WO2001029178A2 - Gene 1 de reponse primaire a l'erythropoietine (epo), gene eprg1 - Google Patents

Gene 1 de reponse primaire a l'erythropoietine (epo), gene eprg1 Download PDF

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WO2001029178A2
WO2001029178A2 PCT/US2000/029072 US0029072W WO0129178A2 WO 2001029178 A2 WO2001029178 A2 WO 2001029178A2 US 0029072 W US0029072 W US 0029072W WO 0129178 A2 WO0129178 A2 WO 0129178A2
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Prior art keywords
polypeptide
seq
identity
polynucleotide
eprgl
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PCT/US2000/029072
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WO2001029178A3 (fr
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Susan B. Dillon
Kenneth A. Lord
Andrew G. King
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Smithkline Beecham Corporation
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Priority to JP2001532164A priority Critical patent/JP2003520577A/ja
Priority to EP00975323A priority patent/EP1224220A2/fr
Publication of WO2001029178A2 publication Critical patent/WO2001029178A2/fr
Publication of WO2001029178A3 publication Critical patent/WO2001029178A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • 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
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agonists, antagonists and/or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides
  • EPRGl (SOCS-3) was isolated from a human hematopoietic cell line which requires EPO for proliferation
  • the expression of EPRG 1 is induced by EPO, and the presence of EPO is required for maintenance of its expression EPRGl is involved in the proliferation of EPO- dependent cells and may be important in the growth and development of erythroid and other hematopoietic lineages
  • Expression of EPRGl is induced to a high level in human bone marrow treated with G-CSF, and a substantial level of EPRGl is also expressed in fetal liver, peripheral blood leukocytes, lung, and in human bone marrow stimulated with EPO
  • expression of EPRGl is induced by EPO and by TPO in the presence of cycloheximide, indicative of a primary genetic response
  • expression of EPRGl is a direct result of activation of signalling pathways following binding of EPO or of TPO to their cognate receptors
  • the present invention relates to EPRGl, in particular EPRGl polypeptides and EPRGl polynucleotides, recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment or prophylaxis of cytopenias, including anemia, neutropenia, and thrombocytopenia
  • the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with EPRGl imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate EPRGl activity or levels.
  • the present invention relates to EPRGl polypeptides.
  • EPRGl polypeptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • polypeptides include those comprising the amino acid of SEQ ID NO:2.
  • peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • Such polypeptides include the polypeptide of SEQ ID NO:2.
  • Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO 1
  • EPRGl polypeptides of the present invention also include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO 4 over the entire length of SEQ ID NO 4
  • Such polypeptides include those composing the amino acid of SEQ ID NO 4
  • peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4
  • polypeptides include the polypeptide of SEQ ID NO 4
  • peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO 3
  • Polypeptides of the present invention are believed to be members of the SOCS family of polypeptides They are therefore of interest because genes in this family modulate activation of signal transduction pathways from cell growth and differentiation factors These properties are hereinafter referred to as "EPRGl activity” or “EPRGl polypeptide activity” or “biological activity of EPRGl " Also included amongst these activities are antigenic and immunogenic activities of said EPRGl polypeptides, in particular the antigenic and immunogenic activities of the polypeptide of SEQ ID NO 2 or 4
  • a polypeptide of the present invention exhibits at least one biological activity of EPRGl
  • polypeptides of the present invention may be in the form of the ' mature' protein or may be a part of a larger protein such as a fusion protein It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production
  • the present invention also includes include variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteristics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are va ⁇ ants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination
  • Polypeptides of the present invention can be prepared in any suitable manner Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood in the art
  • the present invention relates to EPRGl polynucleotides
  • polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of
  • polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred
  • polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO 1 encoding the polypeptide of SEQ ID NO 2
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO 2, over the entire coding region
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO 1 over the entire length of SEQ ID NO 1
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% ldentiy are more highly preferred, and those with at least 99% identity are most highly preferred
  • Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO 1 as well as the polynucleotide of SEQ ID NO 1
  • the invention also provides polynucleotides which are complementary to all the above described polynucleotides
  • the nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 28 to 705) encoding a polypeptide of 225 amino acids, the polypeptide of SEQ ID NO 2
  • the nucleotide sequence encoding the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO 1 or it may be a sequence other than the one contained in SEQ ID NO 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynu
  • the present invention also relates to partial or other polynucleotide and polypeptide sequences which were first identified prior to the determination of the corresponding full length sequences of SEQ ID NO 1 and SEQ ID NO 2
  • the present invention provides for an isolated polynucleotide
  • the present invention provides for an isolated polynucleotide
  • (a) comprises an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO 4 over the entire length of SEQ ID NO 4,
  • (b) has an amino acid sequence which is at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4,
  • (c) comprises the amino acid of SEQ ID NO 4, and
  • (d) is the polypeptide of SEQ ID NO 4, as well as polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO 3
  • the nucleotide sequence of SEQ ID NO 5 is derived from EST (Expressed Sequence
  • polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of human bone marrow and hematopoietic cells, using the expressed sequence tag (EST) analysis (Adams, M D , et al Science ( 1991 ) 252 1651 - 1656, Adams, M D et al , Nature, ( 1992) 355 632-634, Adams, M O , et al , Nature ( 1995) 377 Supp 3- 174) Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • polypeptide variants which comprise the amino acid sequence of SEQ ID NO:2 or 4 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in any combination.
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 , 3 or 5 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to SEQ ID NO: 1 , 3 or 5.
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent.
  • the probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 , 3 or 5, or a fragment thereof; and isolating full- length cDNA and genomic clones containing said polynucleotide sequence.
  • Such hybridization techniques are well known to the skilled artisan.
  • Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC ( 150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. lx SSC at about 65°C.
  • the present invention also includes polynucleotides obtainable by screening an approp ⁇ ate library under stingent hybridization conditions with a labeled probe having the sequence of SEQ ID NO 1 , 3 or 5, or a fragment thereof
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention Introduction of polynucleotides into host cells can be effected by methods desc ⁇ bed in many standard laboratory manuals, such as Davis et al , Basic Methods in Molecular Biology (1986) and Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Sp ⁇ ng Harbor Laboratory Press, Cold
  • expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e g , vectors derived from bacterial plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bactenophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used The appropriate nucle
  • a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested prior to use in the screening assay If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide If produced intracellularly, the cells must first be lysed before the polypeptide is recovered
  • Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification.
  • Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NO 1 , 3 or 5 which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered expression of the gene Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques
  • the present invention also contemplates a method of detecting the presence of or absence of variations in an EPRG 1 polynucleotide in an individual from that of SEQ ID NOS 1 , 3 or 5, comprising the step of comparing an EPRG l polynucleotide sequence contained in a sample obtained from the individual with that of SEQ ID NOS 1 , 3, or 5, respectively
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis RNA or cDNA may also be used in similar fashion
  • Deletions and insertions can be detected by a change in size of the amplified product in compa ⁇ son to the normal genotype
  • Point mutations can be identified by hybridizing amplified DNA to labeled EPRG 1 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures
  • DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (ee, e g , Myers et al , Science ( 1985) 230 1242) Sequence changes at specific locations may also be revealed by nuclease protection assays
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation in the EPRGl gene by the methods described
  • diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods
  • Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present invention relates to a diagonostic kit which comprises
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO 1 , 3 or 5, or a fragment thereof ,
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO 2 or 4, or a fragment thereof, or
  • kits may comprise a substantial component
  • cytopenias including anemia, neutropenia, and thrombocytopenia (including those caused by genetic factors, myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation), polycythemia, cancer, metabolic or genetic growth deficiencies, obesity, infection via immunostimulation, infectious disease or cancer via adjuvant enhancement of vaccine therapy, AIDS, drug-induced anemias, autoimmune diseases, such as rheumatoid arthritis, diabetes, multiple sclerosis, and inflammatory diseases, such as asthma and allergies, amongst others
  • the nucleotide sequences of the present invention are also valuable for chromosome identification
  • the sequence is specifically targeted to, and can hyb ⁇ dize with, a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease
  • genetic map data are found in, for example, V McKusick, Mendehan Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library)
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinhe ⁇ tance of physically adjacent genes)
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed in some or all of the affected individuals but not in any normal individuals
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressing them, can also be used as immunogens to produce antibodies lmmunospecific for polypeptides of the present invention
  • immunogens to produce antibodies lmmunospecific for polypeptides of the present invention
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art
  • Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hyb ⁇ doma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hyb ⁇ doma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hyb ⁇ doma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)
  • antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography
  • Antibodies against polypeptides of the present invention may also be employed to treat the Diseases, amongst others
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE) Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl , where fusion takes place at the hinge region
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases
  • a further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention
  • the vaccine formulation may further comprise a suitable carrier Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection)
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation instonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored
  • Polypeptides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned
  • Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of the polypeptide, or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 ( 1991 ))
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound
  • the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polpypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide Further the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measunng EPRGl activity in the mixture, and comparing the EPRGl activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and EPRGl polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify
  • the interaction of the SH2 domain with a phosphotyrosine peptide is highly specific for a given SH2 domain and its particular peptide substrate
  • the formation of this complex can be used as the basis to configure an in vitro high-throughput screen using ELISA or other suitable means for detection of the association
  • polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, '- ⁇ 1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptors, if any Standard methods for conducting such assays are well understood in the art
  • polypeptide antagonists examples include antibodies or, in some cases, o gonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented
  • antagonists include anti-sense ohgonucleotides (ASO) comp ⁇ sing the sequences of SEQ ID NOS 6 and 7
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for polypeptides of the present invention, or compounds which decrease or enhance the production of such polypeptides, which comprises (a) a polypeptide of the present invention, (b) a recombinant cell expressing a polypeptide of the present invention,
  • polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by
  • the present invention provides methods of treating or preventing abnormal conditions such as, for instance, cytopenias, including anemia, neutropenia, and thrombocytopenia (including those caused by genetic factors, myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation), polycythemia, cancer, metabolic or genetic growth deficiencies, obesity, infection via immunostimulation, infectious disease or cancer via adjuvant enhancement of vaccine therapy, AIDS, drug-induced anemias, autoimmune diseases, such as rheumatoid arthritis, diabetes, multiple sclerosis, and inflammatory diseases, such as asthma and allergies, related to either an excess of, or an under-expression of, EPRGl polypeptide activity
  • abnormal conditions such as, for instance, cytopenias, including anemia, neutropenia, and thrombocytopenia (including those caused by genetic factors, myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation), polycythemia, cancer, metabolic or genetic growth deficiencies, obesity, infection via immunostimulation, infectious disease or cancer via adj
  • an inhibitor compound as heieinabove described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc in competition with endogenous polypeptide may be administered Typical examples of such competitors include fragments of the EPRGl polypeptide
  • expression of the gene encoding endogenous EPRGl polypeptide can be inhibited using expression blocking techniques
  • antisense sequences such as those comprising the sequence of SEQ ID NO 6 or 7, either internally generated or separately administered
  • a gene therapy-based approach can be used to deliver an expression cassette for production of an anti-sense message directly in vivo
  • the anti-sense message can be optimized for effectiveness by size and location, and avoid toxicity issues associated with artificial ohgonucleotides
  • ohgonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res ( 1979) 6 3073, Cooney et al , Science ( 1988) 241 456, Der
  • EPRGl is a negative regulator that acts as a feedback inhibitor
  • ASO anti-sense ohgonucleotides
  • EPRG-1 inhibitor treated hematopoietic cells could be directly transplanted into a patient needing a stem cell transplant or cultured ex vivo in expansion medium containing one or more hematopoietic growth factors (such as G-CSF,
  • cytopenia condition may be genetic or due to myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation
  • In vivo administration of an inhibitor of EPRG- 1 may be effective alone or optimally in combination with cytokine therapy (l e G-CSF, PEG-rHuMGDF , EPO, TPO, IL-1 1 , IL-3, FLT-3, NESP, NEUPOGEN SD, etc )
  • the present invention relates to the EPRGl anti-sense ohgonucleotides
  • anti-sense ohgonucleotides preferably include chemically synthesized phosphorothioate linked ohgonucleotides which contain nucleotide sequences which effectively hybridize with SOCS-3 mRNA and prevent SOCS-3 protein production, and any other ohgonucleotides which can hybridize to SOCS-3 mRNA that have other backbone modifications made to confer nuclease resistance
  • Examples of such chemically synthesized phosphorothioate linked ohgonucleotides are the sequences comprising the sequence of SEQ ID NO 6 or 7,
  • a method for increasing the effectiveness and engraftment of hematopoietic stem cell transplantation by treating human hematopoietic progenitor / stem cells with an inhibitor of EPRG l comprising
  • the instant invention relates to a method for increasing the efficiency of gene therapy transfer into hematopoietic stem cells comprising (a) isolating hematopoietic stem cells or modified stem cell lines and incubating with EPRGl inhibitor before transfection with plasmid vectors containing any corrective gene of interest useful in treating a hereditary genetic diseases (e g ADA deficiency gene therapy)
  • a hereditary genetic diseases e g ADA deficiency gene therapy
  • a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest
  • producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo
  • Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced into the subject
  • polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology This is most easily facilitated by storing the sequence in a computer readable medium and then using the stored data to search a sequence database using well known searching tools, such as GCG Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO 1 , 3 or 5 and/or a polypeptide sequence encoded thereby
  • Antibodies includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library
  • “Isolated” means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or,
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, I e , peptide isosteres
  • Polypeptide refers to both short chains, commonly referred to as peptides, ohgopeptides or oligomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching Cyclic, branched and
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1 , wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence,
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO 1
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 8 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution,
  • n a is the number of amino acid alterations
  • x a is the total number of ammo acids in SEQ ID NO 2
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may include up to a certain integer number of am o acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof
  • EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262]
  • EPRGl is observed as a single 2 4 kb message EPRGl can be detected at low levels in log phase cells proliferating in medium supplemented with 10% FBS and rh-EPO Upon overnight starvation for EPO, message levels decay to undetectable levels EPRGl is rapidly induced upon EPO stimulation, being readily detectable at 1/2 hour, with the maximum level achieved at about 1 hour.
  • EPRGl is induced by EPO in the presence of the protein synthesis inhibitor, cycloheximide, even though there is no induction of EPRG l when cycloheximide alone is added
  • the treatment with EPO+cycloheximide leads to modest supe ⁇ nduction of EPRGl message levels over that obtained with EPO by itself
  • EPRGl is also induced by TPO in human megakaryoblastic leukemia cell lines, M07e and CMK, as well as being superinduced when stimulated in the presence of cycloheximide
  • EPRGl is also expressed as a single 2 4 kb message on northern blots It is induced in in vitro culture by treatment with EPO, but expression is much more strongly induced by stimulation with G-CSF The elevated expression of EPRG l is sustained by the cells in culture for at least 7 days, showing that EPRG l likely serves an important function in the development and maturation of different hematopoietic lineages
  • EPRGl is also expressed in spleen and thymus to a lesser extent
  • the 3 -UTR segment of the RNA transcript should be useful for diagnostic purposes because 3'-UTRs are unique to a gene and sequence specific In addition, the 3'-UTR may be valuable in the development of screening assays to identify agents which modulate RNA stability and turnover rate Decreasing the message's steady state level would decrease the amount of translated protein in the cell, antagonizing the protein's action
  • members of the SOCS family are negative regulators of growth factor signal transduction
  • a SOCS antagonist would function as an agonist of the growth factor
  • One way of achieving this result would be through antisense technology in order to decrease or eliminate the EPRGl message
  • a second means would involve modulating the cellular mechanisms governing turnover of the EPRGl message
  • EPRGl anti-sense ohgonucleotides The utility of EPRGl anti-sense ohgonucleotides is exemplified by the following example Incubation of human bone marrow cells with anti-sense orientation ohgnucleotides of SEQ ID NO 6 and SEQ ID NO 7, but not with sense orientation ohgonucleotides of SEQ ID NO 8 for several hours in serum-free medium results in enhanced responsiveness of bone marrow progenitor / stem cells to optimal and suboptimal concentrations of colony stimulating factors such as G-CSF and EPO As an example, addition of G-CSF at 10 ng/ml resulted in approx 40 colonies / well (50 000 bone marrow cells/well) Marrow cells incubated with EPRGl sense ohgonucleotide of SEQ ID NO 8 resulted in an average of approx 41 colonies / well In contrast bone marrow cells incubated with EPRGl anti-sense ohgonucle

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Abstract

Cette invention, qui a trait à des polypeptides ainsi qu'à des polynucléotides EPRG1, concerne également des procédés permettant de produire ces polypeptides par des techniques de recombinaison. L'invention porte, de plus, sur des méthodes d'utilisation des polypeptides et des polynucléotides EPRG1 en thérapeutique ainsi que sur des méthodes diagnostiques liées à ceux-ci.
PCT/US2000/029072 1999-10-21 2000-10-19 Gene 1 de reponse primaire a l'erythropoietine (epo), gene eprg1 WO2001029178A2 (fr)

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JP2001532164A JP2003520577A (ja) 1999-10-21 2000-10-19 Ｅｐｏ一次応答遺伝子１、ｅｐｒｇ１
EP00975323A EP1224220A2 (fr) 1999-10-21 2000-10-19 Gene 1 de reponse primaire a l'erythropoietine (epo), gene eprg1

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003072778A1 (fr) * 2002-02-27 2003-09-04 Genox Research, Inc. Methode pour etudier une maladie allergique
EP1734982A2 (fr) * 2004-03-04 2006-12-27 Vanderbilt University Polypeptides socs pour inhiber la signalisation induite par la cytokine
US8324148B2 (en) 1994-06-13 2012-12-04 Vanderbilt University Compositions for importing biologically active molecules into cells

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0877030A2 (fr) * 1997-05-07 1998-11-11 Smithkline Beecham Corporation Gène en réponse primaire à l'EPO 1 (EPRG1)
WO1999003993A2 (fr) * 1997-07-18 1999-01-28 Schering Corporation Suppresseurs de signalement de cytokine et reactifs correspondants
WO1999023220A1 (fr) * 1997-11-03 1999-05-14 Incyte Pharmaceuticals, Inc. Suppresseur de la signalisation cytokinaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0877030A2 (fr) * 1997-05-07 1998-11-11 Smithkline Beecham Corporation Gène en réponse primaire à l'EPO 1 (EPRG1)
WO1999003993A2 (fr) * 1997-07-18 1999-01-28 Schering Corporation Suppresseurs de signalement de cytokine et reactifs correspondants
WO1999023220A1 (fr) * 1997-11-03 1999-05-14 Incyte Pharmaceuticals, Inc. Suppresseur de la signalisation cytokinaire

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8324148B2 (en) 1994-06-13 2012-12-04 Vanderbilt University Compositions for importing biologically active molecules into cells
WO2003072778A1 (fr) * 2002-02-27 2003-09-04 Genox Research, Inc. Methode pour etudier une maladie allergique
EP1734982A2 (fr) * 2004-03-04 2006-12-27 Vanderbilt University Polypeptides socs pour inhiber la signalisation induite par la cytokine
EP1734982A4 (fr) * 2004-03-04 2009-08-05 Univ Vanderbilt Polypeptides socs pour inhiber la signalisation induite par la cytokine
US8420096B2 (en) 2004-03-04 2013-04-16 Vanderbilt University Cell-penetrating SOCS polypeptides that inhibit cytokine-induced signaling

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