodies or fragments thereof which specifically bind AGP-1 are also provided. Methods of identifying antagonists of AGP-1 which reduce or eliminate at least one of the biological activities of AGP-1 are also encompassed by the invention. Such antagonists include peptides, proteins, carbohydrates or small molecular weight organic molecules which bind to AGP-1 or to its receptor (s) and interfere with AGP-1 receptor activation.
AGP-1 may be used to treat hematopoeitic disorders characterized by a decrease in cell population of the bone marrow. AGP-1 antagonists may be used to treat inflammatory conditions. AGP-1 antagonists may also be used to treat bone disorders resulting from an increase in bone resorption. Pharmaceutical compositions comprising AGP-1 and AGP-1 antagonists are also encompassed by the invention.
Description of the Figures
Figure 1. cDNA and amino acid sequence of murine AGP-1.
Figure 2. cDNA and amino acid sequence of human AGP-1.
Figure 3. Hematoxylin and Eosin (H&E) stained sections of liver from non-transgenic mouse #12 (A) and HEAGP FI transgenic mouse #75-13 (B) . B illustrates marked proliferative cholangiohepatitis characterized by periportal bile duct hyperplasia and inflammation (arrowheads in B; arrowhead in A points to a normal portal tract for contrast) with scattered foci of hepatocellular necrosis (asterisk in A) . Bars = 50 μm. Figure 4. Myeloperoxidase stained sections of HEAGP FI transgenic (B - mouse #75-13) and non-transgenic (A mouse #12) spleen. B illustrates splenomegaly in the transgenic mouse primarily caused by an expanded red pulp (asterisks) due to increased red pulp myelopoiesis (arrowheads in B illustrate aggregates of myeloperoxidase positive myeloid precursors) in the transgenic spleen as well as by white pulp lymphoid hyperplasia (arrows in B vs. A) . Bars = 250 μm.
Figure 5. TRAP stained sections of bone marrow from a non-transgenic control mouse (A - mouse #12) and an HEAGP FI transgenic mouse (mouse #75-13) illustrating an apparent increase in the number of TRAP+ osteoclasts (arrows) lining bony trabeculae in the transgenic bone marrow (B) vs. the non-transgenic marrow (A) . Bars = 25 μm.
Detailed Description of the Invention
The invention provides for a novel member of the TNF receptor superfamily, termed AGP-1. AGP-1 refers to a polypeptide having an amino acid sequence of mammalian AGP-1 or a derivative thereof and having at least one of the biological activities of AGP-1. In preferred embodiments, AGP-1 is mouse or human AGP-1. cDNA and amino acid sequences of mouse and human AGP-1 are shown in Figures 1 and 2, respectively. The biological activities of AGP-1 include, but are not limited to, involvement in myelopoiesis, inflammation and necrosis, especially in the liver, and bone resorption.
The invention provides for isolated nucleic acids encoding polypeptides having one or more of the biological properties of AGP-1. As used herein, the term nucleic acid represents cDNA, genomic DNA, wholly or partially synthetic DNA or RNA. The nucleic acids of the invention are selected from the group consisting of: a) the nucleic acids as shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) ; b) nucleic acids which hybridize to the polypeptide coding regions of the nucleic acids shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) and remain hybridized to the nucleic acids under high stringency conditions; and c) nucleic acids which are degenerate to the nucleic acids of (a) or (b) .
Nucleic acid hybridizations typically involve a multi-step process comprising a first hybridization step to form nucleic acid duplexes from single strands followed by a second hybridization step carried out under more stringent conditions to selectively retain nucleic acid duplexes having a degree of homology which depends upon the stringency of hybridization during the second step. The conditions of the first hybridization step are generally not crucial, provided they are not of higher stringency than the second hybridization step. Generally, the second hybridization is carried out under conditions of high stringency, wherein "high stringency" conditions refers to conditions of temperature and salt which are about 12-20°C below the melting temperature (Tm) of a perfect hybrid of part or all of the complementary strands corresponding to SEQ. ID. NO: 1 or, alternatively, are about 12-20°C below the Tm of a perfect hybrid of part or all of the complementary strands corresponding to SEQ. ID. NO: 3. In one embodiment, "high stringency" conditions refer to conditions of about 65°C and not more than about IM Na+. It is understood that salt concentration, temperature and/or length of incubation may be varied in either the first or second hybridization steps such that one obtains the hybridizing nucleic acid molecules according to the invention. Conditions for hybridization of nucleic acids and calculations of Tm for nucleic acid hybrids are described in Sambrook et al. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, New York. (1989) .
The nucleic acids of the invention may hybridize to part or all of the polypeptide coding regions of AGP-1 as shown in SEQ ID NO: 1 and SEQ ID NO: 3, and therefore may be truncations or extensions of the nucleic acids in SEQ ID NO: 1 and SEQ ID NO: 3. Truncated or extended nucleic acids are encompassed by the invention provided that they retain one or more of the biological properties of AGP-1, such as stimulating myelopoiesis, bone resorption or an inflammatory response. In one embodiment, the nucleic acid will encode a polypeptide of at least about 10 amino acids. In another embodiment, the nucleic acid will encode a polypeptide of at least about 20 amino acids. In yet another embodiment, the nucleic acid will encode polypeptides of at least about 50 amino acids. The hybridizing nucleic acids may also include noncoding sequences located 5' and/or 3' to the AGP-1 coding regions. Noncoding sequences include regulatory regions involved in AGP-1 expression, such as promoters, enhancer regions, translational initiation sites, transcription termination sites and the like.
In preferred embodiments, the nucleic acids of the invention encode mouse AGP-1 or human AGP-1. Mouse AGP-1 is shown in Figure 1 and SEQ. ID. NO: 2 and human AGP-1 is shown in Figure 2 and SEQ. ID. NO: 4. Nucleic acids may encode a full-length form of AGP-1 which is a membrane-bound or soluble forms of AGP-1 lacking part or all of the transmembrane region. The predicted transmembrane region for human AGP-1 includes residues 16-36 as shown in SEQ. ID. NO: 4. Deletions of part or all these residues would be expected to produce soluble forms of AGP-1.
The nucleic acids of the invention will be linked with DNA sequences so as to express biologically active AGP-1. Sequences required for expression are known to those skilled in the art and include promoters and enhancer sequences for initiation of RNA synthesis, transcription termination sites, ribosome binding sites for the initiation of protein synthesis, and leader sequences for secretion. Sequences directing expression and secretion of AGP-1 may be homologous, i.e., those sequences in the genome involved in AGP-1 expression and secretion, or may be heterologous. A variety of plasmid vectors are available for expressing AGP-1 in host cells. One example is plasmid pDSRα described in
PCT Application No. 90/14363 which may be used for expression in mammalian hosts. AGP-1 coding regions may also be modified by substitution of preferred codons for optimal expression in a given host. Codon usage in bacterial, plant, insect and mammalian host systems is known and may be exploited by one skilled in the art to optimize mRNA translation. In addition, vectors are available for the tissue-specific expression of AGP-1 in transgenic animals. Retroviral and adenovirus-based gene transfer vectors may also be used for the expression of AGP-1 in human cells for in vivo therapy (see PCT Application No. 86/00922) .
Procaryotic and eucaryotic host cells expressing AGP-1 are also provided by the invention. Host cells include bacterial, yeast, plant, insect or mammalian cells. AGP-1 may also be produced in transgenic animals such as mice or goats. Plasmids and vectors containing the nucleic acids of the invention are introduced into appropriate host cells using transfection or transformation techniques known to one skilled in the art. Host cells may contain DNA sequences encoding the full-length AGP-1 gene as shown in Figure 1. Host cells will also process AGP-1 encoded by the full-length gene to the mature form or produce the mature form without processing by expression of DNA sequences encoding same. Examples of mammalian host cells for AGP-1 expression include, but are not limited to COS, CHOd-, 293 and 3T3 cells.
The invention also provides AGP-1 as the product of procaryotic or eucaryotic expression of an exogenous DNA sequence, i.e., AGP-1 is recombinant AGP-1. Exogenous DNA sequences include cDNA, genomic DNA and synthetic DNA sequences. AGP-1 may be the product of bacterial, yeast, plant, insect or mammalian cells expression. AGP-1 produced in bacterial cells will have an N-terminal methionine residue. The invention also provides for a process of producing AGP-1 comprising growing procaryotic or eucaryotic host cells transformed or transfected with nucleic acids encoding AGP-1 and isolating polypeptide expression products of the nucleic acids.
Polypeptides which are mamalian AGP-1 or are derivatives thereof are encompassed by the invention. A derivative of AGP-1 refers to a polypeptide having an addition, deletion, insertion or substitution of one or more amino acids such that the resulting polypeptide has at least one of the biological activities of AGP-1. The derivative may be naturally occurring, such as a polypeptide product of an allelic variant or a mRNA splice variant, or it may be constructed using techniques available to one skilled in the art for manipulating and synthesizing nucleic acids.
AGP-1 polypeptides may be full-length polypeptides or fragments thereof which, in preferred embodiments, are at least about ten amino acids, at least about 20 amino acids, or at least about 50 amino acids in length. AGP-1 full-length polypeptides and fragments preferably have the amino acid sequence in Figure 1 or 2 or a portion thereof. The polypeptides may or may not have an amino terminal methionine residue.
Also included in the invention are AGP-1 polypeptides which have undergone post-translational modifications (e.g., addition of N-linked or O-linked carbohydrate chains, processing of N-terminal or
C-terminal ends) , attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition of an N-terminal methionine residue as a result of procaryotic host cell expression. As mouse and human AGP-1 are encoded as transmembrane proteins, soluble forms of AGP-1 are also envisioned. Such soluble forms may be readily constructed by removal of the transmembrane region of the polypeptide. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
AGP-1 chimeric proteins comprising part or all of an AGP-1 amino acid sequence fused to a heterologous amino acid sequence are also included. The heterologous sequence may be any sequence which allows the resulting fusion protein to retain the activity of AGP-1. The heterologous sequences include, for example, immunoglobulin fusions, such as an Fc region of IgG, which provide dimerization, or fusions to enzymes which provide a label for the polypeptide.
The polypeptides of the invention are isolated and purified from tissues and cell lines which express AGP-1 and from transformed host cells expressing AGP-1, or purified from cell cultures containing the secreted protein. Isolated AGP-1 polypeptide is free from association with human proteins and other cell constituents.
Also provided by the invention are chemically modified derivatives of AGP-1 which provide additional advantages such as increased stability, longer circulating time, or decreased immunogenicity (see for example U.S. Patent No. 4,179,337) . The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxy ethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
A method for the purification of AGP-1 from natural sources (e.g. tissues and cell lines which normally express AGP-1) and from transfected host cells is also encompassed by the invention. The purification process may employ one or more standard protein purification steps in an appropriate order to obtain purified protein. The chromatography steps can include ion exchange, gel filtration, hydrophobic interaction, reverse phase, chromatofocusing, affinity chromatography employing an anti-AGP-1 antibody or biotin-streptavidin affinity complex and the like.
The invention also encompasses AGP-1 antagonists and the methods for obtaining them. An antagonist will reduce or eliminate one or more of the biological activities of AGP-1. As examples, an AGP-1 antagonist may act as an anti-inflammatory agent, or may act to inhibit bone resorption. AGP-1 antagonists include substances which bind to AGP-1 or to AGP-1 receptors in a manner to prevent normal ligand-receptor interaction and substances which regulate the expression of AGP-1. Substances which bind to AGP-1 or to AGP-1 receptors include proteins, peptides, carbohydrates and small molecular weight organic compounds. Examples of protein inhibitors include anti-AGP-1 antibodies, anti-AGP-1 receptor antibodies and soluble forms of AGP-1 receptor comprising part or all of the extraceullular domain of the AGP-1 receptor. Substances which regulate AGP-1 expression typically include nucleic acids which are complementary to nucleic acids encoding AGP-1 or AGP-1 receptors and which act as anti-sense regulators of expression.
Methods for indentifying compounds which interact with AGP-1 are also encompassed by the invention. The method comprises incubating AGP-1 with a compound under conditions which permit binding of the compound to AGP-1 and measuring the extent of binding. The compound may be substantially purified or present in a crude mixture. Binding compounds may be proteins, peptides, carbohydrates or small mo_ecular weight organic compounds. The compounds may be further characterized by their ability to enhance or reduce AGP-1 biological activity and therefore act as AGP-1 agonists or as AGP-1 antagonists. Preferably, the method is used to identify AGP-1 antagonists.
Antibodies specifically binding the AGP-1 polypeptides of the invention are also encompassed by the invention. The antibodies may be produced by immunization with full-length membrane-bound AGP-1, soluble AGP-1, or a peptide fragment thereof, and the antibodies may be polyclonal or monoclonal. In addition, the antibodies of the invention may be recombinant, such as chimeric antibodies wherein the murine constant regions on light and heavy chains are replaced by human sequences, or CDR-grafted antibodies wherein only the complementary determining regions are of murine origin. Antibodies of the invention may also be human antibodies prepared, for example, by immunization of transgenic animals capable of producing human antibodies (see, for example, PCT Application No. W093/12227) . The antibodies are useful for detecting AGP-1 in biological samples, thereby allowing the identification of cells or tissues which produce AGP-1. In addition, antibodies which bind to AGP-1 and prevent receptor interaction may also be useful for blocking the effects of AGP-1.
The invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of the AGP-1 polypeptide of the invention together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. The invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of an AGP-1 antagonist. The term "therapeutically effective amount" means an amount which provides a therapeutic effect for a specified condition and route of administration. The composition may be in a liquid or lyophilized form and comprises a diluent (Tris, acetate or phosphate buffers) having various pH values and ionic strengths, solubilizer such as Tween or Polysorbate, carriers such as human serum albumin or gelatin, preservatives such as thimerosal or benzyl alcohol, and antioxidants such as ascrobic acid or sodium metabisulfite. Also encompassed are compositions comprising AGP-1 modified with water soluble polymers to increase solubility, stability, plasma half-life and bioavailability. Compositions may also comprise incorporation of AGP-1 into liposomes, microemulsions, micelles or vesicles for controlled delivery over an extended period of time. Selection of a particular composition will depend upon a number of factors. including the condition being treated, the route of administration and the pharmacokinetic parameters desired. A more extensive survey of component suitable for pharmaceutical compositions is found in Remington's Pharmaceutical Sciences. 18th ed. A.R. Gennaro, ed. Mack, Easton, PA (1980) .
Compositions of the invention may be administered by injection, either subcutaneous, intravenous or intramuscular, or by oral, nasal, pulmonary or rectal administration. The route of administration eventually chosen will depend upon a number of factors and may be ascertained by one skilled in the art.
The invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of the nucleic acids of the invention together with a pharmaceutically acceptable adjuvant. Nucleic acid compositions will be suitable for the delivery of part or all of the APG-1 coding region and/or flanking regions to cells and tissues as part of ε . anti-sense therapy regimen.
Hepatic expression of AGP-1 in transgenic mice resulted in marked myelopoiesis accompanied by an increase in neutrophils and lymphocytes. Therefore,
AGP-1 may be used to treat hematopoietic disorders that are associated with a decrease in the population of cells in bone marrow. In particular, AGP-1 may be used to treat conditions resulting in low white blood cell levels, particularly reduced levels of neutrophils and lymphocytes. Such conditions may result from disease, injury or exposure to certain environmental agents known to suppress bone marrow levels. It is understood that AGP-1 may be administered alone or in combination with other factors to treat hematopoietic disorders. In one embodiment, AGP-1 is used in conjunction with a therapeutically effective amount of a factor which stimulates hematopoiesis. Such factors include erythropoietin (EPO) , granulocyte colony stimulating factor (G-CSF) , megakaryocyte growth and differentiation factor (MGDF) , granulocyte-macrophage stimulating factor (GM-CSF) , stem cell factor (SCF), interleukin-3 (IL-3) and interleukin-6 (IL-6) .
Hepatic expression of AGP-1 in transgenic mice resulted in increased inflammation and necrosis, especially in the liver. This effect may be the result of a high local concentration of AGP-1 occurring in the liver during transgene expression. Thus, antagonists of AGP-1 may be used as anti-inflammatory agents which are administered to patients susceptible to or suffering from an inflammatory condition. Inflammatory conditions include rhematoid arthritis, systemic lupus erythematosis, psoriasis, systemic and localized amyloidosis, Sjogerns syndrome, sclerodoma, dermatomyositis, glomerulonephritis, and inflammation arising from infections and parasitic diseass. AGP-1 antagonists which reduce or eliminate inflammation may be administered alone or in combination with a therapeutically effective amount of an anti-inflam tory agent such as a corticosteroid, a non-steroidal anti-inflammatory agent (NSAID), or cyclosporin A.
AGP-1 antagonists may also reduce or eliminate necrosis associated with an inflammatory condition.
AGP-1 is also involved in stimulation of osteoclasts which promote bone resorption through mineralization of the bone matrix. Increase in bone resorption rates that exceed rates of bone formation can lead to various bone disorders including osteoporosis, osteomyelitis, hypercalcemia, osteopenia brought on by surgery or steroid administration, Paget's disease, osteonecrosis, bone loss due to rheumatoid arthritis, periodontal bone loss, and osteolytic metastasis. Antagonists of AGP-1 may be administered to patients suffering from disorders brought on by increased osteoclast activity and increased bone resorption. AGP-1 antagonists may be administered alone or in combination with a therapeutically effective amount a bone growth promoting agent including bone morphogenic factors designated BMP-1 to BMP-12, transforming growth factor-β and TGF-β family members, interleukin-1 inhibitors, TNFα inhibitors, parathyroid hormone, E series prostaglandins, bisphosphonates and bone-enhancing minerals such as fluoride and calcium. The following examples are offered to more fully illustrate the invention, but are not construed as limiting the scope thereof.
EXAMPLE 1
Identification and Isolation of Murine and Human AGP-1 Genes
A. Murine AGP-1
Materials and method for cDNA cloning and analysis are described in Sambrook et.al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press (1989) . A cDNA library was constructed using mRNA isolated from 5, 6, and 7 day post-5FU treated bone marrow from C57/B6 female mice. Mice were treated with 150mg/kg 5-fluorouracil (5FU) , intraperitoneally, on each of three consecutive days. On day 5, 6, and 7 post-5FU treatment both femurs and tibias were harvested, and plugs flushed with PBS. Bones were crushed with mortar and pestle and combined with the bone marrow plugs. The poly A+ mRNA was purified using Fast Track mRNA Kit (InVitrogen, San Diego, CA) using the manufacturer's recommended procedures. A random primed cDNA library was prepared using the Superscript Plasmid System (Gibco BRL, Gaithersburg, MD) . A random cDNA primer containing an internal Not I restriction site was used to initiate first strand synthesis and had the following double strand sequence:
5'-CCTCTGCGGCCGCTACANNNNNNNNT-3' (SEQ ID NO: 5) 3'-pGGAGACGCCGGCGA-5 ' (SEQ ID NO: 6)
The first strand cDNA synthesis reaction was assembled using lμg of the mRNA and 150 ng of the Not 1 random primer. After second strand synthesis, the reaction products were extracted with the phenol:chloroform:isoamyl alcohol mixture and ethanol precipitated. The double strand (ds) cDNA products were ligated to the following ds oligonucleotide adapter (Gibco BRL) :
5'-TCGACCCACGCGTCCG-3' (SEQ ID NO: 7) 3'-GGGTGCGCAGGCp-5' (SEQ ID NO: 8)
After ligation the cDNA was digested to completion with Not 1, extracted with phenol:chloroform:isoamyl alcohol (25:24:1 ratio) and ethanol precipitated. The resuspended cDNA was then size fractionated by gel filtration using the premade columns provided with the Superscript Plasmid System (Gibco BRL) as recommended by the manufacturer. The fractions containing the largest cDNA products were ethanol precipitated and then directionally ligated into Not 1 and Sal 1 digested pMOB vector DNA (Strathmann et. al. Science 25Z, 802-808 (1991)) . The ligated cDNA was introduced into electrocompetent XLl-Blue E. coli (Stratagene, LaJolla, CA) by electroporation.
Approximately 20,000 colonies were picked and arrayed into 96 well microtiter plates containing 200 μl of L-broth, 7.5% glycerol, 50 μg/ml ampicillin and 12.5μg/ml tetracycline. The cultures were grown overnight at 37 C, a duplicate set of microtiter plates were made using a sterile 96 pin replicating tool, then o both sets were stored at -80 C for further analysis. To sequence random murine 5FU-treated bone marrow cDNA clones, sequencing template was prepared by PCR amplification of cloned cDNA inserts using vector primers. Glycerol stocks of cDNA clones were thawed, and small aliquots were diluted 1:25 in distilled water. Approximately 3.0 μl of diluted bacterial cultures were added to PCR reaction mixture (Boehringer-Mannheim) containing the following oligonucleotides:
5' TGTAAAACGACGGCCAGT 3' (SEQ ID NO: 9) 5' CAGGAAACAGCTATGACC 3' (SEQ ID NO: 10)
The reactions were incubated in a thermocycler (Perkin-Elmer 9600) with the following cycle conditions: 94°C for 2 minutes; 94°C for 5 seconds, 50°C for
5 seconds and 72 C for 3 minutes for 30 cycles and then a final extension at 72 C for 4 minutes. After incubation in the thermocycler, the reactions were diluted with 2.0 ml of water. The amplified DNA fagments were further purified using Centricon columns (Princeton Separations) using the manufacturer's recommended procedures. In some instances, low primer and deoxynucleoside triphosphate concentrations were used in the amplification reactions, and in those instances, Centricon purification was not necessary.
The PCR reaction products were sequenced on an Applied Biosystems 373A automated DNA sequencer using T3 primer:
5'-CAATTAACCCTCACTAAAGG-3* (SEQ ID NO: 11) Taq dye-terminator reactions (Applied Biosystems) following the manufacturer's recommended procedures.
The resulting 5' nucleotide sequence obtained from randomly picked cDNA clones were translated and then compared to the existing database of known protein sequences using a modified version of the FASTA program (Pearson, et . al. Meth. Enzymol. 18_3_, 63-98 (1990)) . Translated sequences were also analyzed for the presence of specific tumor necrosis factor superfamily motifs, using the sequence profile method of Gribskov, et . al . (Proc. Natl. Acad. Sci. USA £2, 4355-4359 (1987)) as modified by Luethy et al . (Protein Science 2/ 139-146 (1994) ) .
Using the FASTA and Profile search data, an EST designated muAGP-ESTl was identified as a possible new member of the TNF family. The muAGP-ESTl clone contained an 864 bp insert with an open reading frame of about 90 amino acids which was found to have significant homology to pig lymphotoxin-α precursor (TNF-β) and rabbit tumor necrosis factor precursor (TNF-α)
(cachectin) . The region compared showed an overlap of 63 amino acids and a 27% homology to TNF-β and a 71 amino acid overlap and 30% homology to TNF-α. Profile analysis using the TNF family profile yielded a z score of 13.5, indicating that the muAGP-ESTl clone was encoding a possible new member of the TNF family.
To obtain a full-length clone, an internal EST database was searched for overlapping clones and two other murine EST clones were identified. One EST clone designated muAGP-EST2 from a murine irradiated small intestine library gave a sequence which overlapped the sequence obtained from the muAGP-ESTl clone. The muAGP-EST2 clone was subsequently sequenced in its entirety. The insert was 3048 bp and contained an open reading frame of 291 amino acids which was deduced to be the full-length AGP-1 sequence. The nucleotide sequence and deduced amino acid of murine AGP-1 is shown in Figure 1.
B. Human AGP-1 A cDNA library was constructed using RNA from human bladder carcinoma cell line 5637 which had been stimulated with 20nM of PMA for about nine hours. For this library, mRNA was isolated from a membrane bound polysomal fraction of RNA (Mechler Methods in Enzymology 152. 241-248 (1987)) . The poly A+ mRNA fraction was isolated from the total RNA preparation by using the Fast Track mRNA Isolation Kit (InVitrogen) according to the manufacturer's recommended procedure. A directional random primed cDNA library was prepared essentially as described for the 5-FU mouse bone marrow library above. The cDNA inserts were sequenced as described above for the mouse cDNA clones.
The resulting 5' nucleotide sequences obtained from randomly picked cDNA clones were translated and compared to the existing database of known protein sequences using a modified version of the FASTA program (Pearson et al . ibid) . Translated sequences were also analysed for the presence of specific motifs found in the tumor necrosis factor superfamily using the sequence profile method of Gribskov et.al. ibid as modified by Luethy et.al. ibid.
Using the FASTA and Profile search data, an EST from the 5637 cell line cDNA library designated huAGP-ESTl was identified as a possible new member of the TNF family. huAGP-ESTl contained an 446 bp insert with an open reading frame of about 84 amino acids. Translation of the huAGP-ESTl nucleotide sequence gave an amino acid sequence which was 77% identical to the deduced amino acid sequence of murine AGP-1 when compared using FASTA analysis. This high degree of sequence similarity identifies huAGP-ESTl as the human homolog of murine AGP-1.
To obtain a full-length clone, an internal EST database was searched for overlapping clones and one other murine EST clone was identified. This clone, designated huAGP-EST2, was from a human peripheral blood megakaryocyte cDNA library and had an insert of 1028 bp which overlapped the huAGP-ESTl clone. The overlapping clones had an open reading frame of 281 amino acids. The full-length human AGP-1 was obtained as a composite of the sequences from the huAGP-ESTl and huAGP-EST2 clones. The nucleotide sequence and deduced amino acid sequence of human AGP-1 is shown in Figure 2.
EXAMPLE 2
Expression of AGP-1 in transgenic mice
A. PCR and subcloning
The TNFα-related clone muAGP-EST2 was used as template to PCR amplify the coding region for subcloning into an APOE-liver specific expression vector (Simonet et al. J. Clin. Invest. , 1310-1319 (1994), and PCT Application No. W094/11675) . The oligonucleotides used for amplification were:
5'-GAC TAG TCA GAC CTG GAC AGC AGT ATG CCT TC-3' (SEQ ID NO: 12) ; and 5'-ATA AGA ATG CGG CCG CTA AAC TAT GGG TAC TTT AGG GCT GTG TTT G-3' (SEQ ID NO: 13)
The conditions for PCR were: 94°C for 1 minute, followed by 25 cycles of 94 C for 20 sec, 63 C for 30 sec, and 74 C for 1 minute. The PCR reactions contained 1 x PFU buffer, 50 uM dNTPs, 20 pmol of each oligo, 10 ng of DNA template and 2.5 units of PFU enzyme in a total volume of 50 ul. Following amplification, the samples were purified over Qiagen PCR columns and digested overnight with Spel and NotI restriction enzymes. The digested products were extracted and precipitated and subcloned into the ApoE promoter expression vector.
Ligations were transformed into E.. coli strain DH5α and colonies were inipreped for analysis of the insert. Two clones containing the desired size insert were grown in 100ml TB cultures and plasmid DNA was prepared. The two clones were sent to sequencing to verify the authenticity of the insert. One was selected for microinjection to generate transgenic mice. This transgene was designated HE-AGP.
B. Preparation of transgenic mice
For microinjection, the HE-AGP plasmid was purified through two rounds of CsCl . The plasmid was digested with Xhol and Ase I, and the 3.4 kb transgene insert was purified on a 0.8% BRL ultrapure DNA agarose gel by electrophoresis onto NA 45 paper. The purified fragment was diluted to 1 ug/ml in 5 mM Tris, pH 7.4, 0.2 mM EDTA. Single-cell embryos from BDF1 x BDFl-bred mice were injected essentially as described (Brinster et al., 1985), except that injection needles were beveled and siliconized before use. Embryos were cultured overnight in a Cθ2 incubator and 15 to 20 two-cell embryos were transferred to the oviducts of pseudopregnant CD1 female mice.
C. Screening of transgenic founders
Following term pregnancy, 105 offspring were obtained from implantation of microinjected embryos. Of the 105 offspring, 17 were identified as transgenic founders by screening for the HE-AGP transgene in DNA prepared from ear and tail biopsies. The PCR screening involved amplification of a 369 bp region of the human Apo E intron which was included in the expression vector. The oligos used for PCR amplification were:
5'-GCC TCT AGA AAG AGC TGG GAC-3' (SEQ. ID. NO: 14) 5'-CGC CGT GTT CCA TTT ATG AGC-3' (SEQ. ID. NO: 15)
The conditions for PCR were: 94 C for 2 minute, followed by 30 cycles of 94 C for 1 min, 63 C for 20 sec, and 72°C for 30 sec. The PCR reactions contained 1 x Taq buffer, 100 uM each dNTPs, 20 pmol of each oligo, 1 ul of DNA template extract and 0.5 units of taq enzyme in a total volume of 50 ul.
D. Preparation and analysis of total RNA for Northern analysis
At 8-10 weeks of age, 8 of the 17 transgenics (#'s 10, 27, 52, 53, 69, 72, 76 and 77) and 4 control littermates (#'s 55, 56, 57, and 58) were sacrificed for necropsy and pathological analysis (See Example 3) . Liver was isolated from the remaining 9 founders (#'s 25, 42, 44, 45, 48, 50, 67, 74, and 75) by partial hepatectomy. For partial hepatecto y, the mice were anesthetized with avertin and a lobe of liver was surgically removed. Total cellular RNA was isolated from livers of all transgenic founders, and 5 negative control littermates as described (McDonald et al. (1987)) . Northern blot analysis was performed on these samples to assess the level of transgene expression.
Approximately lOug of total RNA from each animal liver was resolved by electrophoresis denaturing gels (Ogden et al. (1987)), then transferred to HYBOND-N nylon membrane (Amersham) , and probed with 32p CTP-labelled pBl.l insert DNA. Hybridization was performed overnight at 42°C in 50% Formamide, 5 x SSPE, 0.5% SDS, 5 x 686 PC17US97/09895
- 24 -
Denhardt's solution, 100 ug/ml denatured salmon sperm DNA and 2-4 x 106 cpm of labeled probe/ml of hybridization buffer. Following hybridization, blots were washed twice in 2 x SSC, 0.1% SDS at room temperature for 5 min each, and then twice in 0.1 x SSC, 0.1% SDS at 55 C for 5-10 min each. Expression of the transgene in founder and control littermates was determined following autoradiography.
The Northern blot data indicate that 13 of the transgenic founders express detectable levels of the transgene mRNA (animal #'s 10, 42, 44, 45, 48, 50, 52, 53, 67, 69, 74, 75 and 76) . The negative control mice expressed no transgene-related mRNA. The highest expressing founders From the group that were necropsied were #'s 52, 69 and 76. The highest expressing animals from the remaining group of founder's were #'s 42, 45, 67, and 75. Six of the founder's that were analyzed by hepatectomy were subsequently bred to generate FI offspring for further analysis.
EXAMPLE 3
Pathology Analysis of Transgenic Mice Expressing AGP- 1
A . Necropsy
Mice from two separate studies were examined. In the first study, five BDF1 female mice which were founder transgenics for the murine AGP-1 molecule targeted to the liver via an apolipoprotein E promoter as well as four male non-transgenic littermate mice were necropsied for phenotypic analysis. In the second study, twelve BDF1 mice (nine females and three males) which were FI transgenics for the murine AGP molecule targeted to the liver via an apolipoprotein E promoter as well as four female non-transgenic littermate mice - 25 -
were necropsied for phenotypic analysis. In both studies, all mice were injected with BrdU one hour prior to harvest and sacrificed. Body and liver, spleen, kidney, stomach, and thymus weights were taken, blood was drawn for hematology and serum chemistries, and liver, spleen, lung, brain, heart, kidney, adrenal, stomach, small intestine, pancreas, cecum, colon, mesenteric lymph node, skin, mammary gland, trachea, esophagus, thyroid, parathyroid, salivary gland, urinary bladder, ovary or testis, uterus or seminal vesicle, bone, and bone marrow were examined were harvested for histologic analysis and BrdU labeling.
B. Histology and Histochemistry Sections of liver, spleen, lung, brain, heart, kidney, adrenal, stomach, small intestine, pancreas, cecum, colon, mesenteric lymph node, skin, mammary gland, trachea, esophagus, thyroid, parathyroid, salivary gland, urinary bladder, ovary or testis, uterus or seminal vesicle, bone, and bone marrow from AGP-1 transgenic and non-transgenic mice were fixed overnight in 10% neutral buffered zinc formalin (Anatech, Battle Creek, Michigan) , paraffin embedded, sectioned at 3 μ\m, and stained with hematoxylin and eosin (H&E) for routine histologic examination. In addition, sections of bone were stained for tartrate resistant acid phosphatase (TRAP) to highlight osteoclasts around bony trabeculae in marrow spaces.
C. Immunohistochemistry
Immunohistochemical staining was done on 4 μm thick paraffin embedded sections using an automated TechMate Immunostainer (BioTek Solutions, Santa Barbara, CA) . For BrdU immunostaining, sections were first digested with 0.1% protease (Sigma Chemical, St. Louis, MO) followed by 2N HCl. BrdU was detected with a rat monoclonal antibody (MAb) to BrdU (Accurate Chemical, Westbury, NY) followed by a biotinylated anti-rabbit/anti-mouse secondary cocktail (BioTek) and an ABC tertiary coupled to alkaline phosphatase (BioTek) . The staining reaction was visualized with BioTek Red chromagen (BioTek) . For myeloperoxidase immunostaining, sections were stained with rabbit polyclonal antisera directed at human myeloperoxidase (Dako, Carpinteria, CA) , followed by a biotinylated anti-rabbit/anti-mouse secondary cocktail (BioTek) and avidin-biotin complex (ABC) tertiary coupled to horseradish peroxidase. The staining reaction was visualized with dia inobenzidine (DAB, Sigma) .
D. Gross Pathology Findings
The livers from two transgenic founder mice(#s 69 and 76) and two FI transgenic mice (#s 75-13 and 75-18) were significantly increased in size and weight (8.42 ± 1.26 SD % of body weight vs. 5.33 ± 0.89 SD % of body weight in non-transgenic control mice) and were pale green-tan and more friable than normal. These four mice also had a significant increase in splenic weight (1.14 ± 0.12 SD % of body weight vs. 0.41 ± 0.09 SD % body weight in non-transgenic control mice. These results are summarized in Table 1.
E. Clinical Pathology Findings
The four transgenic mice with enlarged livers (founder #s 69 and 76 and FI #s 75-13 and 75-18 had marked and significant increases in total serum bilirubin and alkaline phosphatase levels, with moderate but significant increases in hepatic transaminase (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) levels. The four transgenic mice had a mean total bilirubin level of 4.33 ±
5.32 SD mg/dl while non-transgenic control mice had a mean total bilirubin level of 0.16 ± 0.05 SD mg/dl. The mean serum alkaline phosphatase level in these four transgenic mice was 994.5 ± 353.1 SD IU/1 vs. 165.3 ± 53.2 SD IU/1 in non-transgenic control mice. The mean ALT level in these four transgenic mice was 247.3 ± 89.8 SD IU/1 vs. 78.1 ± 43.2 SD IU/1 in non-transgenic control mice while the mean AST level in these four transgenic mice was 350.5 ± 135.6 SD IU/1 vs. 132.5 ± 84.9 SD IU/1 in non-transgenic control mice. All of these results are summarized in table 1.
F. Histopathologic Findings
H&E and BrdU stained sections of liver, spleen, lung, brain, heart, kidney, adrenal, stomach, small intestine, pancreas, cecum, colon, mesenteric lymph node, skin, mammary gland, trachea, esophagus, thyroid, parathyroid, salivary gland, urinary bladder, ovary or testis, uterus or seminal vesicle, bone, and bone marrow were examined from the 17 HE-AGP-1 transgenic mice and 8 non-transgenic control littermates. Myeloperoxidase stained sections of spleen and bone marrow as well as tartrate resistant acid phosphatase (TRAP) stained sections of bone were also examined from all mice. Major histologic changes in the transgenic mice included marked periportal inflammation and bile duct hyperplasia with scattered multifocal to coalescing areas of hepatocellular necrosis in transgenic mice #s 69 and 76 (founders) and #s 75-13 and 75-18 (Fls) (Figure 3) . All four of these transgenic mice also had enlarged spleens primarily due to increased red pulp myelopoiesis and to a lesser extent, lymphoid hyperplasia (Figure 4) . These four transgenic mice also appeared to have increased numbers of TRAP positive osteoclasts lining bony trabeculae in peripheral diaphyseal marrow compared to non-transgenic control mice (Figure 5) . Transgenic mice also exhibited increased intravascular neutrophils, and small atrophic/hypoplastic uteri (only founder transgenics #s 69 and 76) . The two founder transgenic mice (#s 69 and 76) also exhibited moderate peritoneal mixed inflammatory cellular infiltration.
G. Summary of Pathologic Findings in Transgenic Mice Overexpressing AGP-1
Four of the HE-AGP-1 transgenic mice (founder nos. 69 and 76 and FI nos. 75-13 and 75-18) had relatively severe phenotypic alterations, particularly in their livers with marked cholangiohepatitis, bile duct hyperplasia and hepatic necrosis. Accompanying these hepatic histologic abnormalities in these four transgenic mice was evidence of liver dysfunction with marked elevations in total serum bilirubin and alkaline phosphatase with moderate elevations in serum transaminases. In addition to hepatic findings, these four transgenic mice also exhibited increased myelopoiesis, with a less prominent increase in circulating platelets. Founder mouse #69 had a circulating neutrophilia while all transgenic mice had a moderate increase in circulating lymphocytes. Evidence of peritoneal inflammation was also seen in the two founder transgenic mice with marked hepatic inflammation. Two of the other HEAGP founder transgenic mice, #'s 52 and 53, also had evidence of mild cholangiohepatitis, and a mild to moderate increase in myelopoiesis and neutrophilia, suggesting that these two mice were producing the transgenic AGP-1 protein at a lower level than founder mice #s 69 and 76 were. In addition to hepatic findings, at least four of the transgenic mice exhibited a marked increase in splenic myelopoiesis and moderate lymphoid hyperplasia as well as exhibiting an apparent increase in TRAP+ osteoclasts lining bony trabeculae in the bone marrow. All of these findings suggest that the AGP protein plays a role in inflammation, myelopoiesis, and bone resorption (osteoclasis) .
Table 1 Selected Organ Weights and Serum Chemistries in HE-AGP- Transgenic Mice
Figure imgf000031_0001
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Johnson, Merrie Jo Simonet, William S. Danilenko, Dimitry M. (ii) TITLE OF INVENTION: TUMOR NECROSIS FACTOR-RELATED POLYPEPTIDE
(iii) NUMBER OF SEQUENCES: 15 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amgen Inc.
(B) STREET: 1840 Dehavilland Drive
(C) CITY: Thousand Oaks
(D) STATE: California (E) COUNTRY: U.S.A.
(F) ZIP: 91320
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Winter, Robert B.
(C) REFERENCE/DOCKET NUMBER: A-410
(2) INFORMATION FOR SEQ ID NO: 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3048 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 245..1120
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
GTTCATAGAT GGGTTAGATC TCAGAGCGCT GGATCTAGGC TTTCCAGCAC CATCAGGGCG 60 AGCTCTCCTA GCTGGAGGGT TTCTGTGCAC TACGTCCTCG TCACCTTCCT GACTTGCTTA 120
GTTTCACTTT TGGTCTCAAC AGTAAAAAGA AACTGCATGG GCACTCCGCC TTCTAACTGT 180
GACCTTCTCA GGCACTGCTG CTGGGCTGCA AGTCTGCATT GGGAAGTCAG ACCTGGACAG 240
CAGT ATG CCT TCC TCA GGG GCC CTG AAG GAC CTC AGC TTC AGT CAG CAC 289 Met Pro Ser Ser Gly Ala Leu Lys Asp Leu Ser Phe Ser Gin His
1 5 10 15
TTC AGG ATG ATG GTG ATT TGC ATA GTG CTC CTG CAG GTG CTC CTG CAG 337
Phe Arg Met Met Val Ile Cys Ile Val Leu Leu Gin Val Leu Leu Gin
20 25 30 GCT GTG TCT GTG GCT GTG ACT TAC ATG TAC TTC ACC AAC GAG ATG AAG 385
Ala Val Ser Val Ala Val Thr Tyr Met Tyr Phe Thr Asn Glu Met Lys
35 40 45
CAG CTG CAG GAC AAT TAC TCC AAA ATT GGA CTA GCT TGC TTC TCA AAG 433 Gin Leu Gin Asp Asn Tyr Ser Lys Ile Gly Leu Ala Cys Phe Ser Lys
50 55 60
ACG GAT GAG GAT TTC TGG GAC TCC ACT GAT GGA GAG ATC TTG AAC AGA 481
Thr Asp Glu Asp Phe Trp Asp Ser Thr Asp Gly Glu Ile Leu Asn Arg 65 70 75
CCC TGC TTG CAG GTT AAG AGG CAA CTG TAT CAG CTC ATT GAA GAG GTG 529
Pro Cys Leu Gin Val Lys Arg Gin Leu Tyr Gin Leu Ile Glu Glu Val
80 85 90 95
ACT TTG AGA ACC TTT CAG GAC ACC ATT TCT ACA GTT CCA GAA AAG CAG 577
Thr Leu Arg Thr Phe Gin Asp Thr Ile Ser Thr Val Pro Glu Lys Gin
100 105 110 CTA AGT ACT CCT CCC TTG CCC AGA GGT GGA AGA CCT CAG AAA GTG GCA 625
Leu Ser Thr Pro Pro Leu Pro Arg Gly Gly Arg Pro Gin Lys Val Ala
115 120 125
GCT CAC ATT ACT GGG ATC ACT CGG AGA AGC AAC TCA GCT TTA ATT CCA 673 Ala His Ile Thr Gly Ile Thr Arg Arg Ser Asn Ser Ala Leu Ile Pro
130 135 140
ATC TCC AAG GAT GGA AAG ACC TTA GGC CAG AAG ATT GAA TCC TGG GAG 721
Ile Ser Lys Asp Gly Lys Thr Leu Gly Gin Lys Ile Glu Ser Trp Glu 145 150 155
TCC TCT CGG AAA GGG CAT TCA TTT CTC AAC CAC GTG CTC TTT AGG AAT 769
Ser Ser Arg Lys Gly His Ser Phe Leu Asn His Val Leu Phe Arg Asn
160 165 170 175
GGA GAG CTG GTC ATC GAG CAG GAG GGC CTG TAT TAC ATC TAT TCC CAA 817
Gly Glu Leu Val Ile Glu Gin Glu Gly Leu Tyr Tyr Ile Tyr Ser Gin
180 185 190 ACA TAC TTC CGA TTT CAG GAA GCT GAA GAC GCT TCC AAG ATG GTC TCA 865
Thr Tyr Phe Arg Phe Gin Glu Ala Glu Asp Ala Ser Lys Met Val Ser
195 200 205 AAG GAC AAG GTG AGA ACC AAA CAG CTG GTG CAG TAC ATC TAC AAG TAC 913 Lys Asp Lys Val Arg Thr Lys Gin Leu Val Gin Tyr Ile Tyr Lys Tyr 210 215 220 ACC AGC TAT CCG GAT CCC ATA GTG CTC ATG AAG AGC GCC AGA AAC AGC 961 Thr Ser Tyr Pro Asp Pro Ile Val Leu Met Lys Ser Ala Arg Asn Ser 225 230 235
TGT TGG TCC AGA GAT GCC GAG TAC GGA CTG TAC TCC ATC TAT CAG GGA 1009 Cys Trp Ser Arg Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gin Gly 240 245 250 255
GGA TTG TTC GAG CTA AAA AAA AAT GAC AGG ATT TTT GTT TCT GTG ACA 1057 Gly Leu Phe Glu Leu Lys Lys Asn Asp Arg Ile Phe Val Ser Val Thr 260 265 270
AAT GAA CAT TTG ATG GAC CTG GAT CAA GAA GCC AGC TTC TTT GGA GCC 1105 Asn Glu His Leu Met Asp Leu Asp Gin Glu Ala Ser Phe Phe Gly Ala 275 280 285
TTT TTA ATT AAC TAA ATGACCAGTA AAGATCAAAC ACAGCCCTAA AGTACCCAGT 1160 Phe Leu Ile Asn * 290 AATCTTCTAG GTTGAAGGCA TGCCTGGAAA GCGACTGAAC TGGTTAGGAT ATGGCCTGGC 1220
TGTAGAAACC TCAGGACAGA TGTGACAGAA AGGCAGCTGG AACTCAGCAG CGACAGGCCA 1280
ACAGTCCAGC CACAGACACT TTCGGTGTTT CATCGAGAGA CTTGCTTTCT TTCCGCAAAA 1340
TGAGATCACT GTAGCCTTTC AATGATCTAC CTGGTATCAG TTTGCAGAGA TCTAGAAGAC 1400
GTCCAGTTTC TAAATATTTA TGCAACAATT GACAATTTTC ACCTTTGTTA TCTGGTCCAG 1460 GGGTGTAAAG CCAAGTGCTC ACAGGCTGTG TGCAGACCAG GATAGCTATG AATGCAGGTC 1520
AGCATAAAAA TCACAGAATA TCTCACCTAC CAAATCAGAG TGGGTGTGCC CCTGTGTGTA 1580
TATGCGTGTC TGTGTGTGTG TGCATGTATG TGTGTGTGTG TGTGACTGTT CTTTATGG A 1640
ACTGGTTATG TTTTTCTCAA GTGAAAAACA TAACTCTATA CATGATAACA TAA ATCCCA 1700
TCATCAGTGG AACCTTGCCC AAAGAATGTA TGAAATCTCC AGGCAATGAA TGAGGGCAGC 1760 CCAAGAAAGA GGCCCGCAGA GCCATACCAC AGGGCTGCCC CACCCTGCTG GAGCTCAGAT 1820
CCTGCCACTG CTGCAGGCCC TGGGTACCAG GTGTAGAGTT GGAGGAGGTC TTGCCTGTGG 1880
GTCTAGGTCT TTGGTGCCTA CCTCCTTGAT ATGGCCCCAG TCCTCCTTTG CTTGTTTGCT 1940
AGTTTTATCA TGTTTCCCAG GCCGGCCTCA AGTCCAATAT GTAGTCAAGA GTGATCTCTA 2000
ACTGTGCAAC CTCCTGCCTC CAAGATCTGC TGAGATTATA GGCATGTGCC CCCCTGTCTG 2060 ATTTGTGTAG AGCCAGGCTT CTTGTACATG TGACAACCAT GCCACCCTCA GCTCTGTCCC 2120
AGCTCCATTT CTTCCTTTCT GAATGCAAGC ATTTACTTTG TGTCCCTATA TTCTAGAATG 2180 TGCAACAGTG AAGAATTTGC TCTGACTTTC AGGATAAAGT TTGAACTAGG TTCACCATGC 2240
TTGCTTTGTC CAGATTGCGA CTGTCACCCA GTCCTCTGGC TCTTCCATCT GTCTGTCCAC 2300
TCCACCTACC AAGATGTTGA ACACTTGTTC TTTTTAAGAT GTTGGTGCCT GGAGTTTCAT 2360
TAGAGTAACA CAAAACTAAC TAAAACCAAA CAACTCCAAA GGAGCCCATA TGTGTTTTAA 2420
TGAAACATTT TTTAAGCCTA TTGGGGGCCT GAAGAGATTG CTCAGAGGAA AACAGCACTT 2480
CCAGAGGACC CAGGTTCAAT TCTCATCGCT GATGTGATAG TTAACAGCTG TAACTTCAGT 2540
TCCAAGGGGT CTGACTTTCT GCCCTTTGCT TGCAATGCAT GTATGTGATA CACAGACATA 2600 CATTCTGACA AAATATATCC ATACACAAAA GTATTTTTTT AAAAGCTTAT TTGAATGTAA 2660
GAGTATGGCT AGCTGTCACT TCTGATACCC CTTCTTATTT TTTTATGACT CAAGCCCTTA 2720
TAAACTAGCA AATAGAAGTC ACAGCTACCA CTTGAATATA AGCACTTGAA TACCTCCTCT 2780
CACTAGAATA CAACATAGCT TAATAGTAAA AATCTTGCCT TAGTAAAGTA CTTGCATGTC 2840
ATGTCTACAT GAACCAAATG AATGTATTAA TTAATAATAG ACATAATGAT CACATCGGAA 2900 AGGCTGTGAG AAATAATGGA GAACATTTGA AAGCTCAAGA TGGAAGGGAA AGGCACTTGT 2960
CAAAAATCTT GACAACCTGA ATTTGACCTT TGGCAGGGCT GAAAACTAAA CCCAGGGTCT 3020
TACTCCCAGT AGGCATGAAC TCCCCCCT 3048
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 292 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :
Met Pro Ser Ser Gly Ala Leu Lys Asp Leu Ser Phe Ser Gin His Phe 1 5 10 15
Arg Met Met Val Ile Cys Ile Val Leu Leu Gin Val Leu Leu Gin Ala 20 25 30
Val Ser Val Ala Val Thr Tyr Met Tyr Phe Thr Asn Glu Met Lys Gin 35 40 45
Leu Gin Asp Asn Tyr Ser Lys Ile Gly Leu Ala Cys Phe Ser Lys Thr
50 55 60 Asp Glu Asp Phe Trp Asp Ser Thr Asp Gly Glu Ile Leu Asn Arg Pro 65 70 75 80 Cys Leu Gin Val Lys Arg Gin Leu Tyr Gin Leu Ile Glu Glu Val Thr 85 90 95
Leu Arg Thr Phe Gin Asp Thr Ile Ser Thr Val Pro Glu Lys Gin Leu 100 105 110
Ser Thr Pro Pro Leu Pro Arg Gly Gly Arg Pro Gin Lys Val Ala Ala 115 120 125 His Ile Thr Gly Ile Thr Arg Arg Ser Asn Ser Ala Leu Ile Pro lie 130 135 140
Ser Lys Asp Gly Lys Thr Leu Gly Gin Lys Ile Glu Ser Trp Glu Ser 145 150 155 160
Ser Arg Lys Gly His Ser Phe Leu Asn His Val Leu Phe Arg Asn Gly 165 170 175
Glu Leu Val Ile Glu Gin Glu Gly Leu Tyr Tyr Ile Tyr Ser Gin Thr 180 185 190
Tyr Phe Arg Phe Gin Glu Ala Glu Asp Ala Ser Lys Met Val Ser Lys 195 200 205 Asp Lys Val Arg Thr Lys Gin Leu Val Gin Tyr Ile Tyr Lys Tyr Thr 210 215 220
Ser Tyr Pro Asp Pro Ile Val Leu Met Lys Ser Ala Arg Asn Ser Cys 225 230 235 240
Trp Ser Arg Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gin Gly Gly 245 250 255
Leu Phe Glu Leu Lys Lys Asn Asp Arg Ile Phe Val Ser Val Thr Asn 260 265 270
Glu His Leu Met Asp Leu Asp Gin Glu Ala Ser Phe Phe Gly Ala Phe 275 280 285 Leu Ile Asn * 290
(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1060 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 35..880 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GGCTGACTTA CAGCAGTCAG ACTCTGACAG GATC ATG GCT ATG ATG GAG GTC 52
Met Ala Met Met Glu Val 1 5
CAG GGG GGA CCC AGC CTG GGA CAG ACC TGC GTG CTG ATC GTG ATC TTC 100 Gin Gly Gly Pro Ser Leu Gly Gin Thr Cys Val Leu Ile Val Ile Phe 10 15 20
ACA GTG CTC CTG CAG TCT CTC TGT GTG GCT GTA ACT TAC GTG TAC TTT 148 Thr Val Leu Leu Gin Ser Leu Cys Val Ala Val Thr Tyr Val Tyr Phe 25 30 35 ACC AAC GAG CTG AAG CAG ATG CAG GAC AAG TAC TCC AAA AGT GGC ATT 196 Thr Asn Glu Leu Lys Gin Met Gin Asp Lys Tyr Ser Lys Ser Gly Ile 40 45 50
GCT TGT TTC TTA AAA GAA GAT GAC AGT TAT TGG GAC CCC AAT GAC GAA 244 Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu 55 60 65 70
GAG AGT ATG AAC AGC CCC TGC TGG CAA GTC AAG TGG CAA CTC CGT CAG 292 Glu Ser Met Asn Ser Pro Cys Trp Gin Val Lys Trp Gin Leu Arg Gin 75 80 85
CTC GTT AGA AAG ATG ATT TTG AGA ACC TCT GAG GAA ACC ATT TCT ACA 340 Leu Val Arg Lys Met Ile Leu Arg Thr Ser Glu Glu Thr Ile Ser Thr 90 95 100
GTT CAA GAA AAG CAA CAA AAT ATT TCT CCC CTA GTG AGA GAA AGA GGT 388
Val Gin Glu Lys Gin Gin Asn Ile Ser Pro Leu Val Arg Glu Arg Gly
105 110 115 CCT CAG AGA GTA GCA GCT CAC ATA ACT GGG ACC AGA GGA AGA AGC AAC 436 Pro Gin Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn 120 125 130
ACA TTG TCT TCT CCA AAC TCC AAG AAT GAA AAG GCT CTG GGC CGC AAA 484 Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys 135 140 145 150
ATA AAC TCC TGG GAA TCA TCA AGG AGT GGG CAT TCA TTC CTG AGC AAC 532 Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn 155 160 165
TTG CAC TTG AGG AAT GGT GAA CTG GTC ATC CAT GAA AAA GGG TTT TAC 580 Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr 170 175 180
TAC ATC TAT TCC CAA ACA TAC TTT CGA TTT CAG GAG GAA ATA AAA GAA 628 Tyr Ile Tyr Ser Gin Thr Tyr Phe Arg Phe Gin Glu Glu Ile Lys Glu 185 190 195 AAC ACA AAG AAC GAC AAA CAA ATG GTC CAA TAT ATT TAC AAA TAC ACA 676 Asn Thr Lys Asn Asp Lys Gin Met Val Gin Tyr Ile Tyr Lys Tyr Thr 200 205 210 AGT TAT CCT GAC CCT ATA TTG TTG ATG AAA AGT GCT AGA AAT AGT TGT 724 Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys 215 220 225 230 TGG TCT AAA GAT GCA GAA TAT GGA CTC TAT TCC ATC TAT CAA GGG GGA 772 Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gin Gly Gly 235 240 245
ATA TTT GAG CTT AAG GAA AAT GAC AGA ATT TTT GTT TCT GTA ACA AAT 820 Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn 250 255 260
GAG CAC TTG ATA GAC ATG GAC CAT GAA GCC AGT TTT TTC GGG GCC TTT 868 Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe 265 270 275
TTA GTT GGC TAA CTGACCTGGA AAGAAAAAGC AATAACCTCA AAGTGACTAT 920
Leu Val Gly * 280
TCAGTTTTCA GGATGATACA CTATGAAGAT GTTTCAAAAA ATCTGACCAA AACAAACAAA 980
CAGAAAACAG AAAACAAAAA AACCTCTATG CAATCTGAGT AGAGCAGCCA CAACCAAAAT 1040 TGTATACAAC ACACCATGTA 1060
(2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 :
Met Ala Met Met Glu Val Gin Gly Gly Pro Ser Leu Gly Gin Thr Cys 1 5 10 15
Val Leu Ile Val Ile Phe Thr Val Leu Leu Gin Ser Leu Cys Val Ala 20 25 30 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gin Met Gin Asp Lys 35 40 45
Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60
Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gin Val 65 70 75 80
Lys Trp Gin Leu Arg Gin Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95
Glu Glu Thr Ile Ser Thr Val Gin Glu Lys Gin Gin Asn Ile Ser Pro
100 105 110 Leu Val Arg Glu Arg Gly Pro Gin Arg Val Ala Ala His Ile Thr Gly
115 120 125
Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu
130 135 140
Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly
145 150 155 160
His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile 165 170 175
His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gin Thr Tyr Phe Arg Phe 180 185 190
Gin Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gin Met Val Gin 195 200 205 Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220
Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr 225 230 235 240
Ser Ile Tyr Gin Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile 245 250 255
Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala 260 265 270
Ser Phe Phe Gly Ala Phe Leu Val Gly * 275 280 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CCTCTGCGGC CGCTACANNN NNNNNT 26
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AGCGGCCGCA GAGG 14
(2) INFORMATION FOR SEQ ID NO:7 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : TCGACCCACG CGTCCG 16
(2) INFORMATION FOR SEQ ID NO:8 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CGGACGCGTG GG 12 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TGTAAAACGA CGGCCAGT (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 CAGGAAACAG CTATGACC
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CAATTAACCC TCACTAAAGG 20
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GACTAGTCAG ACCTGGACAG CAGTATGCCT TC 32 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ATAAGAATGC GGCCGCTAAA CTATGGGTAC TTTAGGGCTG TGTTT 45
(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GCCTCTAGAA AGAGCTGGGA C 21 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CGCCGTGTTC CATTTATGAG C 21

Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid encoding a polypeptide comprising at least one of the biological activities of AGP-1 wherein the nucleic acid is selected from the group consisting of: a) the nucleic acids shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) ; b) nucleic acids which hybridize to the polypeptide coding regions of the nucleic acids shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) and remain hybridized to the nucleic acids under high stringency conditions; and c) nucleic acids which are degenerate to the nucleic acids of (a) or (b) .
2. The nucleic acid of Claim 1 which is cDNA, genomic DNA, synthetic DNA or RNA.
3. A polypeptide encoded by the nucleic acid of Claim 1.
4. The nucleic acid of Claim 1 including one or more codons preferred for Escherichia coli expression.
5. The nucleic acid of Claim 1 having a detectable label attached thereto.
6. The nucleic acid of Claim 1 comprising the polypeptide-coding region of Figure 2 (SEQ ID NO: 3) .
7. A nucleic acid encoding a polypeptide having the amino acid sequence of SEQ. ID. NO. 2 or SEQ. ID. NO. 4.
8. An expression vector comprising the nucleic acid of Claim 1.
9. The expression vector of Claim 8 wherein the nucleic acid comprises the polypeptide-encoding region as shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) .
10. A host cell transformed or transfected with the expression vector of Claim 8.
11. The host cell of Claim 10 which is a eucaryotic or procaryotic cell.
12. The host cell of Claim 11 which is Escherichia coli.
13. A process for the production of AGP-1 comprising: growing under suitable nutrient conditions host cells transformed or transfected with the nucleic acid of Claim 1; and isolating the polypeptide product of the expression of the nucleic acid.
14. A polypeptide produced by the process of Claim 13.
15. A purified and isolated AGP-1 polypeptide.
16. The polypeptide of Claim 15 which is mammalian AGP-1.
17. The polypeptide of Claim 15 having the amino acid sequence as shown in Figure 2 (SEQ ID NO: 3) .
18. The polypeptide of Claim 17 which has been covalently modified with a water-soluble polymer.
19. The polypeptide of Claim 18 wherein the polymer is polyethylene glycol.
20. An antibody or fragment thereof which specifically binds AGP-1.
21. The antibody of Claim 20 which is a monoclonal antibody.
22. A method for detecting the presence of AGP-1 in a biological sample comprising: incubating the sample with the antibody of Claim 20 under conditions that allow binding of the antiboby to AGP-1; and detecting the bound antibody.
23. A method to assess the ability of a candidate compound to bind AGP-1 comprising: incubating AGP-1 with the candidate compound under conditions that allow binding; and measuring the bound compound.
24. The method of Claim 23 wherein the compound is an antagonist of AGP-1.
25. A method of regulating expression of AGP-1 in an animal comprising administering to the animal a nucleic acid complementary to the nucleic acids as shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) .
26. A pharmaceutical composition comprising a therapeutically effective amount of AGP-1 in a PO7US97/09895
- 44 -
pharmaceutically acceptable carrier, adjuvant, solubilizer, stabilizer and/or anti-oxidant.
27. The composition of Claim 26 wherein AGP-1 is human AGP-1.
28. A method of treating an inflammatory disorder comprising administering a therapeutically effetive amount of an AGP-1 antagonist.
29. The method of Claim 28 further comprising administering a therapeutically effective amount of an anti-inflammatory agent selected from the group consisting of a corticosteroid, a non-steroidal anti-inflammatory agent, and a cyclosporin.
30. A method of treating a hematopoietic disorder comprising adminstering a therapeutically effective amount of AGP-1.
31. The method of Claim 30 further comprising administering a therapeutically effective amount of a hematopoietic factor selected from the group consisting of EPO. G-CSF, MGDF, GM-CSF, SCF, IL-3 and IL-6.
32. A method of treating a bone disorder comprising administering a therapeutically effective amount of an AGP-1 antagonist.
33. The method of Claim 31 further comprising administering a therapeutically effective amount of a bone growth factor selected from the group consisting of: bone morphogenic factors BMP-1 to BMP-12, TGF-β family members, IL-1 inhibitors, TNFα inhibitors, parathyroid hormone, E series prostaglandins, bisphosphonates and bone-enhancing minerals.
PCT/US1997/009895 1996-06-07 1997-06-06 Tumor necrosis factor-related polypeptide WO1997046686A2 (en)

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AU33810/97A AU3381097A (en) 1996-06-07 1997-06-06 Tumor necrosis factor-related polypeptide
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EP97929846A EP0918860A2 (en) 1996-06-07 1997-06-06 Tumor necrosis factor-related polypeptide

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Application Number Priority Date Filing Date Title
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US08/660,562 1996-06-07

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US7972812B2 (en) 1995-06-29 2011-07-05 Immunex Corporation DNA encoding a soluble fragment of TRAIL polypeptide
US7736637B2 (en) 1995-06-29 2010-06-15 Immunex Corporation Use of TRAIL polypeptides to induce apoptosis
US6284236B1 (en) 1995-06-29 2001-09-04 Immunex Corporation Cytokine that induces apoptosis
US6046048A (en) * 1996-01-09 2000-04-04 Genetech, Inc. Apo-2 ligand
US6746668B2 (en) 1996-01-09 2004-06-08 Genentech, Inc. Apo-2 ligand
US6998116B1 (en) 1996-01-09 2006-02-14 Genentech, Inc. Apo-2 ligand
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WO1999007408A1 (en) * 1997-08-04 1999-02-18 Bio Merieux Protein factor associated with a neuro-degenerative or autoimmune and/or inflammatory disease
US6740739B1 (en) 1998-01-15 2004-05-25 Genentech, Inc. Substitutional variants of APO-2 ligand
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US7855066B1 (en) 1999-06-28 2010-12-21 Genentech, Inc. Methods for making Apo-2 ligand using divalent metal ions
EP2311956A1 (en) 1999-06-28 2011-04-20 Genentech, Inc. Methods for making APO-2 ligand using divalent metal ions
EP2339003A2 (en) 1999-06-28 2011-06-29 Genentech, Inc. APO-2 ligand substitutional variants
EP2014303A2 (en) 2000-07-27 2009-01-14 Genentech, Inc. APO-2L receptor agonist and CPT-11 synergism
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CA2256464A1 (en) 1997-12-11

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