WO1999003993A2 - Suppresseurs de signalement de cytokine et reactifs correspondants - Google Patents

Suppresseurs de signalement de cytokine et reactifs correspondants Download PDF

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Publication number
WO1999003993A2
WO1999003993A2 PCT/US1998/014544 US9814544W WO9903993A2 WO 1999003993 A2 WO1999003993 A2 WO 1999003993A2 US 9814544 W US9814544 W US 9814544W WO 9903993 A2 WO9903993 A2 WO 9903993A2
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protein
socs
wds
proteins
seq
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PCT/US1998/014544
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English (en)
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WO1999003993A3 (fr
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James A. Johnson
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Schering Corporation
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Publication of WO1999003993A3 publication Critical patent/WO1999003993A3/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention pertains to compositions related to proteins which function, e.g., in suppressing intracellular signaling pathways, e.g., cytokine signaling.
  • it provides purified genes, proteins, antibodies, and related reagents useful, e.g., to regulate growth hormone-like or cytokine-regulated intracellular processes, including transcription or genes in various cell types, including immune cells.
  • Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment.
  • the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product.
  • cDNA complementary DNA
  • mRNA messenger RNA
  • the carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
  • Lymphokines apparently mediate cellular activities in a variety of ways . They have been shown to support the proliferation, growth, and differentiation of, e.g., pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. Proper and balanced interactions between cellular components are necessary for a healthy developmental or immune response. The different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents.
  • the present invention is based in part upon the discovery of intracellular regulatory molecules which can block signal transduction, e.g., through growth factor- or cytokine-receptor superfamily signaling mechanisms . These proteins exhibit a structural feature designated a SOCS box. See Hilton, et al. (1998) Proc . Nat ' 1 Acad. Sci. USA 95:114-119. Moreover, the SOCS3 protein can block the IL-2 induced signaling via the STAT5, establishing function of the SOCS proteins as suppressors of cytokine signaling.
  • the invention provides a substantially pure or recombinant S0CS14 protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 2 or 6; a natural sequence S0CS14 of SEQ ID NO: 2 or 6; a fusion protein comprising S0CS14 sequence; a substantially pure or recombinant S0CS15 (also designated WDS11) protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 4 or 8; a natural sequence S0CS15 (WDSll) of SEQ ID NO: 4 or 8; a fusion protein comprising S0CS15 (WDSll) sequence; a substantially pure or recombinant S0CS17 protein or peptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 10; a natural sequence SOCS17 of SEQ ID NO: 10; a fusion protein comprising S0CS17 sequence; a substantially pure or recombinant S0CS18 protein or
  • the portion is at least about 25 amino acids.
  • SOCS14 comprises a mature sequence of SEQ ID NO: 2 or 6;
  • SOCS15 (WDSll) comprises a mature sequence of SEQ ID NO: 4 or 8;
  • S0CS17 comprises a mature sequence of SEQ ID NO: 10;
  • SOCS18 comprises a mature sequence of SEQ ID NO: 12;
  • SOCS19 comprises a mature sequence of SEQ ID NO.
  • WDS12 comprises a mature sequence of SEQ ID NO: 16; protein or peptide: is from a warm blooded animal selected from a mammal, including a primate; comprises at least one polypeptide segment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16; exhibits a plurality of portions exhibiting the identity; is a natural allelic variant of SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12 ; has a length at least about 30 amino acids; exhibits at least two non- overlapping epitopes which are specific for a mammalian SOCS14, S0CS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12 ; exhibits identity over a length of at least about 20 amino acids to SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12; exhibits at least two non-overlapping epitopes which are specific for a SOCS14, SOCS
  • compositions comprising: a sterile SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12 protein or peptide; the SOCS14, SOCS15 (WDSll), SOCS17, S0CS18, SOCS19, or WDS12 protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
  • the invention further provides a fusion protein, comprising: mature protein comprising sequence of SEQ ID NO: 2, 6, 4, 8, 10, 12, 14 or 16; a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another SOCS or WDS protein.
  • reagents also make available a kit comprising such a protein or polypeptide, and: a compartment comprising the protein or polypeptide; and/or instructions for use or disposal of reagents in the kit.
  • the invention further provides a binding compound comprising an antigen binding portion from an antibody, which specifically binds to a natural SOCS14, SOCS15 (WDSll), S0CS17, S0CS18, SOCS19, or WDS12 protein, wherein: the protein is a primate protein; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to another chemical moiety; or the antibody: is raised against a peptide sequence of a mature polypeptide comprising sequence of SEQ ID NO: 2, 6, 4, 8, 10, 12, 14 or 16; is raised against a mature SOCS14, SOCS15 (WDSll), SOCS17 , SOCS18, SOCS19, or WDS12; is raised to a purified SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12; is immunoselected; is a polyclonal antibody; binds to a denatured S0CS14, SOCS15 (WDSll), SO
  • compositions comprising: a sterile binding compound, or the binding compound and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
  • the present invention further provides an isolated or recombinant nucleic acid encoding a protein or peptide or fusion protein described above, wherein: the SOCS or WDS family protein is from a mammal, including a primate; or the nucleic acid: encodes an antigenic peptide sequence of SEQ ID NO: 2, 6, 4, 8, 10, 12, 14 or 16; encodes a plurality of antigenic peptide sequences of SEQ ID NO: 2, 6, 4, 8, 10, 12, 14 or 16; exhibits identity to a natural cDNA encoding the segment; is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a mammal, including a primate; comprises a natural full length coding sequence; is a hybridization probe for a gene encoding the SOCS or WDS family protein; or is a PCR primer, PCR product, or mutagenesis primer.
  • the invention provides a cell or tissue comprising such a recombinant nucleic acid.
  • Preferred cells include: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell.
  • kit embodiments include a kit comprising the described nucleic acid, and: a compartment comprising the nucleic acid; a compartment further comprising a S0CS14, SOCS15 (WDSll), SOCS17, SOCS18, S0CS19, or WDS12 protein or polypeptide; and/or instructions for use or disposal of reagents in the kit.
  • the kit is capable of making a qualitative or quantitative analysis .
  • nucleic acid embodiments include those which: hybridize under wash conditions of 50° C and less than 500 mM salt to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15; exhibits identity over a stretch of at least about 30 nucleotides to a SOCS14, S0CS15 (WDSll), SOCS17 , SOCS18, SOCS19 , or WDS12.
  • the wash conditions are at 55° C and/or 300 mM salt; 60° C and/or 150 mM salt; the identity is over a stretch is at least 55 or 75 nucleotides.
  • the invention provides a method of modulating physiology or development of a cell or tissue culture cells comprising introducing into such cell an agonist or antagonist of a SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12.
  • a polynucleotide includes one or more different polynucleotides
  • reference to “a composition” includes one or more of such compositions
  • reference to “a method” include reference to equivalent steps and methods known to a person of ordinary skill in the art, and so forth.
  • cytokines The proliferation, differentiation, and physiological responses of many cell lineages are regulated by secreted proteins, e.g., cytokines . These molecules often exert their biological effects through binding to cell surface receptors that are associated with one or more members of the Janus Kinase (Jak) family of cytoplasmic tyrosine kinases.
  • JAK Janus Kinase
  • cytoplasmic tyrosine kinases members of the Janus Kinase family of cytoplasmic tyrosine kinases.
  • JAK Janus Kinase
  • the JAK and STAT proteins are enzymes which act to transduce a signal from the cell surface to the nucleus, thereby serving as the pathway to signal the cell to respond physiologically to an external signal. These pathways have been shown to involve certain protein phosphorylation or dephosphorylation steps, thereby leading to response or lack of response by the cell. See, e.g., Ihle (1996) Cell 84:331-334; Ivashkiv (1995) Immunity 3:1-4; Ihle, et al. (1995) Ann. Rev. Immunol. 13:369-398; Ihle and Kerr (1995) Trends in Genetics 11:69-74; and Darnell, et al . (1994) Science 264:1415-1421.
  • a primate e.g., human, SOCS14 cDNA fragment and corresponding open reading frame are provided in (SEQ ID NO: 1 and 2) .
  • the translation exhibits significant matching and similarity to other identified SOCS family members .
  • the internal stop codon indicates some errors in the sequence at or near those positions.
  • Additional refined sequence of primate, e.g., human, SOCS14 is provided in SEQ ID NO: 5 and 6.
  • a rodent, e.g., mouse, SOCS15 cDNA fragment and corresponding open reading frame are provided in SEQ ID NO: 3 and 4.
  • the translation exhibits significant matching and similarity to other identified SOCS family members .
  • the internal stop codon indicates some errors in the sequence at or near those positions .
  • a rodent e.g., murine SOCS17 CDNA and corresponding open reading frame are provided in SEQ ID NO: 9 and 10.
  • Nucleotide may be A, C, T, or G at positions: 1680, 1691, 1696, 1704, 1707, 1728, 1740, 1743, 1746, 1755, 1760, 1770, 1773, 1802, 1816, 1817, 1823, 1826, 1827, 1846, 1851, 1857, 1861, 1880, 1885, 1909, 1917, 1920, 1929, 1946, 1953, 1967, 1968, 1980, 1991, 1995, 2001, 2004, 2021, 2033, 2034, 2035, 2036, 2037, 2039, 2040, 2042, 2048, 2051, 2054, 2061, 2075, 2081, 2083, 2084, 2085, 2088, 2105, 2121, 2124, 2132, 2137, 2147, 2149, 2151, 2152, 2160, 2165, 2177, 2179 and 2196; nucleotide may be A or C at position 494; nucleotide may be C or T at positions: 498, 501, 1455,
  • a primate e.g., human, SOCS18 nucleotide and corresponding amino acid sequence are provided in SEQ ID NO: 11 and 12.
  • Nucleotide may be A or C at positions: 740, 797, 2139, and 2184; nucleotide may be G or T at positions: 761, 1313, 1508, and 2226; nucleotide may be C or T at positions 746, 1460, 1499, 2009, 2010, 2199, and 2225; nucleotide may be A or G at positions 788, 863, 1550, 2178, 2188, 2197, and 2211; nucleotide may be G or C at positions: 1163, and 1544; nucleotide may be A or T at positions 2058, and 2128; and nucleotide may be A, C, T, or G at position 2251 (see SEQ ID NO: 27) .
  • a primate e.g., human, SOCS19 nucleotide and corresponding amino acid sequence are provided in SEQ ID NO: 13 and 14.
  • Nucleotide may be A, C, T, or G at positions: 2078, and 2116; and nucleotide may be G or C at position 2063 (see SEQ ID NO: 28) .
  • nucleotide may be A, C, T, or G at positions: 108, and 109; nucleotide may be A or G at positions: 236, 238, and 1258; nucleotide may be G or T at position 233; nucleotide may be G or C at position 234; nucleotide may be C or T at position 237; and nucleotide may be A or T at position 239 (see SEQ ID NO: 29) .
  • SOCS proteins are a family of proteins ranging from approximately 30-60 Kd which inhibit JAK kinase activity.
  • the amino portion of SOCS proteins contain an SH2 binding motif and the carboxy portion of the molecule contains a SOCS box motif which may play a role in dimerization of SOCS proteins .
  • the WDS are closely related in sequence .
  • SOCS3 expression is induced by IL-2 and can be detected by approximately 1 hour after IL-2 activation. Subsequently, SOCS expression is decreased relatively rapidly (e.g., approximately 8 hrs after activation).
  • Western blots show that SOCS3 interacts with IL-2 receptor and JAKl following IL-2 stimulation.
  • binding composition refers to molecules that bind with specificity to SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12 protein, e.g., in an antibody-antigen interaction.
  • other compounds e.g., binding proteins, may also specifically associate with SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12 proteins in contrast to other molecules .
  • the association will be in a natural physiologically relevant protein-protein interaction, either covalent or non-covalent, and may include members of a multiprotein complex, including carrier compounds or dimerization partners.
  • the molecule may be a polymer, or chemical reagent.
  • a functional analog may be a protein with structural modifications, or may be a wholly unrelated molecule, e.g., which has a molecular shape which interacts with the appropriate protein binding determinants .
  • the proteins may serve as agonists or antagonists of the binding partner, see, e.g., Goodman, et al. (eds.) (1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed. ) Pergamon Press, Tarrytown, N.Y.
  • binding agent SOCS or :WDS protein complex
  • binding agent refers to a complex of a binding agent and a SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12 protein that is formed by specific binding of the binding agent to the respective SOCS14, SOCS15 (WDSll), SOCS17, S0CS18, SOCS19, or WDS12 protein.
  • Specific binding of the binding agent means that the binding agent has a specific binding site that recognizes a site on the SOCS14, SOCS15 (WDSll), SOCS17 , SOCS18, SOCS19, or WDS12 protein.
  • antibodies raised to a SOCS14, SOCS15 (WDSll), SOCS17 , SOCS18, SOCS19, or WDS12 protein and recognizing an epitope on the SOCS or WDS protein are capable of forming a binding agent: SOCS or :WDS protein complex by specific binding.
  • a binding agent: SOCS or :WDS protein complex allows the measurement of SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12 protein in a mixture of other proteins and biologies.
  • the term "antibody: SOCS or :WDS protein complex" refers to an embodiment in which the binding agent, e.g., is an antibody.
  • the antibody may be monoclonal, polyclonal, or a binding fragment of an antibody, e.g., an Fv, Fab, or F(ab)2 fragment.
  • the antibody will preferably be a polyclonal antibody for cross-reactivity purposes.
  • "Homologous" nucleic acid sequences when compared, exhibit significant similarity, or identity.
  • the standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison and/or phylogenetic relationship, or based upon hybridization conditions . Hybridization conditions are described in greater detail below.
  • nucleic acid is a nucleic acid, e.g., an RNA, DNA, cDNA, genomic DNA, or a mixed polymer, which is substantially separated from other biologic components which naturally accompany a native sequence, e.g., proteins and flanking genomic sequences from the originating species .
  • the term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs, or analogs biologically synthesized by heterologous systems. Further, the term includes double-stranded or single- stranded embodiments. Where single-stranded, the nucleic acid may be either the "sense" or the "antisense" strand.
  • a substantially pure molecule includes isolated forms of the molecule.
  • An isolated nucleic acid will usually contain homogeneous nucleic acid molecules, but will, in some embodiments, contain nucleic acids with minor sequence heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
  • SOCS SOCS
  • protein or “polypeptide” is meant any chain of amino acids, regardless of length or postranslation modification (e.g., glycosylation or phosphorylation).
  • the term encompasses polypeptides which are pre- or pro-proteins.
  • the invention also embraces a polypeptide which exhibits similar structure to S0CS14, SOCS15 (WDSll), SOCS17, S0CS18, SOCS19, or WDS12 protein, e.g., which interacts with SOCS or WDS protein specific binding components.
  • binding components e.g., antibodies
  • polypeptide or "protein” as used herein includes a significant fragment or segment of a SOCS or WDS protein, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least 10 amino acids, more generally at least 12 amino acids, often at least 14 amino acids, more often at least 16 amino acids, typically at least 18 amino acids, more typically at least 20 amino acids, usually at least 22 amino acids, more usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 28 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids, e.g., 35, 40, 45, 50, 60, 70, 80, etc.
  • the invention encompasses proteins comprising a plurality of distinct, e.g., nonoverlapping, segments of the specified length.
  • the plurality will be at least two, more usually at least three, and preferably 5, 7, or even more. While the length minima are provided, longer lengths, of various sizes, may be appropriate, e.g., one of length 7 , and two of length 12.
  • Features of one of the different genes should not be taken to limit those of another of the genes .
  • a "recombinant" nucleic acid is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by . generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants.
  • products made by transforming cells with any non-naturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oligonucleotide process .
  • Such is o ten done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site.
  • it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms.
  • Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • a similar concept is intended for a recombinant, e.g., fusion, polypeptide.
  • synthetic nucleic acids which, by genetic code redundancy, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various different species variants.
  • Solubility is reflected by sedimentation measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule under particular conditions .
  • the determination of the sedimentation velocity was classically performed in an analytical ultracentrifuge, but is typically now performed in a standard ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d ed. ) W.H. Freeman & Co., San Francisco, CA; and Cantor and Schimmel (1980) Biophysical Chemistry parts 1-3, W.H. Freeman & Co., San Francisco, CA.
  • a sample containing a putatively soluble polypeptide is spun in a standard full sized ultracentrifuge at about 50K rpm for about 10 minutes, and soluble molecules will remain in the supernatant.
  • a soluble particle or polypeptide will typically be less than about 30S, more typically less than about 15S, usually less than about 10S, more usually less than about 6S, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3S. Solubility of a polypeptide or fragment depends upon the environment and the polypeptide. Many parameters affect polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent.
  • the temperature at which the polypeptide is used ranges from about 4° C to about 65° C. Usually the temperature at use is greater than about 18° C and more usually greater than about 22° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37° C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.
  • the size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state.
  • the polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubility, or associated with lipids or detergents in a manner which approximates natural lipid bilayer interactions.
  • the solvent will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological solvent.
  • the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5.
  • a detergent will be added, typically a mild non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3- [3- cholamidopropyl) -dimethylammonio] -1-propane sulfonate) , or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein.
  • Substantially pure in a protein context typically means that the protein is isolated from other contaminating proteins, nucleic acids, and other biologicals derived from the original source organism. Purity, or “isolation” may be assayed by standard methods, and will ordinarily be at least about 50% pure, more ordinarily at least about 60% pure, generally at least about 70% pure, more generally at least about 80% pure, often at least about 85% pure, more often at least about 90% pure, preferably at least about 95% pure, more preferably at least about 98% pure, and in most preferred embodiments, at least 99% pure. Similar concepts apply, e.g., to antibodies or nucleic acids.
  • “Substantial similarity" in the nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least 56%, more generally at least 59%, ordinarily at least 62%, more ordinarily at least 65%, often at least 68%, more often at least 71%, typically at least 74%, more typically at least 77%, usually at least 80%, more usually at least about 85%, preferably at least about 90%, more preferably at least about 95 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides .
  • substantial similarity exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence derived from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15.
  • selective hybridization will occur when there is at least about 55% similarity over a stretch of at least about 30 nucleotides, preferably at least about 65% over a stretch of at least about 25 nucleotides, more preferably at least about 75%, and most preferably at least about 90% over about 20 nucleotides. See Kanehisa (1984) Nuc. Acids Res. 12:203-213.
  • the length of similarity comparison may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 40 nucleotides, preferably at least about 50 nucleotides, and more preferably at least about 75 to 100 or more nucleotides, e.g., 150, 200, etc.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the designated program parameters.
  • Optical alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. APPI . Math. 2:482, by the homology alignment algorithm of Needlman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc . Nat ' 1 Acad. Sci . USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr. , Madison, WI) , or by visual inspection (see generally Ausubel et al., supra).
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment.
  • PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol . 35:351-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-153.
  • the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
  • This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
  • Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology
  • HSPs high scoring sequence pairs
  • T some positive-valued threshold score
  • Altschul, et al . , supra These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment, score can be increased.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5787) .
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a further indication that two nucleic acid sequences of polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions .
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
  • “Stringent conditions”, in referring to homology or substantial similarity in -the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters, typically those controlled in hybridization reactions . The combination of parameters is more important than the measure of any single parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.
  • a nucleic acid probe which binds to a target nucleic acid under stringent conditions is specific for said target nucleic acid.
  • Hybridization under stringent conditions should give a background of at least 2-fold over background, preferably at least 3-5 or more.
  • Such a probe is typically more than 11 nucleotides in length, and is sufficiently identical or complementary to a target nucleic acid over the region specified by the sequence of the probe to bind the target under stringent hybridization conditions.
  • SOCS14, SOCS15 (WDSll), SOCS17, SOCS18, SOCS19, or WDS12 protein from other mammalian species can be cloned and isolated by cross-species hybridization of closely related species. See, e.g., below. Similarity may be relatively low between distantly related species, and thus hybridization of relatively closely related species is advisable. Alternatively, preparation of an antibody preparation which exhibits less species specificity may be useful in expression cloning approaches .
  • the specified antibodies bind to a particular protein and do not significantly bind other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised to the protein immunogen with the amino acid sequence depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 can be selected to obtain antibodies specifically immunoreactive with SOCS or WDS proteins and not with other proteins . These antibodies recognize proteins highly similar to the homologous SOCS or WDS protein.
  • Primate or rodent SOCS or WDS protein is each exemplary of a larger class of structurally and . functionally related proteins . These soluble proteins will serve to transmit signals between different cell types.
  • the preferred embodiments, as disclosed, will be useful in standard procedures to isolate genes from different individuals or other species, e.g., warm blooded animals, such as birds and mammals.
  • Cross hybridization will allow isolation of related genes encoding proteins from individuals, strains, or species.
  • a number of different approaches are available to successfully isolate a suitable nucleic acid clone based upon the information provided herein. Southern blot hybridization studies can qualitatively determine the presence of homologous genes in human, monkey, rat, mouse, dog, cow, and rabbit genomes under specific hybridization conditions.
  • Complementary sequences will also be used as probes or primers . Based upon identification of the likely amino terminus, other peptides should be particularly useful, e.g., coupled with anchored vector or poly-A complementary PCR techniques or with complementary DNA of other peptides.
  • DNA is isolated from a genomic or cDNA library using labeled oligonucleotide probes having sequences identical or complementary to the sequences disclosed herein. Full- length probes may be used, or oligonucleotide probes may be generated by comparison of the sequences disclosed.
  • probes can be used directly in hybridization assays to isolate DNA encoding SOCS or WDS proteins, or .probes can be designed for use in amplification techniques such as PCR, for the isolation of DNA encoding SOCS or WDS proteins .
  • cDNA is prepared from cells which expresses a SOCS or WDS protein.
  • cDNA is prepared from the mRNA and ligated into a recombinant vector.
  • the vector is transfected into a recombinant host for propagation, screening, and cloning. Methods for making and screening cDNA libraries are well known. See Gubler and Hoffman (1983) Gene 25:263-269 and Sambrook, et al.
  • the DNA can be extracted from tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb. The fragments are then separated by gradient centrifugation and cloned in bacteriophage lambda vectors . These vectors and phage are packaged in vitro, as described in Sambrook, et al. Recombinant phage are analyzed by plaque hybridization as described in Benton and Davis (1977) Science 196:180-182. Colony hybridization is carried out as generally described in e.g., Grunstein, et al. (1975) Proc. Natl. Acad. Sci. USA. 72:3961-3965.
  • DNA encoding a SOCS14 or SOCS15 protein can be identified in either cDNA or genomic libraries by its ability to hybridize with the nucleic acid probes described herein, e.g., in colony or plaque hybridization assays .
  • the corresponding DNA regions are isolated by standard methods familiar to those of skill in the art. See, e.g., Sambrook, et al.
  • Various methods of amplifying target sequences such as the polymerase chain reaction, can also be used to prepare DNA encoding SOCS or WDS proteins .
  • Polymerase chain reaction (PCR) technology is used to amplify such nucleic acid sequences directly from mRNA, from cDNA, and from genomic libraries or cDNA libraries.
  • the isolated sequences encoding SOCS or WDS proteins may also be used as templates for PCR amplification.
  • oligonucleotide primers complementary to two 5 ' regions in the DNA region to be amplified are synthesized. The polymerase chain reaction is then carried out using the two primers . See Innis, et al. (eds.) (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA. Primers can be selected to amplify the entire regions encoding a full-length SOCS or WDS protein or to amplify smaller DNA segments as desired. Once such regions are PCR-amplified, they can be sequenced and oligonucleotide probes can be prepared from sequence obtained using standard techniques . These probes can then be used to isolate DNA's encoding SOCS or WDS proteins.
  • Oligonucleotides for use as probes are usually chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Carruthers (1983) Tetrahedron Lett. 22 (20) :1859-1862, or using an automated synthesizer, as described in Needham-VanDevanter, et al. (1984) Nucleic Acids Res. 12:6159-6168. Purification of oligonucleotides is performed e.g., by native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom. 255:137-149.
  • the sequence of the synthetic oligonucleotide can be verified using, e.g., the chemical degradation method of Maxam, A.M. and Gilbert, W. in Grossman, L. and Moldave (eds.) (1980) Methods in Enzvmology 65:499-560 Academic Press, New York.
  • This invention provides isolated DNA or fragments to encode a SOCS or WDS protein.
  • this invention provides isolated or recombinant DNA which encodes a protein or polypeptide which is capable of hybridizing under appropriate conditions, e.g., high stringency, with the DNA sequences described herein.
  • Said biologically active protein or polypeptide can be an intact protein, or fragment, and have an amino acid sequence as disclosed in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, particularly natural embodiments. Preferred embodiments will be full length natural sequences.
  • this invention contemplates the use of isolated or recombinant DNA, or fragments thereof, which encode proteins which are homologous to a SOCS or WDS protein or which were isolated using cDNA encoding a SOCS or WDS protein as a probe.
  • the isolated DNA can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others. Also embraced are methods for making expression vectors with these sequences, or for making, e.g., expressing and purifying, protein products.
  • a DNA which codes for a SOCS or WDS protein will be particularly useful to identify genes, mRNA, and cDNA species which code for related or similar proteins, as well as DNAs which code for homologous proteins from different species. There are likely homologs in other species, including primates, rodents, canines, felines, and birds. Various SOCS or WDS proteins should be homologous and are encompassed herein. However, even proteins that have a more distant evolutionary relationship to the antigen can readily be isolated under appropriate conditions using these sequences if they are sufficiently homologous. Primate SOCS or WDS proteins are of particular interest.
  • Recombinant clones derived from the genomic sequences will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow
  • Antibodies can be raised to various SOCS14 or SOCS15 proteins, including individual, polymorphic, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in their recombinant forms. Additionally, antibodies can be raised to SOCS or WDS proteins in either their active forms or in their inactive forms . Anti-idiotypic antibodies may also be used.
  • a number of immunogens may be used to produce antibodies specifically reactive with SOCS or WDS proteins .
  • Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies .
  • Naturally occurring protein may also be used either in pure or impure form.
  • Synthetic peptides, made using the human SOCS14 or SOCS15 protein sequences described herein, may also used as an immunogen for the production of antibodies to SOCS14 or SOCS15 proteins .
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described herein, and purified as described.
  • Naturally folded or denatured material can be used, as appropriate, for producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.
  • an immunogen preferably a purified protein
  • animals are immunized with the mixture.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the SOCS or WDS protein of interest.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired. See, e.g., Harlow and Lane; or Coligan.
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art.
  • spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein (1976) Eur . J. Immunol . 6:511-519, incorporated herein by reference).
  • Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art.
  • Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
  • Antibodies, including binding fragments and single chain versions, against predetermined fragments of SOCS or WDS protein can be raised by immunization of animals with conjugates of the fragments with carrier proteins as described above.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective SOCS or WDS proteins, or screened for agonistic or antagonistic activity, e.g., effect on cell cycle progression or transcription of specific genes.
  • These monoclonal antibodies will usually bind with at least a KJJ of about 1 mM, more usually at least about 300 ⁇ M, typically at least about 10 ⁇ M, more typically at least about 30 ⁇ M, preferably at least about 10 ⁇ M, and more preferably at least about 3 ⁇ M or better.
  • monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th ed.
  • the population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen.
  • the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
  • the polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies . Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non- covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.
  • Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Patent No. 4,816,567; and Queen, et al . (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.
  • the antibodies of this invention are useful for affinity chromatography in isolating SOCS or WDS protein.
  • Columns can be prepared where the antibodies are linked to a solid support, e.g., particles, such as agarose, SEPHADEX, or the like, where a cell lysate or supernatant may be passed through the column, the column washed, followed by increasing concentrations of a mild denaturant, whereby purified SOCS or WDS protein will be released.
  • the antibodies may also be used to screen expression libraries for particular expression products .
  • the antibodies used in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
  • Antibodies to SOCS or WDS proteins may be used for the identification of cell populations expressing the proteins. By assaying, e.g., by histology or otherwise, probably a disruptive assay which kills that sample of cells, the expression products of cells expressing SOCS or WDS proteins it is possible to diagnose disease, e.g., cancerous conditions.
  • Antibodies raised against each SOCS or WDS protein will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens.
  • a particular protein can be measured by a variety of immunoassay methods .
  • immunoassay methods for a review of immunological and immunoassay procedures in general, see Stites and Terr (eds.) (1991) Basic and Clinical Immunology (7th ed.).
  • the immunoassays of the present invention can be performed in many configurations, which are reviewed extensively in Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Florida; Tijan (1985) "Practice and Theory of Enzyme Immunoassays , " Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V. , Amsterdam; and Harlow and Lane Antibodies, A Laboratory Manual, supra, each of which is incorporated herein by reference.
  • Immunoassays for measurement of SOCS or WDS proteins can be performed by a variety of methods known to those skilled in the art.
  • immunoassays to measure the protein can be either competitive or noncompetitive binding assays.
  • the sample to be analyzed competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface.
  • the capture agent is an antibody specifically reactive with SOCS or WDS proteins produced as described above.
  • the concentration of labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample .
  • the SOCS or WDS protein present in the sample competes with labeled protein for binding to a specific binding agent, for example, an antibody specifically reactive with the SOCS or WDS protein.
  • the binding agent may be bound to a solid surface to effect separation of bound labeled protein from the unbound labeled protein.
  • the competitive binding assay may be conducted in liquid phase and a variety of techniques known in the art may be used to separate the bound labeled protein from the unbound labeled protein. Following separation, the amount of bound labeled protein is determined.
  • the amount of protein present in the sample is inversely proportional to the amount of labeled protein binding.
  • a homogeneous immunoassay may be performed in which a separation step is not needed.
  • the label on the protein is altered by the binding of the protein to its specific binding agent. This alteration in the labeled protein results in a decrease or increase in the signal emitted by label, so that measurement of the label at the end of the immunoassay allows for detection or quantitation of the protein.
  • Qualitative or quantitative analysis of SOCS or WDS proteins may also be determined by a variety of noncompetitive immunoassay methods.
  • a two- site, solid phase sandwich immunoassay may be used.
  • a binding agent for the protein for example an antibody
  • a second protein binding agent which may also be an antibody, and which binds the protein at a different site, is labeled. After binding at both sites on the protein has occurred, the unbound labeled binding agent is removed and the amount of labeled binding agent bound to the solid phase is measured. The amount of labeled binding agent bound is directly proportional to the amount of protein in the sample.
  • Western blot analysis can be used to determine the presence of SOCS or WDS proteins in a sample. Electrophoresis is carried out, for example, on a tissue sample suspected of containing the protein. Following electrophoresis to separate the proteins, and transfer of the proteins to a suitable solid support, e.g., a nitrocellulose filter, the solid support is incubated with an antibody reactive with the protein. This antibody may be labeled, or alternatively may be detected by subsequent incubation with a second labeled antibody that binds the primary antibody.
  • a suitable solid support e.g., a nitrocellulose filter
  • the immunoassay formats described above employ labeled assay components .
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. A wide variety of labels and methods may be used.
  • Non-radioactive labels include proteins which bind to labeled antibodies, fluorophores , chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled protein.
  • the choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation.
  • immunoassays to measure antisera reactive with SOCS or WDS proteins can be either competitive or noncompetitive binding assays .
  • competitive binding assays the sample analyte competes with a labeled analyte for specific binding sites on a capture agent bound to a solid surface.
  • the capture agent is a purified recombinant SOCS or WDS protein produced as described above. Other sources of these proteins, including isolated or partially purified naturally occurring protein, may also be used.
  • Noncompetitive assays include sandwich assays, in which the sample analyte is bound between two analyte-specific binding reagents . One of the binding agents is used as a capture agent and is bound to a solid surface.
  • the second binding agent is labeled and is used to measure or detect the resultant complex by visual or instrument means .
  • a number of combinations of capture agent and labeled binding agent can be used.
  • a variety of different immunoassay formats, separation techniques, and labels can be also be used similar to those described above for the measurement of SOCS or WDS proteins.
  • DNAs which encode a SOCS or WDS protein or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples. Methods for doing so, or making expression vectors are described herein. These DNAs can be expressed in a wide variety of host cells for the synthesis of a full-length protein or fragments which can in turn, e.g., be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified molecules; and for s ructure/function studies. Each SOCS or WDS protein or its fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors.
  • transformed is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant techniques, a DNA molecule that encodes a SOCS or WDS polypeptide.
  • Heterologously expressed SOCS or WDS polypeptides can be substantially purified to be free of protein or cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and/or diluent.
  • the antigen e.g., SOCS or WDS protein, or portions thereof, may be expressed as fusions with other proteins or possessing an epitope tag.
  • Expression vectors are typically self-replicating DNA or RNA constructs containing the desired antigen gene or its fragments, usually operably linked to appropriate genetic control elements that are recognized in a suitable host cell.
  • control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation.
  • Expression vectors also usually contain an origin of replication that allows the vector to replicate independently from the host cell.
  • the vectors of this invention contain DNAs which encode a SOCS or WDS protein, or a fragment thereof, typically encoding, e.g., a biologically active polypeptide, or protein.
  • the DNA can be under the control of a viral promoter and can encode a selection marker.
  • This invention further contemplates use of such expression vectors which are capable of expressing eukaryotic cDNA coding for a SOCS or WDS protein in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the eukaryotic cDNA coding for the protein is inserted into the vector such that growth of the host containing the vector expresses the cDNA in question.
  • expression vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell . It is not always necessary to require that an expression vector replicate in a host cell, e.g., it is possible to effect transient expression of the protein or its fragments in various hosts using vectors that do not contain a replication origin that is recognized by the host cell. It is also possible to use vectors that cause integration of a SOCS or WDS protein gene or its fragments into the host DNA by recombination, or to integrate a promoter which controls expression of an endogenous gene.
  • Vectors contemplate plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host.
  • Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector, but many other forms of vectors which serve an equivalent function are suitable for use herein. See, e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors : A Laboratory Manual Elsevier, N.Y. ; and Rodriguez, et al. (eds.) (1988) Vectors : A Survey of Molecular Cloning Vectors and Their Uses Buttersworth, Boston, MA.
  • Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes.
  • Prokaryotes include both gram negative and gram positive organisms, e.g., E. coli and B. subtilis.
  • Lower eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and species of the genus Dictyostelium.
  • Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents.
  • Prokaryotic host-vector systems include a wide variety of vectors for many different species .
  • E. coli and its vectors will be used generically to include equivalent vectors used in other prokaryotes.
  • a representative vector for amplifying DNA is pBR322 or its derivatives.
  • Vectors that can be used to express these proteins or protein fragments include, but are not limited to, such vectors as those containing the lac promoter (pUC-series) ; trp promoter (pBR322-trp) ; Ipp promoter (the pIN-series) ; lambda-pP or pR promoters (pOTS) ; or hybrid promoters such as ptac (pDR540) .
  • Lower eukaryotes e.g., yeasts and Dictyostelium, may be transformed with SOCS or WDS protein sequence containing vectors.
  • the most common lower eukaryotic host is the baker's yeast, Saccharomyces cerevisiae. It will be used generically to represent lower eukaryotes although a number of other strains and species are also available.
  • Yeast vectors typically consist of a replication origin (unless of the integrating type) , a selection gene, a promoter, DNA encoding the desired protein or its fragments, and sequences for translation termination, polyadenylation, and transcription termination.
  • Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or such indueible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter.
  • Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp-serie ⁇ ) , self-replicating high copy number
  • YEp-series such as the YEp-series
  • integrating types such as the YIp-series
  • mini-chromosomes such as the YCp- series
  • Higher eukaryotic tissue culture cells are typically the preferred host cells for expression of the functionally active SOCS or WDS protein.
  • many higher eukaryotic tissue culture cell lines may be used, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source.
  • mammalian cells are preferred to achieve proper processing, both cotranslationally and posttranslationally. Transformation or transfection and propagation of such cells is routine.
  • Useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS) cell lines.
  • Expression vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA splice sites (e.g., if genomic DNA is used) , a polyadenylation site, and a transcription termination site. These vectors also may contain a selection gene or amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as from adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus . Representative examples of suitable expression vectors include pCDNAl; pCD, see Okaya a, et al. (1985) Mol. Cell Biol.
  • SOCS or WDS proteins need not be glycosylated to elicit biological responses.
  • SOCS or WDS protein polypeptide in a system which provides a specific or defined glycosylation pattern.
  • the usual pattern will be that provided naturally by the expression system.
  • the pattern will be modifiable by exposing the polypeptide, e.g., in unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system.
  • heterologously expressed proteins or polypeptides can also be expressed in plant cells.
  • viral expression vectors e.g., cauliflower mosaic virus and tobacco mosaic virus
  • plasmid expression vectors e.g., Tl plasmid
  • Such cells are available from a wide range of sources (e.g., the American Tissue Type Culture Collection, Rockland, MD; also, see for example, Ausubel, et al. (cur. ed. and Supplements; expression vehicles may be chosen from those provided e.g., in Pouwels, et al. (Cur. ed..) Cloning Vectors, A Laboratory Manual) .
  • a SOCS or WDS protein, or a fragment thereof may be engineered to be phosphatidyl inositol (PI) linked to a cell membrane, but can be removed from membranes by treatment with a phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C.
  • PI phosphatidyl inositol
  • an azide process for example, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester) , a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide (DCCD) /additive process can be used.
  • Solid phase and solution phase syntheses are both applicable to the foregoing processes.
  • the prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, precipitation, electrophoresis and various forms of chromatography, and the like.
  • the SOCS or WDS proteins of this invention can be obtained in varying degrees of purity depending upon its desired use. Purification can be accomplished by use of known protein purification techniques or by the use of the antibodies or binding partners herein described, e.g., in immunoabsorbant affinity chromatography.
  • This immunoabsorbant affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate source cells, lysates of other cells expressing the protein, or lysates or supernatants of cells producing the SOCS or WDS proteins as a result of recombinant DNA techniques, see below.
  • Multiple cell lines may be screened for one .which expresses a SOCS or WDS protein at a high level compared with other cells.
  • Various cell lines e.g., a mouse thymic stromal cell line TA4, is screened and selected for its favorable handling properties.
  • Natural SOCS or WDS proteins can be isolated from natural sources, or by expression from a transformed cell using an appropriate expression vector.
  • Purification of the expressed protein is achieved by standard procedures, or may be combined with engineered means for effective purification at high efficiency from cell lysates or supernatants .
  • Epitope or other tags e.g., FLAG or Hiss segments, can be used for such purification features .
  • This invention also encompasses proteins or peptides having substantial amino acid sequence similarity with an amino acid sequence of a SOCS or WDS protein.
  • Natural variants include individual, polymorphic, allelic, strain, or species variants. Amino acid sequence similarity, or sequence identity, is determined by optimizing residue matches, if necessary, by introducing gaps as required. This changes when considering conservative substitutions as matches . Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Homologous amino acid sequences include natural polymorphic, allelic, and interspecies variations in each respective protein sequence. Typical homologous proteins or peptides will have from 50-100% similarity (if gaps can be introduced) , to 75-100% similarity (if conservative substitutions are included) over fixed stretches of amino acids with the amino acid sequence of the SOCS or WDS protein. Similarity measures will be at least about 50%, generally at least 65%, usually at least 70%, preferably at least 75%, and more preferably at least 90%, and in particularly preferred embodiments, at least 96% or more. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) Time Warps .
  • Stretches of amino acids will be at least about 10 amino acids, usually about 20 amino acids, usually 50 amino acids, preferably 75 amino acids, and in particularly preferred embodiments at least about 100 amino acids. Identity can also be measures over amino acid stretches of about 98, 99, 110, 120, 130, etc.
  • Nucleic acids encoding mammalian SOCS or WDS proteins will typically hybridize to the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 under stringent conditions.
  • nucleic acids encoding human SOCS or WDS proteins will normally hybridize to the nucleic acid of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 under stringent hybridization conditions.
  • stringent conditions are selected to be about 10° C lower than the thermal melting point (Tm) for the probe sequence at a defined ionic strength and pH. The 1m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • stringent conditions will be those in which the salt concentration is about 0.2 molar at pH 7 and the temperature is at least about 50° C.
  • Other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents such as formamide, and the extent of base mismatching.
  • a preferred embodiment will include nucleic acids which will bind to disclosed sequences in 50% formamide and 200 mM NaCl at 42° C.
  • Hybridizing nucleic acids to SOCS nucleic acid of the invention can be used as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe.
  • Hybridizing nucleic acids can be splice variants encoded by one of the SOCS genes described herein.
  • the hybridizing nucleic acids may encode a polypeptide that is shorter or longer than the various forms of SOCS described herein.
  • Hybridizing nucleic acids may also encode proteins that are related to SOCS (e.g., polypeptides encoded by genes that include a portion having a relatively high degree of identity to a SOCS gene described herein) .
  • An isolated SOCS or WDS protein encoding DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and short inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode SOCS or WDS protein antigens, their derivatives, or proteins having highly similar physiological, immunogenic, or antigenic activity.
  • Modified sequences can be used to produce mutant antigens or to enhance expression. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant SOCS or WDS protein derivatives include predetermined or site-specific mutations of the respective protein or its fragments. "Mutant SOCS or WDS protein” encompasses a polypeptide otherwise falling within the homology definition of the human or rodent SOCS or WDS protein as set forth above, but having an amino acid sequence which differs from that of a SOCS or WDS protein as found in nature, whether by way of deletion, substitution, or insertion.
  • site specific mutant SOCS or WDS protein generally includes proteins having significant similarity with a protein having a sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, e.g., natural embodiments, and as sharing various biological activities, e.g., antigenic or immunogenic, with those sequences, and in preferred embodiments contain most or all of the disclosed sequence.
  • the invention encompasses, but is not limited to, SOCS proteins and polypeptides that are functionally related to SOCS encoded by the specific sequence identifiers of the present application.
  • Functionally related proteins and polypeptides include any protein or polypeptide sharing a functional characteristic with SOCS of the present invention e.g., the ability to interact with Janus family tyrosine kinases or the ability to be induced by IL-2 receptor activation.
  • Such functionally related SOCS polypeptides include, but are not limited to, additions or substitutions of amino acid residues within the amino acid sequence encoded by the SOCS sequences described herein which result in a silent change, thus producing a functionally equivalent SOCS polypeptide. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphiphatic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamme; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • While random mutations can be made to SOCS nucleic acid (using well known random mutagenesis techniques) and the resulting SOCS polypeptides can be tested for activity, site-directed mutations of SOCS coding sequences can be engineered (using well known site- directed mutagenesis techniques) to generate mutant SOCS with increased function, e.g. greater inhibition of JANUS kinase activity or greater resistance to degradation.
  • site-directed mutations of SOCS coding sequences can be engineered (using well known site- directed mutagenesis techniques) to generate mutant SOCS with increased function, e.g. greater inhibition of JANUS kinase activity or greater resistance to degradation.
  • conserved residues remain unaltered and that the conformational folding of the SOCS functional sites be preserved.
  • alteration of non-conserved residues are carried out with conservative alterations e.g., a basic amino acid is replaced by a different basic amino acid.
  • conservative alterations e.g., a basic amino acid is replaced by a different basic amino acid.
  • non-conservative changes at variable and or conserved residues it is preferred to make non-conservative changes at variable and or conserved residues . Deletions at conserved and variable residues can also be used to create altered function variants .
  • SOCS or WDS protein mutagenesis can be conducted by making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxyl- terminal fusions, e.g. epitope tags. Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by Ml3 primer mutagenesis or polymerase chain reaction (PCR) techniques. See also, Sambrook, et al.
  • the mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins .
  • a heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner e.g., a marker polypeptide or fusion partner.
  • the polypeptide can be fused to a hexa-histidine tag to facilitate purification or bacterially expressed protein or a hemaglutinin tag to facilitate purification or protein expressed in eukaryotic cells.
  • the fusion product of an immunoglobulin with a SOCS or WDS protein polypeptide is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties derived from each source peptide.
  • a similar concept applies to heterologous nucleic acid sequences .
  • new constructs may be made from combining similar functional domains from other proteins.
  • protein-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham, et al . (1989) Science 243:1330-1336; and O'Dowd, et al . (1988) J. Biol. Chem. 263:15985-15992.
  • new chimeric polypeptides exhibiting new combinations of specificities will result from the functional linkage of protein-binding specificities and other functional domains.
  • the blocking of physiological response to SOCS or WDS protein may result from the inhibition of binding of the protein to its binding partner, e.g., through competitive inhibition.
  • in vitro assays of the present invention will often use isolated protein, membranes from cells expressing a recombinant membrane associated SOCS or WDS protein, soluble fragments comprising binding segments of these proteins, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or protein mutations and modifications, e.g., protein analogs.
  • This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to antigen or binding partner fragments compete with a test compound for binding to the protein. In this manner, the antibodies can be used to detect the presence of a polypeptide which shares one or more antigenic binding sites of the protein and can also be used to occupy binding sites on the protein that might otherwise interact with a binding partner.
  • “Derivatives” of SOCS or WDS protein antigens include amino acid sequence mutants, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.
  • Covalent derivatives can be prepared by linkage of functionalities to groups which are found in SOCS or WDS protein amino acid side chains or at the N- or C- termini, by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N- acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine.
  • Acyl groups are selected from the group of alkyl-moieties including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species . Covalent attachment to carrier proteins may be important when immunogenic moieties are haptens .
  • glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are by exposing the polypeptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes . Deglycosylation enzymes are also contemplated. Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties, including ribosyl groups or cross-linking reagents.
  • phosphorylated amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine
  • a major group of derivatives are covalent conjugates of the SOCS or WDS protein or fragments thereof with other proteins or polypeptides. These derivatives can be synthesized in recombinant culture such as N- or C- terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups. Preferred protein derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.
  • Heterologous polypeptides may be fusions between different surface markers, resulting in, e.g., a hybrid protein exhibiting binding partner specificity.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins.
  • Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a protein, e.g., a segment involved in binding partner interaction, so that the presence or location of the fused protein may be easily determined. See, e.g., Dull, et al., U.S. Patent No.
  • gene fusion partners include bacterial ⁇ - galactosidase, trpE, Protein A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor. See, e.g., Godowski, et al . (1988) Science 241:812-816.
  • the fusion partner can be constructed such that it can be cleaved off such that a protein of substantially natural length is generated.
  • Such polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups.
  • the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity proteins.
  • This invention also contemplates the use of derivatives of SOCS or WDS protein other than variations in amino acid sequence or glycosylation. Such derivatives may involve covalent or aggregative association with chemical moieties . These derivatives generally fall into the three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes.
  • Such covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of proteinss or other binding proteins.
  • a SOCS or WDS protein antigen can be immobilized by covalent bonding to a solid support such as cyanogen bromide- activated SEPHAROSE, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of anti-SOCS or anti-WDS protein antibodies or its respective binding partner.
  • the SOCS or WDS protein can also be labeled with a detectable group, e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
  • a detectable group e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
  • Purification of SOCS or WDS proteins may be effected by immobilized antibodies or binding partner. Isolated SOCS or WDS protein genes will allow transformation of cells lacking expression of corresponding SOCS or WDS protein, e.g., either species types or cells which lack corresponding proteins and exhibit negative background activity. Expression of transformed genes will allow isolation of antigenically pure cell lines, with defined or single specie variants. This approach will allow for more sensitive detection and discrimination of the physiological effects of SOCS or WDS binding proteins. Subcellular fragments, e.g., cytoplasts
  • a SOCS or WDS protein that specifically binds to or that is specifically immunoreactive with an antibody generated against a defined immunogen, such as an immunogen consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 is typically determined in an immunoassay.
  • the immunoassay uses a polyclonal antiserum which was raised to a protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16. This antiserum is selected to have low crossreactivity against other intracellular regulatory proteins and any such crossreactivity is removed by immunoabsorbtion prior to use in the immunoassay.
  • the protein of desired sequence e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, and/or 16 is isolated as described herein.
  • recombinant protein may be produced in a mammalian cell line.
  • An inbred strain of mice such as Balb/c is immunized with the protein of appropriate sequence using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane, supra) .
  • a synthetic peptide, preferably near full length, derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
  • Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 ⁇ or greater are selected and tested for their cross reactivity against other intracellular proteins, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573.
  • a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573.
  • two intracellular proteins are used in this determination in conjunction with the desired SOCS or WDS protein.
  • Immunoassays in the competitive binding format can be used for the crossreactivity determinations .
  • a protein of SEQ ID NO: 2 or 4 can be immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the protein of SEQ ID NO: 2 or 4. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorbtion with the above-listed proteins.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogen protein
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than twice the amount of the protein, e.g., of SEQ ID NO: 2 that is required, then the second protein is said to specifically bind to an antibody generated to the immunogen.
  • SOCS or WDS proteins are members of respective families of homologous proteins that comprise two or more genes .
  • the term refers not only to the amino acid sequences disclosed herein, but also to other proteins that are polymorphic, allelic, non-allelic, or species variants.
  • SOCS14 or SOCS15 protein includes nonnatural mutations introduced by deliberate mutation using conventional recombinant technology such as single site mutation, or by excising short sections of DNA encoding S0CS14 or SOCS15 proteins, or by substituting new amino acids, or adding new amino acids . Such minor alterations should substantially maintain the immunoidentity of the original molecule and/or its biological activity.
  • these alterations include proteins that are specifically immunoreactive with a designated naturally occurring SOCS or WDS protein, for example, the human S0CS14 or SOCS15 protein shown in SEQ ID NO: 2 and 6, or 4 and 8.
  • the biological properties of the altered proteins can be determined by expressing the protein in an appropriate cell line and measuring, e.g., a proliferative effect.
  • Particular protein modifications considered minor would include conservative substitution of amino acids with similar chemical properties, as described above for the SOCS14 or SOCS15 protein as a whole.
  • SOCS or WDS nucleotides e.g., human SOCS14 or SOCS15 DNA or RNA
  • the nucleotide sequences provided may be labeled using, e.g., 2 or biotin and used to probe standard restriction fragment polymorphism blots, providing a measurable character to aid in distinguishing between individuals. Such probes may be used in well-known forensic techniques such as genetic fingerprinting.
  • nucleotide probes made from SOCS or WDS sequences may be used in in situ assays to detect chromosomal abnormalities. For instance, rearrangements in the human chromosome encoding a S0CS14 or S0CS15 gene may be detected via well-known in situ techniques, using S0CS14 or SOCS15 probes in conjunction with other known chromosome markers .
  • Antibodies and other binding agents directed towards SOCS or WDS proteins or nucleic acids may be used to purify the corresponding SOCS or WDS molecule. As described in the Examples below, antibody purification of SOCS or WDS protein components is both possible and practicable. Antibodies and other binding agents may also be used in a diagnostic fashion to determine whether SOCS or WDS protein components are present in a tissue sample or cell population using well-known techniques described herein. The ability to attach a binding agent to a SOCS or WDS protein provides a means to diagnose disorders associated with SOCS or WDS protein misregulation. Antibodies and other SOCS or WDS protein binding agents may also be useful as histological markers. It is likely that specific SOCS or WDS protein expression is limited to specific tissue types. By directing a probe, such as an antibody or nucleic acid to a SOCS14 or SOCS15 protein it is possible to use the probe to distinguish tissue and cell types in situ or in vitro .
  • a probe such as an antibody or nucleic acid
  • This invention also provides reagents with significant therapeutic value.
  • the SOCS or WDS protein naturally occurring or recombinant
  • fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to a SOCS or WDS protein, are useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions. Abnormal proliferation, regeneration, degeneration, and atrophy may be modulated by appropriate therapeutic treatment using the compositions provided herein.
  • a disease or disorder associated with abnormal expression or abnormal signaling by a SOCS or WDS protein is a target for an agonist or antagonist of the protein.
  • the proteins likely play a role in regulation or development of neuronal or hematopoietic cells, e.g., lymphoid cells, which affect immunological responses.
  • SOCS or WDS proteins likely play a role in T cell activation deficiencies in which patients develop clinical manifestations of T cell immunodeficiency such as opportunistic infections, recurrent viral or bacterial infections, diarrhea, autoimmune hemolytic anemia, lymphoid hepatitis and dermatitis, and Hodgkin lymphoma, at various stages of childhood.
  • T cell leukemia/lymphoma is a disease associated with uncontrolled T-cell proliferation and is correlated at the molecular level with the presence of the IL-2 receptor (Schechter, G.P.; "Chronic Lymphocytic Leukemia” in Clinical Immunology; Principles and Practice. Rich (ed. ) Mosby, St. Louis (Curr. ed.)).
  • a model for adult T cell leukemia suggests that the disease may result from constitutive activation of the IL-2 receptor and its subsequent constitutive signaling cascade.
  • Administration of exogenous SOCS to effected T cells may modulate this disease.
  • Recombinant SOCS or WDS protein or SOCS or WDS antibodies can be purified and then administered to a patient.
  • These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients.
  • additional active or inert ingredients e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients.
  • These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations.
  • This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.
  • Drug screening using antibodies or fragments thereof can identify compounds having binding affinity to SOCS or WDS protein, including isolation of associated components. Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of the protein. Likewise, a compound having intrinsic stimulating activity can activate the binding partner and is thus an agonist in that it simulates the activity of a SOCS or WDS protein. This invention further contemplates the therapeutic use of antibodies to SOCS or WDS protein as antagonists. This approach should be particularly useful with other SOCS or WDS protein species variants .
  • Another therapeutic approach included within the invention involves direct administration of reagents or compositions by any conventional administration techniques (for example but not restricted to local injection, inhalation, or administered systemically) , to the subject with an immune, allergic or trauma disorder.
  • the reagents, formulations or compositions included within the bounds and metes of the invention may also be targeted to specific cells by any of the methods described herein.
  • the actual dosage of reagent, formulation or composition that modulates an immune, disorder depends on many factors, including the size and health of an organism, however one of one of ordinary skill in the art can use the following teachings describing the methods and techniques for determining clinical dosages. Spilker (1984) Guide to Clinical Studies and Developing Protocols, Raven Press Books, Ltd., New York, pp.
  • reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman, et al.
  • Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ⁇ M concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar) , and most preferably less than about 1 fM (femtomolar) , with an appropriate carrier.
  • Slow release formulations, or a slow release apparatus will often be utilized for continuous administration.
  • SOCS or WDS protein, fragments thereof, and antibodies to it or its fragments, antagonists, and agonists may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.
  • Therapeutic formulations may be administered in any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman, et al . (eds.) (1990) Goodman and Gilman' s: The Pharmacological Bases of Therapeutics (8th ed. ) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th ed.) Mack Publishing Co., Easton, PA; Avis, et al.
  • Both the naturally occurring and the recombinant forms of the SOCS or WDS proteins of this invention are particularly useful in kits and assay methods which are capable of screening compounds for binding activity to the proteins.
  • Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor, et al. (1991) Science 251:767-773, and other descriptions of chemical diversity libraries, which describe means for testing of binding affinity by a plurality of compounds.
  • the development of suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble SOCS or WDS protein as provided by this invention. For example, antagonists can normally be found once the protein has been structurally defined.
  • the advantages of using a recombinant protein in screening for specific binding partners include: (a) improved renewable source of the SOCS or WDS protein from a specific source; (b) potentially greater number of binding partners per cell giving better signal to noise ratio in assays; and (c) species variant specificity (theoretically giving greater biological and disease specificity) .
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing a SOCS or WDS protein binding counterpart.
  • Cells may be isolated which express a binding counterpart in isolation from any others.
  • Such cells either in viable or fixed form, can be used for standard protein binding assays. See also, Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990) Proc. Nat ' 1 Acad. Sci. USA 87:4007-4011, which describe sensitive methods to detect cellular responses.
  • Viable cells could also be used to screen for the effects of drugs on SOCS or WDS protein mediated functions, e.g., second messenger levels, i.e., cell proliferation; inositol phosphate pool changes, transcription using a luciferase-type assay; and others .
  • second messenger levels i.e., cell proliferation
  • inositol phosphate pool changes transcription using a luciferase-type assay
  • Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system.
  • Another method utilizes membranes from transformed eukaryotic or prokaryotic host cells as the source of a SOCS or WDS protein. These cells are stably transformed with DNA vectors directing the expression of a SOCS or WDS protein, e.g., an engineered membrane bound form. Essentially, the membranes would be prepared from the cells and used in a protein binding assay such as the competitive assay set forth above.
  • Still another approach is to use solubilized, unpurified or solubilized, purified SOCS or WDS protein from transformed eukaryotic or prokaryotic host cells. This allows for a "molecular" binding assay with the advantages of increased specificity, the ability to automate, and high drug test throughput.
  • Another technique for drug screening involves an approach which provides high throughput screening for compounds having suitable binding affinity to a SOCS or WDS protein antibody and is described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984.
  • a solid substrate e.g., plastic pins or some other appropriate surface, see Fodor, et al., supra.
  • all the pins are reacted with solubilized, unpurified or solubilized, purified SOCS or WDS protein antibody, and washed.
  • the next step involves detecting bound SOCS or WDS protein antibody.
  • Rational drug design may also be based upon structural studies of the molecular shapes of the SOCS or WDS protein and other effectors or analogs. See, e.g., Methods in Enzvmology vols 202 and 203. Effectors may be other proteins which mediate other functions in response to protein binding, or other proteins which normally interact with the binding partner.
  • One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., x- ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions . For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystallography Academic Press, NY.
  • a purified SOCS or WDS protein can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to these proteins can be used as capture antibodies to immobilize the respective protein on the solid phase.
  • This invention also contemplates use of SOCS or WDS proteins, fragments thereof, peptides, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of SOCS or WDS protein or a binding partner.
  • the kit will have a compartment containing either a defined SOCS or WDS protein peptide or gene segment or a reagent which recognizes one or the other, e.g., binding partner fragments or antibodies .
  • a kit for determining the binding affinity of a test compound to a SOCS or WDS protein would typically comprise a test compound; a labeled compound, e.g., a binding agent or antibody having known binding affinity for the SOCS or WDS protein; a source of SOCS or WDS protein (naturally occurring or recombinant) ; and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the SOCS or WDS protein.
  • a labeled compound e.g., a binding agent or antibody having known binding affinity for the SOCS or WDS protein
  • a source of SOCS or WDS protein naturally occurring or recombinant
  • a means for separating bound from free labeled compound such as a solid phase for immobilizing the SOCS or WDS protein.
  • a preferred kit for determining the concentration of, for example, a SOCS or WDS protein in a sample would typically comprise a labeled compound, e.g., binding partner or antibody, having known binding affinity for the SOCS or WDS protein, a source of SOCS or WDS protein (naturally occurring or recombinant) , and a means for separating the bound from free labeled compound, for example, a solid phase for immobilizing the SOCS or WDS protein. Compartments containing reagents, and instructions, will normally be provided.
  • Antibodies including antigen binding fragments, specific for the SOCS or WDS protein or fragments thereof are useful in diagnostic applications to detect the presence of elevated levels of SOCS or WDS protein and/or its fragments .
  • Such diagnostic assays can employ lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and further can involve the detection of antigens related to the protein in serum, or the like. Diagnostic assays may be homogeneous (without a separation step between free reagent and antigen-SOCS or -WDS protein complex) or heterogeneous (with a separation step) .
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbentassay
  • EIA enzyme immunoassay
  • EMIT enzyme-multiplied immunoassay technique
  • SFIA substrate-labeled fluorescent immunoassay
  • unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to a SOCS or WDS protein or to a particular fragment thereof.
  • Similar assays have also been extensively discussed in the literature. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press, NY; Chan (ed.) (1987) Immunoassay.
  • Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against a SOCS or WDS protein, as such may be diagnostic of various abnormal states. For example, overproduction of SOCS or WDS protein may result in production of various immunological or other medical reactions which may be diagnostic of abnormal physiological states, e.g., in cell growth, activation, or differentiation.
  • the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay.
  • the protocol, and the label either labeled or unlabeled antibody or binding partner, or labeled SOCS or WDS protein is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like.
  • the kit will also contain instructions for proper use and disposal of the contents after use.
  • the kit has compartments for each useful reagent.
  • the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
  • labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.
  • the protein, test compound, SOCS or WDS protein, or antibodies thereto can be labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups: radiolabels such as 125j. # enzymes (U.S. Pat. No.
  • the SOCS or WDS protein can be immobilized on various matrices followed by washing. Suitable matrices include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the SOCS or WDS protein to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin-avidin.
  • the last step in this approach involves the precipitation of protein/binding partner or antigen/antibody complex by any of several methods including those utilizing, e.g., an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate.
  • Another diagnostic aspect of this invention involves use of oligonucleotide or polynucleotide sequences taken from the sequence of a SOCS or WDS protein. These sequences can be used as probes for detecting levels of the SOCS or WDS protein message in samples from natural sources, or patients suspected of having an abnormal condition, e.g., cancer or developmental problem.
  • the preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature.
  • an oligonucleotide probe should have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes may be up to several kilobases.
  • Various labels may be employed, most commonly radionuclides, particularly 2p_
  • other techniques may also be employed, such as using biotin modified nucleotides for introduction into a polynucleotide.
  • the biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorophores, enzymes, or the like.
  • antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • probes to the novel anti-sense RNA may be carried out using many conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT) , and hybrid arrested translation (HART) . This also includes amplification techniques such as polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • Diagnosis or prognosis may depend on the combination of multiple indications used as markers.
  • kits may test for combinations of markers. See, e.g., Viallet, et al . (1989) Progress in Growth Factor Res. 1:89-97.
  • Standard methods are used to isolate full length genes.
  • the appropriate sequence is selected, and hybridization at high stringency conditions is performed to find a full length corresponding gene. It is noted that the mouse and human protein sequences are virtually identical .
  • Isolation of primate S0CS14 or SOCS15 clones The full length, or appropriate fragments, of human genes are used to isolate a corresponding monkey or other primate gene. Preferably a full length coding sequence is used for hybridization. Similar source materials as indicated above are used to isolate natural genes, including genetic, polymorphic, allelic, or strain variants. Other species variants are also isolated using similar methods . IV. Isolation of an avian S0CS14 or SOCS15 clone
  • An appropriate avian source is selected as above. Similar methods are utilized to isolate a species variant, though the level of similarity will typically be lower for avian protein as compared to a human to mouse sequence .
  • Proteins of interest are immunoprecipitated and affinity purified as described above, e.g., from a natural or recombinant source.
  • the coding sequence is inserted into an appropriate expression vector.
  • This may be in a vector specifically selected for a prokaryote, yeast, insect, or higher vertebrate, e.g., mammalian expression system. Standard methods are applied to produce the gene product, preferably as a soluble secreted molecule, but will, in certain instances, also be made as an intracellular protein. Intracellular proteins typically require cell lysis to recover the protein, and insoluble inclusion bodies are a common starting material for further purificiation.
  • recombinant production means are used, although natural forms may be purified from appropriate sources.
  • the protein product is purified by standard methods of protein purification, in certain cases, e.g., coupled with immunoaffinity methods .
  • Immunoaffinity methods are used either as a purification step, as described above, or as a detection assay to determine the separation properties of the protein.
  • the protein is secreted into the medium, and the soluble product is purified from the medium in a soluble form.
  • inclusion bodies from prokaryotic expression systems are a useful source of material.
  • the insoluble protein is solubilized from the inclusion bodies and refolded using standard methods. Purification methods are developed as described above.
  • the product of the purification method described above is characterized to determine many structural features. Standard physical methods are applied, e.g., amino acid analysis and protein sequencing.
  • the resulting protein is subjected to CD spectroscopy and other spectroscopic methods, e.g., NMR, ESR, mass spectroscopy, etc.
  • the product is characterized to determine its molecular form and size, e.g., using gel chromatography and similar techniques . Understanding of the chromatographic properties will lead to more gentle or efficient purification methods .
  • glycosylation sites may be made, e.g., as reported in Hansen, et al. (1995) Biochem. J. 308:801- 813. However, as intracellular proteins, they are unlikely to be normally glycosylated.
  • the purified protein is also be used to identify other binding partners of SOCS or WDS as described, e.g., in Fields and Song (1989) Nature 340:245-246.
  • polyclonal antiserum is raised using non-purified antigen, though the resulting serum will exhibit higher background levels.
  • the antigen is purified using standard protein purification techniques, including, e.g., affinity chromatography using polyclonal serum indicated above. Presence of specific antibodies is detected using defined synthetic peptide fragments.
  • Polyclonal serum is raised against a purified antigen, purified as indicated above, or using, e.g., a plurality of, synthetic peptides.
  • a series of overlapping synthetic peptides which encompass all of the full length sequence, if presented to an animal, will produce serum recognizing most linear epitopes on the protein.
  • Such an antiserum is used to affinity purify protein, which is, in turn, used to introduce intact full length protein into another animal to produce another antiserum preparation. Similar techniques are used to generate induce monoclonal antibodies to either unpurified antigen, or, preferably, purified antigen.
  • Distribution of the protein or gene products are determined, e.g., using immunohistochemistry with an antibody reagent, as produced above, by Western blotting of cell lysates, or by screening for nucleic acids encoding the respective protein. Either hybridization or PCR methods are used to detect DNA, cDNA, or message content. Histochemistry allows determination of the specific cell types within a tissue which express higher or lower levels of message or DNA. Antibody techniques are useful to quantitate protein in a biological sample, including a liquid or tissue sample. Immunoassays are developed to quantitate protein. Also FACS analysis may be used to evaluate expression in a cell population. Appropriate tissue samples or cell types are isolated and prepared for such detection. Commercial tissue blots are available, e.g., from Clontech (Mountain View, CA) . Alternatively, cDNA library Southern blots can be analyzed.
  • the inhibition of SOCS function may be effected by inhibitors of the specific interaction of these gene products and their respective STAT molecules .
  • compound libraries may be screened for blockage of such interactions.
  • inhibitory action of the SOCS may be blocked with small molecule drug candidates.
  • Tables 1 and 2 show comparison of various SOCS or WDS embodiments .
  • Table 1 shows comparisons of the relevant portions of the gene products, particularly in the region of S0CS14 from Metl68 to Leu293.

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Abstract

La présente invention concerne des gènes purifiés codant des molécules intracellulaires régulatrices d'un être humain, des réactifs de celles-ci comprenant des protéines purifiées, des anticorps spécifiques et des acides nucléiques codant lesdites molécules. La présente invention concerne lesdits réactifs et des kits de diagnostic.
PCT/US1998/014544 1997-07-18 1998-07-17 Suppresseurs de signalement de cytokine et reactifs correspondants WO1999003993A2 (fr)

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Cited By (12)

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EP0877030A2 (fr) * 1997-05-07 1998-11-11 Smithkline Beecham Corporation Gène en réponse primaire à l'EPO 1 (EPRG1)
WO1999023220A1 (fr) * 1997-11-03 1999-05-14 Incyte Pharmaceuticals, Inc. Suppresseur de la signalisation cytokinaire
EP0948522A1 (fr) * 1996-11-01 1999-10-13 The Walter And Eliza Hall Institute Of Medical Research Agents therapeutiques et diagnostiques capables de moduler la receptivite cellulaire aux cytokines
EP0953636A2 (fr) * 1998-03-23 1999-11-03 Smithkline Beecham Plc Régulateurs du signal de cytokines
WO2000037636A1 (fr) * 1998-12-21 2000-06-29 The Walter And Eliza Hall Institute Of Medical Research Boite socs contenant des peptides
WO2001029178A2 (fr) * 1999-10-21 2001-04-26 Smithkline Beecham Corporation Gene 1 de reponse primaire a l'erythropoietine (epo), gene eprg1
GB2373502A (en) * 2000-11-08 2002-09-25 Smithkline Beecham Corp Suppressor of cytokine signalling 4 (SOCS4)
WO2002083705A1 (fr) * 2001-04-13 2002-10-24 The Johns Hopkins University School Of Medicine Methylation du gene socs-1 dans le cancer
WO2002055699A3 (fr) * 2000-12-14 2003-04-03 Pharmacia & Upjohn Company Nouveau socs (suppresseur de la transmission des signaux par les cytokines)
US6905842B1 (en) 1996-11-01 2005-06-14 The Walter And Eliza Hall Institute Of Medical Research Therapeutic and diagnostic agents
US7348139B1 (en) 2001-04-13 2008-03-25 The Johns Hopkins University School Of Medicine SOCS-1 gene methylation in cancer
US8420096B2 (en) 2004-03-04 2013-04-16 Vanderbilt University Cell-penetrating SOCS polypeptides that inhibit cytokine-induced signaling

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
GB9918680D0 (en) * 1999-08-09 1999-10-13 Medical Res Council Polypeptide

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WO1998020023A1 (fr) * 1996-11-01 1998-05-14 The Walter And Eliza Hall Institute Of Medical Research Agents therapeutiques et diagnostiques capables de moduler la receptivite cellulaire aux cytokines

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D.J. HILTON ET AL: "Twenty proteins containing a C-terminal SOCS box form five structural classes" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 95, January 1998, pages 114-119, XP002085497 WASHINGTON US *
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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0948522A1 (fr) * 1996-11-01 1999-10-13 The Walter And Eliza Hall Institute Of Medical Research Agents therapeutiques et diagnostiques capables de moduler la receptivite cellulaire aux cytokines
US7279557B2 (en) 1996-11-01 2007-10-09 The Walter And Eliza Hall Institute Of Medical Research Therapeutic and diagnostic agents
US6905842B1 (en) 1996-11-01 2005-06-14 The Walter And Eliza Hall Institute Of Medical Research Therapeutic and diagnostic agents
EP0948522A4 (fr) * 1996-11-01 2001-09-19 Inst Medical W & E Hall Agents therapeutiques et diagnostiques capables de moduler la receptivite cellulaire aux cytokines
EP0877030A2 (fr) * 1997-05-07 1998-11-11 Smithkline Beecham Corporation Gène en réponse primaire à l'EPO 1 (EPRG1)
EP0877030A3 (fr) * 1997-05-07 2001-05-23 Smithkline Beecham Corporation Gène en réponse primaire à l'EPO 1 (EPRG1)
WO1999023220A1 (fr) * 1997-11-03 1999-05-14 Incyte Pharmaceuticals, Inc. Suppresseur de la signalisation cytokinaire
US6303333B1 (en) 1998-03-23 2001-10-16 Smithkline Beecham P.L.C. SBHWSB2: a new member of the WD40 SOCS box family
EP0953636A2 (fr) * 1998-03-23 1999-11-03 Smithkline Beecham Plc Régulateurs du signal de cytokines
EP0953636A3 (fr) * 1998-03-23 2000-03-29 Smithkline Beecham Plc Régulateurs du signal de cytokines
WO2000037636A1 (fr) * 1998-12-21 2000-06-29 The Walter And Eliza Hall Institute Of Medical Research Boite socs contenant des peptides
US7078174B1 (en) 1998-12-21 2006-07-18 The Walter & Eliza Hall Institute Of Medical Research Screening methods using SOCS box-containing peptides
WO2001029178A3 (fr) * 1999-10-21 2001-09-13 Smithkline Beecham Corp Gene 1 de reponse primaire a l'erythropoietine (epo), gene eprg1
WO2001029178A2 (fr) * 1999-10-21 2001-04-26 Smithkline Beecham Corporation Gene 1 de reponse primaire a l'erythropoietine (epo), gene eprg1
GB2373502A (en) * 2000-11-08 2002-09-25 Smithkline Beecham Corp Suppressor of cytokine signalling 4 (SOCS4)
WO2002055699A3 (fr) * 2000-12-14 2003-04-03 Pharmacia & Upjohn Company Nouveau socs (suppresseur de la transmission des signaux par les cytokines)
WO2002083705A1 (fr) * 2001-04-13 2002-10-24 The Johns Hopkins University School Of Medicine Methylation du gene socs-1 dans le cancer
US7348139B1 (en) 2001-04-13 2008-03-25 The Johns Hopkins University School Of Medicine SOCS-1 gene methylation in cancer
US7749709B2 (en) 2001-04-13 2010-07-06 The Johns Hopkins University School Of Medicine SOCS-1 gene methylation in cancer
US8420096B2 (en) 2004-03-04 2013-04-16 Vanderbilt University Cell-penetrating SOCS polypeptides that inhibit cytokine-induced signaling

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