+

WO2008060540A2 - Procédés d'identification de compositions qui modulent la protéine tyrosine kinase, ptp-bl - Google Patents

Procédés d'identification de compositions qui modulent la protéine tyrosine kinase, ptp-bl Download PDF

Info

Publication number
WO2008060540A2
WO2008060540A2 PCT/US2007/023845 US2007023845W WO2008060540A2 WO 2008060540 A2 WO2008060540 A2 WO 2008060540A2 US 2007023845 W US2007023845 W US 2007023845W WO 2008060540 A2 WO2008060540 A2 WO 2008060540A2
Authority
WO
WIPO (PCT)
Prior art keywords
ptp
stat
polypeptide
cell
cells
Prior art date
Application number
PCT/US2007/023845
Other languages
English (en)
Other versions
WO2008060540A3 (fr
Inventor
Michael J. Grusby
Original Assignee
President And Fellows Of Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Publication of WO2008060540A2 publication Critical patent/WO2008060540A2/fr
Publication of WO2008060540A3 publication Critical patent/WO2008060540A3/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • Cytokines are important regulators of immune responses, and numerous cytokines mediate their biologic function through activation of the signal transducer and activator of transcription (STAT) signaling pathway (O'Shea, J. J., etal. (2002). Cell 109 Suppl, S 121 -31). Ligand binding to the cytokine receptor activates receptor- associated Janus kinases (JAKs), which ultimately results in the tyrosine phosphorylation of latent cytoplasmic STAT proteins and their subsequent translocation into the nucleus where they bind to target genes and effect gene transcription.
  • STAT signal transducer and activator of transcription
  • IL- 12 and IL-4 are the cytokines that are principally responsible for activating the genetic programs necessary for the differentiation of naive CD4+ T helper (Th) cells into ThI and Th2 cells, and they do so by activating STAT4 and STAT6, respectively (Murphy, K. M., and Reiner, S. L. (2002). Nat Rev Immunol 2, 933-944). Indeed, studies on STAT4- and STAT ⁇ -deficient mice confirmed the importance of these signaling molecules for the differentiation of ThI and Th2 cells (Kaplan, M. H., et al. (1996b). Immunity 4, 313-319; Kaplan, M. H., etal. (1996a).
  • suppressor of cytokine signaling (SOCS) proteins bind either to activated JAK proteins resulting in the direct inhibition of JAK activity, or in some cases to the docking site in the cytokine receptor to which the STATs must bind for their activation by JAK kinases (Alexander, W. S., and Hilton, D. J. (2004). Annu Rev Immunol 22, 503-529).
  • PIAS protein inhibitor of activated STAT
  • STAT proteins can be regulated by PTP in either the cytoplasm or nucleus, and individual PTP show some degree of specificity for STAT family members.
  • SHP-2 has been shown to interact with STAT5 (Chen, Y., et al. (2003). J Biol Chem 278, 16520-16527; Chughtai, N., et al. (2002). J Biol Chem 277, 31107-31114) and STATl (Wu, T. R., et al. (2002).
  • SHP-2-deficient mouse embryonic fibroblasts have impaired dephosphorylation of STAT5 mainly in the cytoplasm, and of STATl in nucleus, indicating that SHP-2 is involved in both cytoplasmic and nuclear dephosphorylation of STAT proteins.
  • TC-PTP acts on STAT 1 , and to a lesser degree STAT3, but not STAT5 or STAT6 (Meyer, T., et al. (2003). Genes Dev 7, 1992-2005; ten Hoeve, J., et al. (2002). MoI Cell Biol 22, 5662-5668).
  • STAT4 or STAT6 An understanding of the mechanism(s) by which STAT signaling is regulated and methods for modulating STAT-mediated signaling are lacking in the art. Therefore, the identification of additional PTP necessary to more fully understand the mechanism by which the STAT signaling pathway is regulated during the differentiation of Th cells is needed.
  • the present invention is based, at least in part, on the discovery that the tyrosine phosphatase, PTP-Basophil like (PTP-BL), physically interacts with multiple STAT proteins, e.g., STAT4, STATl, and/or STAT6.
  • PTP-BL PTP-Basophil like
  • STAT4 multiple STAT proteins
  • STAT6 multiple STAT proteins
  • the interaction of PTP-BL with STAT proteins leads to dephosphorylation, e.g., tyrosine dephosphorylation, of these STAT proteins in vitro and in vivo, subsequently resulting in attenuation of STAT-mediated gene activation, e.g., IFN- ⁇ gene activation.
  • the present invention is also based, at least in part, on the generation of Ptp-bT 1' deficient non-human animals.
  • STAT phosphorylation e.g., STAT4 and/or STAT6 phosphorylation
  • cytoplasm and nuclei o ⁇ Ptp-bt 1' deficient cells e.g., CD4+ T cells
  • this correlates with increased levels of Th cell e.g., ThI and/or Th2 cell, differentiation and subsequent cytokine production.
  • the instant invention provides methods of identifying modulators of the interaction of STAT polypeptides and PTP-BL polypeptides, methods of modulating a biological activity mediated by the interaction of STAT polypeptides and PTP-BL polypeptides e.g., modulation of STAT phosphorylation, modulation of STAT signaling, modulation of IFN- ⁇ production, modulation of IL-4 production, modulation of ThI cell differentiation, and modulation of Th cell differentiation, cytokine production, e.g., IFN- ⁇ and/or IL-4 production, and/or an immune response, e.g., a PTP-BL biological activity, as well as PTP-BL Ptp-bt 1' non-human animals, cells derived therefrom, and methods of use thereof.
  • modulation of STAT phosphorylation e.g., modulation of STAT signaling, modulation of IFN- ⁇ production, modulation of IL-4 production, modulation of ThI cell differentiation, and modulation of Th cell differentiation
  • the invention provides a method for identifying a compound which modulates an interaction between a STAT polypeptide and a PTP-BL polypeptide, comprising providing an indicator composition comprising a STAT coiled-coiled domain polypeptide and a PTPase domain polypeptide, contacting in the presence of the compound, the STAT coiled-coiled domain polypeptide and the PTPase domain polypeptide under conditions which allow interaction of the STAT coiled-coiled domain polypeptide and the PTPase domain polypeptide, and detecting the interaction of the STAT coiled-coiled domain polypeptide and the PTPase domain polypeptide to thereby identify a compound which modulates an interaction between a STAT polypeptide and a PTP-BL polypeptide, wherein the ability of the compound to increase an interaction between a STAT polypeptide and a PTP-BL polypeptide is indicated by an increase in the interaction as compared to the amount of interaction in the absence of the compound, and the ability of
  • the STAT polypeptide is full-length STAT. In one embodiment, the PTPase polypeptide is full-length PTP-BL.
  • the STAT polypeptide is STAT4. In another embodiment, the STAT polypeptide is STATl . In yet another embodiment, the STAT polypeptide is STAT6.
  • the interaction of the STAT polypeptide and the PTP-BL polypeptide is determined by measuring the formation of a complex between the STAT coiled-coiled domain polypeptide and the PTPase domain polypeptide.
  • the compound increases the formation or stability of the complex.
  • the compound decreases the formation or stability of the complex.
  • the interaction of the STAT polypeptide and the PTP-BL polypeptide is determined by measuring the phosphorylation of the STAT polypeptide.
  • the indicator composition is a cell comprising a STAT polypeptide a reporter gene responsive to the STAT polypeptide, and the effect of the test compound on the activity of the STAT polypeptide is determined by evaluating the expression of the reporter gene in the presence and absence of the test compound.
  • the reporter gene comprises the IRF-I promoter.
  • the reporter gene is selected from the group consisting of genes that encode: chloramphenicol acetyltransferase, beta-galactosidase, alkaline phosphatase and luciferase.
  • the activity is selected from the group consisting of: modulation of STAT phosphorylation, modulation of STAT signaling, modulation of cytokine production, e.g., IFN- ⁇ and/or IL-4 production, and modulation of ThI cell differentiation.
  • determining the ability of the test compound to modulate the interaction of the STAT polypeptide and the PTP-BL polypeptide comprises determining the ability of the test compound to modulate signaling via a signal transduction pathway involving STAT4. In one embodiment, the ability of the test compound to modulate signaling via a signal transduction pathway involving STAT4 is determined by measuring IFN- ⁇ production.
  • transgenic mouse comprising in its genome an exogenous DNA molecule that functionally disrupts a nucleic acid molecule encoding PTP-BL in said mouse, wherein said mouse exhibits a phenotype characterized by increased phosphorylation of STAT4, sustained STATl, STAT4, and STAT5 phosphorylation, increased amount of total STAT4 protein, increased STAT6 DNA binding activity, and increased ThI and Th2 cell differentiation relative to a wild-type mouse.
  • Yet another aspect of the invention provides an isolated cell from the transgenic mouse. In one embodiment, the cell is selected from the group consisting of fertilized egg cells, embryonic stem cells and lymphoid cells.
  • the invention provides method of modulating a PTP-BL biological activity, comprising contacting a cell with an agent that modulates the interaction of a STAT polypeptide and a PTP-BL polypeptide, to thereby modulate a PTP-BL biological activity.
  • One aspect of the invention provides a method for increasing Th cell differentiation, comprising contacting a cell with an agent that decreases the interaction of PTP-BL and a STAT in the cell such that Th cell differentiation is increased.
  • Another aspect of the invention provides a method for increasing cytokine production, comprising contacting a cell with an agent that decreases the interaction of PTP-BL and a STAT in the cell such that cytokine production is increased.
  • Yet another aspect of the invention provides a method for treating or preventing a disease, disorder, or condition that would benefit from an increased immune response in a subject, comprising contacting a cell from the subject with an agent that decreases the interaction of PTP-BL and a STAT in the cell such that the immune response in the subject is increased.
  • the invention provides a method for decreasing Th cell differentiation, comprising contacting a cell with an agent that increases the interaction of PTP-BL and a STAT in the cell such that Th cell differentiation is decreased.
  • Another aspect of the invention provides a method for decreasing cytokine production, comprising contacting a cell with an agent that decreases the interaction of PTP-BL and a STAT in the cell such that cytokine production is decreased.
  • Yet another aspect of the invention provides a method for treating or preventing a disease, disorder, or condition, that would benefit from a decreased immune response in a subject, comprising contacting a cell from the subject with an agent that decreases the interaction of PTP-BL and a STAT in the cell such that the immune response in the subject is decreased.
  • the step of contacting occurs in vitro. In another embodiment, the step of contacting occurs in vivo.
  • the agent is selected from the group consisting of: a nucleic acid molecule that is antisense to a PTP-BL molecule, a nucleic acid molecule that is antisense to a STAT molecule, a PTP-BL siRNA molecule, a STAT siRNA molecule, a dominant negative PTP-BL molecule, a dominant negative STAT molecule, or combinations thereof.
  • the agent is selected from the group consisting of: a nucleic acid molecule encoding a PTP-BL polypeptide, a nucleic acid molecule encoding a STAT polypeptide, a PTP-BL polypeptide, a STAT polypeptide, or combinations thereof.
  • the STAT is STAT4. In another embodiment, the STAT is STATl . In yet another embodiment, the STAT is STAT6.
  • the cell is a T cell.
  • Figure IA shows that PTP-BL interacts with STAT4.
  • A Schematic diagram of the leucine zipper motif of STAT4 used as bait in a yeast two-hybrid screen for STAT- interacting proteins.
  • Figure IB shows that PTP-BL interacts with STAT4 independent of tyrosine phosphorylation.
  • B Expression vectors for FLAG-tagged STAT4 wild type (WT) or Y693F mutant (1.0 ⁇ g each) were co-transfected into 293 T cells together with those for c-Myc-tagged PTP-BL frame shift (FS) or C-terminal PTP domain deleted (AC) mutants (11 ( ⁇ g each) as indicated.
  • TCLs Total cell lysates prepared from the transfected cells stimulated with IFN- ⁇ (1000 U/ml) for 30 minutes, or left unstimulated, were immunoprecipitated (IP) with anti- c-Myc and subjected to immunoblot with anti- STAT4.
  • FIGS 2A-D show that PTP-BL dephosphorylates STAT4 through its PTPase domain.
  • PTP-BL impairs STAT4 phosphorylation.
  • An expression vector for FLAG- tagged STAT4 (0.6 ⁇ g) was cotransfected into 293T cells together with those for c-Myc- tagged PTP-BL (FS, WT or AC) (12 ⁇ g each).
  • TCLs from cells treated with IFN- ⁇ (1000 U/ml) for 30 minutes were immunoprecipitated with anti-FLAG and immunobloted with anti-phospho STAT4.
  • B PTP-BL inhibits STAT4-mediated transactivation.
  • IRF-I luciferase reporter construct 0.2 ⁇ g
  • an expression vector for FLAG-tagged STAT4 0.2 ⁇ g
  • Cells were stimulated with IFN- ⁇ (1000 U/ml) for 6 hours and cell lysates were subjected to luciferase assay.
  • C Purification of GST-tagged PTPase domain of PTP-BL.
  • HighFive insect cells were infected with recombinant baculo virus that encodes either glutathoine S-transferase (GST) or GST-tagged PTPase domain of PTP-BL for 40 hours.
  • GST-tagged proteins were prepared by affinity purification and visualized by coomassie staining.
  • PTP- BL can directly dephosphorylate STAT4.
  • Phosphorylated STAT4 was immunoprecipitated with anti-FLAG from IFN- ⁇ -stimulated 293 T transfectants and incubated with 0.2 ⁇ g / ⁇ l recombinant proteins. Immunoprecipitates were subjected to immunoblot with anti-phospho-tyrosine (PY20).
  • FIGS. 3A-D show that PTP-BL inhibits IL- 12 signaling at the level of STAT4 phosphorylation.
  • A Generation of 2D6 cell transfectants that stably overexpress c- Myc-PTP-BL (FS or WT).
  • B PTP-BL impairs IL-12-induced STAT4 phosphorylation.
  • IL-12-starved 2D6 cell transfectants expressing c-Myc-PTP-BL were stimulated with IL-12 (5 ng/ml) for 30 minutes.
  • TCLs were immunoblotted with anti- phospho STAT4.
  • PTP-BL does not impair IL-12-induced Tyk2 phosphorylation.
  • EL- 12-starved 2D6 transfectants were stimulated with IL-12 (5 ng/ml) for 15 minutes. TCLs were immunoblotted with anti-phospho Tyk2.
  • D PTP-BL overexpression impairs IL- 12-induced IFN-y production.
  • IL-12-starved 2D6 cell clones (2 xlO 5 cells/well) were cultured without or with IL-12 (5 ng/ml) or PMA (50 ng/ml) plus Ionomycin (1 ⁇ g /ml) in 96-well culture plates. After 24 hours, supernatants were harvested and IFN- ⁇ production was measured by ELISA.
  • FIGS 4A-D show that PTP-BL dephosphorylates multiple STAT family members.
  • PTP-BL impairs STAT6 phosphorylation.
  • An expression vector for FLAG-tagged STAT6 (0.6 ⁇ g) was cotransfected into 293T cells together with those for c-Myc-tagged PTP-BL (FS, WT or AC) (12 ug each).
  • TCLs from cells treated with IL-4 (10 ng/ml) for 30 minutes were immunoprecipitated with anti-FLAG and subjected to immunoblot with anti-phospho STAT6.
  • B PTP-BL inhibits STAT6-mediated transactivation.
  • a STAT6-responsive luciferase reporter construct (TPU474) (0.2 ⁇ g) and a STAT6 expression vector (0.2 ⁇ g) were cotransfected into 293T cells together with increasing amounts (0.25, 0.5, 1.0 and 2.0 ⁇ g) of c-Myc-tagged PTP-BL expression vectors (WT or AC).
  • the total amount of transfected PTP-BL vector in each sample was adjusted to 2.0 ⁇ g by the addition of PTP-BL (FS). Twenty-four hours after transfection, cells were stimulated with IL-4 (10 ng/ml) for 6 hours and lysates were subjected to luciferase assay.
  • C PTP-BL can dephosphorylate STAT6 in vitro.
  • Phosphorylated STAT6 was immunoprecipitated with anti-FLAG from IL-4-stimulated 293 T transfectants and incubated with 0.25 ⁇ g /ul recombinant proteins. Immunoprecipitates were probed with anti-phospho tyrosine (PY20). Equal loading was verified by reprobing with anti-STAT6.
  • PTP-BL does not inhibit NF- ⁇ B-mediated transactivation.
  • a luciferase reporter construct which contains 5 copies of NF- ⁇ B- binding sequence (0.2 ⁇ g) and an NF- ⁇ B p65 expression vector (0.2 ⁇ g) were cotransfected into 293 T cells together with c-Myc-tagged PTP-BL expression vectors (WT or AC). The total amount of transfected PTP-BL vector in each sample was adjusted to 2.0 ⁇ g by the addition of PTP-BL (FS). Thirty hours after transfection, cells were harvested and lysates were subjected to luciferase assay.
  • Figures 5A-D show the generation o ⁇ Ptp-bt ' mice and PTP-BL expression in CD4+ T cells.
  • A Gene targeting construct for disruption o ⁇ Ptp-bl. Restriction enzymes are the following: Ec, EcoRI; Xb, Xbal; Xh, Xhol.
  • B Northern blot analysis of total RNA isolated from the kidneys of wild type or Ptp-bl 1' mice. 18S and 28S RNAs are shown to verify equal loading in each lane.
  • C PTP-BL expression in CD4+ T cells from wild type and Ptp-bT' ' rak,t.
  • TCLs were prepared from freshly isolated CD4+ T cells and subjected to immunoblot with anti-PTP-BL.
  • D Subcellular localization of PTP-BL.
  • CD4+ T cells from wild type and Ptp-bt 1' mice were differentiated under ThI or Th2 cell conditions for 5 days. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblot with anti-PTP-BL.
  • FIGS 6A-C show that STAT activation is increased and prolonged in PTP-BL deficient CD4+ T cells.
  • STAT4 phosphorylation is increased mPtp-bT 1' CD4+ T cells. Freshly isolated CD4+ T cells were stimulated with immobilized anti-CD3 (1 ⁇ g/ml for coating), 1 ⁇ g /ml anti-CD28, 100 U/ml human IL-2, 5 ng/ml murine IL- 12 and 10 ⁇ g /ml anti-IL-4 for 15 hours. Cytoplasmic and nuclear extracts were subjected to immunoblot as indicated.
  • STAT4 phosphorylation is prolonged in Ptp-b& CD4+ T cells.
  • Freshly isolated CD4+ T cells were stimulated with IFN- ⁇ (3000 U/ml) for 30 minutes, followed by staurosporine chase (0.5 ( ⁇ M) for another 15, 30 or 45 minutes. Cytoplasmic and nuclear extracts were subjected to immunoblot as indicated.
  • C Prolonged STAT6 DNA binding activity in nuclei o ⁇ Ptp-bT x' CD4+ T cells. Freshly isolated WT and Ptp-bt 1' CD4+ T cells were stimulated with BL-4 (40 ng/ml) for 30 minutes, followed by staurosporine chase (0.5 ⁇ M) for another 15 or 30 minutes. Nuclear extracts (1.3 ⁇ g) were subjected to EMSA using STAT6 and SpI consensus probes.
  • FIGS 7A-C show that there is enhanced ThI and Th2 Cell differentiation in Ptp-bf ' CD4+ T cells.
  • A Increased IFN- ⁇ and IL-4 production by Ptp-bT u Th and Th2 cells after secondary stimulation.
  • CD4+ T cells isolated from WT and Ptp-bl x' mice were differentiated under unskewed (UNSK), ThI- and Th2-skewing conditions for 5 days and then re-stimulated with plate-bound anti-CD3. IFN- ⁇ and IL-4 production was measured by ELISA.
  • B Enhanced ThI and Th2 cell differentiation in Ptp-bt 1' CD4+ T cells. The generation of ThI and Th2 cells was assessed by intracellular cytokine staining.
  • the present invention is based, at least in part, on the discovery that the tyrosine phosphatase, PTP-Basophil like (PTP-BL), physically interacts with multiple STAT proteins, e.g., STAT4, STATl, and/or STAT6.
  • PTP-BL PTP-Basophil like
  • STAT4 multiple STAT proteins
  • STAT6 multiple STAT proteins
  • the interaction of PTP-BL with STAT proteins leads to dephosphorylation, e.g., tyrosine dephosphorylation, of these STAT proteins in vitro and in vivo, subsequently resulting in attenuation of STAT-mediated gene activation, e.g., IFN- ⁇ gene activation.
  • the present invention is also based, at least in part, on the generation of Ptp-bt 1' deficient non-human animals.
  • STAT phosphorylation e.g., STAT4 and/or STAT6 phosphorylation
  • cytoplasm and nuclei oi Ptp-bt 1' deficient cells e.g., CD4+ T cells
  • Th cell e.g., ThI and/or Th2 cell
  • differentiation and subsequent cytokine production e.g., IL-4 and/or STAT6 phosphorylation.
  • PTP-BL deficiency results in improved host defense against bacterial infection, e.g., intrapulmonary K. pneumoniae, infection.
  • the instant invention provides methods of identifying modulators of the interaction between a STAT polypeptides and PTP-BL polypeptides, as well as Ptp-bt 1' non-human animals, cells derived therefrom, methods of modulating cytokine gene activation, e.g., IFN- ⁇ gene activation, Th cell differentiation, cytokine production, e.g., IFN- ⁇ and IL-4, and/or an immune response, and methods of use thereof.
  • cytokine gene activation e.g., IFN- ⁇ gene activation
  • Th cell differentiation e.g., cytokine production
  • IFN- ⁇ and IL-4 e.g., IFN- ⁇ and IL-4
  • an element means one element or more than one element.
  • STAT signal transducers and activators of transcription
  • STATs function as latent cytoplasmic transcriptional activators that become activated by tyrosine phosphorylation in response to the engagement of various cytokine receptors.
  • Phosphorylated STAT proteins dimerize and subsequently move to the cell nucleus, where they activate transcription by binding to specific DNA elements.
  • Members of the STAT family contain conserved structural features commonly found in transcription factors, e.g., coiled-coil domains, heptad leucine repeats, a helix-turn-helix motif, and SH2 and SH3 domains.
  • SH2 domains specifically recognize short sequence motifs flanking a tyrosine phosphorylated residue and play a crucial role in signal transduction.
  • SH3 domains are involved in the targeting of signaling components to specific subcellular locations (see, e.g., Schindler (2002) J Clin Invest 109:1133, the entire contents of which are incorporated by reference).
  • Exemplary STATs include, STATl, STAT4, and STAT6.
  • the nucleotide and amino acid sequences of human STAT4 are known and can be found in, for example, GenBank accession No.: gi:21618332 (SEQ ID NO:3); the nucleotide and amino acid sequences of mouse STAT4 are known and can be found in, for example, GenBank accession No.: gi:6755669 (SEQ ID NO:4); the nucleotide and amino acid sequences of human STATl, isoform alpha, are known and can be found in, for example, GenBank accession No.: gi:21536299 (SEQ ID NO: 5); the nucleotide and amino acid sequences of human STATl, isoform beta, are known and can be found in, for example, GenBank accession No.: gi:21536300 (SEQ ID NO:6); the nucleotide and amino acid sequences of mouse STATl are known and can be found in, for example, GenBank accession No.: gi:31543777
  • STAT4 is one of seven mammalian STAT family members and is activated following stimulation by IL-12 or IFN- ⁇ (Nguyen, K. B. et al. (2002) Science 297, 2063-2066). STAT4 is essential for IL-12-mediated differentiation of naive Th cells into IFN ⁇ -secreting ThI cells as evidenced by the phenotype of STAT4-deficient mice (Kaplan, M. H., et al. (1996) Nature 382, 174-177; Thierfelder, W. E. et al. (1996) Nature 382, 171-174).
  • STATl -alpha and STATl -beta are activated in response to IFN receptor engagement.
  • Animals deficient in STATl are susceptible to viral disease. Tissues isolated from these animals are unresponsive to stimulation with IFN but remain responsive to other cytokines.
  • STAT6 also referred to as EL-4 STAT mediates signaling via IL-3, IL-4, and IL- 13 (Brierley MM and Fish EN. (2005) J Interferon Cytokine Res. 25(12):733-44; Hebenminister, D, etal. (2006) Cytokine Growth Factor Rev. 17(3): 173-88).
  • tyrosine specific protein phosphatases or “protein tyrosine phosphatases” are enzymes that catalyse the removal of a phosphate group attached to a tyrosine residue. These enzymes are very important in the control of cell growth, proliferation, differentiation and transformation and contain a "PTPase” catalytic domain at their carboxyl-terminus.
  • PTP-Basophil like or "PTP-BL”, also referred to as “protein tyrosine phosphatase, non-receptor type 13" (“PTPN13").
  • PTP-BL is known to be involved in multiple regulatory functions including, for example, negative regulation of FAS-induced apoptosis and NGFR-mediated pro-apoptotic signalling, regulation of the phophorylation status of ephrinB receptors in neurons, regulation of cytokinesis in epithelial cells, and the regulation of negative proliferation signals in breast cancer cells (Me, S. et al. (1999) FEBS Lett. 460:191-8; Palmer A. , et al.
  • PTP-BL possess at least seven potential protein-protein interaction domains including a kinase noncatalytic C-lobe (KIND) domain, a Four-point- one/Ezrin/Radixin/Moesin (FERM) domain and five PSD-95/Dorosophila discs large/Zonula occludens (PDZ) domains, and many potential interacting proteins have been identified (Erdmann, K. S. (2003). Eur J Biochem 270(24), 4789-4798).
  • PTP-BL also contains a leucine zipper (LZ) domain in the N-terminus of the molecule (Chida, D., et al. (1995).
  • the nucleotide and amino acid sequences of human PTP-BL, isoform 1 are known and can be found in, for example, GenBank accession No.: gi:18375645 (SEQ ID NO:10); the nucleotide and amino acid sequences of human PTP-BL, isoform 2, are known and can be found in, for example, GenBank accession No.: gi:5453991 (SEQ ID NO:11); the nucleotide and amino acid sequences of human PTP-BL, isoform 3, are known and can be found in, for example, GenBank accession No.: gi: 18375647 (SEQ ID NO: 12); the nucleotide and amino acid sequences of human PTP-BL, isoform 4, are known and can be found in, for example, GenBank accession No.: gi: 18375649 (SEQ ID NO: 13); and the nucleotide and amino acid sequences of mouse PTP-BL are
  • a yeast two-hybrid screen using a polypeptide comprising a portion of the coiled-coil domain of STAT which comprises a leucine zipper motif ⁇ i.e., LX 6 LX 6 LX 6 LXeL, a motif know to mediate hetero and homo-dimerization of DNA-binding proteins (Turner and Tjian (1989) Science 243:1689-1694)) of STAT4, ⁇ i.e., amino acid residues 250-310 of SEQ ID NO:4) identified PTP-BL as a STAT4 interacting molecule.
  • STAT polypeptide refers to a polypeptide that comprises a PTP-interacting domain
  • PTP-BL polypeptide refers to a polypeptide with a STAT-interacting domain.
  • a STAT polypeptide comprises a full-length STAT polypeptide, e.g., a full-length STAT4 (/. e., any one of SEQ ID NOs:3 or 4), a full- length STATl ⁇ i.e., any one of SEQ ID NOs:5 or 6 or 7), and/or a full-length STAT6 polypeptide ⁇ i.e., any one of SEQ ID NOs: 8 or 9).
  • a STAT polypeptide comprises a PTPase-interacting domain, e.g., a coiled-coil domain (i.e., at least about amino acid residues 135-315 of any one of SEQ ED NOs; 3-9) and is referred to as a "STAT coiled-coil domain polypeptide".
  • a polypeptide is a chimeric polypeptide (i.e., comprising a coiled-coil domain amino acid sequence and a sequence derived from a second, non-STAT polypeptide).
  • such a polypeptide consists of a full-length STAT polypeptide.
  • the STAT coiled-coil domain comprises a leucine zipper motif (i.e., amino acid residues 250-310 of SEQ ID NO:4 or 5).
  • a PTP-BL polypeptide comprises a full-length PTP-BL polypeptide, i.e., any one of SEQ ID NOs: 10-14.
  • a PTP-BL polypeptide comprises a STAT-interacting domain, e.g., a PTPase domain, i.e., amino acid residues 2238-2475 of SEQ ID NO: 10; amino acid residues 2219-2456 of SEQ ID NO:11; amino acid residues 2047-2284 of SEQ ID NO; 12; amino acid residues 2243-2480 of SEQ ID NO; 13; and/or amino acid residues 2204-2441 of SEQ ID NO: 14, referred to herein as a "PTPase domain polypeptide".
  • a PTPase domain i.e., amino acid residues 2238-2475 of SEQ ID NO: 10; amino acid residues 2219-2456 of SEQ ID NO:11; amino acid residues 2047-2284 of SEQ ID NO; 12; amino acid residues 2243-2480 of SEQ ID NO; 13; and/or amino acid residues 2204-2441 of SEQ ID NO: 14, referred to herein as a "PTPase domain poly
  • such a polypeptide is a chimeric polypeptide (i.e., comprising a PTPase domain amino acid sequence and a sequence derived from a second, non-PTP-BL polypeptide).
  • such a polypeptide consists of a full-length PTP-BL polypeptide.
  • a PTP-BL polypeptide does not comprise a PTPase domain, (i.e., a PTP-BL polypeptide comprises amino acid residues 1-2237 of SEQ ID NO: 10, amino acid residues 1-2218 of SEQ ID NO: 11; amino acid residues 1-2046 of SEQ ID NO: 12; amino acid residues 1-2242 of SEQ ID NO: 13; and/or amino acid residues 1-2203 of SEQ ED NO: 14) and is referred to herein as a "PTP-BL PTPase domain deleted polypeptide".
  • the various forms of the term “modulate” includes stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
  • stimulation e.g., increasing or upregulating a particular response or activity
  • 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 or coimmunoprecipitation.
  • the term 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 a compound
  • contacting is intended to include incubating the compound and the cell together in vitro (e.g., adding the compound to cells in culture) or administering the compound to a subject such that the compound and cells of the subject are contacted in vivo.
  • the term "compound” includes any agent, e.g., nucleic acid molecules, antisense nucleic acid molecule, peptide, peptidomimetic, small molecule, or other drug, which modulates an interaction between a STAT polypeptide and a PTP-BL polypeptide.
  • the agents of the invention can directly or indirectly modulate, i.e., increase or decrease, an interaction between a STAT polypeptide and a PTP-BL polypeptide.
  • exemplary agents are described herein or can be identified using screening assays that select for such compounds, e.g., as described in detail below.
  • the "test compound or agent" screened includes molecules that are not known in the art to modulate an interaction between a STAT polypeptide and a PTP-BL polypeptide as described herein.
  • a plurality of agents are tested using the instant methods.
  • library of test compounds is intended to refer to a panel comprising a multiplicity of test compounds.
  • the agent or test compound is a compound that directly interacts with PTP-BL or directly interacts with a molecule with which PTP-BL interacts (e.g., a compound that inhibits or stimulates the interaction between PTP-BL and a STAT polypeptide).
  • a compound that inhibits or stimulates the interaction between PTP-BL and a STAT polypeptide e.g., a compound that inhibits or stimulates the interaction between PTP-BL and a STAT polypeptide.
  • Such compounds can be identified using screening assays that select for such compounds, e.g., as described in detail below.
  • target molecule or "binding partner” is a molecule with which PTP-BL and/or STAT binds or interacts in nature, and which interaction results in a biological response resulting from such an interaction, e.g., modulation of STAT phosphorylation, modulation of cytokine production, e.g., EFN- ⁇ and/or IL-4 production, modulation of STAT signaling, modulation of Th cell differentiation, and/or an immune response, e.g., a "PTP-BL biological activity”.
  • the term "indicator composition” refers to a composition that includes a protein of interest (e.g., a STAT polypeptide and/or a PTP-BL polypeptide), for example, a cell that naturally expresses the protein, a cell that has been engineered to express the protein by introducing an expression vector encoding the protein into the cell, or a cell free composition that contains the protein (e.g., purified naturally- occurring protein or recombinantly-engineered protein).
  • the term “cell free composition” refers to an isolated composition which does not contain intact cells. Examples of cell free compositions include cell extracts and compositions containing isolated proteins.
  • reporter gene includes genes that express a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable. The reporter gene may also be included in a construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta- galactosidase; firefly luciferase (deWet, et al. (1987), MoI. Cell. Biol.
  • CAT chloramphenicol acetyl transferase
  • nucleic acid is intended to include fragments or equivalents thereof (e.g., fragments or equivalents thereof PTP-BL and/or STAT).
  • equivalent is intended to include nucleotide sequences encoding functionally equivalent PTP-BL and/or STAT proteins, i.e., proteins which have the ability to bind to the natural binding partner(s) of the PTP-BL and/or STAT antigen.
  • a functionally equivalent PTP-BL protein has the ability to bind STAT, e.g., STAT4, STATl, and/or STAT6.
  • a functionally equivalent STAT polypeptide has the ability to bind a PTP-BL polypeptide.
  • 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 1 and 3 1 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” polypeptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the PTP-BL and/or STAT polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of PTP-BL and/or STAT polypeptide in which the polypeptide 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, e.g., by standard recombinant DNA techniques.
  • a nucleic acid molecule 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 rep Ii cation 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 rep heated 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 retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae 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), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • PTP-BL and/or STAT polypeptides can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al. (1983)M?/. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid molecule of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman, et al. (1987) EMBO J. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of 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.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid molecule preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton ( ⁇ 9SS) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (19S9) EMBO J.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • 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 one embodiment, a host cell is a murine cell.
  • misexpression includes a non-wild-type pattern of gene expression.
  • Expression includes transcriptional, post transcriptional, e.g., mRNA stability, translational, and post translational stages. Misexpression includes: expression at non-wild-type levels, i.e., over or under expression; a pattern of expression that differs from wild-type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild-type) at a predetermined developmental period or stage; a pattern of expression that differs from wild-type in terms of decreased expression (as compared with wild-type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild-type in terms of the splicing of the mRNA, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild-type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene,
  • Misexpression includes any expression from a transgenic nucleic acid. Misexpression includes the lack or non-expression of a gene or transgene, e.g., that can be induced by a deletion of all or part of the gene or its control sequences.
  • knockout refers to an animal or cell therefrom, in which the insertion of a transgene disrupts an endogenous gene in the animal or cell therefrom. This disruption can essentially eliminate, for example, PTP-BL, in the animal or cell.
  • misexpression of the gene encoding the PTP-BL protein is caused by disruption of the PTP-BL gene.
  • the PTP-BL gene can be disrupted through removal of DNA encoding all or part of the protein, for example, as described in the appended Examples.
  • disruption of a gene refers to a change in the gene sequence, e.g., a change in the coding region. Disruption includes: insertions, deletions, point mutations, and rearrangements, e.g., inversions.
  • the disruption can occur in a region of the native PTP-BL DNA sequence ⁇ e.g., one or more exons) and/or the promoter region of the gene so as to decrease or prevent expression of the gene in a cell as compared to the wild-type or naturally occurring sequence of the gene.
  • the "disruption” can be induced by classical random mutation or by site directed methods. Disruptions can be transgenically introduced. The deletion of an entire gene is a disruption. Preferred disruptions reduce PTP-BL levels to about 50% of wild-type, in heterozygotes or essentially eliminate PTP-BL in homozygotes.
  • transgenic cell refers to a cell containing a transgene.
  • a transgenic animal refers to a non-human animal, preferably a mammal, more preferably a mouse, in which one or more of the cells of the animal includes a "transgene”.
  • transgene refers to exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, for example directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • the term "cells deficient in PTP-BL” is intended to include cells of a subject that are naturally deficient in PTP-BL, as wells as cells of a non-human PTP-BL deficient animal, e.g., a mouse, that have been altered such that they are deficient in PTP-BL.
  • the term "cells deficient in PTP-BL” is also intended to include cells isolated from a non-human PTP-BL deficient animal or a subject that are cultured in vitro.
  • non-human PTP-BL deficient animal refers to a non- human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal, such that the endogenous PTP-BL gene is altered, thereby leading to either no production of PTP-BL or production of a mutant form of PTP-BL having deficient PTP-BL activity.
  • the activity of PTP-BL is entirely blocked, although partial inhibition of PTP-BL activity in the animal is also encompassed.
  • non-human PTP-BL deficient animal is also intended to encompass chimeric animals (e.g., mice) produced using a blastocyst complementation system, such as the RAG-2 blastocyst complementation system, in which a particular organ or organs (e.g., the lymphoid organs) arise from embryonic stem (ES) cells with homozygous mutations of the PTP-BL gene.
  • a blastocyst complementation system such as the RAG-2 blastocyst complementation system
  • a particular organ or organs e.g., the lymphoid organs
  • ES embryonic stem
  • T cell i.e., T lymphocyte
  • T lymphocyte is intended to include all cells within the T cell lineage, including thymocytes, immature T cells, mature T cells and the like, from a mammal (e.g., human).
  • T cells include mature T cells that express either CD4 or CD8, but not both, and a T cell receptor.
  • the various T cell populations described herein can be defined based on their cytokine profiles and their function.
  • immune response includes T cell mediated and/or B cell mediated immune.
  • exemplary immune responses include T cell responses, e.g., cytokine production, and cellular cytotoxicity.
  • immune response includes antibody production (humoral responses) and activation of cells of the innate immune system, e.g., cytokine responsive cells such as macrophages.
  • an immune response is a response of the innate immune system.
  • innate immune system includes natural or native immune mechanisms, i.e., mechanisms that exist before infection, are capable of rapid responses to microbes, and react in essentially the same way to repeated infections.
  • adaptive immune system or “specific immune system” includes immune mechanisms that are stimulated by exposure of infectious agents and increase in magnitude and defensive capabilities with each successive exposure to a particular microbe.
  • T helper type 1 response refers to a response that is characterized by the production of one or more cytokines selected from IFN- ⁇ , IL-2, TNF, and lymphtoxin (LT) and other cytokines produced preferentially or exclusively by ThI cells rather than by Th2 cells.
  • Th2 response refers to a response by CD4 + T cells that is characterized by the production of one or more cytokines selected from IL-4, IL-5, IL-6 and IL-IO, and that is associated with efficient B cell "help” provided by the Th2 cells (e.g., enhanced IgGl and/or IgE production).
  • small molecules can be used as test compounds.
  • the term "small molecule” is a term of the art and includes molecules that are less than about 7500, less than about 5000, less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., Cane et al. 1998. Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic. For example, a small molecule is preferably not itself the product of transcription or translation.
  • the invention provides methods (also referred to herein as “screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptidomimetics, small molecules or other drugs) which modulate, for example an interaction between a STAT polypeptide and a PTP-BL polypeptide, or for testing or optimizing the activity of such agents.
  • modulators i.e., candidate or test compounds or agents (e.g., peptidomimetics, small molecules or other drugs) which modulate, for example an interaction between a STAT polypeptide and a PTP-BL polypeptide, or for testing or optimizing the activity of such agents.
  • the subject assays can be used to identify, e.g., agents that alter an interaction between a PTP-BL polypeptide and a STAT polypeptide, such as, but not limited to, STAT4, STATl, and/or STAT6, or modulate, e.g., increase or decrease, the stability of such an interaction, and/or modulate a PTP-BL biological activity.
  • a STAT polypeptide such as, but not limited to, STAT4, STATl, and/or STAT6
  • modulate e.g., increase or decrease, the stability of such an interaction, and/or modulate a PTP-BL biological activity.
  • the subject screening assays can measure the interaction between a PTP-BL polypeptide and a STAT polypeptide directly (e.g., formation of a complex and/or phosphorylation), or can measure a downstream event controlled by modulation of an interaction between a PTP-BL polypeptide and a STAT polypeptide (e.g., STAT phosphorylation, cytokine production, e.g., IFN- ⁇ and/or IL-4 production, STAT signaling, Th cell differentiation, and/or an immune response), i.e., a biological activity resulting from an interaction between a PTP-BL polypeptide and a STAT polypeptide, e.g., a PTP-BL biological activty.
  • STAT polypeptide e.g., STAT phosphorylation, cytokine production, e.g., IFN- ⁇ and/or IL-4 production, STAT signaling, Th cell differentiation, and/or an immune response
  • the subject screening assays employ indicator compositions. These indicator compositions comprise the components required for performing an assay that detects and/or measures a particular event.
  • the indicator compositions of the invention provide a reference readout and changes in the readout can be monitored in the presence of one or more test compounds. A difference in the readout in the presence and the absence of the compound indicates that the test compound is a modulator of the interaction of the molecule(s) present in the indicator composition.
  • the indicator composition used in the screening assay can be a cell that expresses a PTP-BL polypeptide and/or a STAT polypeptide.
  • a cell that naturally expresses or, more preferably, a cell that has been engineered to express the protein by introducing into the cell an expression vector encoding the protein may be used.
  • the cell is a mammalian cell, e.g., a human cell.
  • the cell is a T cell.
  • the cell is a non-T cell.
  • the indicator composition can be a cell-free composition that includes the protein(s) (e.g., a cell extract or a composition that includes e.g., either purified natural or recombinant protein(s)).
  • the indicator composition comprises a full- length STAT polypeptide, e.g., a full-length STAT4 (i.e., any one of SEQ ID NOs:3 or 4), a full-length STATl (i.e., any one of SEQ ED NOs:5 or 6 or 7), and/or a full-length STAT6 polypeptide (i.e., any one of SEQ ED NOs:8 or 9).
  • the indicator composition comprises a STAT coiled-coil domain polypeptide (i.e., at least about amino acid residues 135-315 of any one of SEQ ID NOs; 3-9).
  • such a polypeptide is a chimeric polypeptide (i.e., comprising a coiled-coil domain amino acid sequence and a sequence derived from a second, non-STAT polypeptide).
  • such a polypeptide consists of a full-length STAT.
  • the STAT coiled-coil domain polypeptide comprises a leucine zipper motif (i.e., amino acid residues 250-310 of SEQ ID NO:4 or 5).
  • the indicator composition comprises a PTP-BL polypeptide comprises a full-length PTP-BL polypeptide, i.e., any one of SEQ ID NOs: 10-14.
  • the indicator composition comprises a PTPase domain polypeptide, i.e., amino acid residues 2238-2475 of SEQ ID NO: 10; amino acid residues 2219-2456 of SEQ ID NO:11; amino acid residues 2047-2284 of SEQ ID NO;12; amino acid residues 2243-2480 of SEQ ID NO;13; and/or amino acid residues 2204-2441 of SEQ ID NO: 14.
  • a polypeptide is a chimeric polypeptide (i.e., comprising a coiled-coil domain amino acid sequence and a sequence derived from a second, non-PTP-BL polypeptide).
  • such a polypeptide consists of a full-length PTP-BL.
  • the indicator composition comprises a PTP-BL
  • PTPase domain deleted polypeptide i.e., a PTP-BL polypeptide comprises amino acid residues 1-2237 of SEQ ID NO:10, amino acid residues 1-2219 of SEQ ID NO:11; amino acid residues 1-1046 of SEQ ID NO: 12; amino acid residues 1-2242 of SEQ ID NO:13; and/or amino acid residues 1-2203 of SEQ ID NO:14.
  • secondary assays can be used to confirm that the modulating agent affects an interaction between a PTP-BL polypeptide and a STAT polypeptide in a specific manner.
  • compounds identified in a primary screening assay can be used in a secondary screening assay to determine whether the compound affects a PTP-BL biological activity, e.g., a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide.
  • a PTP-BL biological activity e.g., a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide.
  • 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, e.g., to detect binding, and the ability of the agent to modulate the interaction can be confirmed using a biological read-out to measure, e.g., STAT phosphorylation, cytokine production, DNA binding abiltity, Th cell differentiation, and/or an immune response, in vitro or in vivo.
  • a biological read-out to measure, e.g., STAT phosphorylation, cytokine production, DNA binding abiltity, Th cell differentiation, and/or an immune response, in vitro or in vivo.
  • a modulator of an interaction between PTP-BL and STAT identified as described herein may be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such a modulator.
  • a modulator identified as described herein may be used in an animal model to determine the mechanism of action of such a modulator.
  • the screening assays of the invention are high throughput or ultra high throughput (e.g., Fernandes, P.B., Curr Opin Chem Biol. 1998 2:597; Sundberg, SA, Curr Opin Biotechnol. 2000, 11:47).
  • the indicator compositions of the invention may be cells that express a PTP-BL polypeptide and/or a STAT polypeptide.
  • a cell that naturally expresses endogenous polypeptide, or, more preferably, a cell that has been engineered to express one or more exogenous polypeptides, e.g., by introducing into the cell an expression vector encoding the protein may be used in a cell based assay.
  • the cells used in the instant assays can be eukaryotic or prokaryotic in origin.
  • the cell is a bacterial cell.
  • the cell is a fungal cell, e.g., a yeast cell.
  • the cell is an insect cell.
  • the cell is a vertebrate cell, e.g., an avian or a mammalian cell (e.g., a murine cell, rhesus monkey, or a human cell).
  • the cell is a human cell.
  • a cell line which expresses low levels of endogenous PTP-BL polypeptide and/or STAT polypeptide and is then engineered to express recombinant protein.
  • cDNA is first introduced into a recombinant expression vector using standard molecular biology techniques.
  • a cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library.
  • PCR polymerase chain reaction
  • 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 can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can 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 retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid molecule in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression and the level of expression desired, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell, those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) or those which direct expression of the nucleotide sequence only under certain conditions (e.g., inducible regulatory sequences). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma virus, adenovirus, cytomegalovirus and Simian Virus 40.
  • mammalian expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman, et al (1987), EMBO J. 6: 187-195).
  • pCDM8 Seed, B., (1987) Nature 329:840
  • pMT2PC Kaufman, et al (1987), EMBO J. 6: 187-195.
  • a variety of mammalian expression vectors carrying different regulatory sequences are commercially available.
  • a preferred regulatory element is the cytomegalovirus promoter/enhancer.
  • inducible regulatory systems for use in mammalian cells are known in the art, for example systems in which gene expression is regulated by heavy metal ions (see e.g., Mayo, et al. (1982) Cell 29:99-108; Brinster, et al. (19S2) Nature 296:39-42; Searle, et al. (1985) MoI. Cell. Biol. 5:1480-1489), heat shock (see e.g., Nouer, et al. (1991) in Heat Shock Response, e.d. Nouer, L., CRC, Boca Raton , FL, pp 167-220), hormones (see e.g., Lee, et al.
  • heavy metal ions see e.g., Mayo, et al. (1982) Cell 29:99-108; Brinster, et al. (19S2) Nature 296:39-42; Searle, et al. (1985) MoI. Cell. Biol. 5:14
  • tissue-specific regulatory sequences are known in the art, including the albumin promoter (liver-specific; Pinkert, et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji, et al.
  • Vector DNA may be introduced into mammalian cells via conventional transfection techniques.
  • transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into mammalian host cells, including calcium phosphate co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transfecting 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 manuals.
  • 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 can be introduced into a host cell on a separate vector from that encoding a PTP-BL polypeptide and/or STAT polypeptide, or on the same vector.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • within the expression vector coding sequences are operatively linked to regulatory sequences that allow for constitutive expression of the molecule in the indicator cell (e.g., viral regulatory sequences, such as a cytomegalovirus promoter/enhancer, may be used).
  • regulatory sequences e.g., viral regulatory sequences, such as a cytomegalovirus promoter/enhancer, may be used.
  • Use of a recombinant expression vector that allows for constitutive expression of the genes in the indicator cell is preferred for identification of compounds that enhance or inhibit the interaction between molecules and/or the activity of the molecule.
  • the coding sequences are operatively linked to regulatory sequences of the endogenous gene (i.e., the promoter regulatory region derived from the endogenous gene).
  • the endogenous gene i.e., the promoter regulatory region derived from the endogenous gene.
  • an indicator cell can be transfected with an expression vector comprising a PTP-BL polypeptide and/or an expression vector comprising a STAT polypeptide, incubated in the presence and in the absence of a test compound, and the effect of the compound on an interaction between a PTP-BL polypeptide and a STAT polypeptide or on a PTP-BL biological activity, e.g., a biological response regulated by the interaction of the PTP-BL polypeptide with the STAT polypeptide, e.g., a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide, can be determined.
  • the biological activities resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide include activities determined in vivo, or in vitro, according to standard techniques.
  • Activity can be a direct activity, such as binding to DNA, or an association with or enzymatic activity, e.g., phosphorylation, e.g., tyrosine phosphorylation, on a target molecule (e.g., STAT, e.g., STAT4, STATl, and/or STAT6).
  • an activity may be an indirect activity, such as, for example, a cellular signaling activity occurring downstream of the interaction of the protein with a target molecule or a biological effect occurring as a result of the signaling cascade triggered by that interaction, such as STAT signaling, cytokine production, e.g., IFN- ⁇ and/or IL-4 production, and/or Th cell differentiation, e.g., ThI and/or Th2 cell differentiation, and/or an immune response.
  • a cellular signaling activity occurring downstream of the interaction of the protein with a target molecule or a biological effect occurring as a result of the signaling cascade triggered by that interaction, such as STAT signaling, cytokine production, e.g., IFN- ⁇ and/or IL-4 production, and/or Th cell differentiation, e.g., ThI and/or Th2 cell differentiation, and/or an immune response.
  • cytokine production e.g., IFN- ⁇ and/or IL-4 production
  • Th cell differentiation
  • Compounds that modulate a PTP-BL biological activity may be identified using various "read-outs.”
  • a variety of reporter genes are known in the art and are suitable for use in the screening assays of the invention.
  • suitable reporter genes include those which encode chloramphenicol acetyltransferase, beta-galactosidase, alkaline phosphatase or luciferase. Standard methods for measuring the activity of these gene products are known in the art.
  • the ability of the test compound to modulate an interaction between a PTP-BL polypeptide and a STAT polypeptide can be accomplished, for example, by determining the ability of the molecules to be coimmunoprecipitated or by coupling the target molecule, e.g., a STAT polypeptide, with a radioisotope or enzymatic label such that binding of the target molecule to a PTP-BL polypeptide can be determined, e.g., by detecting the labeled molecule in a complex.
  • PTP-BL can be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate PTP-BL binding to a target molecule in a complex, e.g., a STAT polypeptide.
  • Determining the ability of the test compound to bind to a PTP-BL polypeptide and/or a STAT polypeptide can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound can be determined by detecting the labeled compound in a complex.
  • targets can be labeled with 12 ⁇ I, 3 ⁇ S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • compounds can be labeled, e.g., with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • fluorescence technologies can be used, e.g., fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer (Selvin, PR, Nat. Struct. Biol. 2000 7:730; Hertzberg RP and Pope AJ, Curr Opirt Chem Biol. 2000 4:445).
  • a microphysiometer may be used to detect the interaction of a compound with a PTP-BL polypeptide and/or a STAT polypeptide without the labeling of either the compound or the molecule (McConnell, H. M., et al. (1992) Science 257:1906-1912).
  • a "microphysiometer” ⁇ e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • a PTP-BL polypeptide and/or a STAT polypeptide may be used as "bait protein" e.g., 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.
  • binding proteins proteins which bind to or interact with a PTP-BL polypeptide and/or a STAT polypeptide
  • binding proteins or "bp"
  • Such PTP-BL polypeptide- and/or STAT polypeptide -binding proteins are also likely to be involved in the propagation of signals by the PTP-BL and/or STAT proteins.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs. For example, in one construct, the gene that codes for a PTP-BL polypeptide (or a STAT polypeptide) is fused to a gene encoding the DNA binding domain of a known transcription factor ⁇ e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • 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) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the PTP-BL and/or STAT polypeptides.
  • a reporter gene ⁇ e.g., LacZ
  • agents that modulate an interaction between a PTP-BL polypeptide and a STAT polypeptide and/or a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide can be identified by operably linking the upstream regulatory sequences ⁇ e.g., the full length promoter and enhancer) of a STAT to a reporter gene such as chloramphenicol acetyltransferase (CAT) or luciferase and introducing in into host cells.
  • CAT chloramphenicol acetyltransferase
  • a reporter gene comprises an "IFN regulatory factor- 1" (IRF-I") promoter (see, e.g., Galon, Jerome, et al. (1999) J Immunol 162:7256-7262, the contents of which are incorporated by reference.
  • the IRF-I promoter comprises the nucleic acid sequence: (5 I -AGCTTCAGCCTGATTTCCCCGAAATGACGGA-3 I ) (SEQ ID NO: 15).
  • the terms "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 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 level of expression of the reporter gene in the indicator cell in the presence of the test compound is higher than the level of expression of the reporter gene in the indicator cell in the absence of the test compound and the test compound is identified as a compound that stimulates the expression of a STAT gene and/or increases a PTP-BL biological activity.
  • the level of expression of the reporter gene in the indicator cell in the presence of the test compound is lower than the level of expression of the reporter gene in the indicator cell in the absence of the test compound and the test compound is identified as a compound that inhibits the expression of a STAT gene and/or decreases a PTP-BL biological activity.
  • protein expression may be measured.
  • standard techniques such as Western blotting or in situ detection can be used.
  • the ability of a compound to modulate cytokine production can be determined.
  • Cytokine production e.g., IFN- ⁇ and/or IL-4 production
  • JJFN- ⁇ and/or BL-4 can also be detected using an ELISA assay or in a bioassay, e.g., employing cells which are responsive to IFN- ⁇ and/or IL-4 ⁇ e.g., cells which proliferate in response to the cytokine or which survive in the presence of the cytokine) using standard techniques.
  • the effect of a compound on a STAT signaling pathway can be determined.
  • STAT phosphorylation/activation by JAK kinases leads to their translocation to the nucleus where they activate transcription of various cytokine genes by binding to specific DNA elements.
  • the effect of a compound on the ability of STAT to bind to DNA can be determined by, for example, an elctrophoretic mobility shift assay (EMSA).
  • ESA elctrophoretic mobility shift assay
  • Activated STATs are also known to stimulate transcription of SOCS (suppressors of cytokine signaling) genes which bind phosphorylated JAKs and their receptors to prevent further phosphorylation of STATs and thus serve as a negative feedback regulatory loop.
  • SOCS suppressors of cytokine signaling
  • Other exemplary molecules in a STAT signaling pathway include but are not limited to, Ras, EGFR and PDGFR, and TGF- ⁇ (reviewed, in, for example, Rawlings, J. S. (2004) Journal of Cell Science 117:1281-1283). Accordingly, to determine the effect of a compound on a STAT signal transduction pathway, the ability of the compound to modulate the activation status of various molecules in the signal transduction pathway can be determined using standard techniques. In one embodiment, the expression of SOCS is determined.
  • the phosphorylation of SOCS is determined.
  • modulation of the effect of the compound on the phosphorylation status of a STAT polypeptide e.g., STAT4, STATl, and/or STAT6, can be determined by, for example, Western blotting, as described in the Examples herein, or by immunoblotting with antibodies specific to the phosphorylation status of a STAT polypeptide.
  • the compound modulates the tyrosine phosphorylation of a STAT polypeptide.
  • a downstream effect of an interaction of a PTP-BL polypeptide with a STAT polypeptide may be used as an indicator of modulation of a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide.
  • Th cell differentiation can be monitored directly (e.g. by microscopic examination of the cells), or indirectly, e.g., by monitoring one or more markers of Th cells (e.g., by FACs analysis and/or an increase in mRNA for a gene product associated with Th cells) or the expression of a cell surface marker.
  • Standard methods for detecting mRNA of interest such as reverse transcription- polymerase chain reaction (RT-PCR) and Northern blotting, are known in the art.
  • Standard methods for detecting protein secretion in culture supernatants such as enzyme linked immunosorbent assays (ELISA), are also known in the art. Proteins can also be detected using antibodies, e.g., in an immunoprecipitation reaction or for staining and FACS analysis. iL Cell-free assays
  • the indicator composition can be a cell-free composition that includes a PTP-BL and/or a STAT polypeptide, e.g., a cell extract from a cell expressing the protein or a composition that includes purified either natural or recombinant protein.
  • the indicator composition is a cell free composition.
  • Polypeptides expressed by recombinant methods in a host cells or culture medium can be isolated from the host cells, or cell culture medium using standard methods for protein purification. For example, ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies may be used to produce a purified or semi-purified protein that may be used in a cell free composition.
  • a lysate or an extract of cells expressing the protein of interest can be prepared for use as cell-free composition.
  • Cell extracts with the appropriate post-translation modifications of proteins can be prepared using commercially available resources found at, for example Promega, Inc., and include but are not limited to reticulocyte lysate, wheat germ extract and E. coli S30 extract.
  • compounds that specifically modulate an interaction between a PTP-BL polypeptide and a STAT polypeptide can be identified.
  • Suitable assays are known in the art that allow for the detection of protein-protein interactions ⁇ e.g., immunoprecipitations and the like) or that allow for the detection of interactions between a DNA binding protein and a target DNA sequence ⁇ e.g., electrophoretic mobility shift assays (EMSA), DNAse I footprinting assays and the like). By performing such assays in the presence and absence of test compounds, these assays may be used to identify compounds that modulate ⁇ e.g., inhibit or enhance) the interaction between a PTP-BL polypeptide and a STAT polypeptide.
  • ESA electrophoretic mobility shift assays
  • the complete, i.e., full-length, PTP-BL polypeptide and/or the complete STAT polypeptide may be used in the method, or, alternatively, only portions of the polypeptides may be used.
  • an isolated coiled-coil domain and/or an isolated PTPase domain, and/or an isolated lecuine zipper motif may be used as described herein.
  • An assay may be used to identify test compounds that either stimulate or inhibit the interaction between the PTP- BL polypeptide and the STAT polypeptide.
  • a test compound that stimulates the interaction between the protein and a target molecule is identified based upon its ability to increase the degree of interaction between the two molecules being tested ⁇ e.g., a PTP-BL polypeptide and a STAT polypeptide) as compared to the degree of interaction in the absence of the test compound and such a compound would be expected to increase a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide in the cell.
  • test compound that inhibits the interaction between the two molecules being tested is identified based upon its ability to decrease the degree of interaction between the protein and a target molecule as compared to the degree of interaction in the absence of the compound and such a compound would be expected to decrease a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide.
  • the amount of binding of a PTP-BL polypeptide with a STAT polypeptide in the presence of the test compound is greater than the amount of binding in the absence of the test compound, in which case the test compound is identified as a compound that enhances binding of the PTP-BL polypeptide to the STAT polypeptide and/or a PTP-BL biological activity.
  • the amount of binding of the PTP-BL polypeptide with the STAT polypeptide in the presence of the test compound is less than the amount of binding of PTP-BL polypeptide with the STAT polypeptide in the absence of the test compound, in which case the test compound is identified as a compound that inhibits binding of the PTP-BL polypeptide to the STAT polypeptide and/or a PTP-BL biological activity.
  • binding of the test compound to a PTP-BL polypeptide and/or a STAT polypeptide can be determined either directly or indirectly as described above. Determining the ability of a PTP-BL polypeptide and/or a STAT polypeptide to bind to a test compound can also be accomplished using a technology such as real-time
  • BIOA Biomolecular Interaction Analysis
  • SPR surface plasmon resonance
  • the ability of a compound to modulate the ability of a STAT polypeptide to be acted on by an enzyme (e.g., a PTP-BL polypeptide) or to act on a substrate can be measured.
  • an enzyme e.g., a PTP-BL polypeptide
  • immunoblotting to determine the phosphorylation status of a STAT polypeptide is used to detect the ability of a PTP-BL polypeptide to phosphorylate a STAT polypeptide.
  • binding to a surface can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided in which a domain that allows one or both of the proteins to be bound to a matrix is added to one or more of the molecules.
  • glutathione-S-transferase fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed PTP-BL or STAT protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix is immobilized in the case of beads, and complex formation is determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity determined using standard techniques.
  • proteins may be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, DL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies which are reactive with protein or target molecules but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and unbound PTP-BL or STAT polypeptide is trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with a PTP-BL polypeptide or a STAT polypeptide, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the PTP-BL protein STAT.
  • the invention provides methods for identifying compounds that modulate a biological effect of PTP-BL using cells deficient in PTP-BL (or e.g., a STAT).
  • PTP-BL or e.g., a STAT
  • inhibition of PTP-BL activity e.g., by disruption of the PTP-BL gene
  • results e.g., in prolonged STAT phosphorylation, in increased cytokine production, e.g., increased IFN- ⁇ production and increased IL-4 production, in enhanced Th cell development, and/or an immune response.
  • cells deficient in PTP-BL or STAT can be used identify agents that modulate a biological response regulated by PTP-BL by means other than modulating PTP-BL itself (i.e., compounds that "rescue” the PTP-BL deficient phenotype).
  • a "conditional knock-out" system in which the gene is rendered nonfunctional in a conditional manner, can be used to create deficient cells for use in screening assays.
  • a tetracycline-regulated system for conditional disruption of a gene as described in WO 94/29442 and U.S. Patent No.
  • 5,650,298 can be used to create cells, or animals from which cells can be isolated, be rendered deficient in PTP-BL (or a molecule with which PTP-BL interacts e.g., STAT) in a controlled manner through modulation of the tetracycline concentration in contact with the cells.
  • the exon encoding amino acids 452-526 of PTP-BL can be replaced, e.g., with a neomycin cassette, resulting in an allele that produces no PTP-BL protein. This embodiment is described in the appended examples.
  • Cells deficient in PTP-BL or a STAT can be obtained from a non-human animals created to be deficient in PTP-BL or a STAT.
  • Preferred non-human animals include monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep.
  • the deficient animal is a mouse.
  • Mice deficient in PTP-BL or a STAT can be made using methods known in the art. One example of such a method and the resulting PTP-BL heterozygous and homozygous animals is described in the appended examples.
  • lymphoid cells e.g., thymic, splenic and/or lymph node cells
  • purified cells such as T cells, B cells
  • a cell from a PTP-BL deficient animal is a fertilized oocyte or an embryonic stem cell into which PTP-BL-coding sequences have been introduced.
  • Such cells can then be used to create non-human transgenic animals in which exogenous PTP-BL sequences have been introduced into their genome or homologous recombinant animals in which endogenous PTP-BL sequences have been altered.
  • cells deficient in PTP-BL or a STAT can be contacted with a test compound and a PTP-BL biological activity, e.g., a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide, can be monitored.
  • a PTP-BL biological activity e.g., a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide
  • Modulation of the response in cells deficient in PTP-BL or a STAT (as compared to an appropriate control such as, for example, untreated cells or cells treated with a control agent) identifies a test compound as a modulator of the PTP-BL regulated response.
  • the test compound is administered directly to a non-human knock out animal, preferably a mouse (e.g., a mouse in which the PTP-BL gene or a STAT is disrupted, or conditionally disrupted by means described above, or a chimeric mouse in which the lymphoid organs are deficient in PTP-BL or a STAT as described above), to identify a test compound that modulates the in vivo responses of cells deficient in PTP-BL, e.g., by acting on a non-PTP-BL target in a cell.
  • a mouse e.g., a mouse in which the PTP-BL gene or a STAT is disrupted, or conditionally disrupted by means described above, or a chimeric mouse in which the lymphoid organs are deficient in PTP-BL or a STAT as described above
  • cells deficient in PTP-BL are isolated from the non-human PTP-BL deficient animal or a STAT deficient animal, and contacted with the test compound ex vivo to identify a test compound that modulates a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide regulated by PTP- BL in the cells
  • compositions tested for their ability to modulate a biological response regulated by PTP-BL or a STAT are contacted with deficient cells by administering the test compound to a non-human deficient animal in vivo and evaluating the effect of the test compound on the response in the animal.
  • the test compound can be administered to a non-knock out animal as a pharmaceutical composition.
  • Such compositions typically comprise the test compound and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions are described in more detail below.
  • compounds that modulate a biological response regulated by PTP-BL or a STAT are identified by contacting cells deficient in PTP-BL or a STAT ex vivo with one or more test compounds, and determining the effect of the test compound on a read-out.
  • PTP-BL deficient cells contacted with a test compound ex vivo can be readministered to a subject.
  • cells deficient e.g., in PTP-BL, and/or STAT
  • a test compound e.g., T cells
  • cells can be isolated from e.g., PTP-BL, and/or STAT, deficient animals by standard techniques.
  • the cells are isolated form animals deficient in one or more of PTP-BL, and/or STAT.
  • cells deficient in PTP-BL and STAT e.g., STAT4
  • STAT e.g., STAT4
  • the effect of the test compound on the biological response regulated by PTP-BL or a STAT can be determined by any one of a variety of suitable methods, such as those set forth herein, e.g., including light microscopic analysis of the cells, histochemical analysis of the cells, production of proteins, induction of certain genes, e.g., cytokine genes, such as IFN- ⁇ , IL-4, STAT phosphorylation, DNA binding activity, as described herein.
  • Non-human animals e.g., mice, deficient in PTP-BL and/or or a STAT can be made using methods known in the art. One example of such a method and the resulting animals is described in the appended examples.
  • Non-human animals deficient in a particular gene product typically are created by homologous recombination.
  • a homologous recombinant animal is created by preparing a vector which contains at least a portion of a PTP-BL gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the PTP- BL gene.
  • the PTP-BL gene can be a murine gene (e.g., SEQ ID NO: 14), or a homologue or orthologue thereof.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous PTP-BL gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock-out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous PTP-BL gene is mutated or otherwise altered but still encodes functional polypeptide (e.g.
  • the upstream regulatory region can be altered to thereby alter the expression of the endogenous PTP-BL polypeptide).
  • the altered portion of the PTP-BL gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the PTP-BL gene to allow for homologous recombination to occur between the exogenous PTP-BL gene carried by the homologous recombination nucleic acid molecule and an endogenous PTP-BL gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking PTP-BL nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking PTP- BL both at the 5 1 and 3 1 ends
  • flanking PTP- BL both at the 5 1 and 3 1 ends
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced PTP-BL gene has homologously recombined with the endogenous PTP-BL gene are selected (see e.g., Li, E., et al.
  • the selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • homologous recombination nucleic acid molecules e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos. : WO 90/11354 by Le Mouellec, et al. ; WO 91/01140 by Smithies, et al; WO 92/0968 by Zijlstra, et al; and WO 93/04169 by Berns, et al
  • the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous PTP-BL protein).
  • the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell.
  • the additional flanking nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • flanking DNA both at the 5' and 3' ends
  • test compound includes any reagent or test agent which is employed in the assays of the invention and assayed for its ability to influence the interaction of a PTP-BL polypeptide with a STAT polypeptide, and/or a biological activity resulting from the interaction between a PTP-BL polypeptide and a STAT polypeptide, e.g., STAT phosphorylation, STAT signaling, production, expression and/or activity of cytokines, e.g., JFN-y and/or IL-4 production and/or Th cell differentiation.
  • STAT phosphorylation e.g., STAT phosphorylation, STAT signaling, production, expression and/or activity of cytokines, e.g., JFN-y and/or IL-4 production and/or Th cell differentiation.
  • More than one compound e.g., a plurality of compounds, can be tested at the same time for their ability to modulate cytokine production, expression and/or activity in a screening assay.
  • screening assay preferably refers to assays which test the ability of a plurality of compounds to influence the readout of choice rather than to tests which test the ability of one compound to influence a readout.
  • the subject assays identify compounds not previously known to have the effect that is being screened for.
  • high throughput screening may be used to assay for the activity of a compound.
  • the compounds to be tested can be derived from libraries (i.e., are members of a library of compounds). While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin, et al.
  • the 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 1 library method, and synthetic library methods using affinity chromatography selection.
  • biological libraries are 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).
  • Other exemplary methods for the synthesis of molecular libraries can be found in the art, for example in: Erb, et al (1994). Proc. Natl.
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries.
  • Candidate/test compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K.S., et al. (1991) Nature 354:82-84; Houghten, R., et al. (1991) Nature 354:84-86) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z., et al.
  • peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K.S., et al. (1991) Nature 354:82-84; Houghten, R., et al. (19
  • antibodies e.g., antibodies (e.g., intracellular, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')2, Fab expression library fragments, and epitope-binding fragments of antibodies);
  • small organic and inorganic molecules e.g., molecules obtained from combinatorial and natural product libraries);
  • enzymes e.g., endoribonucleases, hydrolases, nucleases, proteases, synthatases, isomerases, polymerases, kinases, phosphatases, oxido-reductases and ATPases
  • mutant forms of molecules e.g., dominant negative mutant forms of PTP-BL or STAT.
  • 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 1 library method; and synthetic library methods using affinity chromatography selection.
  • biological libraries are 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).
  • Compounds identified in the subject screening assays may be used, e.g., in methods of modulating a PTP-BL biological activity, e.g., STAT signaling, cytokine production, e.g., IFN- ⁇ and/or IL-4 production, and/or Th cell differentiation and/or an immune response. It will be understood that it may be desirable to formulate such compound(s) as pharmaceutical compositions (described supra) prior to contacting them with cells.
  • test compound that directly or indirectly modulates, e.g., an activity of or an interaction between a PTP-BL polypeptide and a STAT polypeptide, by one of the variety of methods described herein
  • the selected test compound can then be further evaluated for its effect on cells, for example by contacting the compound of interest with cells either in vivo (e.g., by administering the compound of interest to a subject) or ex vivo (e.g., by isolating cells from the subject and contacting the isolated cells with the compound of interest or, alternatively, by contacting the compound of interest with a cell line) and determining the effect of the compound of interest on the cells, as compared to an appropriate control (such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response).
  • an appropriate control such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response.
  • the instant invention also pertains to compounds identified in the subject screening assays.
  • Yet another aspect of the invention pertains to methods of modulating a PTP-BL expression and/or activity in a cell.
  • the modulatory methods of the invention involve contacting the cell with an agent that modulates PTP-BL expression and/or activity such that PTP-BL expression and/or activity in the cell is modulated.
  • the cell is contacted with a modulatory agent in an amount sufficient to modulate the expression and/or activity of PTP-BL.
  • the modulatory methods of the invention are performed in vitro.
  • the modulatory methods of the invention are performed in vivo, e.g., in a subject having a disorder or condition that would benefit from modulation of PTP-BL expression and/or activity.
  • the agent may act by modulating the activity of PTP-BL polypeptide in the cell, (e.g., by contacting a cell with an agent that, e.g., interferes with the interaction of a
  • PTP-BL polypeptide with a STAT polypeptide changes the binding specificity of T-bet, or post-translationally modifies PTP-BL) and/or the expression of PTP-BL, (e.g., by modulating transcription of the PTP-BL gene or translation of the PTP-BL mRNA).
  • the invention features methods for modulating one or more biological responses regulated by PTP-BL by contacting the cells with a modulator of PTP-BL expression and/or activity such that the biological response is modulated.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease, disorder, or condition or having a disease, disorder, or condition associated with an aberrant PTP-BL biological activity, e.g., an aberrant interaction between a PTP-BL polypeptide and a STAT polypeptide, as a result of, for example, aberrant expression and/or activity of a PTP-BL and/or a STAT polypeptide.
  • a disease, disorder, or condition that would benefit from increased or decreased Th cell differentiation, cytokine production, and/or an immune response, such as, for example, an infection, e.g., a bacterial infection, and/or cancer.
  • 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, such that a disease, disorder, or condition, is prevented or, alternatively, delayed in its progression.
  • a PTP-BL and/or STAT antagonist or agonist agent can be used for treating a subject.
  • the appropriate agent can be determined based on screening assays described herein.
  • Another aspect of the invention pertains to methods of modulating a PTP-BL biological activity for therapeutic purposes. PTP-BL activity can be modulated in order to modulate Th cell differentiation, cytokine production, and/or an immune response.
  • decreased PTP-BL activity increases Th cell differentiation, cytokine production, and/or an immune response, therefore decreasing PTP-BL expression and/or activity results in increased Th cell differentiation, cytokine production, and/or an immune response.
  • increasing PTP-BL expression and/or activity would result in decreased STAT signaling, Th cell differentiation, cytokine production, and/or an immune response.
  • Modulatory methods of the invention involve contacting a cell (e.g., a T cell, B cell) with an agent that modulates the expression and/or biological activity of PTP-BL, e.g., an interaction between a PTP-BL polypeptide and a STAT polypeptide.
  • a cell e.g., a T cell, B cell
  • an agent that modulates the expression and/or biological activity of PTP-BL e.g., an interaction between a PTP-BL polypeptide and a STAT polypeptide.
  • An agent that modulates PTP-BL activity can be an agent as described herein, such as a PTP-BL peptide (e.g., the agent may be a peptide comprising a PTP-BL polypeptide as describer herein, a peptide that binds to PTP-BL, or a small molecule), a STAT polypeptide, as describer herein, a nucleic acid molecule encoding one of the aforementioned peptides, a PTP-BL or STAT agonist or antagonist, a peptidomimetic of a PTP-BL or STAT agonist or antagonist, a PTP-BL or STAT peptidomimetic, or other small molecule identified using the screening methods described herein.
  • a PTP-BL peptide e.g., the agent may be a peptide comprising a PTP-BL polypeptide as describer herein, a peptide that binds to PTP-BL, or a small molecule
  • Additional agents include, but are not limited to a nucleic acid molecule that is antisense to a PTP-BL molecule, a nucleic acid molecule that is antisense to a STAT molecule, a PTP-BL siRNA molecule, a STAT siRNA molecule, a dominant negative PTP-BL molecule, a dominant negative STAT molecule, or combinations thereof.
  • 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 disease, condition, or disorder that would benefit from up- or down- modulation of a PTP-BL activity, e.g., a disorder characterized by an unwanted, insufficient, or aberrant Th cell differentiation, cytokine production, and/or an immune response would be beneficial.
  • 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) PTP-BL activity, as described herein.
  • PTP-BL activity is desirable in situations in which PTP-BL is abnormally upregulated and/or in which decreased PTP-BL activity is likely to have a beneficial effect, for example in a situation when increased Th cell differentiation, cytokine production, and/or an immune response is desirable
  • exemplary disorders that would benefit from modulation of PTP-BL expression and/or activity are set forth herein.
  • agents for use in upmodulating PTP-BL include, e.g., nucleic acid molecules encoding a PTP-BL polypeptide, a STAT polypeptide, and compounds that stimulate the interaction of a PTP-BL polypeptide and a STAT polypetide, for example (e.g., compounds identified in the subject screening assays).
  • agents for use in downmodulating PTP-BL include agents that inhibit the activity of PTP-BL in cell, for example, nucleic acid molecules that are antisense to a PTP-BL or a STAT, or PTP-BL or STAT siRNA molecules, a dominant negative PTP-BL or STAT molecule, or combinations thereof (e.g., compounds identified in the subject screening assays).
  • a PTP-BL polypeptide to thereby upregulate or promote Th cell differentiation, cytokine production, and/or an immune response.
  • Downregulating the activity of PTP-BL can be in the form of promoting or increasing prior to development of a condition (e.g., in a subject diagnosed as likely to develop a condition that would benefit from an increased immune response) or may involve promoting the induction of, e.g., an immune response to treat, for example a bacterial infection and/or cancer, or ameliorate symptoms of an ongoing condition.
  • PTP-BL activity can be inhibited by contacting a cell which expresses PTP-BL with an agent that inhibits the expression or activity of PTP-BL.
  • an agent can be a compound identified by the screening assays described herein.
  • the agent is a peptide.
  • Agents that inhibit a PTP-BL 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.
  • a molecule which inhibits the activity of PTP-BL e.g., by blocking the interaction of PTP-BL with a STAT, in T cells (such as a PTP-BL and/or a STAT peptide or a small molecule) alone or in conjunction with another downmodulatory agent can increase STAT signaling, Th cell differentiation, cytokine production, and/or an immune response.
  • Exemplary Inhibitory Compounds Since inhibition of PTP-BL activity is associated with increased Th cell differentiation, cytokine production, and/or an immune response, to increase Th cell differentiation, cytokine production, and/or an immune response, cells (e.g., T cells) are contacted with an agent that inhibits PTP-BL activity.
  • the 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.
  • the methods of the invention using PTP-BL inhibitory compounds can be used in the treatment of disorders in which increased Th cell differentiation, cytokine production, and/or an immune response, 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 PTP-BL.
  • 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, siRNA molecules, peptidic compounds that inhibit the interaction of PTP-BL with a target molecule ⁇ e.g., a STAT) and chemical agents that specifically inhibit PTP-BL activity.
  • an inhibitory compound of the invention is an antisense nucleic acid molecule that is complementary to a gene encoding PTP-BL, a gene encoding a STAT, or a molecule in a signal transduction pathway involving PTP-BL, or to a portion of said genes, or a recombinant expression vector encoding said antisense nucleic acid molecules.
  • an antisense nucleic acid molecule that is complementary to a gene encoding PTP-BL, a gene encoding a STAT, or a molecule in a signal transduction pathway involving PTP-BL, or to a portion of said genes, or a recombinant expression vector encoding said antisense nucleic acid molecules.
  • the below-mentioned exemplary antisense and siRNA molecules will refer to PTP-BL antisense and siRNA molecules.
  • antisense and siRNA molecules of the above-mentioned molecules e.g., a STAT, a molecule in a signal transduction pathway involving PTP- BL, or a portion of said genes, are also included in the invention.
  • the use of 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 1 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 PTP-BL mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of PTP-BL mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PTP-BL 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
  • one or more antisense oligonucleotides can be used.
  • 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 PTP-BL 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.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol in 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- methylribo nucleotide (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 compound that mediates RNAi.
  • RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target gene or genomic sequence, e.g., PTP-BL, and/or a STAT, or a fragment thereof, "short interfering RNA” (siRNA), "short hairpin” or “small hairpin RNA” (shRNA), and small molecules which interfere with or inhibit expression of a target gene by RNA interference (RNAi).
  • siRNA short interfering RNA
  • shRNA small hairpin RNA
  • RNA interference is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containing the same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287, 2431-2432 (2000); Zamore, P.D., et al. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197 (1999)).
  • dsRNA double-stranded RNA
  • mRNA messenger RNA
  • Tuschl T. et al. Genes Dev. 13, 3191-3197 (1999)
  • siRNAs small interfering RNAs
  • RNAi The smaller RNA segments then mediate the degradation of the target mRNA.
  • Kits for synthesis of RNAi are commercially available from, e.g. New England Biolabs and Ambion. In one embodiment one or more of the chemistries described above for use in antisense RNA can be employed.
  • 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 PTP-BL mRNA transcripts to thereby inhibit translation of PTP-BL mRNA.
  • a ribozyme having specificity for a PTP-BL-encoding nucleic acid can be designed based upon the nucleotide sequence of , for example, SEQ ID NO : 16.
  • 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 PTP-BL-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.
  • PTP-BL 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 PTP-BL (e.g., the PTP-BL promoter and/or enhancers) to form triple helical structures that prevent transcription of the PTP-BL gene in target cells.
  • PTP-BL e.g., the PTP-BL promoter and/or enhancers
  • the PTP-BL 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 PTP-BL 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 PTP-BL 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., Sl 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 PTP-BL 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 PTP-BL 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 1 PNA segment (Peterser, K.H. et al, 1975, Bioorganic Med Chem. Lett. 5: 1119-11124).
  • modified nucleoside analogs e.g., 5'-(4-
  • 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, ScL US. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad ScL USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • 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, ScL US. 86:6553-6556; Lemaitre et al, 1987, Proc
  • 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.
  • RNA interference is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containing the same sequence as the dsRNA (Sharp, P. A. and Zamore, P.D. 287, 2431-2432 (2000); Zamore, P.D., et al. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197 (1999); Cottrell TR, and Doering TL.
  • dsRNA double-stranded RNA
  • mRNA messenger RNA
  • RNAs or siRNAs are commercially available from, e.g. New England Biolabs or Ambion.
  • Kits for synthesis of RNAi are commercially available from, e.g. New England Biolabs or Ambion.
  • one or more of the chemistries described above for use in antisense RNA can be employed in molecules that mediate RNAi.
  • Non- limiting exemplary siRNA molecules of the invention are listed below.
  • siRNA molecules specific for human PTP-BL for example, transcript variant 1 (SEQ ID NO: 16) are shown below: Beginning at position 1217:
  • Sense strand siRNA AAGAAACUUCAGGUUCUGAUU
  • Antisense strand siRNA UCAGAACCUGAAGUUUCUUUU
  • Sense strand siRNA GCCAGGAGACCGUUUGAUAUU
  • Antisense strand siRNA UAUCAAACGGUCUCCUGGCUU
  • Sense strand siRNA ACAUAUCAAAUUCGACCUGUU
  • Antisense strand siRNA C AGGUCGAAUUUGAUAUGUUU
  • siRNA molecules specific for human STAT e.g., STATl, e.g., variant alpha (SEQ ID NO: 5) are shown below: Beginning at position 1132: Sense strand siRNA: CUGGUUCACUAUAGUUGCGUU Antisense strand siRNA: CGCAACUAUAGUGAACCAGUU
  • Sense strand siRNA GAGGUCUCAAUGUGGACCAUU
  • Antisense strand siRNA UGGUCCACAUUGAGACCUCUU
  • Sense strand siRNA GUGAAAAACAUCCAGAUACUU
  • Antisense strand siRNA GUAUCUGGAUGUUUUUCACUU
  • siRNA molecules specific for human STAT4 are shown below: Beginning at position 370: Sense strand siRNA: AAUCAAGACUGGGAGGCAGUU Antisense strand siRNA: CUGCCUCCCAGUCUUGAUUUU Beginning at position 707:
  • Sense strand siRNA ACAGUGUGCAGAUGACAGAUU
  • Antisense strand siRNA UCUGUCAUCUGCACACUGUUU
  • Sense strand siRNA CUAAACUAUCAGGUAAAGGUU
  • Antisense strand siRNA CCUUU ACCUGAUAGUUUAGUU
  • siRNA molecules specific for human STAT6 are shown below:
  • Sense strand siRNA GUCCCAGAUCAUGUCUCUGUU
  • Antisense strand siRNA C AGAGAC AUGAUCUGGGACUU
  • Sense strand siRNA CCUGAAGUUCAUGGCUGAGUU
  • Antisense strand siRNA CUCAGCCAUGAACUUCAGGUU
  • Sense strand siRNA AUUUGGGAGGGUGAGACACUU
  • Antisense strand siRNA GUGUCUCACCCUCCCAAAUUU
  • an inhibitory compound of the invention is a peptidic compound derived from the PTP-BL amino acid sequence.
  • the below-mentioned exemplary peptidic compounds will refer to peptidic compound derived from the PTP-BL amino acid sequence.
  • exemplary peptidic compounds of the above-mentioned molecules e.g., a STAT, or a portion of said genes, are also included in the invention.
  • the inhibitory compound comprises a portion of PTP-BL (or a mimetic thereof) that mediates interaction of PTP-BL with a target molecule such that contact of PTP-BL with this peptidic compound competitively inhibits the interaction of PTP-BL with the target molecule, e.g., a STAT.
  • the peptide compound is designed based on the region of PTP-BL that mediates interaction of PTP-BL with, for example, a STAT polypeptide as described herein.
  • the peptidic compounds of the invention can be made intracellularly in immune cells by introducing into the 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, for example, SEQ ID NO: 10.
  • 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).
  • Other inhibitory agents that can be used to specifically inhibit the activity of an
  • PTP-BL protein are chemical compounds that directly inhibit PTP-BL activity or inhibit the interaction between PTP-BL and a STAT. Such compounds can be identified using screening assays that select for such compounds, as described in detail above.
  • B. Upregulation of PTP-BL Biological Activities Stimulation of PTP-BL activity as a means of downmodulating Th cell differentiation, cytokine production, and/or an immune response is also useful in therapy. For example, decreasing or inhibiting Th cell differentiation, cytokine production, and/or an immune response by enhancing PTP-BL is beneficial in diseases, disorders, or conditions in which there is inappropriate activation of T cells that are reactive against self tissue and that promote the production of cytokines and autoantibodies in, for example, an autoimmune disease
  • Th cell differentiation, cytokine production, and/or an immune response can be decreased in a patient by removing cells from the patient, contacting cells in vitro with an agent (e.g., a small molecule) that enhances PTP-BL activity, and reintroducing the in v/Yro-stimulated cells into the patient.
  • an agent e.g., a small molecule
  • a method of enhancing Th cell differentiation, cytokine production, and/or an immune response involves isolating cells from a patient, transfecting them with a nucleic acid molecule encoding a PTP-BL molecule and reintroducing the transfected cells into the patient.
  • Th cell differentiation in performing any of the methods described herein, it is within the scope of the invention to inhibit Th cell differentiation, cytokine production, and/or an immune response by administering one or more additional agents.
  • a method of upregulating Th cell differentiation, cytokine production, and/or an immune response involves transfecting cells with a nucleic acid molecule encoding a PTP-BL molecule with a mutation or a peptide that enhances, for example, PTP-BL-STAT interaction, such that the cells express the PTP- BL 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.
  • a compound that specifically stimulates PTP-BL activity and/or expression can be used to inhibit Th cell differentiation, cytokine production, and/or an immune response.
  • a subject is treated with a stimulatory compound that stimulates expression and/or activity of a PTP-BL molecule.
  • the methods of the invention using PTP-BL stimulatory compounds can be used in the treatment of disorders in which the enhancement of Th cell differentiation, cytokine production, and/or an immune response is desirable.
  • stimulatory compounds include active PTP-BL protein or a molecule that interacts with PTP-BL, e.g., a STAT, expression vectors encoding PTP- BL and chemical agents that specifically stimulate PTP-BL activity.
  • exemplary stimulatory compounds will refer to PTP-BL stimulatory compounds.
  • exemplary stimulatory compounds of the above-mentioned molecules e.g., a STAT, a molecule in a signal transduction pathway involving PTP-BL, or a portion of said genes, are also included in the invention.
  • a preferred stimulatory compound is a nucleic acid molecule encoding PTP-BL, 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 PTP-BL protein in the cells of the subject.
  • a PTP-BL cDNA (full length or partial PTP-BL cDNA sequence) is cloned into a recombinant expression vector and the vector is transfected into the immune cell using standard molecular biology techniques.
  • the PTP-BL cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library.
  • PCR polymerase chain reaction
  • the nucleotide sequences of PTP-BL cDNA is describer herein 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 PTP-BL in the form suitable for expression of the PTP-BL 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 PTP-BL in a cell is a chemical compound that specifically stimulates the expression or activity of endogenous PTP-BL in the cell. Such compounds can be identified using screening assays that select for compounds that stimulate the expression or activity of PTP-BL as described herein.
  • the method of the invention for modulating PTP-BL 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 PTP-BL.
  • 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 differentiation of cells in the subject.
  • a stimulatory or inhibitory compound is administered to a subject in vivo.
  • stimulatory or inhibitory agents that comprise nucleic acids (e.g., recombinant expression vectors encoding PTP-BL, antisense RNA, or PTP-BL-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 (1991) Nature 332:815-818: Wolff 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. 267:963-967; and U.S. Patent No. 5, 166,320).
  • a cation such as polylysine
  • 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 retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271).
  • a recombinant retrovirus can be constructed having a nucleotide sequences of interest incorporated into the retroviral genome. Additionally, portions of the retroviral 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).
  • Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral El and E3 genes but retain as much as 80 % of the adenoviral genetic material.
  • 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
  • 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).
  • 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.
  • the invention features a method of diagnosing a subject for a disorder associated with aberrant biological activity of PTP-BL (e.g., that would benefit from modulation of, e.g., modulation of the interaction of a STAT polypeptide and a PTP-BL polypeptide, modulation of, STAT signaling, STAT phosphorylation, Th cell differentiation, cytokine production, and/or an immune response.
  • a disorder associated with aberrant biological activity of PTP-BL e.g., that would benefit from modulation of, e.g., modulation of the interaction of a STAT polypeptide and a PTP-BL polypeptide, modulation of, STAT signaling, STAT phosphorylation, Th cell differentiation, cytokine production, and/or an immune response.
  • the invention comprises identifying the subject as one that would benefit from modulation of PTP-BL activity.
  • expression of PTP-BL or a STAT can be detected in cells of a subject suspected of having a disorder associated with aberrant biological activity of PTP-BL.
  • the expression of PTP-BL or a STAT in cells of said subject could then be compared to a control and a difference in expression of PTP-BL or a STAT in cells of the subject as compared to the control could be used to diagnose the subject as one that would benefit from modulation of PTP-BL activity.
  • the "change in expression” or “difference in expression” of PTP-BL or a STAT in cells of the subject can be, for example, a change in the level of expression of PTP-BL or a STAT in cells of the subject as compared to a previous sample taken from the subject or as compared to a control, which can be detected by assaying levels of, e.g., PTP-BL mRNA, for example, by isolating cells from the subject and determining the level of PTP-BL mRNA expression in the cells by standard methods known in the art, including Northern blot analysis, microarray analysis, reverse-transcriptase PCR analysis and in situ hybridizations.
  • a biological specimen can be obtained from the patient and assayed for, e.g., expression or activity of PTP-BL or a STAT.
  • a PCR assay could be used to measure the level of PTP-BL in a cell of the subject.
  • a level of PTP-BL higher or lower than that seen in a control or higher or lower than that previously observed in the patient indicates that the patient would benefit from modulation of a signal transduction pathway involving PTP-BL.
  • the level of expression of PTP-BL or a STAT in cells of the subject can be detected by assaying levels of, e.g., PTP-BL, for example, by isolating cells from the subject and determining the level of PTP-BL or a molecule in a signal transduction pathway involving PTP-BL protein expression by standard methods known in the art, including Western blot analysis, immunoprecipitations, enzyme linked immunosorbent assays (ELISAs) and immunofluorescence.
  • Antibodies for use in such assays can be made using techniques known in the art.
  • a change in expression of PTP-BL or a STAT in cells of the subject results from one or more mutations (i.e., alterations from wildtype), e.g., the PTP-BL gene and mRNA leading to one or more mutations (i.e., alterations from wildtype) in the amino acid sequence of the protein.
  • the mutation(s) leads to a form of the molecule with increased activity (e.g., partial or complete constitutive activity).
  • the mutation(s) leads to a form of the molecule with decreased activity (e.g., partial or complete inactivity).
  • the mutation(s) may change the level of expression of the molecule for example, increasing or decreasing the level of expression of the molecule in a subject with a disorder.
  • the mutation(s) may change the regulation of the protein, for example, by modulating the interaction of the mutant protein with one or more targets e.g., resulting in a form of PTP-BL that cannot interact with a PTP-BL binding partner, e.g., a STAT.
  • Mutations in the nucleotide sequence or amino acid sequences of proteins can be determined using standard techniques for analysis of DNA or protein sequences, for example for DNA or protein sequencing, RFLP analysis, and analysis of single nucleotide or amino acid polymorphisms.
  • mutations can be detected using highly sensitive PCR approaches using specific primers flanking the nucleic acid sequence of interest.
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 : 1077-1080; and Nakazawa et al (1994) PNAS 91 :360-364).
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, DNA) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically amplify a sequence under conditions such that hybridization and amplification of the sequence (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, DNA
  • the complete nucleotide sequence for PTP-BL or a STAT can be determined.
  • Particular techniques have been developed for determining actual sequences in order to study polymorphism in human genes. See, for example, Proc. Natl. Acad. Sci. U.S.A. 85, 544-548 (1988) and Nature 330, 384-386 (1987); Maxim and Gilbert. 1977. PNAS 74:560; Sanger 1977. PNAS 74:5463.
  • any of a variety of automated sequencing procedures can be utilized when performing diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
  • Restriction fragment length polymorphism mappings are based on changes at a restriction enzyme site.
  • polymorphisms from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of a specific ribozyme cleavage site.
  • Another technique for detecting specific polymorphisms in particular DNA segment involves hybridizing DNA segments which are being analyzed (target DNA) with a complimentary, labeled oligonucleotide probe.
  • target DNA DNA segments which are being analyzed
  • a complimentary, labeled oligonucleotide probe See Nucl. Acids Res. 9, 879-894 (1981). Since DNA duplexes containing even a single base pair mismatch exhibit high thermal instability, the differential melting temperature can be used to distinguish target DNAs that are perfectly complimentary to the probe from target DNAs that only differ by a single nucleotide.
  • This method has been adapted to detect the presence or absence of a specific restriction site, U.S. Pat. No. 4,683,194. The method involves using an end- labeled oligonucleotide probe spanning a restriction site which is hybridized to a target DNA.
  • the hybridized duplex of DNA is then incubated with the restriction enzyme appropriate for that site.
  • Reformed restriction sites will be cleaved by digestion in the pair of duplexes between the probe and target by using the restriction endonuclease.
  • the specific restriction site is present in the target DNA if shortened probe molecules are detected.
  • Other methods for detecting polymorphisms in nucleic acid sequences include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA hetero duplexes (Myers et al. (1985) Science 230: 1242).
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Sl nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels. See, for example, Cotton et al. (l9SS) Proc. NatlAcadSci USA 85:4397; Saleeba ef ⁇ /. (1992) Methods Enzymol. 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • alterations in electrophoretic mobility can be used to identify polymorphisms.
  • single strand conformation polymorphism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad Sd USA: 86:2766, see also Cotton (1993) MutatRes 285:125-144; and Hayashi (1992) Genet Anal TechAppl 9:73-79).
  • Single-stranded DNA fragments of sample and control nucleic acids can be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of nucleic acid molecule comprising polymorphic sequences in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985)
  • DGGE denaturing gradient gel electrophoresis
  • DNA can be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265: 12753).
  • oligonucleotide primers may be prepared in which the polymorphic region is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different polymorphisms when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • primer extension process which consists of hybridizing a labeled oligonucleotide primer to a template RNA or DNA and then using a DNA polymerase and deoxynucleoside triphosphates to extend the primer to the 5 1 end of the template. Resolution of the labeled primer extension product is then done by fractionating on the basis of size, e.g., by electrophoresis via a denaturing polyacrylamide gel. This process is often used to compare homologous DNA segments and to detect differences due to nucleotide insertion or deletion. Differences due to nucleotide substitution are not detected since size is the sole criterion used to characterize the primer extension product.
  • nucleotide analogs can be used to identify changes since they can cause an electrophoretic mobility shift. See, U.S. Pat. No. 4,879,214.
  • This assay is based on the ability of DNA ligase to distinguish single nucleotide differences at positions complementary to the termini of co-terminal probing oligonucleotides (see, e.g., Nickerson et al. 1990. Proc. Natl. Acad. Sci. USA 87:8923.
  • a modification of this approach termed coupled amplification and oligonucleotide ligation (CAL) analysis, has been used for multiplexed genetic typing (see, e.g., Eggerding 1995 PCR Methods Appl. 4:337); Eggerding et al. 1995 Hum. Mutat. 5:153).
  • genetic bit analysis GBA
  • GBA genetic bit analysis
  • microchip electrophoresis can be used for high-speed SNP detection (see e.g., Schmalzing et al. 2000. Nucleic Acids Research, 28).
  • matrix-assisted laser desorption/ionization time-of-flight mass (MALDI TOF) mass spectrometry can be used to detect SNPs (see, e.g., Stoerker et al. Nature Biotechnology 18:1213).
  • a difference in a biological activity of PTP-BL between a subject and a control can be detected.
  • an activity of PTP-BL or a STAT can be detected in cells of a subject suspected of having a disorder associated with aberrant biological activity of PTP-BL.
  • the activity of PTP-BL or a STAT in cells of the subject could then be compared to a control and a difference in activity of PTP-BL or a STAT in cells of the subject as compared to the control could be used to diagnose the subject as one that would benefit from modulation of an PTP-BL activity.
  • Activities of PTP-BL or a STAT can be detected using methods described herein or known in the art.
  • the diagnostic assay is conducted on a biological sample from the subject, such as a cell sample or a tissue section (for example, a freeze- dried or fresh frozen section of tissue removed from a subject).
  • a biological sample from the subject such as a cell sample or a tissue section (for example, a freeze- dried or fresh frozen section of tissue removed from a subject).
  • the level of expression of PTP-BL or a STAT in cells of the subject can be detected in vivo, using an appropriate imaging method, such as using a radiolabeled antibody.
  • the level of expression of PTP-BL or a STAT in cells of the test subject may be elevated ⁇ i.e., increased) relative to the control not associated with the disorder or the subject may express a constitutively active (partially or completely) form of the molecule.
  • This elevated expression level of, e.g., PTP-BL or a STAT can be used to diagnose a subject for a disorder associated with increased PTP-BL activity.
  • the level of expression of PTP-BL or a STAT in cells of the subject may be reduced (i.e., decreased) relative to the control not associated with the disorder or the subject may express an inactive (partially or completely) mutant form of PTP-BL.
  • This reduced expression level of PTP-BL or expression of an inactive mutant form of PTP-BL can be used to diagnose a subject for a disorder, such as immunodeficiency disorders characterized by insufficient cytokine production.
  • the level of expression of gene whose expression is regulated by PTP-BL can be measured (e.g., IFN-y and/or IL4).
  • an assay diagnosing a subject as one that would benefit from modulation of PTP-BL expression, post-translational modification, and/or activity (or a STAT) is performed prior to treatment of the subject.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe/primer nucleic acid or other reagent (e.g., antibody), which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving PTP-BL or a STAT.
  • 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 modulating agent.
  • subject is intended to include living organisms in which an immune response or bone formation and mineralization can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof, including but not limited to a transgenic PTP-BL and/or transgenic STAT mouse as described herein.
  • 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 modulating agent may vary according to factors such as the disease state, age, sex, reproductive state, and weight of the individual, and the ability of the agent 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, or delivered on a surface, e.g., a biocompatible surface, for example on the surface of a surgically implanted device, e.g., as, for example, a putty, for the stabilization, replacement, etc., of a bone, joint, tooth, etc.
  • Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation.
  • the PTP-BL modulator can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • PTP-BL 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 PTP-BL modulator can be in a composition which aids in delivery into the cytosol of a cell.
  • the agent may be conjugated with a carrier moiety such as a liposome that is capable of delivering the peptide into the cytosol of a cell.
  • the PTP-BL modulator can be modified to include specific transit peptides or fused to such transit peptides which are capable of delivering the PTP-BL modulator into a cell.
  • the agent 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.
  • PTP- BL 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 PTP-BL modulator are to be administered orally.
  • Such formulations are preferably encapsulated and formulated with suitable carriers in so Hd 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 proteolytic 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. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a PTP-BL modulator may be therapeutically administered by implanting into patients vectors or cells capable of producing a biologically-active form of PTP-BL or a precursor of PTP-BL, i.e. a molecule that can be readily converted to a biological-active form of PTP-BL by the body.
  • cells that secrete PTP-BL may be encapsulated into semipermeable membranes for implantation into a patient.
  • the cells can be cells that normally express PTP-BL or a precursor thereof or the cells can be transformed to express PTP-BL or a biologically active fragment thereof or a precursor thereof. It is preferred that the cell be of human origin and that the PTP-BL polypeptide be human PTP-BL 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 PTP-BL protein can be applied not only in basic drug screening, but also in clinical trials.
  • the effectiveness of an agent determined by a screening assay as described herein to increase PTP-BL gene expression, protein levels, or upregulate PTP-BL activity can be monitored in clinical trials of subjects exhibiting decreased PTP-BL gene expression, protein levels, or downregulated PTP-BL activity.
  • the effectiveness of an agent determined by a screening assay to decrease PTP-BL gene expression, protein levels, or downregulate PTP-BL activity can be monitored in clinical trials of subjects exhibiting increased PTP-BL gene expression, protein levels, or upregulated PTP-BL activity.
  • the expression or activity of a PTP-BL 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 PTP-BL, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates PTP-BL activity can be identified.
  • an agent e.g., compound, drug or small molecule
  • PTP-BL 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 PTP-BL and other genes implicated in the PTP- BL 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 PTP-BL 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 PTP-BL 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 PTP-BL protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the PTP-BL protein, mRNA, or genomic DNA in the pre-administration sample with the PTP-BL protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the
  • increased administration of the agent may be desirable to increase the expression or activity of PTP-BL 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 PTP-BL to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • PTP-BL 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 PTP-BL modulating agent to modulate Th cell differentiation, cytokine production, and/or an immune response in a cell of a subject that would benefit from modulation of the expression and/or activity of PTP-BL can be measured by detecting an improvement in the condition of the patient after the administration of the agent.
  • the ability of a PTP-BL modulating agent to modulate Th cell differentiation, cytokine production, and/or an immune response in a subject that would benefit from modulation of the expression and/or activity of PTP-BL 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.
  • PTP-BL can be administered exogenously and it would likely be desirable to achieve certain target levels of PTP-BL 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 PTP-BL 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 PTP-BL and, in some circumstances, also monitoring levels of a STAT, for example. Accordingly, the present invention also provides methods for detecting the presence of PTP-BL in a sample from a patient.
  • kits for carrying out the screening assays, modulatory methods or diagnostic assays of the invention can include an indicator composition comprising PTP-BL or a STAT, means for measuring a readout (e.g., protein secretion) and instructions for using the kit to identify modulators of biological effects of PTP-BL.
  • a kit for carrying out a screening assay of the invention can include cells deficient in PTP-BL or a STAT, means for measuring the readout and instructions for using the kit to identify modulators of a biological effect of PTP-BL.
  • the invention provides a kit for carrying out a modulatory method of the invention.
  • the kit can include, for example, a modulatory agent of the invention (e.g., PTP-BL inhibitory or stimulatory agent) in a suitable carrier and packaged in a suitable container with instructions for use of the modulator to modulate a biological effect of PTP-BL.
  • a modulatory agent of the invention e.g., PTP-BL inhibitory or stimulatory agent
  • kits for diagnosing a disorder associated with a biological activity of PTP-BL in a subject can include a reagent for determining expression of PTP-BL (e.g., a nucleic acid probe for detecting PTP-BL mRNA or an antibody for detection of PTP-BL protein), 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 PTP-BL e.g., a nucleic acid probe for detecting PTP-BL mRNA or an antibody for detection of PTP-BL protein
  • the leucine zipper bait was generated by subcloning a portion of the murine STAT4 cDNA encoding amino acids 250-310 in-frame into pEG202 (Clontech).
  • Yeast strain EGY48 was sequentially transformed with bait construct, pSH17 reporter plasmid (Clontech) and pJG4-5 (Clontech) expression vector containing a cDNA library from a mouse ThI cell clone stimulated with anti-CD3 for 5 hours. Positive colonies were screened on dropout and X-gal plates and subjected to sequencing analysis. Plasmid Constructs and Reagents
  • FLAG-tagged murine STATl, STAT4 (WT and Y693F) and STAT6 were cloned into pcDNA3 (Invitrogen).
  • c-Myc tagged PTP-BL (WT, FS and AC) were generated in pCMV-Myc (Clontech).
  • Anti-phospho STAT4 (71-7900; Zymed Laboratory), anti- phospho STAT6 (#9361; Cell Signaling Technology), anti-phospho STATl (#9171; Cell Signaling Technology), anti-STAT6 (M-20) (sc-981; Santa Cruz Biotechnology), anti- STAT4 (C-20) (sc-486; Santa Cruz Biotechnology), anti-Spl (PEP2) (sc-59; Santa Cruz Biotechnology), anti-HSP90(H-114) (sc-7947; Santa Cruz Biotechnology), anti-c-Myc (9E10) (sc-40; Santa Cruz Biotechnology), anti-FLAG M2 agarose beads (A2220; Sigma), anti-phospho tyrosine antibody (PY20) (PI 1120; BD Transduction Laboratories), HRP-rabbit anti-mouse IgG (H+L) (61-6520; Zymed) and HRP-goat anti- rabbit IgG (H+L) (81-6120; Zymed) were purchased from the indicated companies.
  • Staurosporine was purchased from Calbiochem (#569396).
  • Immun-Star HRP substrate kit (170-5040; BIO-RAD) or ECL western blotting detection reagent (RPN2106; Amersham) were used.
  • 293 T cells were maintained in DMEM supplemented with 10% FCS.
  • 2D6 cells were maintained in RPMI supplemented with 10% FCS in the presence of 500 pg/ml mouse IL-12.
  • BAC-TO-BAC Baculovirus Expression System (Invitrogen) was used to generate recombinant baculovirus using the manufacture's protocol. Briefly, glutathione S- transferase (GST) protein and GST-tagged PTPase domain were cloned into pF ASTBACl donor plasmid. The recombinant plasmids were transformed into bacmid- carrying E. coli strain (DHIOBAC) to obtain the recombinant bacmids by transposition in E. coli. The resultant recombinant bacmids were transfected into Sf9 insect cells to get the recombinant baculovirus.
  • GST glutathione S- transferase
  • DHIOBAC E. coli strain
  • Recombinant proteins were purified from HighFive cells (Invitrogen) using GST bind resin (70541-3; Novagen). When lysates were prepared from HighFive cells, a 3x higher amount of Complete Mini protease inhibitor cocktail (#11836153001; Roche) than usual was used, along with 1 mM phenylmethylsulfonyl fluoride (PMSF).
  • PMSF phenylmethylsulfonyl fluoride
  • phosphorylated FLAG-tagged STAT4 or FLAG-tagged STAT6 were immunoprecipitated from IFN- ⁇ - or EL-4- stimulated 293 T transfectants, respectively. Immunoprecipitates were incubated with GST-tagged proteins in 25 mM HEPES, pH 7.5, 5mM EDTA, 10 mM DTT at 37 0 C for 3 hours and subjected to immunoblot with anti-phospho-tyrosine (PY20). Immunoprecipitation and Immunoblot Analysis
  • TCLs Total cell lysates
  • 2x lysis buffer 50 mM Tris-HCL pH8.0, 300 mM NaCl, 1% TritonX-100, 2 mM Na3VO4, 20 mM NaF
  • Complete Mini protease inhibitor cocktail 50 mM Tris-HCL pH8.0, 300 mM NaCl, 1% TritonX-100, 2 mM Na3VO4, 20 mM NaF
  • TCLs in Ix lysis buffer were incubated with Protein A/G agarose beads (sc-2003; Santa Cruz Biotechnology) conjugated with antibodies as indicated and subjected to SDS- PAGE followed by immunoblot analysis.
  • Protein A/G agarose beads sc-2003; Santa Cruz Biotechnology
  • Murine Ptp-bl was isolated from a C57BL/6 genomic library. To disrupt Ptp-bl, we constructed a targeting vector in which the neomycin resistance gene cassette was inserted into the exon encoding amino acids 452-526. The targeting vector was electroporated into C57BL/6 ES cells, followed by G418 selection. Colonies containing the disrupted allele were identified by Southern blot analysis. Targeted ES cell clones were injected into C57BL/6 blastocysts and implanted into pseudopregnant females to obtain chimeric mice that were then bred for germline transmission. Preparation of Cytoplasmic and Nuclear Extracts
  • Cytoplasmic and nuclear extracts were prepared using hypotonic buffer (2OmM HEPES-NaOH pH7.9, 1OmM KC 1 , 0. ImM EGTA, 2mM MgC 12, ImM Na3 VO4, 2OmM NaF, 0.1% Nonidet P-40, ImM DTT) and hypertonic buffer (2OmM HEPES-NaOH pH7.9, 0. ImM EGTA, 2mM MgC12, ImM Na3VO4, 2OmM NaF, 42OmM NaCl, 20% glycerol, ImM DTT), respectively. These buffers were supplemented with proteinase inhibitor cocktail. Electrophoretic Mobility Assays (EMSA)
  • ESA Electrophoretic Mobility Assays
  • Binding reaction was performed in a total volume of 20 ⁇ l in the following buffer: 20 mM HEPES-NaOH (pH 7.9), 5 mM MgC12, 50 mM KCl, 1 mM DTT and 6% glycerol. Each reaction, also containing 2 ⁇ g of poly (dl-dC) and 32 P end-labeled probe (60000 cpm/reaction), was initiated by the addition of 1.3 ⁇ g nuclear extract and was allowed to incubate at room temperature for 30 minutes before electrophoretic analysis on a 4% native PAGE in 0.5x Tris-borate-EDTA (TBE) buffer.
  • TBE Tris-borate-EDTA
  • the STAT5/6 consensus oligonucleotide probe (S'-GTATTTCCCAGAAAAGGAAC-ST; SC-2567) (SEQ ID NO:1) and SpI consensus oligonucleotide probe (5 1 - ATTCGATCGGGGCGGGGCGAGC-S 1 ; sc-2502) (SEQ ID NO:2) were purchased from Santa Cruz Biotechnology.
  • SEQ ID NO:1 The STAT5/6 consensus oligonucleotide probe (S'-GTATTTCCCAGAAAAGGAAC-ST; SC-2567) (SEQ ID NO:1) and SpI consensus oligonucleotide probe (5 1 - ATTCGATCGGGGCGGGGCGAGC-S 1 ; sc-2502) (SEQ ID NO:2) were purchased from Santa Cruz Biotechnology.
  • Murine CD4+ T cells were isolated by MACS (Miltenyi Biotec) and stimulated for 4 days with immobilized anti-CD3 (1 ⁇ g/ml for coating) in the presence of 1 ( ⁇ /ml anti-CD28 (37.51; BD Pharmingen) and 100 U/ml human IL-2.
  • murine IL- 12 5 ng/ml
  • anti-IL-4 10 ( ⁇ g/ml) were added to the culture media
  • Th2 cell differentiation murine IL-4 (10 ng/ml) was added.
  • the cells were cultured with just cytokines for another day, harvested, washed and re- stimulated with immobilized anti-CD3 (0.2 ng/ml for coating). After 24 hours, culture supernatants were harvested for ELISA measurement of cytokine production.
  • ThI or Th2-polarlized CD4+ T cells were obtained as described above and re- stimulated with immobilized anti-CD3 (5 ⁇ g/ml for coating) for 5.5 hours in the presence of 3 nM Monensin (Sigma) for the last 2.5 hours. These cells were stained with PE-labeled anti-mouse IL-4 (#554435; BD Pharmingen), FITC- labeled anti-mouse IFN- ⁇ (#554411; BD Pharmingen) and their isotype controls. Flow cytometric analysis was performed on a FACSCalibur (Becton Dickinson).
  • mice (7 week old C57BL/6 males) were infected by intratracheal instillation as previously described (Jones et al. (2005) J Immunol 175:7530). After anesthesia by ketamine (50 mg/kg) and xylazine (5 mg/kg) i.p., trachea were surgically exposed and an angiocatheter was placed in the left bronchus, through which mice received 50 ⁇ l of 4x10 4 colony-forming units (CFU)AnI Klebsiella pneumoniae (43816 from the American Type Culture Collection; Manassas, VA). After 48 hours, mice were euthanized with an overdose of halo thane.
  • CFU colony-forming units
  • Lungs were homogenized in 10 ml sterile water, serially diluted, and plated on agar for overnight growth at 37 0 C. Bacterial burdens were expressed as CFU/ml lung homogenate. Densiometric Analysis Immunoblot signals were quantified using ImageJ (http://rsb.info.nih.gov/ij).
  • EXAMPLE 1 PTP-BL Interacts with STAT4 STAT4 is one of seven mammalian STAT proteins and is required for the differentiation of ThI cells from naive CD4+ T cells. Aside from the action of PIASx, which was reported to inhibit STAT4-mediated transcription by functioning as a corepressor (Arora, T., et al. (2003). JBiol Chem 278, 21327-21330), little is known about how the activity of this particular STAT protein is regulated. Thus, proteins that interact with STAT4 and modulate its function were identified to further understand the regulatory mechanisms underlying STAT4 signaling.
  • amino acids 250 to 310 in the STAT4 coiled-coil domain encode a leucine zipper motif (Yamamoto, K., et al. (1994). MoI Cell Biol 14, 4342-4349). Therefore a bait construct containing the leucine zipper region of STAT4 was made for use in a yeast two-hybrid screen for STAT4-interacting proteins ( Figure IA).
  • Figure IA A cDNA library prepared from a mouse ThI cell clone was screened, and 7 cDNAs were isolated from 3 x 10 independent yeast transformants.
  • STAT4 could be coimmunoprecipitated with PTP-BL (AC) independent of cytokine stimulation.
  • PTP-BL AC
  • Y693F mutant of STAT4 which cannot be tyrosine phosphorylated upon cytokine stimulation, was used instead of WT STAT4, this mutant could be also coimmunoprecipitated with PTP-BL.
  • a FLAG-tagged STAT4 expression vector was cotransfected with three forms of c-Myc-tagged PTP-BL expression vectors (frame shift mutant: (FS), wild type: (WT), C-terminal PTPase domain-deleted: (AC)) into 293T cells. Twenty-four hours after transfection, cells were stimulated with IFN- ⁇ for 30 minutes, or left unstimulated, and total cell lysates were immunoprecipitated with FLAG antibody and analyzed by immunoblot using anti-phospho-STAT4.
  • FS frame shift mutant
  • WT wild type
  • AC C-terminal PTPase domain-deleted
  • EXAMPLE 3 PTP-BL Inhibits IL-12 Signaling At the Level of STAT4 Phosphorylation
  • the ThI- like cell line 2D6 was stably transfected with PTP-BL (FS) or (WT) expression vectors ( Figure 3A).
  • FS PTP-BL
  • WT WT expression vectors
  • Figure 3B IL-12-induced STAT4 phosphorylation was markedly decreased in 2D6 cells overexpressing PTP-BL (WT) ( Figure 3B).
  • the receptor-associated JAK kinase Tyk2 becomes tyrosine phosphorylated prior to its phosphorylation of STAT4 (Karaghiosoff, M., et al.
  • EXAMPLE 4 PTP-BL Dephosphorylates Multiple STAT Family Members The specificity of PTP-BL for another STAT family member, STAT6 was also examined. 293T cells were cotransfected with expression vectors for FLAG-STAT6 and the three forms of c-Myc-tagged PTP-BL as in Figure 2 A. Transfected cells were stimulated with DL-4, and total cell lysates were immunoprecipitated with FLAG antibody followed by immunoblot with anti-phospho STAT6.
  • Ptp-bT 1' mice have normal numbers of splenic lymphocyte subsets including CD4+ T cells, CD8+ T cells, B cells, macrophages, dendritic cells and NK cells and a normal ratio of CD4+/CD8+ cells in the thymus.
  • the expression of activation markers CD69, CD62L and CD25
  • WT CD4+ T cells WT CD4+ T cells.
  • the proliferation of Ptp-bT 1' CD4+ T cells after anti-CD3, IL-2 or IL-4 stimulation was also no different from WT cells.
  • EXAMPLE 6 The Effect of PTP-BL Deficiency on STAT Activation in CD4+ T Cells
  • TCR-triggering plus IL- 12 stimulation resulted in the phosphorylation of STAT4 in both cytoplasmic and nuclear compartments of WT CD4+ T cells (Figure 6A).
  • increased STAT4 phosphorylation was observed in both subcellular compartments prepared from Ptp-bT 1' CD4+ T cells.
  • the total amount of STAT4 protein in the nucleus was also increased mPtp-bV 1' CD4+ T cells following IL- 12 stimulation, consistent with its increased phosphorylation status.
  • the balance of phosphorylation and dephosphorylation activities determines the total level of phosphorylated STAT4 protein. IfPTP-BL deficiency leads to an increase in the amount of phosphorylated STAT4 protein due to a decrease in dephosphorylation activity, prolonged phosphorylation of STAT4 following cytokine stimulation would be observed. This possibility was examined by performing a staurosporine chase experiment. CD4+ T cells were stimulated with IFN- ⁇ to activate STAT4 (Nguyen, K. B., et al. (2002). Science 297, 2063-2066) and then treated with staurosporine, a protein kinase inhibitor, to inhibit further STAT phosphorylation by JAK kinases.
  • Cytoplasmic and nuclear extracts were prepared from cells harvested at various time points and subjected to immunoblot analysis to monitor the phosphorylation state of STAT4.
  • STAT4 phosphorylation diminished over time in both the cytoplasmic and nuclear compartments of WT cells following staurosporine treatment.
  • Ptp-bT 1' cells had STAT4 phosphorylation that was sustained in both subcellular compartments for longer periods of time than that seen in WT cells. Similar results were obtained when STATl and STAT5 phosphorylation were examined by staurosporine chase.
  • a concomitant increase in the amount of total STAT4 protein was also observed (Figure 6B).
  • EXAMPLE 7 Increased ThI and Th2 Differentiation in Ptp-bl ' " CD4+ T Cells Because STAT4 and STAT6 have been shown to play critical roles in the differentiation of Th cells (Murphy, K. M., and Reiner, S. L. (2002). Nat Rev Immunol 2, 933-944), the effect of PTP-BL deficiency on this process was observed.
  • CD4+ T cells isolated from WT and Ptp-bt' ' mice were differentiated under unskewed (UNSK), ThI, or Th2 culture conditions in vitro. Following re-stimulation with anti-CD3, ELISA of culture supernatants was performed to measure the production of IFN- ⁇ and IL-4.
  • Ptp-bt 1' CD4+ T cells Increased production of IFN- ⁇ under Thl-skewing conditions, and enhanced production of BL-4 under Th2 culture conditions was observed in Ptp-bt 1' CD4+ T cells as compared to WT cells ( Figure 7A). The status of T helper cell differentiation was also observed by intracellular cytokine staining, and enhanced Th cell differentiation was confirmed in Ptp-bt 1' CD4+ T cells ( Figure 7B). Thus, Ptp-bt 1' CD4+ T cells have an enhanced potential to develop into ThI and Th2 cells, which can be ascribed to increased and/or prolonged STAT4 and STAT6 activation.
  • EXAMPLE 8 Effect of PTP-BL Deficiency on Lung Host Defense

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Veterinary Medicine (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne, au moins partiellement, la découverte que la tyrosine phosphatase, du type PTP-Basophile (PTP-BL), interagit physiquement avec de multiples protéines STAT, par exemple, STAT4, STAT1 et/ou STAT6. L'invention concerne également, au moins partiellement, la génération d'animaux non humains déficients en Ptp-bt1-. En conséquence, l'invention met en oeuvre des procédés pour identifier des modulateurs de l'interaction des polypeptides STAT et des polypeptides PTP-BL ainsi que des animaux non humains déficients en Ptp-bt1- et des cellules dérivés de ces polypeptides et leurs procédés d'utilisation.
PCT/US2007/023845 2006-11-13 2007-11-13 Procédés d'identification de compositions qui modulent la protéine tyrosine kinase, ptp-bl WO2008060540A2 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US85861506P 2006-11-13 2006-11-13
US60/858,615 2006-11-13
US90049307P 2007-02-09 2007-02-09
US60/900,493 2007-02-09
US90122707P 2007-02-13 2007-02-13
US60/901,227 2007-02-13

Publications (2)

Publication Number Publication Date
WO2008060540A2 true WO2008060540A2 (fr) 2008-05-22
WO2008060540A3 WO2008060540A3 (fr) 2008-10-16

Family

ID=39313222

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/023845 WO2008060540A2 (fr) 2006-11-13 2007-11-13 Procédés d'identification de compositions qui modulent la protéine tyrosine kinase, ptp-bl

Country Status (1)

Country Link
WO (1) WO2008060540A2 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002042489A1 (fr) * 2000-11-27 2002-05-30 The Hospital For Sick Children Methodes faisant intervenir la proteine tyrosine phosphatase de lymphocyte t
WO2002096412A1 (fr) * 2001-05-31 2002-12-05 The Cleveland Clinic Foundation Inhibiteurs de ptpase et leur methode d'utilisation
US20040033979A1 (en) * 1999-04-12 2004-02-19 Dean Nicholas M. Antisense modulation of Fas mediated signaling
US20040224337A1 (en) * 2003-03-04 2004-11-11 Erik Foehr Use of biomolecular targets in the treatment and visualization of tumors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040033979A1 (en) * 1999-04-12 2004-02-19 Dean Nicholas M. Antisense modulation of Fas mediated signaling
WO2002042489A1 (fr) * 2000-11-27 2002-05-30 The Hospital For Sick Children Methodes faisant intervenir la proteine tyrosine phosphatase de lymphocyte t
WO2002096412A1 (fr) * 2001-05-31 2002-12-05 The Cleveland Clinic Foundation Inhibiteurs de ptpase et leur methode d'utilisation
US20040224337A1 (en) * 2003-03-04 2004-11-11 Erik Foehr Use of biomolecular targets in the treatment and visualization of tumors

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HAQUE S J ET AL: "Roles of protein-tyrosine phosphatases in Stat1 alpha-mediated cell signaling." THE JOURNAL OF BIOLOGICAL CHEMISTRY 27 OCT 1995, vol. 270, no. 43, 27 October 1995 (1995-10-27), pages 25709-25714, XP002489749 ISSN: 0021-9258 *
NAKAHIRA MASAKIYO ET AL: "Regulation of signal transducer and activator of transcription signaling by the tyrosine phosphatase PTP-BL" IMMUNITY, vol. 26, no. 2, February 2007 (2007-02), pages 163-176, XP002489752 ISSN: 1074-7613 *
SATO ET AL: "Nuclear retention of STAT3 through the coiled-coil domain regulates its activity" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 336, no. 2, 21 October 2005 (2005-10-21), pages 617-624, XP005165755 ISSN: 0006-291X *
WURSTER ANDREA L ET AL: "Stat6 and IRS-2 cooperate in interleukin 4 (IL-4)-induced proliferation and differentiation but are dispensable for IL-4-dependent rescue from apoptosis." MOLECULAR AND CELLULAR BIOLOGY JAN 2002, vol. 22, no. 1, January 2002 (2002-01), pages 117-126, XP002489750 ISSN: 0270-7306 *
ZHU WEI ET AL: "Arginine methylation of STAT1 regulates its dephosphorylation by T cell protein tyrosine phosphatase." THE JOURNAL OF BIOLOGICAL CHEMISTRY 27 SEP 2002, vol. 277, no. 39, 27 September 2002 (2002-09-27), pages 35787-35790, XP002489751 ISSN: 0021-9258 *

Also Published As

Publication number Publication date
WO2008060540A3 (fr) 2008-10-16

Similar Documents

Publication Publication Date Title
EP1874811B1 (fr) Procédé permettant de modeler la formation et la minéralisation osseuses par modulation de l'activité de KRC
CA2496897C (fr) Procedes et compositions permettant la modulation de l'activite xbp-1
US20110142799A1 (en) Modulation of neurodegenerative disease by modulating xbp-1 activity
US20090048117A1 (en) Modulation of immune system function by modulation of polypeptide arginine methyltransferases
US6548734B1 (en) Methods relating to modulation of cartilage cell growth and/or differentiation by modulation of NFATp activity
US20060063187A1 (en) Modulation of XBP-1 activity for treatment of metabolic disorders
US7615380B2 (en) Methods for modulating an immune response by modulating KRC activity
US20060246543A1 (en) Slim compositions and methods of use thereof
US9745589B2 (en) Methods for modulating skeletal remodeling and patterning by modulating SHN2 activity, SHN3 activity, or SHN2 and SHN3 activity in combination
AU2006252636A1 (en) Modulation of T cell recruitment
US20120141539A1 (en) Methods for modulating toll-like receptor mediated activation of cells of the innate system by modulating xbp-1 activity
JP2010528586A (ja) 骨形成及び鉱化作用を調節する化合物の同定のためのアッセイ
JP2008528005A (ja) Ｔ−ｂｅｔによるＴＨ２系譜コミットメントの調節
WO2008060540A2 (fr) Procédés d'identification de compositions qui modulent la protéine tyrosine kinase, ptp-bl
US20020133844A1 (en) Regulation of Th2 cell activity by modulation of NFATp and NFAT4 activity
US20060035301A1 (en) Method of identifying protein kinase modulators and uses therefore
WO2006031930A2 (fr) Modulation de l'activite de l'xbp-1 pour le traitement de troubles metaboliques
US7592147B2 (en) Modulation of IL-2 production by T-bet

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07867433

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07867433

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载