EP0918860A2 - Tumor necrosis factor-related polypeptide - Google Patents
Tumor necrosis factor-related polypeptideInfo
- Publication number
- EP0918860A2 EP0918860A2 EP97929846A EP97929846A EP0918860A2 EP 0918860 A2 EP0918860 A2 EP 0918860A2 EP 97929846 A EP97929846 A EP 97929846A EP 97929846 A EP97929846 A EP 97929846A EP 0918860 A2 EP0918860 A2 EP 0918860A2
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- European Patent Office
- Prior art keywords
- agp
- seq
- polypeptide
- nucleic acid
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Humanized animals, e.g. knockin
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- 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
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- 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]
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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
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- 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
Abstract
Description
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US66056296A | 1996-06-07 | 1996-06-07 | |
US660562 | 1996-06-07 | ||
PCT/US1997/009895 WO1997046686A2 (en) | 1996-06-07 | 1997-06-06 | Tumor necrosis factor-related polypeptide |
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DE102006017226A1 (en) * | 2006-04-12 | 2007-10-18 | Schaeffler Kg | Guide carriage roller for linear guide roller, has inner ring provided with depression to receive screw head, where countersunk head of countersunk screw is absorbed in conical depression |
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US6284236B1 (en) | 1995-06-29 | 2001-09-04 | Immunex Corporation | Cytokine that induces apoptosis |
WO1997001633A1 (en) * | 1995-06-29 | 1997-01-16 | Immunex Corporation | Cytokine that induces apoptosis |
US6998116B1 (en) | 1996-01-09 | 2006-02-14 | Genentech, Inc. | Apo-2 ligand |
US6046048A (en) * | 1996-01-09 | 2000-04-04 | Genetech, Inc. | Apo-2 ligand |
US6030945A (en) * | 1996-01-09 | 2000-02-29 | Genentech, Inc. | Apo-2 ligand |
FR2766713B1 (en) * | 1997-08-04 | 1999-09-24 | Bio Merieux | PROTEIN FACTOR ASSOCIATED WITH NEURO-DEGENERATIVE AND / OR AUTOIMMUNE AND / OR INFLAMMATORY DISEASE |
DE69939732D1 (en) | 1998-01-15 | 2008-11-27 | Genentech Inc | APO-2 LIGAND |
EP1941905A1 (en) | 1998-03-27 | 2008-07-09 | Genentech, Inc. | APO-2 Ligand-anti-her-2 antibody synergism |
WO2000047740A2 (en) * | 1999-02-12 | 2000-08-17 | Amgen Inc. | Tnf-related proteins |
AU3923000A (en) * | 1999-04-16 | 2000-11-02 | Amgen, Inc. | Agp-1 fusion protein compositions and methods |
EP1873244A3 (en) * | 1999-06-02 | 2008-04-02 | Genentech, Inc. | Methods and compositions for inhibiting neoplastic cell growth |
IL147029A0 (en) | 1999-06-28 | 2002-08-14 | Genentech Inc | Method for making apo-2 ligand using divalent metal ions |
ES2317924T3 (en) | 2000-07-27 | 2009-05-01 | Genentech, Inc. | SEQUENTIAL ADMINISTRATION OF CPT-11 AND APO-2L POLYPEPTIDE. |
RU2609871C1 (en) * | 2015-08-10 | 2017-02-06 | Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) | Antitumor agent |
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US4179337A (en) * | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
WO1997001633A1 (en) * | 1995-06-29 | 1997-01-16 | Immunex Corporation | Cytokine that induces apoptosis |
US6030945A (en) * | 1996-01-09 | 2000-02-29 | Genentech, Inc. | Apo-2 ligand |
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