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WO2002090595A9 - Procedes de modulation d'une reponse immune par modulation de l'activite krc - Google Patents

Procedes de modulation d'une reponse immune par modulation de l'activite krc

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Publication number
WO2002090595A9
WO2002090595A9 PCT/US2002/014166 US0214166W WO02090595A9 WO 2002090595 A9 WO2002090595 A9 WO 2002090595A9 US 0214166 W US0214166 W US 0214166W WO 02090595 A9 WO02090595 A9 WO 02090595A9
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WIPO (PCT)
Prior art keywords
krc
cell
polypeptide
molecule
traf
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Ceased
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PCT/US2002/014166
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English (en)
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WO2002090595A1 (fr
Inventor
Laurie H Glimcher
Mohamed Oukka
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Harvard University
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Harvard University
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Publication of WO2002090595A1 publication Critical patent/WO2002090595A1/fr
Publication of WO2002090595A9 publication Critical patent/WO2002090595A9/fr
Priority to US10/701,401 priority Critical patent/US7615380B2/en
Anticipated expiration legal-status Critical
Priority to US10/578,402 priority patent/US20070224653A1/en
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • Transcription factors are a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes.
  • ZAS zinc finger-acidic domain structures
  • DNA binding protein family is involved in the regulation of gene transcription, DNA recombination, and signal transduction (Mak, C.H., et al. 1998. Immunogenetics 48: 32- 39).
  • Zinc finger proteins are identified by the presence of highly conserved Cys2His2 zinc fingers (Mak, C.H., et al. 1998. Immunogenetics 48: 32-39).
  • the zinc fingers are an integral part of the DNA binding structure called the ZAS domain.
  • the ZAS domain is comprised of a pair of zinc fingers, a glutamic acid/aspartic acid-rich acidic sequence and a serine/threonine rich sequence (Mak, C.H., et al. 1998. Immunogenetics 48: 32- 39).
  • the ZAS domains have been shown to interact with the kB like cz ' s-acting regulatory elements found in the promoter or enhancer regions of genes.
  • the ZAS proteins recognize nuclear factor kB binding sites which are present in the enhancer sequences of many genes, especially those involved in immune responses (Bachmeyer, et al. 1999. Nuc. Acid Res. 27, 643-648).
  • the ZAS DNA binding proteins have been shown to be transcription regulators of these target genes (Bachmeyer, et al. 1999. Nuc. Acid Res. 27, 643-648; u et al. 1998. Science 281, 998-1001).
  • the zinc finger transcription factor Kappa Recognition Component (“KRC”) is a member of the ZAS DNA binding family of proteins (Bachmeyer, et al. 1999. Nuc. Acid Res. 27, 643-648; Wu et al. 1998. Science 281, 998-1001).
  • KRC The KRC gene was identified as a DNA binding protein for the heptameric consensus signal sequences involved in somatic V(D)J recombination of the immune receptor genes (Mak, C. H., et al. 1994. Nuc.Acid Res. 22: 383-390).
  • KRC is a substrate for epidermal growth factor receptor kinase and p34cdc2 kinase in vitro (Bachmeyer, et al. 1999. Nuc. Acid Res. 27, 643-648).
  • Gene-specific transcription factors provide a promising class of targets for novel therapeutics because they provide substantial specificity and are known to be involved in human disease.
  • a number of extremely effective presently marketed drugs act, at least indirectly, by modulating gene transcription.
  • the LDL receptor is pathogenically down-regulated at the level of transcription by intracellular sterol levels.
  • the drug compactin an inhibitor of HMG CoA reductase, functions by up-regulating transcription of the LDL receptor gene which leads to clearance of cholesterol from the blood stream.
  • transcription factors can be modulated to regulate an immune response.
  • autoimmune diseases self-tolerance is lost and the immune system attacks "self tissue as if it were a foreign target.
  • Many autoimmune diseases are presently known, such as multiple sclerosis (MS), rheumatoid arthritis, insulin- dependent diabetes mellitus, hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barre syndrome, psoriasis, glornerulonephritis, autoimmune hepatitis, multiple sclerosis, etc.
  • MS multiple sclerosis
  • rheumatoid arthritis insulin- dependent diabetes mellitus
  • hemolytic anemias rheumatic fever
  • Crohn's disease Guillain-Barre syndrome
  • psoriasis glornerulonephritis
  • autoimmune hepatitis multiple sclerosis, etc.
  • inhibiting the immune response is desirable.
  • inhibiting the body's immune response is beneficial
  • Urgently needed are efficient methods of identifying pharmacological agents or drugs which are active at the level of gene transcription. Specifically, agents for use modulating such cellular processes in T cells are needed to regulate the immune response. Agents and methods of using such agents in modulation of cell survival, proliferation, differentiation and/or motility would be of great benefit.
  • the present invention is based, at least in part, on the discovery that KRC molecules have multiple important functions as modulating agents in regulating a wide variety of cellular processes.
  • the invention is based, at least in part, on the discovery that KRC inhibits NFkB transactivation, increases TNF-alpha induced apoptosis, inhibits JNK activation, inhibits endogenous TNF-alpha expression, promotes immune cell proliferation and immune cell activation (e.g., in T cells (such as Thl cells), B cells, or macrophages), activates IL-2 expression e.g., by activating the AP-1 transcription factor, activates the Ras and Rac oncogenes, regulates PKC theta activity and increases actin polymerization.
  • the present invention also demonstrates that KRC interacts with TRAF.
  • the interaction between KRC and TRAF involves the C domain of TRAF and amino acid residues 204 to 1055 of KRC.
  • one embodiment of the present invention provides a method for modulating inflammation comprising contacting an immune cell with a compound that modulates KRC activity such that inflammation modulated.
  • a compound that modulates KRC activity such that inflammation modulated.
  • the cell is a T cell.
  • the agent is selected from the group comprised of: a KRC nucleic acid molecule and a KRC peptide.
  • the agent is selected from the group comprised of: an intracellular antibody, a nucleic acid molecule that is complementary to at least about 7 contiguous nucleotide bases of SEQ ID NO: 1 and a dominant negative KRC molecule.
  • KRC activity is enhanced.
  • KRC activity is inhibited.
  • the KRC activity is selected from the group consisting of: activation of JNK signaling pathway, activation of a NFkB signaling pathway, activation of AP-1, activation of Ras and/or Rac, enhancement of actin polymerization and regulation of PKC theta expression.
  • cytokine gene expression in an immune cell is inhibited.
  • Another embodiment of the invention provides a method for modulating apoptosis in a cell comprising contacting a cell with a compound that modulates KRC activity such that apoptosis in the cell is modulated.
  • the method may be performed either in vivo or in vitro.
  • the cell is a T cell.
  • the agent is selected from the group comprised of: a KRC nucleic acid molecule and a KRC peptide. In another embodiment, the agent is selected from the group comprised of: an intracellular antibody, a nucleic acid molecule that is complementary to at least about 7 contiguous nucleotide bases of SEQ ID NO:l and a dominant negative KRC molecule.
  • KRC activity is enhanced. In another embodiment, KRC activity is inhibited. In yet another embodiment, the KRC activity is selected from the group consisting of: activation of JNK signaling pathway, activation of a NFkB signaling pathway, activation of AP-1, activation of Ras and Rac, enhancement of actin polymerization and regulation of PKC theta expression. In a preferred embodiment, apoptosis is inhibited.
  • Another embodiment of the invention provides a method for modulating immune cell proliferation comprising contacting a cell with a compound that modulates KRC activity such that immune cell proliferation is modulated.
  • the method may be performed either in vivo or in vitro.
  • the cell is an immune cell.
  • the cell is a Thl cell.
  • the agent is selected from the group comprised of: a KRC nucleic acid molecule and a KRC peptide.
  • the agent is selected from the group consisting of: an intracellular antibody, a nucleic acid molecule that is complementary to at least about 7 contiguous nucleotide bases of SEQ ID NO: 1 and a dominant negative KRC molecule.
  • KRC activity is enhanced.
  • KRC activity is inhibited.
  • the KRC activity is selected from the group consisting of: activation of JNK signaling pathway, activation of a NFkB signaling pathway, activation of AP-1, activation of Ras and Rac, enhancement of actin polymerization and regulation of PKC theta expression.
  • immune cell proliferation is increased.
  • Another embodiment of the invention provides a method for modulating immune cell activation comprising contacting a cell with a compound that modulates KRC activity such that immune cell activation is modulated.
  • the method may be performed either in vivo or in vitro.
  • the cell is a T cell.
  • the cell is a Thl cell.
  • the agent is selected from the group comprised of: a KRC nucleic acid molecule and a KRC peptide. In another embodiment, the agent is selected from the group comprised of: an intracellular antibody, a nucleic acid molecule that is antisense to a KRC molecule and a dominant negative KRC molecule.
  • KRC activity is enhanced. In another embodiment, KRC activity is inhibited. In yet another embodiment, the KRC activity is selected from the group consisting of: activation of JNK signaling pathway, activation of a NFkB signaling pathway, activation of AP-1, activation of Ras and Rac, enhancement of actin polymerization and regulation of PKC theta expression.
  • immune cell activation is increased. In a further preferred embodiment, IL-2 expression is increased.
  • Another embodiment of the invention provides a method for modulating inflammation in a cell comprising contacting a cell with a compound that modulates the interaction between a TRAF molecule and KRC such that inflammation in the cell is modulated.
  • the method may be performed either in vivo or in vitro.
  • the cell is a T cell.
  • the agent is selected from the group consisting of: a KRC nucleic acid molecule, a TRAF nucleic acid molecule, a KRC peptide, and a TRAF peptide.
  • the agent is selected from the group consisting of: an intracellular antibody, a nucleic acid molecule that is antisense to a TRAF molecule, a nucleic acid molecule that is antisense to a KRC molecule, a dominant negative KRC molecule, and a dominant negative TRAF molecule.
  • the interaction between a TRAF molecule and KRC in enhanced. In another embodiment, the interaction between a TRAF molecule and KRC in inhibited. In a preferred embodiment, cytokine gene expression is inhibited.
  • Another embodiment of the invention provides a method for modulating apoptosis in a cell comprising contacting a cell with an agent that modulates the interaction between a TRAF molecule and KRC such that apoptosis in the cell is modulated.
  • the method may be performed either in vivo or in vitro.
  • the cell is a T cell.
  • the agent is selected from the group consisting of: a KRC nucleic acid molecule, a TRAF nucleic acid molecule, a KRC peptide, and a TRAF peptide.
  • the agent is selected from the group consisting of: an intracellular antibody, a nucleic acid molecule that is antisense to a TRAF molecule, a nucleic acid molecule that is antisense to a KRC molecule, a dominant negative KRC molecule, and a dominant negative TRAF molecule.
  • the interaction between a TRAF molecule and KRC in enhanced. In another embodiment, the interaction between a TRAF molecule and KRC in inhibited. In a preferred embodiment, apoptosis is inhibited.
  • Another embodiment of the invention provides a method for identifying a compound which modulates an interaction between KRC and a TRAF polypeptide comprising: contacting a cell having a first polypeptide comprising a TRAF-interacting portion of a KRC molecule and a second polypeptide comprising a KRC-interacting portion of a TRAF molecule in the presence and the absence of a test compound; and determining the degree of interaction between the first and the second polypeptide in the presence and the absence of the test compound to thereby identify a compound which modulates an interaction between KRC and a TRAF polypeptide.
  • the first polypeptide comprises amino acid residues 204 to 1055 of KRC.
  • the first polypeptide comprises at least one KRC zinc finger domain.
  • the first polypeptide is derived from an exogenous source.
  • the second polypeptide comprises a TRAF C domain.
  • the second polypeptide is a TRAFl polypeptide.
  • the second polypeptide is a TRAF2 polypeptide.
  • the second polypeptide is derived from an exogenous source.
  • the cell is a yeast cell.
  • determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the compound to modulate growth of the yeast cell on nutritionally selective media.
  • determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the compound to modulate expression of a reporter gene in the yeast cell.
  • determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the test compound to modulate the coimmunoprecipitation of the first polypeptide and the second polypeptide. In a preferred embodiment, determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the test compound to modulate apoptosis in the cell.
  • determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the test compound to modulate cytokine gene expression in the cell.
  • determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the test compound to modulate signaling via a signal transduction pathway in the cell.
  • NFkB-dependent transactivation or JNK phosphorylation is measured.
  • Another embodiment of the invention provides a method for identifying a compound which modulates the interaction of KRC and a TRAF molecule comprising: contacting a first polypeptide comprising a TRAF-interacting portion of a KRC molecule and a second polypeptide comprising a KRC-interacting portion of a TRAF molecule in the presence and the absence of a test compound; and determining the degree of interaction between the first and the second polypeptide in the presence and the absence of the test compound to thereby identify a compound which modulates an interaction between KRC and TRAF polypeptide.
  • the first polypeptide comprises amino acid residues 204 to 1055 of KRC. In another embodiment, the first polypeptide comprises at least one KRC zinc finger domain. In yet another embodiment, the first polypeptide is derived from an exogenous source. In another preferred embodiment the second polypeptide comprises a TRAF C domain. In another embodiment, the second polypeptide is a TRAFl polypeptide. In a further preferred embodiment, the second polypeptide is a TRAF2 polypeptide. In a yet further embodiment, the second polypeptide is derived from an exogenous source.
  • the binding of first and second polypeptide is inhibited. In another embodiment, the binding of first and second polypeptide is stimulated. In yet another embodiment, determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the test compound to modulate the coimmunoprecipitation of the first polypeptide and the second polypeptide. In a preferred embodiment, the NFkB-dependent transactivation or JNK phosphorylation is measured.
  • Figure 1A - IE depict the interaction of amino acid residues 204 to 1055 of KRC ("KRC tr") (SEQ ID NO: 5) with TRAF family members.
  • Figure 1A depicts the schema of KRC constructs used.
  • Figure IB upper panel depicts the interaction of KRC tr with TRAFs in mammalian cells. 293 T cells were cotransfected with the indicated FLAG- TRAFs and MYC-tagged KRC tr, and immunoprecipitated with anti-MYC antibody, followed by blotting with anti-FLAG antibody.
  • Figure IB /ower panel depicts the direct western blot of overexpressed TRAFS and KRC tr with anti-FLAG or anti-MYC.
  • Figure 1C depicts the differential interaction of KRC tr with TRAF proteins.
  • the coimmunoprecipitation experiments were performed in the presence of 300 mM NaCl instead of 137 mM NaCl.
  • Figure ID depicts KRC tr interacting with TRAF2 lacking the Ring finger domain.
  • 293 T cells were transfected with MYC-KRC tr and with FLAG tagged TRAF2 or with FLAG-tagged TRAF2 (87-501).
  • Figure IE depicts the interaction of KRC tr with endogenous TRAF2 but not with endogenous TRAF5 or TRAF6.
  • 293T were transfected with an expression vector encoding an MYC-tagged KRC tr, or empty plasmid.
  • TRAF2 ( Figure 2A), TRAF5 ( Figure 2B) and TRAF6 ⁇ Figure 2 mediated activation of NFkB by ectopically expressed KRC.
  • 293 T cells (3 X10 " ) were transfected with 25 ng of NFkB luciferase reporter plasmid, 50 ng of CMV ⁇ Gal and 1 ⁇ g of each indicated plasmid and 24 hours post transfection cells were harvested. Data from at least five experiments normalized for ⁇ galactosidase activity are shown. Vec refers to the empty MYC vector without the addition of TRAFs.
  • Figure 3 depicts KRC and KRC tr inhibit while antisense and dominant negative KRC increase TNF ⁇ -driven NFKB transactivation.
  • 293 T cells (3 X10 ) were transfected with 25 ng of NFKB luciferase reporter plasmid, 50 ng of CMV ⁇ Gal and 1 ⁇ g of each indicated plasmid and 24 hours post transfection cells were stimulated for 4 hours with 10 ng/ml of TNF ⁇ .
  • KRC and KRC tr B
  • dominant negative and antisense KRC C
  • Data from at least five experiments normalized for ⁇ galactosidase activity are shown.
  • Figure 4 depicts IKK ⁇ overexpression overcomes KRC inhibition of NFKB-dependent transactivation.
  • 293 T cells (3 X10 ) were transfected with 25 ng of NFKB luciferase reporter plasmid, with 50 ng of CMV ⁇ Gal, 200 ng of IKK ⁇ expression vector when indicated and 1 ⁇ g of each indicated plasmid and cells harvested 24 hours post transfection. Data from two experiments normalized for ⁇ galactosidase activity are shown.
  • FIG. 5 depicts KRC increases TNF ⁇ -induced apoptosis.
  • 3T3 cells were cotransfected with CMV lacZ vector (300 ng per plate) and either empty expression vector or the expression vectors indicated (2 ⁇ g of each).
  • Half of the transfected cultured cells were treated with TNF ⁇ (20 ng/ml) at 12 hours after the transfection and the other half left untreated. All the cells were fixed and stained at 36 hours after the transfection. The number of blue cells in each transfection was determined by counting six different fields. A representative experiment of three performed is presented.
  • FIG. 6 depicts KRC prevents TRAF2 and TNF ⁇ -dependent JNK activation.
  • A 293 T cells were transfected with 400 ng of TRAF2 and 2 ⁇ g of the indicated expression vector. Twenty-four hours after the transfection, the cells were harvested and lysed, and the endogenous JNK was precipitated with 5 ⁇ g of GST-cJUN (1-79) for 4 hours. JNK activity was determined by using GST-cJUN (1-79) as a substrate.
  • T cells were cotransfected with vectors encoding HA-tagged JNK2 (500ng) and the indicated expression vector (2 ⁇ g). Twenty-four hours after the transfection cells were stimulated for 10 min with 10 ng/ml of TNF ⁇ and cells harvested at varying time points. JNK activity was assayed with GST-cJUN (1-79) as substrate.
  • Figure 7 depicts KRC is a negative regulator of endogenous TNF ⁇ expression.
  • Northern blotting analysis was performed using total RNA made from RAW cell lines transfected with an empty vector as a control and from a panel of 9 independent RAW clones stably transfected with full-length KRC (upper) and 3 RAW clones stably transfected with dominant negative KRC (lower). The blot was probed with a TNF ⁇ cDNA and with HPRT as loading control.
  • FIG 8 depicts KRC is present in both cytosol and nucleus.
  • GFP-tagged KRC was stably transfected into NIH 3T3 cells, and cells examined by fluorescence microscopy immediately after trypsinization (left panel) or after adherence to glass slides (right panel).
  • Figure 9 depicts that KRC is Thl -specific. RT-PCR analysis of KRC expression in primary T cells was performed. KRC expression was measured at 24 hours and 72 hours. The results demonstrate that KRC expression is rapidly lost in Th2 cells at 72 hours whereas KRC expression in Thl cells is maintained at 72 hours.
  • Figure 10 depicts KRC activates T cells. KRC was transfected into Jurkat T cells and CD69 expression was measured by FACS analysis. The results show that KRC overexpression increases CD69 expression in Jurkat T cells.
  • Figure 11 depicts KRC increases IL-2 gene transcription in the presence of PMA/Ionomycin and does so primarily through activating AP-1 with no contribution from NFAT.
  • Figure 11(A) shows IL-2 promoter transactivation by KRC in Jurkat T cells activated by PMA/Ionomycin.
  • Figure 11(B) shows transactivation of a composite NFAT- API reporter by KRC.
  • Figure 11(C) shows tranactivation of an AP-1 reporter by KRC.
  • Figure 12 depicts KRC increases IL-2 gene transcription in the presence of B cell antigen presenting cells and superantigen SEE and does so primarily through activating AP-1 with no contribution from NFAT.
  • Figure 12(A) shows IL-2 promoter transactivation by KRC in Jurkat T cells activated by the Raji B cell APC line and the superantigen SEE.
  • Figure 12(B) shows transactivation of a composite NFAT- API reporter by KRC.
  • Figure 12(C) shows transactivation of an AP-1 reporter by KRC.
  • Figure 13 depicts increased IL-2 production in Jurkat T cells stably expressing
  • FIG. 13(A) shows increased IL-2 protein by transfection of KRC in Jurkat T cells.
  • Figure 13(B) shows increased IL-2 protein by retroviral transduction of KRC into primary CD4 T cells.
  • Figure 14 depicts KRC transactivation of AP-1 response element depends on Ras, Raf and PKC-theta signaling molecules.
  • Figure 14(A) shows KRC transactivation of the AP-1 reporter is blocked by dominant negative Ras and Raf.
  • Figure 14(B) shows KRC transactivation of the AP-1 reporter is blocked by dominant negative PKC-theta and by the specific PKC-theta inhibitor Rottlerin.
  • FIG 15 depicts KRC controls PKC-theta and IL-2 expression.
  • RT-PCR of KRC transfected Jurkat clones was performed. The results show increased PKC-theta expression.
  • Figure 16 depicts KRC increases actin polymerization. Immunofluorescence of F-actin upon KRC overexpression in Jurkat T cells was performed. The results show the reorganization of F-actin filaments in KRC transfected Jurkat T cells.
  • the present invention is based, at least in part, on the discovery that KRC molecules regulate a wide variety of cellular processes, including inhibiting NFkB transactivation, increasing TNF-alpha induced apoptosis, inhibiting JNK activation, inhibiting endogenous TNF-alpha expression, activating immune cell proliferation and immune cell activation (e.g., in Thl cells), activating IL-2 expression e.g., by activating the AP-1 transcription factor, activating the Ras and Rac oncogenes, regulating PKC theta activity and increasing actin polymerization.
  • the present invention also demonstrates that that KRC interacts with TRAF molecules.
  • the interaction between KRC and TRAF involves the C domain of TRAF and amino acid residues 204 to 1055 of KRC.
  • the KRC protein The KRC protein:
  • the KRC protein (for KB binding and putative recognition component of the V(D)J Rss) is a DNA binding protein comprised of 2282 amino acids. KRC has been found to be present in T cells, B cells, and macrophages.
  • the KRC cDNA sequence is set forth in SEQ ID NO:l .
  • the amino acid sequence of KRC is set forth in SEQ ID NO:2.
  • KRC is a member of a family of zinc finger proteins that bind to the kB motif (Bachmeyer, C, et al, 1999. Nuc. Acids. Res. 27(2):643-648).
  • Zinc finger proteins are divided into three classes represented by KRC and the two MHC Class I gene enhancer binding proteins, MBP1 and MBP2 (Bachmeyer, C, et al., 1999. Nuc. Acids. Res. 27(2):643-648).
  • Zinc finger proteins are identified by the presence of highly conserved Cys2His2 zinc fingers.
  • the zinc fingers are an integral part of the DNA binding structure called the ZAS domain.
  • the ZAS domain is comprised of a pair of zinc fingers, a glutamic acid/aspartic acid-rich acidic sequence and a serine/threonine rich sequence.
  • the ZAS domains have been shown to interact with the kB like czs-acting regulatory elements found in the promoter or enhancer regions of genes.
  • the genes targeted by these zinc finger proteins are mainly involved in immune responses.
  • the KRC ZAS domain in particular, has a pair of Cys2-His2 zinc fingers followed by a glutamic acid/aspartic acid-rich acidic sequence and five copies of the serine/threonine-proline-X-arginine/lysine sequence.
  • Southwestern blotting experiments, EMS A and methylation interference analysis has also demonstrated that KRC recombinant proteins bind to the KB motif as well as to the Rss sequence (Bachmeyer, et al. 1999. Nuc. Acid Res. 27, 643-648; Wu et al. 1998. Science 281, 998-1001) and do so in highly ordered complexes (Mak, C. H., et al. 1994. Nuc.AcidRes. 22, 383-390..; Wu et al. 1998. Science 281, 998-1001).
  • KRC has recently been shown to regulate transcription of the mouse metastasis-associated gene, sl00A4/mtsl*, by binding to the Sb element (a kB like sequence) of the gene. (Hjelmsoe, I., et al. 2000. J Biol. Chem. 275(2): 913-920). KRC is regulated by post- translational modification as evidenced by the fact that pre-B cell nuclear protein kinases phosphorylate KRC proteins on serine and tyrosine residues.
  • Phosphorylation increases DNA binding, providing a mechanism by which KRC may respond to signals transmitted from the cell surface (Bachmeyer, C, et al., 1999. Nuc. Acids. Res. 27(2):643-648).
  • PKA cyclic AMP-dependent protein kinase A
  • PKC protein kinase C
  • MAP mitogen-activated protein
  • KRC Calcium-dependent protein kinase
  • CaM-kinase II Calcium-dependent protein kinase II
  • c-raf-protooncogene KRC is known to be a substrate for epidermal growth factor receptor kinase and p34cdc2 kinase in vitro.
  • TRAFs for tumor necrosis factor receptor associated factors, which participate in the TNFR signal transduction cascade.
  • TRAFs polypeptide factors
  • Six members of the TRAF family of proteins have been identified in mammalian cells (reviewed in Arch, R.H., et al. 1998. Genes Dev. 12, 2821-2830). All TRAF proteins, with the exception of TRAFl, contain an amino terminal RING finger domain with a characteristic pattern of cysteines and histidines that coordinate the binding of Zn2+ ions (Borden, K. L. B., et al. 1995. EMBO J 14, 1532-1521), which is followed by a stretch of multiple zinc fingers.
  • TRAFs share a highly conserved carboxy-terminal domain (TRAF-C domain) which is required for receptor binding and can be divided into two parts, a highly conserved domain which mediates homo and heterodimerization of TRAF proteins and also the association of the adapter proteins with their associated receptors and an amino-terminal half that displays a coiled-coil configuration.
  • TRAF molecules have distinct patterns of tissue distribution, are recruited by different cell surface receptors and have distinct functions as revealed most clearly by the analysis of TRAF-deficient mice (see Lomaga, M. A., et al. 1999. Genes Dev. 13, 1015-24; Nakano, H., et al. 1999. Proc. Natl. Acad. Sci.
  • Tumor necrosis factor is a cytokine produced mainly by activated macrophages which elicits a wide range of biological effects. These include an important role in endotoxic shock and in inflammatory, immunoregulatory, proliferative, cytotoxic, and anti-viral activities (reviewed by Goeddel, D. V. et al., 1986. Cold Spring Harbor Symposia on Quantitative Biology 51 : 597-609; Beutler, B. and Cerami, A.,
  • TNFR1 approximately 55 kDa receptor termed TNFR1
  • TNFR2 approximately 75 kDa receptor
  • Human and mouse cDNAs corresponding to both receptor types have been isolated and characterized (Loetscher, H. et al., 1990. Cell 61:351; Schall, T. J. et al., 1990. Cell 61: 361; Smith, C. A.
  • TNF ⁇ binds to two distinct receptors, TNFRl and TNFR2, but in most cell types NFKB activation and JNK/SAPK activation occur primarily through TNFRl .
  • TNFRl is known to interact with TRADD which functions as an adaptor protein for the recruitment of other proteins including RIP, a serine threonine kinase, and TRAF2.
  • TRADD functions as an adaptor protein for the recruitment of other proteins including RIP, a serine threonine kinase, and TRAF2.
  • TRAF2 TRAF5 and TRAF6 have all been linked to NFKB activation (Cao, Z., et al. 1996. Nature 383: 443-6; Rothe, M., et al. 1994. Cell 78: 681-692; Nakano, H., et al. 1996. J. Biol. Chem.
  • TRAF2 in particular has been linked to activation of the JNK/SAPK proteins as shown unequivocally by the failure of TNF ⁇ to activate this MAP kinase in cells lacking TRAF2 or expressing a dominant negative form of TRAF2 (Yeh, W. C, et al. 1997. Immunity 7: 715-725; Lee, S. Y. duplicate et al. 1997. Immunity 7.T-20).
  • KRC refers to KB binding and putative recognition component of the V(D)J Rss.
  • the nucleotide sequence of KRC is set forth in SEQ ID NO:l and the amino acid sequence of KRC is set forth in SEQ ID NO:2.
  • the amino acid sequence of the ZAS domain of KRC is set forth in SEQ ID NO: 3.
  • the amino acid sequence of KRC tr (amino acid residues 204 to 1055 of KRC) is set forth is as SEQ ID NO:5.
  • KRC unless specifically used to refer a specific SEQ ID NO, will be understood to refer to a KRC family polypeptide as defined below.
  • KRC family polypeptide is intended to include proteins or nucleic acid molecules having a KRC structural domain or motif and having sufficient amino acid or nucleotide sequence identity with a KRC molecule as defined herein.
  • family members can be naturally or non-naturally occurring and can be from the same or different species.
  • a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or, alternatively, can contain homologues of non-human origin.
  • Preferred members of a family may also have common functional characteristics.
  • Preferred KRC polypeptides comprise one or more of the following KRC characteristics: a pair of Cys2-His2 zinc fingers followed by a Glu- and Asp-rich acidic domain and five copies of the ser/Thr-Pro-X-Arg/Lys sequence thought to bind DNA.
  • KRC activity or "activity of a KRC polypeptide” includes the ability to modulate an immune response (e.g., by inhibiting or enhancing immune cell activation and/or proliferation, such as by modulating cytokine gene expression), cell survival (e.g., by modulating apoptosis), and/or the ability to modulate a signaling pathway (e.g., an NFkB signaling pathway, a JNK signaling pathway), the ability to modulate activity and/or expression of Ras and Rac oncogenes, the ability to modulate actin polymerization, and/or the ability to modulate PKC theta activity.
  • an immune response e.g., by inhibiting or enhancing immune cell activation and/or proliferation, such as by modulating cytokine gene expression
  • cell survival e.g., by modulating apoptosis
  • a signaling pathway e.g., an NFkB signaling pathway, a JNK signaling pathway
  • the term "modulate” includes alteration, e.g., by increasing or decreasing the particular parameter being described, e.g., KRC activity. Modulation includes both upmodulation and downmodulation.
  • KRC increases immune cell activation and cytokine production.
  • NFkB and JNK signaling pathways Inhibition of these pathways is associated with cellular inflammatory and apoptotic responses.
  • the KRC activity is a direct activity, such as an association with a KRC-target molecule or binding partner.
  • a "target molecule” or “binding partner” is a molecule with which a KRC protein binds or interacts in nature, such that KRC mediated function is achieved.
  • the term "TRAF” refers to TNF Receptor Associated Factor (See e.g. , Wajant et al, 1999, Cytokine Growth Factor Rev 10:15-26).
  • the "TRAF” family includes a family of cytoplasmic adapter proteins that mediate signal transduction from many members of the TNF-receptor superfamily and the interleukin-1 receptor (see e.g., Arch, R.H. et al., 1998, Genes Dev. 12:2821-2830).
  • the amino acid sequence comparison of TRAFl, TRAF2, TRAF3, TRAF4, TRAF5 AND TRAF6 is set forth in SEQ ID NO:8.
  • the term "TRAF C domain” refers to the highly conserved sequence motif found in TRAF family members. The amino acid sequence of the TRAF C domain is set forth in SEQ ID NO: 7.
  • the term "TRAF interacting portion of a KRC molecule” includes a region of KRC that interacts with TRAF.
  • a region of KRC that interacts with TRAF is amino acid residues 204- 1055 of KRC (SEQ ID NO:5)
  • the term "KRC interacting portion of a TRAF molecule” includes a region of TRAF that interacts with KRC.
  • a region of TRAF that interacts with KRC is the TRAF C domain.
  • modulate are intended to include stimulation (e.g. , increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
  • interact as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a yeast two hybrid assay.
  • interact is also meant to include "binding" interactions between molecules. Interactions may be protein-protein or protein-nucleic acid in nature.
  • the term "contacting" i.e., contacting a cell e.g. an immune cell, with an compound
  • contacting is intended to include incubating the compound and the cell together in vitro (e.g., adding the compound to cells in culture) and administering the compound to a subject such that the compound and cells of the subject are contacted in vivo.
  • the term "contacting” is not intended to include exposure of cells to a KRC modulator that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).
  • test compound includes a compound that has not previously been identified as, or recognized to be, a modulator of KRC activity and/or expression and/or a modulator of cell growth, survival, differentiation and/or migration.
  • library of test compounds is intended to refer to a panel comprising a multiplicity of test compounds.
  • cell free composition refers to an isolated composition which does not contain intact cells.
  • cell free compositions include cell extracts and compositions containing isolated proteins.
  • an "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • immune response includes immune cell-mediated (e.g., T cell and/or B cell-mediated) immune responses that are influenced by modulation of immune cell activation.
  • exemplary immune responses include B cell responses (e.g., antibody production), T cell responses (e.g., cytokine production and cellular cytotoxicity), and activation of cytokine responsive cells, e.g., macrophages.
  • an immune response is T cell mediated.
  • the term "downregulation" with reference to the immune response includes a diminution in any one or more immune responses, preferably T cell responses, while the term “upregulation” with reference to the immune response includes an increase in any one or more immune responses, preferably T cell responses. It will be understood that upregulation of one type of immune response may lead to a corresponding downregulation in another type of immune response. For example, upregulation of the production of certain cytokines (e.g., IL-10) can lead to downregulation of cellular immune responses.
  • cytokines e.g., IL-10
  • immune cell includes cells that are of hematopoietic origin and that play a role in the immune response, immune cells include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • T cell includes CD4 + T cells and CD8 + T cells.
  • T cell also includes both T helper 1 (Thl) type T cells and T helper 2 (Th2) type T cells.
  • antigen presenting cell include professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, and Langerhans cells) as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).
  • professional antigen presenting cells e.g., B lymphocytes, monocytes, dendritic cells, and Langerhans cells
  • other antigen presenting cells e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes.
  • the term "receptor” includes immune cell receptors that bind antigen, complexed antigen (e.g., in the context of MHC molecules), or antibodies.
  • activating receptors include T cell receptors (TCRs), B cell receptors (BCRs), cytokine receptors, LPS receptors, complement receptors, and Fc receptors.
  • T cell receptors are present on T cells and are associated with CD3 molecules. T cell receptors are stimulated by antigen in the context of MHC molecules (as well as by polyclonal T cell activating reagents).
  • T cell activation via the TCR results in numerous changes, e.g., protein phosphorylation, membrane .lipid changes, ion fluxes, cyclic nucleotide alterations, RNA transcription changes, protein synthesis changes, and cell volume changes.
  • the term "dominant negative” includes KRC molecules (e.g., portions or variants thereof) that compete with native (i.e., wild-type) KRC molecules, but which do not have KRC activity. Such molecules effectively decrease KRC activity in a cell.
  • the term "inflammation” includes a response to injury which results in a dilation of the blood capillaries, a decrease in blood flow and an accumulation of leucocytes at the site of injury.
  • apoptosis includes programmed cell death which can be characterized using techniques which are known in the art. Apoptotic cell death can be characterized, e.g., by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation. Cells undergoing apoptosis also display a characteristic pattern of internucleosomal DNA cleavage.
  • the term “modulating apoptosis” includes modulating programmed cell death in a cell, such as a epithelial cell.
  • the term “modulates apoptosis” includes either up regulation or down regulation of apoptosis in a cell. Modulation of apoptosis is discussed in more detail below and can be useful in ameliorating various disorders, e.g., neurological disorders.
  • NFkB signaling pathway refers to any one of the signaling pathways known in the art which involve activation or deactivation of the transcription factor NFkB, and which are at least partially mediated by the NFkB factor (Karin, 1998, Cancer Jfrom Scientific American, 4:92-99; Wallach et al, 1999, Ann Rev of Immunology, 17:331-367).
  • NFkB signaling pathways are responsive to a number of extracellular influences e.g. mitogens, cytokines, stress, and the like.
  • the NFkB signaling pathways involve a range of cellular processes, including, but not limited to, modulation of apoptosis.
  • These signaling pathways often comprise, but are by no means limited to, mechanisms which involve the activation or deactivation via phosphorylation state of an inhibitor peptide of NFkB (IkB), thus indirectly activating or deactivating NFkB.
  • JNK signaling pathway refers to any one of the signaling pathways known in the art which involve the Jun amino terminal kinase (JNK) (Karin, 1998, Cancer Jfrom Scientific American, 4:92-99; Wallach et al, 1999, Ann Rev of Immunology, 17:331-367).
  • JNK Jun amino terminal kinase
  • This kinase is generally responsive to a number of extracellular signals e.g. mitogens, cytokines, stress, and the like.
  • the JNK signaling pathways mediate a range of cellular processes, including, but not limited to, modulation of apoptosis.
  • JNK activation occurs through the activity of one or more members of the TRAF protein family (See, e.g., Wajant et al, 1999, Cytokine Growth Factor Rev 10:15-26).
  • nucleic acid is intended to include fragments or equivalents thereof (e.g., fragments or equivalents thereof of KRC or TRAF).
  • equivalent is intended to include nucleotide sequences encoding functionally equivalent KRC proteins, i.e., proteins which have the ability to bind to the natural ligand(s) of the KRC antigen.
  • a functionally equivalent KRC protein has the ability to bind TRAF in the cytoplasm of an immune cell, e.g., a T cell.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an “isolated” nucleic acid molecule is free of sequences which naturally flank the nucleic acid molecule (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) in the genomic DNA of the organism from which the nucleic acid molecule is derived.
  • an "isolated protein” or “isolated polypeptide” refers to a protein or polypeptide that is substantially free of other proteins, polypeptides, cellular material and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the KRC protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of KRC protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the nucleic acids of the invention can be prepared by standard recombinant DNA techniques.
  • a nucleic acid of the invention can also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which has been automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retro viruses, adeno viruses and adeno-associated viruses), which serve equivalent functions.
  • a host cell is intended to refer to a cell into which a nucleic acid molecule of the invention, such as a recombinant expression vector of the invention, has been introduced.
  • the terms "host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell is a mammalian cell, e.g., a human cell. In particularly preferred embodiments, it is a epithelial cell.
  • antibody is intended to include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which binds (immunoreacts with) an antigen, such as Fab and F(ab')2 fragments, single chain antibodies, intracellular antibodies, scFv, Fd, or other fragments.
  • antibodies of the invention bind specifically or substantially specifically to KRC or TRAF molecules (i.e., have little to no cross reactivity with non-KRC or non-TRAF molecules).
  • monoclonal antibodies and “monoclonal antibody composition”, as used herein, refer to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen
  • polyclonal antibodies and “polyclonal antibody composition” refer to a population of antibody molecules that contain multiple species of antigen binding sites capable of interacting with a particular antigen.
  • a monoclonal antibody compositions thus typically display a single binding affinity for a particular antigen with which it immunoreacts.
  • KRC associated disorder includes disorders in which KRC activity is aberrant or in which a non-KRC activity that would benefit from modulation of a KRC activity is aberrant.
  • KRC associated disorders involve aberrant proliferation of cells, e.g., excessive or unwanted proliferation of cells or deficient proliferation of cells.
  • KRC associated disorders are disorders such as inflammation. Examples of KRC associated disorders include: disorders involving aberrant or unwanted proliferation of cells, e.g., inflammation, neoplasia, apoptosis, or necrosis.
  • KRC associated disorders include carcinomas, adenocarcmomas, and other neoplasias.
  • KRC disorders may also include disorders that have been linked generally to aberrant TNF receptor activity or function, including Crohn's Disease (Baert and Rutgeerts, 1999, Int J Colorectal Dis, 14:47-51) and certain cardiovascular diseases (Ferrari, 1999, Pharmacol Res, 40:97-105). They may also include disorders characterized by uncontrolled or aberrant levels of apoptosis, for example myelokathexis (Aprikyan et al.
  • the nucleotide sequence of a DNA or RNA molecule coding for a KRC polypeptide of the invention can be used to derive the KRC amino acid sequence, using the genetic code to translate the DNA or RNA molecule into an amino acid sequence.
  • corresponding nucleotide sequences that can encode KRC protein can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence).
  • description and/or disclosure herein of a KRC nucleotide sequence should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
  • KRC amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
  • One aspect of the invention pertains to isolated nucleic acid molecules that encode KRC proteins" or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify KRC -encoding nucleic acids (e.g., KRC mRNA) and fragments for use as PCR primers for the amplification or mutation of KRC nucleic acid molecules. It will be understood that in discussing the uses of KRC nucleic acid molecules, e.g., as shown in SEQ.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single- stranded or double-stranded, but preferably is double-stranded DNA.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or portion of the nucleic acid sequence of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide as a hybridization probe, KRC nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E..F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide respectively.
  • Nucleic acid sequences encoding other KRC family polypeptides can be identified based on nucleic acid and/or amino acid identity with KRC, possession of KRC domains, and/or possession of a KRC activity as defined herein.
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to KRC nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NOT a nucleic acid molecule encoding another KRC family polypeptide.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide or a portion of any of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO or a nucleotide sequence encoding another KRC family polypeptide is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide respectively, such that it can hybridize to the nucleotide sequence shown in SEQ ID NO or a nucleotide sequence encoding another KRC family polypeptide respectively, thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide or a portion thereof , e.g.
  • nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NOT a nucleic acid molecule encoding another KRC family polypeptide for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a KRC protein.
  • the nucleotide sequence determined from the cloning of the KRC genes allows for the generation of probes and primers designed for use in identifying and/or cloning yet other KRC family members, as well as KRC family homologues from other species.
  • the probe/primer typically comprises a substantially purified oligonucleotide.
  • the oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, 75, or 100 consecutive nucleotides of a sense sequence of SEQ ID NO or a nucleotide sequence encoding another KRC family polypeptide or of a naturally occurring allelic variant or mutant of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide.
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide or the complement thereof.
  • a nucleic acid molecule of the invention comprises at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or more contiguous nucleotides of SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide.
  • a nucleic acid molecule of the invention has at least 70% identity, more preferably 80% identity, and even more preferably 90% identity with a nucleic acid molecule comprising: at least about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or about 1500 nucleotides of SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide.
  • Probes based on the KRC nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues, particularly epithelial cells or tissues, particularly epithelial cells or tissues, which misexpress a KRC protein, such as by measuring a level of a KRC -encoding nucleic acid in a sample of cells from a subject e.g., detecting KRC mRNA levels or determining whether a genomic KRC gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of a KRC protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide which encodes a polypeptide having a KRC biological activity (e.g., the ability to modulate proliferation, apoptosis, and/or signaling via an NFkB or JNK signaling pathway), expressing the encoded portion of the KRC protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the KRC protein.
  • a KRC biological activity e.g., the ability to modulate proliferation, apoptosis, and/or signaling via an NFkB or JNK signaling pathway
  • Nucleic acid molecules that differ from SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide due to degeneracy of the genetic code, and thus encode the same KRC protein as that encoded by SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide are encompassed by the invention. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence of another KRC family polypeptide.
  • KRC nucleotide sequence shown in SEQ ID NO or a nucleotide sequence encoding another KRC family polypeptide DNA sequence polymorphisms that lead to changes in the amino acid sequences of the KRC proteins may exist within a population (e.g., the human population). Such genetic polymorphism in the KRC genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a KRC protein, preferably a mammalian KRC protein, and can further include non-coding regulatory sequences, and introns.
  • Such natural allelic variations include both functional and non-functional KRC proteins and can typically result in 1-5% variance in the nucleotide sequence of a KRC gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in KRC genes that are the result of natural allelic variation and that do not alter the functional activity of a KRC protein can be used in the claimed methods.
  • nucleic acid molecules encoding other KRC family members and, thus, which have a nucleotide sequence which differs from the KRC family sequence of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide are intended to be within the scope of the invention.
  • nucleic acid molecules encoding KRC proteins from different species, and thus which have a nucleotide sequence which differs from the KRC sequence of SEQ ID NO: 1 or a nucleotide sequence encoding another KRC family polypeptide can be used in the claimed methods.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the KRC molecules of the invention can be isolated, e.g., based on their homology to the KRC nucleic acids disclosed herein using the cDNAs disclosed herein, or portions thereof, as a hybridization probe according to standard hybridization techniques.
  • a KRC DNA can be isolated from a human genomic DNA library using all or portion of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., et al. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of a KRC gene can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide.
  • mRNA can be isolated from cells (e.g., by the guanidinium- fhiocyanate extraction procedure of Chirgwin et al, 1979, Biochemistry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence shown in SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide.
  • a nucleic acid molecule of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to a KRC nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention can be identified based on shared nucleotide sequence identity using a mathematical algorithm. Such algorithms are outlined in more detail below.
  • an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide or its complement.
  • the nucleic acid molecule is at least 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non- limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO or a nucleic acid molecule encoding another KRC family polypeptide or its complement corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleotide sequence that occurs in nature e.g., encodes a natural protein.
  • DNA sequence polymorphisms that lead to minor changes in the nucleotide or amino acid sequences of a KRC may exist within a population.
  • Such genetic polymorphism in a KRC gene may exist among individuals within a population due to natural allelic variation.
  • Such natural allelic variations can typically result in 1-2 % variance in the nucleotide sequence of the gene.
  • Such nucleotide variations and resulting amino acid polymorphisms in a KRC that are the result of natural allelic variation and that do not alter the functional activity of a KRC polypeptide are within the scope of the invention.
  • allelic variants of KRC sequences that may exist in the population, the skilled artisan will further appreciate that minor changes may be introduced by mutation into nucleotide sequences, e.g., of SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide, thereby leading to changes in the amino acid sequence of the encoded protein, without altering the functional activity of a KRC protein.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues may be made in the sequence of SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a KRC nucleic acid molecule (e.g., the sequence of SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide) without altering the functional activity of a KRC molecule.
  • Exemplary residues which are non-essential and, therefore, amenable to substitution can be identified by one of ordinary skill in the art by performing an amino acid alignment of KRC-related molecules and determining residues that are not conserved. Such residues, because they have not been conserved, are more likely amenable to substitution. Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding KRC proteins that contain changes in amino acid residues that are not essential for a KRC activity. Such KRC proteins differ in amino acid sequence from SEQ ID NO: 2 or an amino acid sequence of another KRC family polypeptide yet retain an inherent KRC activity.
  • An isolated nucleic acid molecule encoding a non-natural variant of a KRC protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO: 1 or a nucleic acid molecule encoding another KRC family polypeptide by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g. , threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • a nonessential amino acid residue in a KRC is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a KRC coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for their ability to bind to DNA and/or activate transcription, to identify mutants that retain functional activity.
  • the encoded a KRC mutant protein can be expressed recombinantly in a host cell and the functional activity of the mutant protein can be determined using assays available in the art for assessing a KRC activity.
  • Yet another aspect of the invention pertains to isolated nucleic acid molecules encoding a KRC fusion proteins.
  • nucleic acid molecules comprising at least a first nucleotide sequence encoding a full-length KRC protein, polypeptide or peptide having a KRC activity operatively linked to a second nucleotide sequence encoding a non- KRC protein, polypeptide or peptide, can be prepared by standard recombinant DNA techniques.
  • a mutant KRC protein can be assayed for KRC activity as described herein.
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire KRC coding strand, or only to a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding KRC.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding KRC.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of KRC mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of KRC mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of KRC mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- meth
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a KRC protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, 1988, Nature 334:585-591)) can be used to catalytically cleave KRC mRNA transcripts to thereby inhibit translation of KRC mRNA.
  • a ribozyme having specificity for a KRC -encoding nucleic acid can be designed based upon the nucleotide sequence of SEQ ID NO or 3 a nucleic acid molecule encoding another KRC family polypeptide.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a KRC- encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 ; and Cech et al. U.S. Patent No. 5,116,742.
  • KRC mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W., 1993, Science 261:1411-1418.
  • gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of KRC (e.g., the KRC promoter and/or enhancers) to form triple helical structures that prevent transcription of the KRC gene in target cells.
  • KRC e.g., the KRC promoter and/or enhancers
  • the KRC nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al., 1996, Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. , 1996, supra; Perry-O'Keefe et al, 1996, Proc. Natl Acad. Sci. USA 93: 14670-675.
  • PNAs of KRC nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of KRC nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA- directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B., 1996, supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al, 1996, supra; Perry- O'Keefe supra).
  • PNAs of KRC can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of KRC nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B., 1996, supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B., 1996, supra and Finn P.J. et al, 1996, Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al, 1989, Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al, 1996, supra). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al, 1975, Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • modified nucleoside analogs e.g., 5'-(4-me
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl Acad. Sci. US. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al, 1989, Proc. Natl Acad. Sci. US. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sci.
  • oligonucleotides can be modified with hybridization- triggered cleavage agents (See, e.g., Krol et al, 1988, Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • Antisense polynucleotides may be produced from a heterologous expression cassette in a transfectant cell or transgenic cell.
  • the antisense polynucleotides may comprise soluble oligonucleotides that are administered to the external milieu, either in the culture medium in vitro or in the circulatory system or in interstitial fluid in vivo. Soluble antisense polynucleotides present in the external milieu have been shown to gain access to the cytoplasm and inhibit translation of specific mRNA species.
  • Isolated KRC Proteins, Fragments Thereof, and Anti-KRC Antibodies Isolated KRC proteins, and biologically active portions thereof can also be used as modulating agents, as well as polypeptide fragments suitable for use as immunogens to raise anti-KRC antibodies.
  • native KRC proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • KRC proteins are produced by recombinant DNA techniques.
  • a KRC protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques. It will be understood that in discussing the uses of KRC proteins (e.g., as shown in SEQ. ID NO.
  • KRC polypeptides that are not full length KRC polypeptides (e.g., that comprise one or more KRC domains, e.g. a domain comprising amino acid residues corresponding to residues 204-1055 of SEQ ID NO:2) are included.
  • the KRC proteins comprise the amino acid sequence encoded by SEQ ID NOT or a nucleotide sequence encoding another KRC family polypeptide or a portion thereof.
  • the protein comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence of another KRC family polypeptide or a portion thereof.
  • the protein has at least 50%, at least 60 % amino acid identity, more preferably 70% amino acid identity, more preferably 80%, and even more preferably, 90% or 95% amino acid identity with the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence of another KRC family polypeptide or a portion thereof, e.g., the consensus domains set forth above.
  • KRC polypeptide molecules are biologically active, i.e., encode a portion of the KRC polypeptide having the ability to modulate cell survival, proliferation, differentiation and/or motility.
  • the cell is a T cell, e.g., a Thl cell.
  • Biologically active portions of a KRC protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the KRC protein, which include less amino acids than the full length KRC proteins, and exhibit at least one activity of a KRC protein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence.
  • the residues at corresponding positions are then compared and when a position in one sequence is occupied by the same residue as the corresponding position in the other sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which are introduced for optimal alignment of the two sequences.
  • amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology”.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm utilized for comparison of sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad.
  • Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Research 25(17):3389.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Another preferred, non-limiting algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • Another non-limiting example of a mathematical algorithm utilized for the alignment of protein sequences is the Lipman-Pearson algorithm (Lipman and Pearson, 1985, Science 227:1435). When using the Lipman-Pearson algorithm, a PAM250 weight residue table, a gap length penalty of 12, a gap penalty of 4, and a Kutple of 2 can be used.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the alignment of nucleic acid sequences is the Wilbur-Lipman algorithm (Wilbur and Lipman, 1983, Proc. Natl. Acad. Sci.
  • the percent identity between two amino acid sequences is determined using the GAP program in the GCG software package, using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • Protein alignments can also be made using the Geneworks global protein alignment program (e.g., version 2.5.1) with the cost to open gap set at 5, the cost to lengthen gap set at 5, the minimum diagonal length set at 4, the maximum diagonal offset set at 130, the consensus cutoff set at 50% and utilizing the Pam 250 matrix.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to 5 for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. , 1990, J Mol Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the nucleotide sequences of the invention can be analyzed using the default BLASTN matrix 1-3 with gap penalties set at: existence 11 and extension 1.
  • the amino acid sequences of the invention can be analyzed using the default settings: the Blosum62 matrix with gap penalties set at existence 11 and extension 1. See http://www.ncbi.nlm.nih.gov.
  • KRC chimeric or fusion proteins As used herein, a KRC "chimeric protein” or “fusion protein” comprises a KRC polypeptide operatively linked to a non- KRC polypeptide.
  • An " KRC polypeptide” refers to a polypeptide having an amino acid sequence corresponding to KRC polypeptide
  • a “non- KRC polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the KRC protein, e.g., a protein which is different from the KRC protein and which is derived from the same or a different organism.
  • the KRC polypeptide can correspond to all or a portion of a KRC protein.
  • a KRC fusion protein comprises at least one biologically active portion of a KRC protein, e.g., a KRC consensus domain.
  • a KRC consensus domain e.g., a KRC consensus domain.
  • the term "operatively linked" is intended to indicate that the KRC polypeptide and the non-KRC polypeptide are fused in-frame to each other.
  • the non-KRC polypeptide can be fused to the N-terminus or C-terminus of the KRC polypeptide.
  • the fusion protein is a GST-KRC member fusion protein in which the KRC member sequences are fused to the C-terminus of the GST sequences.
  • the fusion protein is a KRC -HA fusion protein in which the KRC member nucleotide sequence is inserted in a vector such as pCEP4- HA vector (Herrscher, R.F. et al, 1995, Genes Dev. 9:3067-3082) such that the KRC member sequences are fused in frame to an influenza haemagglutinin epitope tag.
  • pCEP4- HA vector Herrscher, R.F. et al, 1995, Genes Dev. 9:3067-3082
  • Such fusion proteins can facilitate the purification of a recombinant KRC member.
  • Fusion proteins and peptides produced by recombinant techniques may be secreted and isolated from a mixture of cells and medium containing the protein or peptide. Alternatively, the protein or peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a cell culture typically includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. Protein and peptides can be isolated from cell culture media, host cells, or both using techniques known in the art for purifying proteins and peptides. Techniques for transfecting host cells and purifying proteins and peptides are known in the art.
  • a KRC fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide or an HA epitope tag).
  • a KRC encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the KRC protein.
  • the fusion protein is a KRC protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of KRC can be increased through use of a heterologous signal sequence.
  • the KRC fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • Use of KRC fusion proteins may be useful therapeutically for the treatment of disorders, e.g., as soluble antagonists of the KRC ligand. Disorders that would benefit from such treatment include, e.g. cancer or Alzheimer's disease.
  • Such Fc fusion proteins can be used as soluble antagonists of the KRC ligand.
  • the KRC-fusion proteins of the invention can be used as immunogens to produce anti- KRC antibodies in a subject.
  • a KRC chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a KRC- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the KRC protein.
  • a KRC-Fc fusion protein can be made using techniques that are known in the art.
  • a soluble KRC-Fc fusion protein can be constructed by joining the cDNA sequence encoding the extracellular region of KRC to the hinge-C H 2-C H 3 regions of human immunoglobulin (Ig). Any isotype may be used in making such a construct, for example, Fc ⁇ l, ⁇ 2, ⁇ 3, ⁇ or ⁇ .
  • Cells can be transfected with a plasmid carrying the KRC-Ig construct, cultured, and conditioned medium harvested. The fusion protein can then be purified, e.g., using a column of immobilized protein A.
  • allotypic variants of Fc sequences could be used to construct Fc fusion proteins.
  • mutations which block effector functions such as, for example, complement and Fc receptor binding (Armour et al, 1999, Eur. J. Immunol, 29:2613; Morgan et al, 1995, Immunology 86: 319; Lund et al, 1991, J. Immunol. 147:2657) could be incorporated into a fusion protein.
  • the present invention also pertains to variants of the KRC proteins which function as either KRC agonists (mimetics) or as KRC antagonists.
  • Variants of the KRC proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a KRC protein.
  • An agonist of the KRC proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a KRC protein.
  • An antagonist of a KRC protein can inhibit one or more of the activities of the naturally occurring form of the KRC protein by, for example, competitively modulating a cellular activity of a KRC protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the KRC protein.
  • the invention pertains to derivatives of KRC which may be formed by modifying at least one amino acid residue of KRC by oxidation, reduction, or other derivatization processes known in the art.
  • variants of a KRC protein which function as either KRC agonists (mimetics) or as KRC antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a KRC protein for KRC protein agonist or antagonist activity.
  • a variegated library of KRC variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of KRC variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential KRC sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of KRC sequences therein.
  • a degenerate set of potential KRC sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of KRC sequences therein.
  • methods which can be used to produce libraries of potential KRC variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential KRC sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A., 1983, Tetrahedron 39:3; Itakura et al, 1984, Annu. Rev. Biochem. 53:323; Itakura et al, 1984, Science 198:1056; Ike et al, 1983, Nucleic Acid Res. 11 :477).
  • libraries of fragments of a KRC protein coding sequence can be used to generate a variegated population of KRC fragments for screening and subsequent selection of variants of a KRC protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a KRC coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the KRC protein.
  • REM Recursive ensemble mutagenesis
  • cell based assays can be exploited to analyze a variegated KRC library.
  • a library of expression vectors can be transfected into a cell line which ordinarily synthesizes and secretes KRC.
  • the transfected cells are then cultured such that KRC and a particular mutant KRC are secreted and the effect of expression of the mutant on KRC activity in cell supematants can be detected, e.g., by any of a number of enzymatic assays.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of KRC activity, and the individual clones further characterized.
  • KRC polypeptides consisting only of naturally-occurring amino acids
  • KRC peptidomimetics are also provided.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics” or “peptidomimetics” (Fauchere, J., 1986, Adv. Drug Res. 15: 29; Veber and Freidinger, 1985, TINS p.392; and Evans et al, 1987, J. Med. Chem 30: 1229, which are incorporated herein by reference) and are usually developed with the aid of computerized molecular modeling.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling.
  • a spacer e.g., an amide group
  • Such non- interfering positions generally are positions that do not form direct contacts with the macromolecules(s) to which the peptidomimetic binds to produce the therapeutic effect.
  • Derivitization (e.g., labeling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.
  • Systematic substitution of one or more amino acids of a KRC amino acid sequence with a D-amino acid of the same type may be used to generate more stable peptides.
  • constrained peptides comprising a KRC amino acid sequence or a substantially identical sequence variation may be generated by methods known in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61: 387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • KRC polypeptides identified herein will enable those of skill in the art to produce polypeptides corresponding to KRC peptide sequences and sequence variants thereof.
  • Such polypeptides may be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding a KRC peptide sequence, frequently as part of a larger polypeptide. Alternatively, such peptides may be synthesized by chemical methods.
  • Peptides can be produced, typically by direct chemical synthesis, and used e.g., as agonists or antagonists of a KRC/KRC binding protein interaction. Peptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terminus.
  • either the carboxy-terminus or the amino-terminus, or both are chemically modified.
  • the most common modifications of the terminal amino and carboxyl groups are acetylation and amidation, respectively.
  • Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, may be incorporated into various embodiments of the invention.
  • Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others.
  • Peptides may be used therapeutically to treat disease, e.g., by altering the process of cell proliferation, differentiation or apoptosis in a cell population of a patient.
  • KRC protein can also be used as an immunogen to generate antibodies that bind KRC using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length KRC protein can be used or, alternatively, the invention provides antigenic peptide fragments of KRC for use as immunogens.
  • the antigenic peptide of KRC comprises at least 8 amino acid residues and encompasses an epitope of KRC such that an antibody raised against the peptide forms a specific immune complex with KRC.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • an antigenic peptide fragment of a KRC polypeptide can be used as the immunogen.
  • An antigenic peptide fragment of a KRC polypeptide typically comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence of another KRC family polypeptide and encompasses an epitope of a KRC polypeptide such that an antibody raised against the peptide forms an immune complex with a KRC molecule.
  • Preferred epitopes encompassed by the antigenic peptide are regions of KRC that are located on the surface of the protein, e.g., hydrophilic regions.
  • an antibody binds substantially specifically to a KRC molecule.
  • an antibody binds specifically to a KRC polypeptide.
  • the antigenic peptide comprises at least about 10 amino acid residues, more preferably at least about 15 amino acid residues, even more preferably at least 20 about amino acid residues, and most preferably at least about 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of a KRC polypeptide that are located on the surface of the protein, e.g., hydrophilic regions, and that are unique to a KRC polypeptide.
  • such epitopes can be specific for a KRC proteins from one species, such as mouse or human (i.e., an antigenic peptide that spans a region of a KRC polypeptide that is not conserved across species is used as immunogen; such non conserved residues can be determined using an alignment such as that provided herein).
  • a standard hydrophobicity analysis of the protein can be performed to identify hydrophilic regions.
  • a KRC immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, a recombinantly expressed KRC protein or a chemically synthesized KRC peptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic KRC preparation induces a polyclonal anti- KRC antibody response.
  • another aspect of the invention pertains to anti- KRC family polypeptide antibodies and methods of their use. Such antibodies can be used as agonists and/or antagonists of KRC family polypeptides.
  • antibodies specifically recognize KRC and not another KRC family polypeptide.
  • Polyclonal anti-KRC antibodies can be prepared as described above by immunizing a suitable subject with a KRC immunogen.
  • the anti-KRC antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized a KRC polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against a KRC polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (Kohler and Milstein, 1975, Nature 256:495-497) (see also, Brown et al, 1981, J Immunol 127:539-46; Brown et ⁇ /., 1980, JBiol Chem 255:4980-83; Yeh et al, 1976, Proc. Natl. Acad. Sci USA 76:2927-31; and Yeh et al, 1982, Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supematants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds specifically to a KRC polypeptide.
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or S ⁇ 2/O-Agl4 myeloma lines.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind a KRC molecule, e.g., using a standard ELISA assay.
  • a monoclonal anti-KRC antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a KRC to thereby isolate immunoglobulin library members that bind a KRC polypeptide.
  • Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene S ⁇ r ⁇ APTM . Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791 ; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al International Publication No. WO 92/01047; Garrard et al International Publication No.
  • WO 92/09690 Ladner et al International Publication No. WO 90/02809; Fuchs et al , 1991, Bio/Technology 9:1370-1372; Hay et al, 1992, Hum Antibod Hybridomas 3:81- 85; Huse et al, 1989, Science 246:1275-1281; Griffiths et al, 1993, EMBOJ 12:725- 734; Hawkins et al, 1992, JMolBiol 226:889-896; Clarkson et al, 1991, Nature 352:624-628; Gram et al, 1992, Proc. Natl Acad.
  • recombinant anti- KRC antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Patent Publication PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT Application WO 86/01533; Cabilly et al U.S.
  • Patent 5,225,539 Jones et al, 1986, Nature 321:552-525; Verhoeyan et al, 1988, Science 239:1534; and Beidler et al, 1988, J. Immunol. 141:4053-4060.
  • humanized antibodies can be made according to standard protocols such as those disclosed in US patent 5,565,332.
  • antibody chains or specific binding pair members can be produced by recombination between vectors comprising nucleic acid molecules encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicable generic display package and vectors containing nucleic acid molecules encoding a second polypeptide chain of a single binding pair member using techniques known in the art, e.g., as described in US patents 5,565,332, 5,871,907, or 5,733,743.
  • An anti- KRC antibody e.g. , monoclonal antibody
  • KRC polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.
  • Anti- KRC antibodies can facilitate the purification of natural KRC polypeptides from cells and of recombinantly produced KRC polypeptides expressed in host cells.
  • an anti- KRC antibody can be used to detect a KRC protein (e.g., in a cellular lysate or cell supernatant). Detection may be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • an anti- KRC antibody of the invention is labeled with a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ - galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125 I, 13 T, 35 S or 3 H.
  • anti-KRC antibodies can be used, e.g., intracellularly to inhibit protein activity.
  • intracellular antibodies to inhibit protein function in a cell is known in the art (see e.g., Carlson, J. R, 1988, Mol Cell. Biol. 8:2638-2646; Biocca, S. et al, 1990, EMBO J. 9:101-108; Werge, T.M. et al, 1990, FEBS Letters 274:193-198; Carlson, J.R., 1993, Proc. Natl. Acad. Sci. USA
  • a recombinant expression vector is prepared which encodes the antibody chains in a form such that, upon introduction of the vector into a cell, the antibody chains are expressed as a functional antibody in an intracellular compartment of the cell.
  • an intracellular antibody that specifically binds the KRC protein is expressed in the cytoplasm of the cell.
  • antibody light and heavy chain cDNAs encoding antibody chains specific for the target protein of interest, e.g., KRC are isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the KRC protein.
  • Hybridomas secreting anti- KRC monoclonal antibodies, or recombinant anti- KRC monoclonal antibodies can be prepared as described above.
  • a monoclonal antibody specific for KRC protein e.g., either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library
  • DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques.
  • light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening.
  • cDNA encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated during the library screening process.
  • Nucleotide sequences of antibody light and heavy chain genes from which PCR primers or cDNA library probes can be prepared are known in the art. For example, many such sequences are disclosed in Kabat, E.A., et al, 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and in the "Vbase" human germline sequence database.
  • an intracellular antibody expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly. In another embodiment, the vector encodes a full-length light chain but only the VH/CH1 region of the heavy chain such that a Fab fragment is expressed intracellularly.
  • the vector encodes a single chain antibody (scFv) wherein the variable regions of the light and heavy chains are linked by a flexible peptide linker (e.g., (Gly4Ser)3) and expressed as a single chain molecule.
  • a single chain antibody e.g., (Gly4Ser)3
  • the expression vector encoding the anti- KRC intracellular antibody is introduced into the cell by standard transfection methods, as discussed herein.
  • an antibody or antibody portion of the invention can be derivatized or linked to another functional molecule (e.g., a peptide or polypeptide). Accordingly, the antibodies and antibody portions of the invention are intended to include derivatized and otherwise modified forms of the anti-KRC antibodies described herein, including, e.g., antibodies conjugated to other molecules (e.g., antibodies or polypeptides which bind to other cell markers).
  • an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., to create a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • another antibody e.g., to create a bispecific antibody or a diabody
  • a detectable agent e.g., to create a bispecific antibody or a diabody
  • cytotoxic agent e.g., to create a bispecific antibody or a diabody
  • a pharmaceutical agent e.g., to create a bispecific antibody or a diabody
  • a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such
  • One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
  • Such linkers are available from Pierce Chemical Company, Rockford, IL.
  • Useful detectable agents with which an antibody or antibody portion of the invention may be derivatized include fluorescent compounds.
  • Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5- dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like.
  • An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable.
  • An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • Certain KRC modulating agents e.g., nucleic acid molecules
  • KRC modulating agents can be expressed by incorporating a KRC gene described herein into an expression vector and introducing the expression vector into an appropriate host cell. Accordingly, the invention further pertains to the use of expression vectors containing a nucleic acid encoding a KRC peptide and to host cells into which such expression vectors have been introduced.
  • An expression vector of the invention typically includes nucleotide sequences encoding the KRC peptide operably linked to at least one regulatory sequence.
  • operably linked and “operatively linked” are intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence in a host cell (or by a cell extract). Regulatory sequences are art-recognized and can be selected to direct expression of the desired protein in an appropriate host cell.
  • regulatory sequence is intended to include promoters, enhancers, polyadenylation signals and other expression control elements. Such regulatory sequences are known to those skilled in the art and are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transfected and/or the type and/or amount of protein desired to be expressed.
  • the KRC nucleic acid molecules are operably linked to regulatory sequences which allow their expression to be controlled by the addition or removal of an exogenous compound, e.g., tetracycline, as described herein in the Example section.
  • an exogenous compound e.g., tetracycline
  • Methods and sequences relating to the use of tetracycline controlled regulatory sequences can be found, for example, in Gossen, M. and Bujard, H (1992) Proc. Natl. Acad. Sci. USA 89(12):5547-51; Gossen, M. et al (1993) Trends Biochem. Sci. 18(12):471-5; Gossen, M. et al. (1994) Curr. Opin.
  • An expression vector of the invention can be used to transfect cells, either prokaryotic or eukaryotic (e.g., mammalian, insect or yeast cells) to thereby produce peptides encoded by nucleotide sequences of the vector. Expression in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters. Certain E.
  • coli expression vectors are designed to add a number of amino acid residues to the expressed recombinant protein, usually to the amino terminus of the expressed protein.
  • fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the target recombinant protein; and 3) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification.
  • fusion expression vectors examples include pGEX (Amrad Corp., Melbourne, Australia; also available from Pharmacia Corp.) and pMAL (New England Biolabs, Beverly, MA) which fuse glutathione S-transferase and maltose E binding protein, respectively, to the target recombinant protein. Accordingly, a KRC gene may be linked to additional coding sequences in a prokaryotic fusion vector to aid in the expression, solubility or purification of the fusion protein.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the target recombinant protein to enable separation of the target recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and tlieir cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Inducible non-fusion expression vectors include pTrc (Amann et_al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from the T7 gnlO-lac 0 fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize expression of recombinant KRC peptide in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy would be to alter the nucleotide sequence of the KRC peptide to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al, (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences are encompassed by the invention and can be carried out by standard DNA synthesis techniques.
  • a KRC peptide in another embodiment, can be expressed in a eukaryotic host cell, such as mammalian cells (e.g., T cells such as Jurkat cells, COS cells, Chinese hamster ovary cells (CHO) or NS0 cells), insect cells (e.g., using a baculovirus vector) or yeast cells.
  • a eukaryotic host cell is a Jurkat T cell.
  • Other suitable host cells may be found in Goeddel, (1990) supra or are known to those skilled in the art.
  • Eukaryotic, rather than prokaryotic, expression of a KRC peptide may be preferable since expression of eukaryotic proteins in eukaryotic cells can lead to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulf ⁇ de bonds of a recombinant protein.
  • the expression vector's control functions are often provided by viral material.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • COS cells Gluzman, Y, (1981) Cell 23:175-182
  • pCDM8 Seed, B., (1987) Nature 329:840
  • CHO dhfr" Chinese Hamster Ovary
  • pMT2PC Kaufman et al. (1987), EMBO J. 6:187-195
  • a preferred cell line for production of recombinant protein is the NS0 myeloma cell line available from the ECACC (catalog #85110503) and described in Galfre, G.
  • yeast examples include pYepSecl (Baldari. et al, (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al, (1983) Mol Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M.D., (1989) Virology 170:31 -39).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker may be introduced into a host cell on the same plasmid as the gene of interest or may be introduced on a separate plasmid.
  • Cells containing the gene of interest can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). The surviving cells can then be screened for production of KRC peptides by, for example, Western blotting or immunoprecipitation from cell supernatant with an anti- KRC monoclonal antibody.
  • the invention also features methods of producing KRC peptides. For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding a KRC peptide can be cultured in a medium under appropriate conditions to allow expression of the protein to occur.
  • a recombinant expression vector containing DNA encoding a KRC peptide is produced.
  • Peptides produced by recombinant technique may be secreted and isolated from a mixture of cells and medium containing the protein.
  • the protein may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a cell culture typically includes host cells, media and other byproducts. Suitable mediums for cell culture are well known in the art. Protein can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins.
  • a KRC peptide can be synthesized chemically using standard peptide synthesis techniques known in the art.
  • Modulators of KRC activity can be known (e.g., dominant negative inhibitors of KRC activity, antisense KRC intracellular antibodies that interfere with KRC activity, peptide inhibitors derived from KRC) or can be identified using the methods described herein.
  • the invention provides methods (also referred to herein as a "screening assay") for identifying other modulators, i.e., candidate or test compounds or agents (e.g., peptidomimetics, small molecules or other drugs) which modulate KRC activity and for testing or optimizing the activity of other agents.
  • the invention provides assays for screening candidate or test compounds which modulate the activity of the KRC polypeptide.
  • the invention provides assays for screening candidate or test compounds which have a stimulatory or inhibitory effect on KRC activity.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • test modulating agent can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with upstream or downstream elements.
  • Methods and services for high-throughput drug screening can be found commercially, for example, from companies such as GPC Biotech (Martinsried, Germany; Cambridge, MA; and Princeton, NJ), Upstate Discovery (Dundee, Scotland), Beckman Coulter, Inc. (Fullerton, CA), Transtech Pharma (High Point, NC), Morphochem (Monmouth Junction, NJ), Pall Corporation (Fishers, IN), Torcon Instruments, Inc.
  • Assays can be used to screen for modulating agents which are either agonists or antagonists of the normal cellular function of the subject KRC polypeptides, e.g., those that modulate KRC activity e.g., by modulating the interaction between KRC and
  • the invention provides a method in which an indicator composition is provided which has a KRC peptide having a KRC activity.
  • the KRC peptide can be a full-length KRC polypeptide, amino acid residues 204-1055 of KRC (KRCtr), a fragment of a KRCtr, or a peptide consisting solely of a zinc finger DNA binding motif.
  • the KRC peptide can also be a peptide containing more than one KRCtr domain. Such a peptide may be useful, for example, in identifying compounds that bind to the KRCtr domain because they may increase the effective concentration of KRCtr domains available for the compound to bind to.
  • the indicator composition may also comprise a TRAF peptide, e.g., a full-length TRAF peptide or a fragment thereof, for example TRAF-C domain.
  • the indicator composition can be contacted with a test compound.
  • the effect of the test compound on KRC activity, as measured by a change in the indicator composition, can then be determined to thereby identify a compound that modulates the activity of a KRC protein.
  • a statistically significant change such as a decrease or increase, in the level of KRC activity or in the level of interaction between KRC and TRAF in the presence of the test compound
  • the agent binds to the amino acid residues 204-1055 of KRC.
  • the indicator composition can be, for example, a cell or a cell extract.
  • KRC activity is assessed as described in the appended Examples.
  • KRC activity is determined by the ability of the KRC peptide to bind to TRAF via the TRAF- C domain.
  • KRC activity is determined by the ability of KRC to modulate inflammation and apoptosis.
  • the modulating agent of interest is contacted with TRAF.
  • a composition containing a KRC peptide is then added to the mixture of the modulating agent and the interactor molecule.
  • KRC e.g., amino acid residues 204-1055 of KRC
  • an assay is a cell-based assay in which a cell (e.g., a T cell) which expresses a KRC polypeptide or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate KRC activity is determined.
  • a cell e.g., a T cell
  • determining the ability of the test compound to modulate KRC activity interaction can be accomplished by monitoring, for example, the ability of KRC to inhibit NFkB activation or JNK activation. Determining the ability of the test compound to modulate KRC activity can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to KRC can be determined by detecting the labeled test compound in a complex.
  • KRC could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate KRC binding to the test compound in a complex.
  • Determining the ability of the test compound to bind KRC can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to KRC can be determined by detecting the labeled KRC compound in a complex.
  • a radioisotope or enzymatic label such that binding of the compound to KRC can be determined by detecting the labeled KRC compound in a complex.
  • compounds can be labeled with 125 I, 35 S, 1 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the ability of a test compound to modulate KRC binding to TRAF can be determined by measuring, in the absence or the presence of the compound, the amount of TRAF bound to KRC by immunoprecipitation, as described herein in the Example section.
  • a microphysiometer can be used to detect the interaction of a compound with KRC without the labeling of either the compound or the KRC (McConnell, H. M. et al. (1992) Science 257:1906-1912).
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of KRC. Determining the ability of the test compound to modulate the activity of KRC can be accomplished, for example, by determining the ability of a KRC peptide to, for example, increase apoptosis, inhibit NFkB or JNK activation. Determining the ability of the KRC peptide, or a biologically active fragment thereof (e.g., to amino acid residues 204-1055 of KRC), to bind to or interact with TRAF, can be accomplished by one of the methods described herein for determining direct binding.
  • determining the ability of the KRC peptide to bind to or interact with TRAF can be accomplished by determining the activity of KRC.
  • the binding of TRAF to KRC upregulates KRC activity (and thus inhibits NFkB activation and increases apoptosis).
  • the activity of KRC can be determined by detecting T cell activation (using methods known in the art or described herein).
  • T cell activation can be determined by measuring T cell proliferation, using standard methods.
  • T cell activation can be determined by measuring cytokine production (e.g., IL-2 production) using a standard cytokine ELISA, a Western blot, or other methods known in the art.
  • T cell activation can be determined by detecting the induction of a reporter gene (comprising a target-responsive regulatory element such as the IL-2 promoter/enhancer region operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase).
  • a reporter gene comprising a target-responsive regulatory element such as the IL-2 promoter/enhancer region operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase.
  • determining the ability of the test compound to modulate the activity of a KRC peptide can be accomplished by determining the ability of the test compound to modulate the activity of a molecule that functions downstream of or concomitantly with KRC, e.g., a T cell receptor (TCR).
  • TCR T cell receptor
  • levels of second messengers, the activity of the interacting molecule on an appropriate target, or the binding of the interactor to an appropriate target can be determined as previously described.
  • TCR associated tyrosine kinase activity can be determined.
  • Other methods for determining the activity of a T cell receptor are known in the art.
  • the KRC peptides can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify compounds (e.g., small molecules or other polypeptides) which can modulate the activity of KRC
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. If the "bait" and the “prey” polypeptides are able to interact, in vivo, forming a KRC-test compound complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ or a gene which confers survival on nutritionally selective media) which is operably linked to a transcriptional regulatory site responsive to the transcription factor.
  • a reporter gene e.g., LacZ or a gene which confers survival on nutritionally selective media
  • a screening assay of the present invention utilizes the yeast cells such as those described in Example 1, wherein the amino acid residues 204- 1055 of KRC is used as the "bait".
  • the "bait" may also comprise any fragment of amino acid residues 204-1055 of KRC.
  • yeast cells containing the KRC bait are cultured under conditions that allow for interaction of the bait and the prey (e.g., as described in the Example section). The cells are then contacted with a compound and the ability of the compound to modulate the interaction of the bait and the prey is determined. In one embodiment, interaction of the bait and prey is determined by the level growth on nutritionally selective media.
  • interaction of the bait and prey is determined by expression of a LacZ reporter gene.
  • a LacZ reporter gene it will be understood by those skilled in the art that when using nutritional selection as a readout of the assay, compounds that inhibit the inhibit the activity of KRC will prevent growth of the cells. Accordingly, a compound identified as being a modulator of KRC activity under such conditions should also be tested for the ability to inhibit the growth of the cells under non-selective conditions, so that compounds that generally inhibit yeast growth will not be chosen for further study.
  • an assay of the present invention is a cell-free assay in which a KRC peptide or biologically active portion thereof (e.g., to amino acid residues 204-1055 of KRC) is contacted with a test compound and the ability of the test compound to bind to the KRC polypeptide or biologically active portion thereof is determined.
  • Preferred biologically active portions of the KRC polypeptides to be used in assays of the present invention include fragments which participate in interactions with TRAF, e.g., at least a portion of amino acid residues 204-1055 of KRC which bind to TRAF.
  • a biologically active portion of KRC comprises the amino acid residues 204-1055 of KRC.
  • a biologically active portion of KRC comprises at least one or more zinc finger domains. Binding of the test compound to the KRC peptide can be determined either directly or indirectly as described above.
  • the assay includes contacting the KRC peptide or biologically active portion thereof with a test compound, and determining the ability of the test compound to interact with a KRC peptide, wherein determining the ability of the test compound to interact with a KRC peptide comprises determining the ability of the test compound to preferentially bind to KRC or biologically active portion thereof (e.g., to amino acid residues 204-1055 of KRC).
  • Determining the ability of the KRC polypeptide to bind to TRAF can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705).
  • BIOA Biomolecular Interaction Analysis
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • determining the ability of the test compound to modulate the activity of a KRC polypeptide can be accomplished by determining the ability of the KRC peptide to modulate the activity of TRAF.
  • the activity of the TRAF on an appropriate target e.g., the ability of TRAF to activate NFkB
  • the binding of the TRAF to an appropriate target can be determined as previously described.
  • the cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of polypeptides (e.g., KRC peptides or biologically active portions thereof).
  • polypeptides e.g., KRC peptides or biologically active portions thereof.
  • a membrane-bound form a polypeptide e.g., a cell-surface KRC
  • non-ionic detergents such as
  • a fusion protein can be provided which adds a domain that allows one or both of the polypeptides to be bound to a matrix.
  • glutathione-S- transferase/KRC fusion proteins can be adsorbed onto glutathione sepharose beads
  • the complexes can be dissociated from the matrix, and the level of KRC and test compound binding or activity determined using standard techniques.
  • the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the KRC sequence, a second protein for which antibodies are readily available (e.g. from commercial sources).
  • the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
  • Other useful epitope tags include myc-epitopes (e.g., see Ellison et al, 1991, J. Biol. Chem.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell-free assay, and the ability of the agent to modulate KRC activity can be confirmed in vivo, e.g., in an animal such as an animal model for an autoimmune or inflammatory disease or an organ transplant.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., an agent that can modulate KRC activity
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above- described screening assays for treatments as described herein.
  • KRC peptides can also be used for rational drug design of candidate KRC modulating agents (e.g., molecules useful for downregulating KRC activity, and thus, downregulating immune responses).
  • candidate KRC modulating agents e.g., molecules useful for downregulating KRC activity, and thus, downregulating immune responses.
  • Potential therapeutic drugs may be designed rationally on the basis of structural information thus provided.
  • such drugs are designed to prevent or enhance formation of a KRC polypeptide:TRAF complex.
  • such drugs are designed to bind to amino acid residues 204-1055 of KRC.
  • the present invention may be used to design drugs, including drugs with a capacity to inhibit or promote binding of KRC to
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with an aberrant cell proliferation or survival.
  • an immune system disorder or condition associated with an undesirable immune response such as an unwanted or excessive inflammatory response, an autoimmune disorder, graft-versus-host disease (GVHD), an allogeneic transplant
  • an immune system disorder or condition that would benefit from an enhanced immune response e.g. an immunosuppressed individual.
  • the invention provides a method for preventing in a subject, a disease or condition 1 associated with an aberrant or unwanted immune response or, alternatively, an abnormally low immune response, by administering to the subject an agent which downmodulates the activity of KRC.
  • Subjects at risk for such disorders can be identified by, for example, any or a combination of diagnostic or prognostic assays known in the art.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrant immune response, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a KRC antagonist or agonist agent can be used for treating a subject.
  • the appropriate agent can be determined based on screening assays described herein.
  • the agent may be a peptide comprising the amino acid residues 204-1055 of KRC, a peptide that binds to KRC, a KRC ZAS domain and a small molecule.
  • KRC activity can be modulated in order to modulate the immune response. Because KRC upregulates immune responses, enhanced KRC activity and/or expression results in upregulation of immune responses, whereas inhibition of KRC activity results in downregulation of immune responses.
  • Modulatory methods of the invention involve contacting a cell (e.g., a T cell) with a agent that modulates the activity of KRC.
  • An agent that modulates KRC activity can be an agent as described herein, such as a KRC peptide (e.g., the agent may be a peptide comprising the amino acid residues 204-1055 of KRC, a peptide that binds to KRC, a KRC ZAS domain or a small molecule), a nucleic acid molecule encoding one of the aforementioned peptides, a KRC agonist or antagonist, a peptidomimetic of a KRC agonist or antagonist, a KRC peptidomimetic, or other small molecule identified using the screening methods described herein.
  • modulatory methods can be performed in vitro (e.g., by contacting the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a condition or disorder that would benefit from up- or down-modulation of a KRC polypeptide, e.g., a disorder characterized by an unwanted, insufficient, or aberrant immune response.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) KRC activity.
  • Inhibition of KRC activity is desirable in situations in which KRC is abnormally upregulated and/or in which decreased KRC activity is likely to have a beneficial effect, for example in a situation of an excessive or unwanted immune response.
  • Such situations include conditions, disorders, or diseases such as an autoimmune disorder (e.g., rheumatoid arthritis, my asthenia gravis, autoimmune thyroiditis, systemic lupus erythematosus, type I diabetes mellitus, Grave's disease, or multiple sclerosis), a transplant (e.g., a bone marrow transplant, a stem cell transplant, a heart transplant, a lung transplant, a liver transplant, a kidney transplant, a cornea transplant, or a skin transplant), graft versus host disease (GVHD), an allergy, or in inflammatory disorder.
  • an autoimmune disorder e.g., rheumatoid arthritis, my asthenia gravis, autoimmune thyroiditis, systemic lupus erythematosus
  • KRC agonists include, e.g., nucleic acid molecules encoding KRC polypeptides, KRC peptides, and compounds that stimulate the interaction of KRC with TRAF (e.g., compounds identified in the subject screening assays).
  • agents for use in downmodulating KRC include agents that inhibit the activity of KRC in an immune cell (e.g., compounds identified in the subject screening assays).
  • Downregulation can be in the form of inhibiting or blocking an immune response already in progress, or may involve preventing the induction of an immune response.
  • the functions of activated immune cells can be inhibited by downregulating immune cell responses or by inducing specific anergy in immune cells, or both.
  • KRC activity can be inhibited by contacting a cell which expresses KRC with an agent that inhibits KRC.
  • an agent can be a compound identified by the screening assays described herein.
  • the agent is a peptide.
  • the agent can interact with the amino acid residues 204-1055 of KRC to inhibit KRC activity.
  • An immune response can be further inhibited by the use of an additional agent that can thereby downmodulate the immune response, as described further herein.
  • Agents that inhibit a KRC activity can be identified by their ability to inhibit immune cell proliferation and/or effector function, or to induce anergy when added to an in vitro assay.
  • a number of art-recognized readouts of cell activation can be employed to measure, e.g., cell proliferation or effector function (e.g., cytokine production or phagocytosis) in the presence of the activating agent.
  • the ability of a test agent to block this activation can be readily determined by measuring the ability of the agent to effect a decrease in proliferation or effector function being measured.
  • immune responses can be downregulated in a subject by removing immune cells from the patient, contacting the immune cells in vitro with an agent (e.g., a small molecule) that downregulates KRC activity, and reintroducing the in w ' tro-stimulated immune cells into the patient.
  • an agent e.g., a small molecule
  • Downregulating immune responses by inhibiting KRC activity is useful in downmodulating the immune response, e.g., in situations of tissue, skin and organ transplantation, in graft-versus-host disease (GVHD), or allergies, or in autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis.
  • GVHD graft-versus-host disease
  • autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis.
  • blockage of immune cell function results in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by immune cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits the activity of KRC e.g., by blocking the interaction of KRC with TRAF in immune cells (such as a KRC or TRAF peptide or a small molecule) alone or in conjunction with another downmodulatory agent can inhibit the generation of an immune response.
  • immune cells such as a KRC or TRAF peptide or a small molecule
  • inhibition of KRC activity by inhibition of KRC-TRAF interaction may also be sufficient to anergize the immune cells, thereby inducing tolerance in a subject.
  • FK506, cyclosporin, rapamycin, steroids immunosuppressive drugs
  • KRC activity may also be useful in treating autoimmune disease.
  • Many autoimmune disorders are the result of inappropriate activation of immune cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
  • Preventing the activation of autoreactive immune cells may reduce or eliminate disease symptoms.
  • Administration of agents that inhibit an activity of KRC may lead to long- term relief from the disease.
  • co-administration of agents which block costimulation of immune cells by disrupting receptor-ligand interactions may be useful in inhibiting immune cell activation to prevent production of autoantibodies or cytokines which may be involved in the disease process.
  • the efficacy of reagents in preventing or alleviating autoimmune disorders can be determined using a number of well- characterized animal models of human autoimmune diseases.
  • Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
  • Inhibition of immune cell activation is useful fherapeutically in the treatment of allergies and allergic reactions, e.g., by inhibiting IgE production.
  • An agent that inhibits KRC activity can be administered to an allergic subject to inhibit immune cell-mediated allergic responses in the subject.
  • Inhibition of KRC activity can be accompanied by exposure to allergen in conjunction with appropriate MHC molecules. Allergic reactions can be systemic or local in nature, depending on the route of entry of the allergen and the pattern of deposition of IgE on mast cells or basophils. Thus, immune cell-mediated allergic responses can be inhibited locally or systemically by administration of an agent that inhibits KRC activity. Downregulation of immune cell activation through inhibition of KRC activity may also be important therapeutically in pathogenic infections of immune cells (e.g., by viruses or bacteria). For example, in the acquired immune deficiency syndrome (AIDS), viral replication is stimulated by immune cell activation. Inhibition of KRC activity may result in inhibition of viral replication and thereby ameliorate the course of AIDS.
  • AIDS acquired immune deficiency syndrome
  • Downregulation of immune cell activation via inhibition of KRC activity interaction may also be useful in treating inflammatory disorders and in promoting the maintenance of pregnancy when there exists a risk of immune-mediated spontaneous abortion.
  • cells e.g., T cells
  • an agent that inhibits KRC activity e.g., T cells
  • the immune cells may be contacted with the agent in vitro and then the cells can be administered to a subject or, alternatively, the agent may be administered to the subject (e.g., directly to an articular site at which T growth and/or differentiation is desired).
  • the methods of the invention using KRC inhibitory compounds can be used in the treatment of disorders in which the immune response is diminished, blocked, inhibited, downregulated or the like.
  • Inhibitory compounds of the invention can be, for example, intracellular binding molecules that act to specifically inhibit the expression or activity of KRC.
  • intracellular binding molecule is intended to include molecules that act intracellularly to inhibit the expression or activity of a protein by binding to the protein or to a nucleic acid (e.g. , an mRNA molecule) that encodes the protein.
  • nucleic acid e.g. , an mRNA molecule
  • intracellular binding molecules include antisense nucleic acids, intracellular antibodies, peptidic compounds that inhibit the interaction of KRC with a target molecule (e.g., calcineurin) and chemical agents that specifically inhibit KRC activity.
  • an inhibitory compound of the invention is an antisense nucleic acid molecule that is complementary to a gene encoding KRC, or to a portion of said gene, or a recombinant expression vector encoding said antisense nucleic acid molecule.
  • antisense nucleic acids to downregulate the expression of a particular protein in a cell is well known in the art (see e.g., Weintraub, H. et al, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986; Askari, F.K. and McDonnell, W.M. (1996) N Eng. J. Med. 334:316- 318; Bennett, M.R.
  • An antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to the coding strand of another nucleic acid molecule (e.g., an mRNA sequence) and accordingly is capable of hydrogen bonding to the coding strand of the other nucleic acid molecule.
  • Antisense sequences complementary to a sequence of an mRNA can be complementary to a sequence found in the coding region of the mRNA, the 5' or 3' untranslated region of the mRNA or a region bridging the coding region and an untranslated region (e.g. , at the junction of the 5' untranslated region and the coding region).
  • an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element.
  • an antisense nucleic acid is designed so as to be complementary to a region preceding or spanning the initiation codon on the coding strand or in the 3' untranslated region of an mRNA.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of a KRC mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of a KRC mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a KRC mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymetl ⁇ ylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5
  • an antisense nucleic acid can be produced biologically using an expression vector into which all or a portion of KRC cDNA has been subcloned in an antisense orientation (i. e. , nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the expression of the antisense RNA molecule in a cell of interest, for instance promoters and/or enhancers or other regulatory sequences can be chosen which direct constitutive, tissue specific or inducible expression of antisense RNA.
  • the antisense expression vector is prepared according to standard recombinant DNA methods for constructing recombinant expression vectors, except that the KRC cDNA (or portion thereof) is cloned into the vector in the antisense orientation.
  • the antisense expression vector can be in the form of, for example, a recombinant plasmid, phagemid or attenuated virus.
  • the antisense expression vector is introduced into cells using a standard transfection technique.
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a KRC protein to thereby inhibit expression of the protein, e.g. , by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of an antisense nucleic acid molecule of the invention includes direct injection at a tissue site.
  • an antisense nucleic acid molecule can be modified to target selected cells and then administered systemically.
  • an antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen.
  • the antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave KRC mRNA transcripts to thereby inhibit translation of KRC mRNAs.
  • a ribozyme having specificity for a KRC-encoding nucleic acid can be designed based upon the nucleotide sequence of the KRC cDNA.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a KRC-encoding mRNA. See, e.g. , Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742.
  • KRC mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • KRC gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an KRC gene (e.g. , an KRC promoter and/or enhancer) to form triple helical structures that prevent transcription of an KRC gene in target cells.
  • an KRC gene e.g. , an KRC promoter and/or enhancer
  • KRC gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an KRC gene (e.g. , an KRC promoter and/or enhancer) to form triple helical structures that prevent transcription of an KRC gene in target cells.
  • an KRC promoter and/or enhancer e.g., an KRC promoter and/or enhancer
  • a recombinant expression vector which encodes the antibody chains in a form such that, upon introduction ' of the vector into a cell, the antibody chains are expressed as a functional antibody in an intracellular compartment of the cell.
  • an intracellular antibody that specifically binds the transcription factor is expressed within the nucleus of the cell.
  • Nuclear expression of an intracellular antibody can be accomplished by removing from the antibody light and heavy chain genes those nucleotide sequences that encode the N- terminal hydrophobic leader sequences and adding nucleotide sequences encoding a nuclear localization signal at either the N- or C-terminus of the light and heavy chain genes (see e.g., Biocca, S. et al. (1990) EMBO J. 9:101-108; Mhashilkar, A. M. et al. (1995) EMBO J. 14: 1542-1551).
  • a preferred nuclear localization signal to be used for nuclear targeting of the intracellular antibody chains is the nuclear localization signal of SV40 Large T antigen (see Biocca, S. et al. (1990) EMBO J.
  • antibody light and heavy chain cDNAs encoding antibody chains specific for the target protein of interest e.g., KRC protein
  • KRC protein a target protein of interest
  • Preparation of antisera against KRC protein has been described in the art (see e.g., Rao et al, U.S. patent 5,656,452).
  • Anti-KRC protein antibodies can be prepared by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with a KRC protein immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinanfly expressed KRC protein or a chemically synthesized KRC peptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory compound.
  • Antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al. (1981) J Immunol 127:539-46; Brown et al (1980) J Biol Chem 255 :4980-83 ; Yeh et al.
  • an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a KRC protein immunogen as described above, and the culture supematants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds specifically to the KRC protein.
  • lymphocytes typically splenocytes
  • Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-KRC protein monoclonal antibody (see, e.g., G. Galfre et al. (1911) Nature 266:550-52; Gefter et al.
  • the immortal cell line e.g., a myeloma cell line
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody that specifically binds the maf protein are identified by screening the hybridoma culture supematants for such antibodies, e.g., using a standard ELISA assay.
  • a monoclonal antibody that binds to a KRC can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the protein, or a peptide thereof, to thereby isolate immunoglobulin library members that bind specifically to the protein.
  • Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and compounds particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al International Publication No.
  • DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques.
  • hybridoma derived antibodies light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening.
  • cDNA encoding the light and heavy chains can be recovered from the display package (e.g., phage) isolated during the library screening process.
  • Nucleotide sequences of antibody light and heavy chain genes from which PCR primers or cDNA library probes can be prepared are known in the art. For example, many such sequences are disclosed in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and in the "Vbase" human germline sequence database.
  • the antibody light and heavy chain sequences are cloned into a recombinant expression vector using standard methods.
  • sequences encoding the hydrophobic leaders of the light and heavy chains are removed and sequences encoding a nuclear localization signal (e.g., from SV40 Large T antigen) are linked in-frame to sequences encoding either the amino- or carboxy terminus of both the light and heavy chains.
  • the expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly.
  • the vector encodes a full-length light chain but only the VH/CHl region of the heavy chain such that a Fab fragment is expressed intracellularly.
  • the vector encodes a single chain antibody (scFv) wherein the variable regions of the light and heavy chains are linked by a flexible peptide linker (e.g., (Gly4Ser)3) and expressed as a single chain molecule.
  • scFv single chain antibody
  • the expression vector encoding the KRC- specific intracellular antibody is introduced into the cell by standard transfection methods as described hereinbefore.
  • an inhibitory compound of the invention is a peptidic compound derived from the KRC amino acid sequence.
  • the inhibitory compound comprises a portion of KRC (or a mimetic thereof) that mediates interaction of KRC with a target molecule such that contact of KRC with this peptidic compound competitively inhibits the interaction of KRC with the target molecule.
  • the peptide compound is designed based on the region of KRC that mediates interaction of KRC with TRAF.
  • amino acid residues 204- 1055 of the KRC protein mediate the interaction of the KRC proteins with TRAF and peptides spanning the region inhibit the ability of TRAF to bind to and phosphorylate KRC proteins, without affecting the phosphatase activity of TRAF against other substrates.
  • peptides spanning this region inhibit KRC dephosphorylation, nuclear translocation and KRC-mediated gene expression in response to stimulation, thereby inhibiting KRC-dependent functions.
  • a KRC inhibitory compound is a peptidic compound, which is prepared based on a TRAF-interacting region of KRC.
  • a peptide can be derived from the TRAF-interacting region of KRC having an amino acid sequence that comprises the amino acid residues 204-1055 of KRC.
  • longer or shorter regions of human KRC can be used such as a peptide.
  • the peptidic compounds of the invention can be made intracellularly in immune cells by introducing into the immune cells an expression vector encoding the peptide.
  • expression vectors can be made by standard techniques, using, for example, oligonucleotides that encode the amino acid sequences of SEQ ID NO: 2.
  • the peptide can be expressed in intracellularly as a fusion with another protein or peptide (e.g., a GST fusion).
  • the peptides can be made by chemical synthesis using standard peptide synthesis techniques. Synthesized peptides can then be introduced into cells by a variety of means known in the art for introducing peptides into cells (e.g., liposome and the like).
  • inhibitory agents that can be used to specifically inhibit the activity of an KRC protein are chemical compounds that directly inhibit KRC activity or inhibit the interaction between KRC and target molecules. Such compounds can be identified using screening assays that select for such compounds, as described in detail above.
  • Stimulation of KRC activity as a means of upregulating immune responses is also useful in therapy.
  • Upregulation of immune responses can be in the form of enhancing an existing immune response or eliciting an initial immune response.
  • enhancing an immune response through enhancing of KRC activity is useful in cases of infections with microbes, e.g., bacteria, viruses, or parasites.
  • an agent that enhances KRC activity e.g., a. small molecule or a KRC peptide, is therapeutically useful in situations where upregulation of antibody and cell- mediated responses, resulting in more rapid or thorough clearance of a virus, would be beneficial.
  • These conditions include viral skin diseases such as Herpes or shingles, in which case such an agent can be delivered topically to the skin.
  • systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of such agents systemically.
  • it may be desirable to further administer other agents that upregulate immune responses for example, agents that transduce signals via costimulatory receptors, in order further augment the immune response.
  • immune responses can be enhanced in an infected patient by removing immune cells from the patient, contacting immune cells in vitro with an agent (e.g., a small molecule) that enhances KRC activity, and reintroducing the in vitro- stimulated immune cells into the patient.
  • an agent e.g., a small molecule
  • a method of enhancing immune responses involves isolating infected cells from a patient, e.g., virally infected cells, transfecting them with a nucleic acid molecule encoding a form of KRC that is more active than the wild type KRC, such that the cells express all or a portion of the KRC molecule on their surface, and reintroducing the transfected cells into the patient.
  • the transfected cells may be capable of preventing an inhibitory signal to, and thereby activating, immune cells in vivo.
  • An agent that enhances KRC activity can be used prophylactically in therapy against various polypeptides, e.g., polypeptides derived from pathogens for vaccination. Immunity against a pathogen, e.g., a virus, can be induced by vaccinating with a viral polypeptide along with an agent that enhances KRC activity.
  • Nucleic acid vaccines can be administered by a variety of means, for example, by injection (e.g., intramuscular, intradermal, or the biolistic injection of DNA-coated gold particles into the epidermis with a gene gun that uses a particle accelerator or a compressed gas to inject the particles into the skin (Haynes et al. (1996) J. Biotechnol. 44:37)).
  • nucleic acid vaccines can be administered by non-invasive means.
  • pure or lipid- formulated DNA can be delivered to the respiratory system or targeted elsewhere, e.g., Peyers patches by oral delivery of DNA (Schubbert (1997) Proc. Natl Acad. Sci. USA 94:961). Attenuated microorganisms can be used for delivery to mucosal surfaces (Sizemore et al. (1995) Science 270:29).
  • Stimulation of an immune response to tumor cells can also be achieved by enhancing KRC activity by treating a patient with an agent that for example, enhancing KRC -TRAF interaction.
  • agents include, e.g., and compounds identified in the subject screening assays and peptides.
  • the immune response can be stimulated by enhancing of
  • KRC activity such that preexisting tolerance is overcome.
  • immune responses against antigens to which a subject cannot mount a significant immune response e.g., tumor-specific antigens
  • KRC activity can be induced by administering an agent that stimulates the activity of KRC activity.
  • Other KRC agonists can be used as adjuvants to boost responses to foreign antigens in the process of active immunization.
  • immune cells are obtained from a subject and cultured ex vivo in the presence of an agent that that enhances KRC activity to expand the population of immune cells.
  • the immune cells are then administered to a subject, immune cells can be stimulated to proliferate in vitro by, for example, providing the immune cells with a primary activation signal and a costimulatory signal, as is known in the art.
  • Various forms of KRC polypeptides or agents that enhance KRC activity can also be used to costimulate proliferation of immune cells.
  • immune cells are cultured ex vivo according to the method described in PCT Application No. WO 94/29436.
  • the agent can be soluble, attached to a cell membrane or attached to a solid surface, such as a bead.
  • an immune response by administering one or more additional agents.
  • agents known to stimulate the immune response such as cytokines, adjuvants, or stimulatory forms of costimulatory molecules or their ligands can be used in conjunction with an agent that enhances KRC activity.
  • a method of upregulating immune responses involves transfecting them with a nucleic acid molecule encoding a KRC molecule with a mutation or a peptide that enhances KRC-TRAF interaction (e.g., a TRAF-C domain), such that the cells express the KRC molecule (e.g., in the cell membrane) or the peptide (e.g., in the cytoplasm), and reintroducing the transfected cells into the patient.
  • the ability of the transfected cells to be activated can thus be increased.
  • immunomodulating reagents examples include antibodies that provide a costimulatory signal, (e.g., agonists of CD28 or ICOS), stimulating antibodies against immune cell markers , and/or cytokines and the like. 4.
  • costimulatory signal e.g., agonists of CD28 or ICOS
  • a compound that specifically stimulates KRC activity and/or expression can be used to enhance or upmodulate an immune response.
  • a subject is treated with a stimulatory compound that stimulates expression and/or activity of a KRC molecule.
  • the methods of the invention using KRC stimulatory compounds can be used in the treatment of disorders in which the immune response is enhanced, promoted, stimulated, upregulated or the like.
  • stimulatory compounds include active KRC protein, expression vectors encoding KRC and chemical agents that specifically stimulate KRC activity.
  • a preferred stimulatory compound is a nucleic acid molecule encoding KRC, wherein the nucleic acid molecule is introduced into the subject (e.g., T cells of the subject) in a form suitable for expression of the KRC protein in the cells of the subject.
  • a KRC cDNA full length or partial KRC cDNA sequence
  • the KRC cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library.
  • the nucleotide sequences of KRC cDNA is known in the art and can be used for the design of PCR primers that allow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA library using standard hybridization methods.
  • Nucleic acid molecules encoding KRC in the form suitable for expression of the KRC in a host cell can be prepared as described above using nucleotide sequences known in the art.
  • the nucleotide sequences can be used for the design of PCR primers that allow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA library using standard hybridization methods.
  • Another form of a stimulatory compound for stimulating expression of KRC in a cell is a chemical compound that specifically stimulates the expression or activity of endogenous KRC in the cell. Such compounds can be identified using screening assays that select for compounds that stimulate the expression or activity of KRC as described herein.
  • the method of the invention for modulating aberrant KRC activity in a subject can be practiced either in vitro or in vivo (the latter is discussed further in the following subsection).
  • cells e.g., T cells
  • a stimulatory or inhibitory compound of the invention to stimulate or inhibit, respectively, the activity of KRC.
  • Methods for isolating immune cells are known in the art.
  • Cells treated in vitro with either a stimulatory or inhibitory compound can be administered to a subject to influence the growth and/or differentiation of immune cells in the subject.
  • immune cells can be isolated from a subject, expanded in number in vitro by enhancing KRC activity in the cells using an enhancing agent (thereby promoting the proliferation of the cells), and then the immune cells can be readministered to the same subject, or another subject tissue compatible with the donor of the immune cells.
  • the modulatory method of the invention comprises culturing immune cells in vitro with a KRC modulator and further comprises administering the immune cells to a subject to thereby modulate T growth and/or differentiation in a subject. Upon culture in vitro, the immune cells can differentiate into mature immune cells and thus the methods encompass administering this mature immune cells to the subject.
  • a stimulatory or inhibitory compound is administered to a subject in vivo, such as directly to an articulation site of a subject.
  • stimulatory or inhibitory agents that comprise nucleic acids (e.g. , recombinant expression vectors encoding KRC, antisense RNA, intracellular antibodies or KRC-derived peptides)
  • the compounds can be introduced into cells of a subject using methods known in the art for introducing nucleic acid (e.g., DNA) into cells in vivo. Examples of such methods include: Direct Injection: Naked DNA can be introduced into cells in vivo by directly injecting the DNA into the cells (see e.g., Acsadi et al.
  • a delivery apparatus e.g., a "gene gun" for injecting DNA into cells in vivo
  • a delivery apparatus e.g., a "gene gun” for injecting DNA into cells in vivo
  • Such an apparatus is commercially available (e.g., from BioRad).
  • Receptor-Mediated DNA Uptake Naked DNA can also be introduced into cells in vivo by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, CH. (1988) J. Biol. Chem. 263:14621; Wilson et al. (1992) J Biol. Chem.
  • Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis.
  • a DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl Acad. Sci. USA 90:2122-2126).
  • Retroviruses Defective retro viruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 16:211).
  • a recombinant retrovirus can be constructed having a nucleotide sequences of interest incorporated into the retro viral genome. Additionally, portions of the retro viral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
  • retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am.
  • Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc.
  • Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell.
  • Adenoviruses The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68: 143-155.
  • Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482- 6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl Acad. Sci. USA 89:2581 -2584).
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj- Ahmand and Graham (1986) J Virol. 57:267).
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • AAV Adeno-associated virus
  • Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb.
  • An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad.
  • DNA introduced into a cell can be detected by a filter hybridization technique (e.g. , Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • the gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product, such as an enzymatic assay.
  • KRC modulating agents of the invention or KRC modulating agents identified in screening assays of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human.
  • Such compositions typically comprise the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a small molecule, nucleic acid molecule, or peptide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules used in the methods of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a. slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • Viral vectors include, for example, recombinant retroviruses, adenovirus, adeno- associated virus, and herpes simplex virus- 1.
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans.
  • viruses can be used for introducing exogenous genes ex vivo into immune cells in culture.
  • These vectors provide efficient delivery of genes into immune cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host cell.
  • a major prerequisite for the use of viruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
  • the development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271).
  • recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) is replaced by a gene of interest rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • Suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am.
  • retroviral-based vectors by modifying the viral packaging proteins on the surface of the viral particle.
  • strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) Proc. Natl. Acad. Sci. USA 86:9079-9083; Mm et al. (1992) J. Gen. Virol.
  • Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single-chain antibody/er ⁇ v fusion proteins).
  • viral particles containing a nucleic acid molecule containing a gene of interest operably linked to appropriate regulatory elements are modified for example according to the methods described above, such that they can specifically target subsets of liver cells.
  • the viral particle can be coated with antibodies to surface molecule that are specific to certain types of liver cells. This method is particularly useful when only specific subsets of liver cells are desired to be transfected.
  • Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors.
  • the genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) Biotechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and
  • adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells. Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity.
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et ⁇ l. cited supra; Haj-Ahmand and Graham (1986) J Virol. 51:261).
  • adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80 % of the adenoviral genetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner et al, supra; and Graham et al. in Methods in Molecular Biology, E.J. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127).
  • Expression of the gene of interest comprised in the nucleic acid molecule can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
  • MLP major late promoter
  • Yet another viral vector system useful for delivery of a nucleic acid molecule comprising a gene of interest is the adeno-associated virus (AAV).
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics Microbiol. Immunol. (1992) 158:97-129).
  • Adeno-associated viruses exhibit a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963- 1973).
  • Vectors containing as few as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb.
  • An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol 5:3251-3260 can be used to introduce DNA into immune cells.
  • AAV vectors see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. (1985) Mol. Cell. Biol 4:2072- 2081; Wondisford et al. (1988) Mol. Endocrinol 2:32-39; Tratschin et al. (1984) J
  • compositions can be included in a container, pack, or dispenser together with instructions for administration. VIII. Administration of KRC Modulating Agents
  • KRC modulating agents of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo to either enhance or suppress immune responses (e.g., T cell mediated immune responses).
  • biologically compatible form suitable for administration in vivo is meant a form of the protein to be administered in which any toxic effects are outweighed by the therapeutic effects of the protein.
  • subject is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • Administration of an agent as described herein can be in any pharmacological form including a therapeutically active amount of an agent alone or in combination with a pharmaceutically acceptable carrier.
  • a therapeutically active amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of a KRC modulating agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to elicit a desired response in the individual.
  • Dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • compositions of the present invention can be administered by any suitable route known in the art including for example intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that a KRC polypeptide be administered to cells in the central nervous system, administration can be with one or more agents capable of promoting penetration of KRC polypeptide across the blood-brain barrier.
  • CSF cerebrospinal fluid
  • KRC can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • KRC can be coupled to any substance known in the art to promote penetration or transport across the blood- brain barrier such as an antibody to the transferrin receptor, and administered by intravenous injection.
  • KRC can be stably linked to a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties.
  • a polymer such as polyethylene glycol
  • the KRC polypeptide can be in a composition which aids in delivery into the cytosol of a cell.
  • the peptide may be conjugated with a carrier moiety such as a liposome that is capable of delivering the peptide into the cytosol of a cell.
  • a carrier moiety such as a liposome that is capable of delivering the peptide into the cytosol of a cell.
  • the KRC polypeptide can be modified to include specific transit peptides or fused to such transit peptides which are capable of delivering the KRC polypeptide into a cell.
  • the polypeptide can be delivered directly into a cell by microinjection.
  • compositions are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art.
  • One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated.
  • Supplementary active compounds can also be incorporated into the compositions. It may also be desirable that a suitable buffer be present in the composition. Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
  • the primary solvent can be aqueous or alternatively non-aqueous.
  • KRC can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requiring treatment.
  • the carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
  • excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion.
  • Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used. It is also provided that certain formulations containing the KRC polypeptide or fragment thereof are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
  • Suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, olyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
  • the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
  • the formulations can also contain substances that diminish proteorytic degradation and/or substances which promote absorption such as, for example, surface active agents.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the specific dose can be readily calculated by one of ordinary skill in the art, e.g., according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in assay preparations of target cells. Exact dosages are determined in conjunction with standard dose-response studies.
  • the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50%) of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • a KRC polypeptide may be therapeutically administered by implanting into patients vectors or cells capable of producing a biologically-active form of KRC or a precursor of KRC, i.e. a molecule that can be readily converted to a biological-active form of KRC by the body.
  • cells that secrete KRC may be encapsulated into semipermeable membranes for implantation into a patient.
  • the cells can be cells that normally express
  • KRC or a precursor thereof or the cells can be transformed to express KRC or a biologically active fragment thereof or a precursor thereof. It is preferred that the cell be of human origin and that the KRC polypeptide be human KRC when the patient is human.
  • the formulations and methods herein can be used for veterinary as well as human applications and the term "patient” or "subject” as used herein is intended to include human and veterinary patients.
  • Monitoring the influence of agents (e.g., drugs or compounds) on the expression or activity of a KRC protein can be applied not only in basic drug screening, but also in clinical trials.
  • agents e.g., drugs or compounds
  • the effectiveness of an agent determined by a screening assay as described herein to increase KRC gene expression, protein levels, or upregulate KRC activity can be monitored in clinical trials of subjects exhibiting decreased KRC gene expression, protein levels, or downregulated KRC activity.
  • the effectiveness of an agent determined by a screening assay to decrease KRC gene expression, protein levels, or downregulate KRC activity can be monitored in clinical trials of subjects exhibiting increased KRC gene expression, protein levels, or upregulated KRC activity.
  • the expression or activity of a KRC gene, and preferably, other genes that have been implicated in a disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes, including KRC, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates KRC activity can be identified.
  • an agent e.g., compound, drug or small molecule
  • KRC activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of KRC and other genes implicated in the KRC associated disorder, respectively.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of KRC or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a KRC protein, mRNA, or genomic DNA in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the KRC protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the KRC protein, mRNA, or genomic DNA in the pre- administration sample with the KRC protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g.,
  • increased administration of the agent may be desirable to increase the expression or activity of KRC to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of KRC to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • KRC expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the ability of a KRC modulating agent to modulate inflammation or apoptosis in a epithelial cell of a subject that would benefit from modulation of the expression and/or activity of KRC can be measured by detecting an improvement in the condition of the patient after the administration of the agent. Such improvement can be readily measured by one of ordinary skill in the art using indicators appropriate for the specific condition of the patient. Monitoring the response of the patient by measuring changes in the condition of the patient is preferred in situations were the collection of biopsy materials would pose an increased risk and/or detriment to the patient.
  • compositions containing KRC can be administered exogenously and it would likely be desirable to achieve certain target levels of KRC polypeptide in sera, in any desired tissue compartment or in the affected tissue. It would, therefore, be advantageous to be able to monitor the levels of KRC polypeptide in a patient or in a biological sample including a tissue biopsy sample obtained form a patient and, in some cases, also monitoring the levels of KRC and, in some circumstances, also monitoring levels of TRAF or another KRC- interacting polypeptide. Accordingly, the present invention also provides methods for detecting the presence of KRC in a sample from a patient. IX.
  • kits of the Invention Another aspect of the invention pertains to kits for carrying out the screening assays, modulatory methods or diagnostic assays of the invention.
  • a kit for carrying out a screening assay of the invention can include a cell comprising a KRC polypeptide, means for determining KRC polypeptide activity and instructions for using the kit to identify modulators of KRC activity.
  • a kit for carrying out a screening assay of the invention can include an composition comprising a KRC polypeptide, means for determining KRC activity and instructions for using the kit to identify modulators of KRC activity.
  • the invention provides a kit for carrying out a modulatory method for the invention.
  • the kit can include, for example, a modulatory agent of the invention (e.g. , a KRC inhibitory or stimulatory agent) in a suitable carrier and packaged in a suitable container with instructions for use of the modulator to modulate KRC activity.
  • a modulatory agent of the invention e.g. , a KRC inhibitory or stimulatory agent
  • kits for diagnosing a disorder associated with aberrant KRC expression and/or activity in a subject can include a reagent for determining expression of KRC (e.g. , a nucleic acid probe(s) for detecting KRC mRNA or one or more antibodies for detection of KRC proteins), a control to which the results of the subject are compared, and instructions for using the kit for diagnostic purposes.
  • a reagent for determining expression of KRC e.g. , a nucleic acid probe(s) for detecting KRC mRNA or one or more antibodies for detection of KRC proteins
  • the human embryonic kidney cell line HEK293, the NIH/3T3 fibroblast cells and the macrophage cell line RAW were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.
  • HEK293 cells 4 X 510 5 per well were seeded in 6 well plates, and 12 h later cells were transfected with EFECTENE ( Qiagen) with 25 ng of a 2XNF ⁇ B-luciferase (Luc) gift of ?reporter gene plasmid (gift of Dr. Ranjan Sen), and 0.5 ⁇ g of the indicated TRAF and KRC expression vectors. Total amounts of transfected DNA were kept constant by supplementing with control empty expression vector plasmids as needed. Cell extracts were prepared 24 h after transfection, and reporter gene activity was determined via the luciferase assay system (PROMEGA).
  • PRSV- ⁇ Gal vector 50 ng was used to normalize for transfection efficiency by measuring ⁇ galactosidase activity using the Galacton-PLUS substrate system (TROPIX, Inc.). Whenever indicated, the cells were treated for 4 hours with TNF ⁇ or IL1 (lOng/ml). To generate stable transfectants, effectene mediated transfection of the RAW cell line was performed and clones were selected and maintained in complete medium supplemented with G418 (2 mg/ml).
  • the yeast strain EGY48 containing the reporter genes for LEU and ⁇ galactosidase activity under the control of an upstream LexA-binding site was used as a host for the two hybrid screen.
  • the KRC fragment from amino acid 204 to 1055 (KRC tr) (Fig 2a) was fused in frame to the LexA DNA binding domain and a yeast strain expressing the LexA-KRC tr fusion protein was transfected with a mouse Thl clone cDNA library Szabo et al. fused to the GAL4 transcriptional activation domain. Transformants were plated on agar selection media lacking uracil, tryptophan, leucine and histidine.
  • Plasmid DNA was purified from colonies that were Leu ⁇ gal and used for retransformation of a yeast strain expressing a heterologous bait to determine the specificity of interaction.
  • the cell lysates were precleared with 30 ⁇ l of protein A/G-Sepharose beads and then incubated for 4h with 25 ⁇ l of anti MYC antibody directly conjugated to sepharose beads.
  • the immunoprecipitates were then washed 5 times with the lysis buffer, resuspended in SDS sample buffer, and heated at 95 C for 5 min. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitocellulose membrane (Schleicher & Schuell) and western blotting performed by probing with primary antibodies followed by horseradish peroxidase-conjugated goat anti-rabbit IgG and enhanced chemiluminescence according to the manufacturer's instructions (Amersham).
  • In vitro Kinase Assay Anti-HA or anti FLAG immunoprecipitates were used for immune complex kinase assays that were performed at 30 C for 30 min with 1 ⁇ g of substrate, 10 ⁇ Ci of ⁇ 32 P ATP, and 10 ⁇ M ATP in 30 ⁇ l of kinase buffer ( 20 mM HEPES, pH 7.4, 10 mM MgC12, 25 mM b-glycerophosphate, 50 ⁇ mNA3VO4, and 50 ⁇ m DTT).
  • the substrate was GST-c-JUN.
  • Apoptosis Assay ⁇ galactosidase cotransfection assays for determination of cell death were performed as described Hsu et al.. Transfected NIH 3T3 cells were washed with PBS, fixed in PBS containing 3% paraformaldehyde for 10 min at 4 C, and washed with PBS. Fixed cells were stained overnight with XGal. The number of blue-stained cells was determined microscopically. The average number from one representative experiment of three is shown.
  • EXAMPLE 1 INTERACTION OF KRC WITH TRAF FAMILY MEMBERS IN YEAST (A).
  • a yeast two-hybrid interaction trap was used to select a T cell cDNA library for sequences encoding polypeptides that specifically interacted with a KRC-LexA fusion protein.
  • KRC sequences encoding amino acids 204 to 1055 were used which include the third zinc finger domain, one of the three acidic domains and the putative NLS sequence, expressed in the pEG202 vector (Fig la).
  • TRAFl One class of interactors encoding a fusion protein with apparently high affinity for the KRC- LexA bait as exhibited by high level of ⁇ -galactosidase activity and ability to confer leucine prototrophy was isolated and upon sequencing proved to be the C terminal segment of TRAFl.
  • the interaction with TRAFl was specific since no interaction was detected with control plasmids that encode KRC, c-Maf or relA fusion proteins or with the control vector alone (not shown).
  • B The ability of TRAF proteins to interact specifically with KRC in vivo was tested in mammalian cells.
  • KRC sequences 204-1055 were subcloned into a mammalian expression vector which fuses the coding region to an N-terminal epitope tag from a myc peptide, and the expression of the protein confirmed by direct western blot analysis with anti-MYC antibody (Fig lb, right panel).
  • This tagged construct was then cotransfected with TRAF-FLAG-tagged expression plasmids into 293T cells and lysates prepared for immunoprecipitation with an anti-MYC antibody.
  • a STAT4-FLAG- tagged expression construct was used as negative control.
  • TRAF6-induced NFKB dependent gene expression using transfection assays in 293T human embryonic kidney cells was tested.
  • the results show that overexpression of both the full-length KRC and the KRC 204-1055 (KRC truncated, tr) in the absence of exogenous TRAFs blocked NF- ⁇ B-dependent transactivation in a manner comparable in strength to the inhibition observed with a dominant negative form of TRAF2 (Fig 2a).
  • the results also show that both the KRC tr and the full length KRC blocked TRAF2- induced NFKB activation (Fig 2b) while NFKB activation induced by TRAF 5 and TRAF6 were substantially but not completely affected (Fig 2c,d).
  • I l l EXAMPLE 3 ANTISENSE AND DOMINANT NEGATIVE KRC INCREASE CYTOKINE DRIVEN NFkB TRANSACTIVATION WHILE SENSE KRC INHIBIT.
  • KRC 204-1055 tr construct, full length KRC, ZAS2 expressing construct and the antisense KRC were cotransfected into 293 cells together with TRAF2, and JNK activity measured 24 hours after transfection. Both the KRC tr and the full length KRC blocked TRAF2-dependent JNK activation (Fig 6a). Full length KRC blocked JNK activation only partially, likely due to the approximately 10 fold lower expression of this construct as compared to KRC tr. The results also show a dramatic increase of TRAF2 dependent JNK activation with expression of both the antisense KRC as well with the dominant negative ZAS2 expressing construct.
  • the immediate target of TRAF2 in TNF-induced JNK/SAPK activation may be the MAP3 kinase ASK1 or members of the GCK family of kinases.
  • TNF ⁇ was tested.
  • Overexpressed KRC or dominant negative KRC was transfected in the RAW macrophage cell line and levels of TNF ⁇ in a panel of transfectant clones were analyzed.
  • RAW transfectants stably overexpressing KRC displayed a substantial decrease of baseline TNF ⁇ mRNA transcripts when compared to control vector transfected RAW cells while RAW transfectants expressing the dominant negative version had substantial increase in TNF ⁇ expression (Fig 7).
  • KRC (originally decried as a nuclear protein) physiologically interacts with the predominantly cytosolic TRAF2 to affect gene activation was tested.
  • a full-length KRC was fused to GFP and its cellular localization upon transfection into 3T3 cells was examined.
  • KRC was mainly localized to the cytosol while in 3T3 cells that had adhered to the glass slide, KRC was primarily present in the nucleus (Fig 8),.
  • TRAF2 has recently been described to translocate from cytosol to nucleus as well (Min et al, 1998). Thus KRC and TRAF2 may well interact in both subcellular compartments.
  • EXAMPLE 9 KRC IS TH1 SPECIFIC
  • KRC expression in primary T cells was measured.
  • RT-PCR analysis of KRC expression in primary T cells was performed. KRC expression was measured at 24 hours and 72 hours. The results demonstrate that KRC expression is rapidly lost in Th2 cells at 72 hours whereas KRC expression in Thl cells is maintained at 72 hours. These results demonstrate that KRC is Thl specific.
  • KRC was transfected into Jurkat T cells and CD69 expression was measured by FACS analysis.
  • CD69 a T cell activation marker
  • FIG. 11(A) shows IL-2 promoter transactivation by KRC in Jurkat T cells activated by PMA Ionomycin.
  • Figure 11(B) shows transactivation of a composite NFAT- API reporter by KRC.
  • Figure 11(C) shows tranactivation of an AP- 1 reporter by KRC .
  • EXAMPLE 12 KRC INCREASES IL-2 GENE TRANSCRIPTION TNTHE PRESENCE OF B CELLANTIGENPRESENTING CELLS
  • Figure 12(A) shows IL-2 promoter transactivation by KRC in Jurkat T cells activated by the Raji B cell APC line and the superantigen SEE.
  • Figure 12(B) shows transactivation of a composite NFAT- API reporter by KRC.
  • Figure 12(C) shows transactivation of an AP- 1 reporter by KRC.
  • EXAMPLE 13 KRC INCREASES IL-2 PROTEIN PRODUCTION IN JURKAT T
  • FIG. 13(A) shows increased IL-2 protein by transfection of KRC in Jurkat T cells.
  • Figure 13(B) shows increased IL-2 protein by retroviral transduction of KRC into primary CD4 T cells.
  • results demonstrate that KRC controls PKC-theta and IL-2 expression.
  • RT-PCR of KRC transfected Jurkat clones was performed.
  • the results show increased PKC-theta expression upon KRC transfection.
  • the results demonstrate that KRC increases actin polymerization.
  • Immunofluorescence of F-actin upon KRC overexpression in Jurkat T cells was performed.
  • the results show the reorganization of F-actin filaments in KRC transfected Jurkat T cells.

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Abstract

L'invention a pour but de démontrer que les molécules KRC possèdent de multiples fonctions importantes en tant qu'agents modulateurs dans la régulation d'une grande variété de processus cellulaires, à savoir dans l'inhibition de la transactivation de NFkB, dans l'augmentation de l'apoptose induite par TNF-alpha, dans l'inhibition de l'activation de JNK, dans l'inhibition de l'expression de TNF-alpha endogène dans la stimulation de la prolifération de cellules immunes et dans l'activation de cellules immunes (p.ex. dans les cellules Th1), dans l'activation de l'expression IL-2, p.ex. par l'activation du facteur de transcription AP-1, dans l'activation des encogènes Ras et Rac, dans la régulation de l'activité de PKC theta et dans l'augmentation de la polymérisation de l'actine. L'invention vise également à démontrer que KRC entre en interaction avec TRAF. Font également l'objet de cette invention des procédés d'identification de modulateurs de l'activité de KRC ainsi que des procédés de modulation d'une réponse immune faisant appel à des agents modulant l'activité de KRC.
PCT/US2002/014166 2001-05-03 2002-05-03 Procedes de modulation d'une reponse immune par modulation de l'activite krc Ceased WO2002090595A1 (fr)

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