WO2004070344A2 - Method of screening for agents which modulate the activity of tlr9 - Google Patents

Method of screening for agents which modulate the activity of tlr9 Download PDF

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Publication number
WO2004070344A2
WO2004070344A2 PCT/EP2004/000641 EP2004000641W WO2004070344A2 WO 2004070344 A2 WO2004070344 A2 WO 2004070344A2 EP 2004000641 W EP2004000641 W EP 2004000641W WO 2004070344 A2 WO2004070344 A2 WO 2004070344A2
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protein
same
polynucleotide encoding
polynucleotide
protem
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PCT/EP2004/000641
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French (fr)
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WO2004070344A3 (en
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Ningshu Liu
Shinichi Watanabe
Lin Ni
Kevin Bacon
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Bayer Healthcare Ag
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Publication of WO2004070344A3 publication Critical patent/WO2004070344A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5052—Cells of the immune system involving B-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to methods for identifying and evaluating reagents that modulate the activity of TLR9 (Toll-like receptor 9).
  • the invention further relates to the use of reagents that modulate the activity of TLR9 in the manufacture of medicaments for the treatment of cancer, immunological diseases such as allergy, asthma, chronic obstructive pulmonary disease (COPD), autoimmune diseases, viral and parasitic infection, transplantation rejection, and conditions where 10 TLR9 signal transduction is disordered.
  • immunological diseases such as allergy, asthma, chronic obstructive pulmonary disease (COPD), autoimmune diseases, viral and parasitic infection, transplantation rejection, and conditions where 10 TLR9 signal transduction is disordered.
  • COPD chronic obstructive pulmonary disease
  • T cells can produce either T H 1 or T H 2 cytokines depending on the initial signal and environment. These initially secreted cytokines are thought to provide permissive signals that favor na ⁇ ve CD4 + helper T cells polarization towards the T H 1 or T H 2
  • T H 1 gene regulator T box expressed in T cells
  • GATA3 and STAT6 transcription factors GATA3 and STAT6
  • T H 2 gene regulator the transcription factors GATA3 and STAT6 (signal transducer and activator of transcription 6)
  • IFN /STAT1, IL-12/STAT4 and IL-4/STAT6 results in the differentiation to effector T cells 3 .
  • Differentiated T H .1 and T H 2 have distinct functions and cytokine profiles.
  • Dysregulated T H 1 or T H 2 responses can cause diseases such as asthma which is characterized by T ⁇ 2-associated pulmonary inflammation. Therefore, redirecting immune responses with therape ⁇ tic reagents is an important strategy for disease treatments. This includes the redirection of T H 2 mediated diseases which include allergies like asthma and T H 1 mediated diseases which 25 include autoimmune disease like rheumatoid arthritis and multiple sclerosis.
  • TLR Toll-like receptor
  • Mammalian TLRs comprise a large family with extracellular leucine-rich repeats and a cytoplasmic Toll/interleukin-lR homology domain.
  • Adapter protein MyD88 interacts with TLRs through TIR domain.
  • MyD88 recruits IL-1 receptor associated kinase (IRAK) and TRAF6 to the receptors, and results in the activation of IKK-NF- ⁇ B and JNK-AP-1 signaling pathways.
  • IRAK IL-1 receptor associated kinase
  • TRAF6 twp MyD88-dependent pathways are common for all known TLRs and are well demonstrated by MyD88-def ⁇ cient mice 4"6 . So far 10 members have been identified.
  • Oligonucleotides containing CpG motifs induce the expression of T H 1 cytokines 7 .
  • TLR9 has been identified as the receptor for CpG oligonucleotides (WOO 1/32877), the CpG/TLR9 signaling pathway and the mechanism of action that leads to the unique protective effects against allergic inflammation are unknown. Since T cells do not express TLR9, it is not clear how CpG achieves its regulatory effects on T helper cells. Additionally, the inhibition of IgE production by CpG has been shown in vivo and in vitro in cultured peripheral blood mononuclear cells (PBMCs). The underlying mechanism still awaits to be discovered.
  • PBMCs peripheral blood mononuclear cells
  • CpG oligonucleotides are useful modulators of various immune responses, there is considerable interest to understand the mechanisms of actions of CpG and the TLR9 signaling t . pathway.
  • the signaling pathway needs to be understood in order to be able to provide assays for the identification of reagents that mimic the effect of CpG oligonucleotides and which are useful as therapeutics for diseases such as autoimmune diseases, allergic diseases including asthma and COPD.
  • TLR9 signaling pathway in order to identify novel therapeutic targets for the treatment of diseases such as autoimmune diseases, allergic diseases including asthma and COPD and to monitor and evaluate the efficacy of novel therapeutics.
  • the present invention relates to methods for identifying and evaluating reagents that modulate the activity of TLR9 using at least one of the following as a marker: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii)
  • NF- ⁇ B protein or a polynucleotide encoding the same (iv) p38 protein or a polynucleotide encoding the same; (v) IL-12 protein or a polynucleotide encoding the same; (vi) STAT4 protem or a polynucleotide encoding the same; (vii) IgG2a protein or a germline transcript of the same, (viii) IgGl protein or a germline transcript of the same, (ix) IgE protem or a germline transcript of the same, x) TCF2, protein or a polynucleotide encoding the same, xi) BATF protein or a polynucleotide encoding the same, xii) PLAUR protein or a polynucleotide encoding the same,
  • FIG1 interleukin-four induced gene 1 protein or a polynucleotide encoding the same
  • MHC2TA class II transactivator
  • HCST hematopoietic cell signal transducer
  • FceR2A Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same, xxxiv) Id2 protein or a polynucleotide encoding the same, xxxv) CD74 protein or a polynucleotide encoding the same.
  • IKKs, NF- ⁇ B, p38, IL-12, and STAT4 are indicators for the activation of TLR9.
  • Fc ⁇ R2A, CD74 are the output signal molecules of NF- B, STAT4, T-bet, TCF2, C/EBP ⁇ , C/EBP ⁇ , NOP5/NOP58, BATF, LM04, Id2, and MHC2TA activation or repression. Any combination of two or more markers selected from the above can also be used.
  • the present invention also relates to the use of a reagent that alters the expression, amount, activity or phosphorylation, in a cell or tissue, of (i) T-bet protein or a polynucleotide encoding the
  • IKKs protein or polynucleotides encoding the same;
  • IgG2a protein or a germline transcript of the same (viii) IgGl protein or a germline transcript of the same and
  • a polynucleotide encoding the same a polynucleotide encoding the same, xvii) C/EBP ⁇ protem or a polynucleotide encoding the same, xviii) TACSTD1 (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same, xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same, xx) SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the "same, xxi) ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same, xxii) UBEIC (ubiquitin-activating enzyme E1C) protein or a polynucleotide encoding the same, xxiii) FNBP3 (
  • the present invention is based on the discovery that inhibition or activation of TLR9 activity can be detected by determining the amount, expression, activity or the phosphorylation of signal molecules which can lead to the activation of KB binding site and/or STAT4-binding site containing promoters or the T-bet, or gene vii-xxxv promoter in a tissue.
  • the present invention is further based on the discovery that immune responses resulting from CpG-TLR9 interaction can be mimicked by increasing or diminishing the amount of mRNA or protein of T-bet or gene vii- xxxv, or the amount of mRNA or protein of the members, which can lead to the regulation of NF- KB and/or STAT4 activities, and/or output signal molecules of T-bet or gene vii-xxxv.
  • One embodiment of the invention provides a method for monitoring the effect of TLR-9 activation or inhibition by determining the difference in the level relative to a test sample of T-bet, IKKs, or NF-kB or gene x-xxxv mRNA Q ⁇ protein, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a germline transcripts or protein, IgGl germline transcripts or protein, or IgE germline transcripts or protein in a tissue.
  • Level used herein includes, but not limited to, the amount of a protein, expression amount of mRNA, a gene activity, a protein activity, and the amount of phosphorylation.
  • the invention relates to methods for identifying and evaluating reagents that activate or inhibit TLR9-activtiy comprising determining the difference in the amount, expression, activity or phosphorylation relative to a test sample of at least one of the following: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF- ⁇ B- protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL-12 protein or a polynucleotide encoding the same; (vi) STAT 4 protein or a polynucleotide encoding the same; (vii) IgG2a protein or a germline transcript of the same, (viii) IgGl protein or,,a germline transcript of the same and (ix) IgE
  • such method comprises determining the difference in the amount relative to a test sample of at least two, at least three, of each of (i) to (xxxv) as defined supra.
  • the difference in the amount relative to a test sample of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays.
  • the difference in the amount relative to a test sample of protein is determined, while in still other embodiment the difference in the amount relative to a test sample of mRNA and protem is determined.
  • STAT4 when the difference in the amount relative to a test sample of mRNA and protem is determined.
  • the amount of STAT4 protein be determined.
  • the amount determined can be the total amount of the STAT protein present in a sample or, alternatively, can be the amount of phosphorylated STAT protein present in the sample.
  • the difference in the amount relative to a test sample of mRNA is determined, and can, for example,
  • the difference in the amount relative to a test sample of protem is determined, while in still other embodiments, the difference in the amount relative to a test sample of mRNA and protein is determined.
  • the present invention relates to a method for identifying or evaluating reagents that modulate the activity of TLR9, said method comprises: (a) contacting cell expressing 0 TLR9 receptor or CpG recognizing receptor with a test compound; (b) determining the difference in the level relative to a test sample of at least one of the following: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF- ⁇ B protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL-12 protein or a polynucleotide encoding the same; (vi) STAT 4 protein
  • protem or a polynucleotide encoding the same
  • xv NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same
  • TACSTD1 Tuor-associated calcium signal transducer 1 protein or a polynucleotide encoding the same
  • xix LM04 (LIM ONLY4) protein or a polynucleotide encoding the same
  • ESP 15 epidermal growth factor receptor pathway -
  • substrate 15 protein or a polynucleotide encoding the same
  • UBEl ' C ubiquitin-activating 5 enzyme E1C
  • FNBP3 formin binding protein 3
  • CHUK conserved helix-loop- helix ubiquitous kinase
  • SELL Selectin lymphocyte
  • such methods comprise determining the amount of at least two, at least three, at least four, at least five, or each of (i) to (xxxv) present in a cell expressing TLR9 receptor or CpG recognizing receptor
  • Cell expressing TLR9 receptor or CpG recognizing receptor includes, but not limited to, a B cell or a myeloid cell.
  • the difference in the amount relative to a test sample of mRNA is determined, in other embodiments, the difference in the amount relative to a test sample of protein, 30 or .protein modification is determined, while in still other embodiments, the difference in the amount relative to a test sample of mRNA and protein is determined.
  • STAT4 when the amount of STAT4 is being determined, it is preferable that the . amount of STAT4 protem, or phosphorylation of STAT4 protein be determined. In any such embodiment wherein a STAT protein amount is determined, the amount determined can be the total amount of the STAT protein present in a sample or, alternatively, can be the amount of phosphorylated STAT protem present in the sample.
  • said method comprises: (a) contacting cell samples expressing TLR9 or CpG- recognizing receptors with a test compound; (b) determining the amount of STAT4 mRNA or STAT4 protein or phosphorylated STAT4 present in the sample; and (c) comparing the amounts in (b) to those present in a corresponding control cell samples not bearing TLR9 or CpG-recognizing receptors that has not been contacted with the test compound, so that if the amount of STAT4 mRNA, or protem, or phospho-STAT4 is decreased or the amount of STAT4 mRNA, or protein, or phospho-STAT4 is increased relative to the amount in the control sample, a compound to be tested for an ability to modifying TLR9 or CpG recognizing receptors.
  • the present invention further relates to a pharmaceutical composition.
  • the composition comprises: (a) a reagent I that modulates the activity of a TLR9 polypeptide or polynucleotide; and b) a reagent II that modulates the activity of an IL-12 receptor polypeptide or polynucleotide, and c) a pharmaceutically acceptable carrier.
  • Rreagent I can be a TLR 9 agonist or antagonist and the reagent II can be a IL-12 receptor agonist or antagonist.
  • the composition can be used to treat the diseases such as an allergic disease when TLR9 agonist and IL-12 agonist are included in the composition.
  • the composition can be used to treat an autoimmune disease when TLR9 antagonist and IL-12 antagonist are included in the composition.
  • the present invention further relates to a pharmaceutical composition.
  • the composition comprisses: (a) a reagent I that modulates the activity of a TLR9 polypeptide or polynucleotide; and b) a reagent II that modulates the activity of a CD40 polypeptide or polynucleotide, and c) a pharmaceutically acceptable carrier.
  • Rreagent I can be a TLR 9 agonist or antagonist and the reagent II can be a CD40 agonist or antagonist.
  • the composition can be used to treat the diseases such as an allergic disease when TLR9 agonist and CD40 agonist are included in the composition.
  • the composition can be used to treat an autoimmune disease when TLR9 antagonist and CD40 antagonist are included in the composition.
  • Figure 1 shows the induction of T-bet in mouse splenocytes.
  • Spleen cells from 8-week old C57BL/6 mice were cultured at a density of 2.0xl0 6 /ml in RPMIl 640/10%FCS with or without indicated stimuli.
  • the concentration of IFN- ⁇ , LPS, CpG and GpC are 50 ng/ml, 50 ng/ml, 3 ⁇ M, and 3 ⁇ M respectively.
  • Cells were collected at 3 (A) or 6 (B) hours post stimulation and the mRNA levels of T-bet were measured by real time LightCycler PCR. Data are representative of three independent experiments (mean + SD).
  • FIG. 2 shows Stimuli-induced STATl ⁇ / ⁇ phosphorylation in splenocytes.
  • Spleen cells from 8-week old C57BL/6 mice were treated with indicated stimuli (using the concentrations shown in Figure 1) and cell lysates were made after incubation for 2, 5, and 18 hours.
  • Phosphorylation of STAT1 was assessed by immunoblotting with antibodies against phospho-STATl ⁇ / ⁇ (pY701). Data are representative of three separate experiments.
  • FIG. 3 shows T-bet transactivation and STAT1 phosphorylation in MyD88- or TLR9- deficient mice.
  • Splenocytes from 129/C57BL/6/MyD88 + + and 129/C57BL/6/MyD88 ' ' , 129/C57BL/6/TLR9 +/" and 1129/C57BL/6/TLR9 " ' " mice were isolated and treated with indicated stimuli as described in Figure 1 and methods.
  • Figure 3A shows T-bet induction with 3h (A), 6h
  • FIG. 4 shows the cells response to CpG in T-bet and IFN- ⁇ expression.
  • a and B splenocytes were isolated from C57BL/6 mice and incubated in medium with or without CpG (3 ⁇ M) for 6 hours. Thereafter, T cells were obtained by positive selection with anti-CD90 microbeads and B cells were purified by negative selection (B Cell Isolation Kit). "Non-T, non-B” represents the remaining cells after T and B cell depletion.
  • the expression of T-bet (A) and IFN- ⁇ (B) was assessed by LightCycler RT-PCR. Data are from one experiment, representative of three separate experiments.
  • T-bet expression was measured in purified mouse splenic B cells treated with indicated stimuli for 6 hours (C) and in purified human peripheral B cells cultured in the presence or absence of ODN 2006 (CpG) or negative ODN 2006 (GpC) for 18 hours (D). 'Induction' indicates the ratio to non-stimulated cells. Data in (C) are from. one experiment, representative of four independent experiments.
  • FIG. 5 shows the CpG-induced IFN- ⁇ /STATl-independent T-bet expression in purified B ,_. cells.
  • Splenic B cells isolated by negative selection
  • non-B the remaining splenocytes
  • Data are from single experiments, representative of three.
  • B cells were negatively purified from STAT1 + " and STATl " ' " spleens and treated with indicated stimuli for 6 hours. Thereafter, T- bet mRNA was measured by LightCycler PCR (B). Data presented are the mean + SD of three independent experiments.
  • Figure 7 shows the inhibition of IL-4 and CD40-ligation-induced IgGl, IgG2a and IgE production.
  • B cells were isolated from splenocytes by negative selection and treated as described in Materials and Methods.
  • Ig production was determined by ELISA and cell purity was checked by FACS with PE-labeled anti-B220 antibody. Data represent four animals in each group.
  • Germline transcripts were measured by RT-PCR with primer sets corresponding to the sequences of the I ⁇ l-C ⁇ l, I ⁇ 2a-C ⁇ 2a and I ⁇ -C ⁇ exon-hinge region, respectively.
  • the housekeeping gene GAPDH was used as a control. Shown is a single experiment, representative of three.
  • Figure 8 shows the expression of Id2 in resting B cells and B cells stimulated with CpG, IL-4, Poly( C) and LPS.
  • the invention is based on the discovery that CpG is engaged in the transcriptional regulation of transcription factors T-bet, TCF2, C/EBP ⁇ , C/EBP ⁇ , BATF, LM04, Id2, and MHC2TA. Furthermore, T-bet TCF2, C/EBP ⁇ , C/EBP ⁇ , BATF, LM04, Id2, and C2TA mRNA is specifically induced after binding of CpG to TLR9. LPS, the ligand of TLR4 is incapable of inducing T-bet expression.
  • CpG can induce the expression of T-bet, TCF2, C/EBP ⁇ , C/EBP ⁇ , BATF, LM04, Id2, and MHC2TA which results in the inhibition of IL-4/CD- 40 'induced IgGl and IgE class switching by direct acting on B-cells.
  • the present invention is also based on the, discovery of the signalling pathway and the molecules therin that mediates the CpG-induced upregulation of T-bet mRNA.
  • the invention provides methods for identifying and evaluating reagents that modulate the activity of TLR9 using at least one of the following as markers: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF- ⁇ B protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL- ⁇ *, ⁇ .
  • polynucleotide encoding the same xv) N0P5/N0P58 (nucleolar protein 5) protein or a polynucleotide encoding the same, xvi) GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same, xvii) C/EBP ⁇ protein or a polynucleotide encoding the same, xviii) TACSTD1 (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same, xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same, xx)
  • protem or a polynucleotide encoding the same xxv) SELL (Selectin lymphocyte) protem or a polynucleotide encoding the same, xxvi) GNPNAT1 (glutamine repeat protein 1) protein or a polynucleotide encoding the same, xxvii) BAP29 (B-cell receptor-associated protein 29) protem or a polynucleotide encoding the same, xxviii) PML (promyelocytic leukemia) protem or a polynucleotide encoding the same, xxix) MMKROX2R (mouse mRNA for KROX-20 protein
  • FIG1 interleukin- four induced gene 1 protein or a polynucleotide encoding the same
  • MHC2TA class II transactivator
  • HCST hematopoietic cell signal signal transducer
  • FceR2A Fc receptor, IgE, low affinity II, alpha polypeptide protem or a polynucleotide encoding the same
  • Id2 protein or a polynucleotide encoding the same
  • CD74 protein or a polynucleotide encoding the same.
  • Inhibition or activation of TLR9 activity can be detected by determining the amount of mRNA or protein of at least one of the following members of signal transduction pathway present within a tissue; T-bet, IKKs, NF- ⁇ B, p 8, IL-12, STAT4, Igs, and gene x - xxxv. Specifically, the following members of signal transduction pathway present within a tissue; T-bet, IKKs, NF- ⁇ B, p 8, IL-12, STAT4, Igs, and gene x - xxxv. Specifically, the following members of signal transduction pathway present within a tissue; T-bet, IKKs, NF- ⁇ B, p 8, IL-12, STAT4, Igs, and gene x - xxxv. Specifically, the following members of signal transduction pathway present within a tissue; T-bet, IKKs, NF- ⁇ B, p 8, IL-12, STAT4, Igs, and gene x - xxxv
  • 35 inhibition or activation of TLR9 activity can be detected by determining the amount, expression, or activity of at least one of the following: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF- ⁇ B protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL- lr ⁇ , 12 "protein or a polynucleotide encoding the same; (vi) STAT 4 protein or a polynucleotide
  • TCF2 protein or a polynucleotide encoding the same
  • BATF protein or a polynucleotide encoding the same
  • PLAUR protein or a polynucleotide encoding the same
  • C/EBP ⁇ protein or a polynucleotide encoding the same
  • SCS GTP-specific succinyl-COA synthetase beta subunit
  • ESP 15 epidermal growth factor receptor pathway substrate 15
  • UBEIC ubiquitin-activating enzyme E1C
  • FNBP3 formin binding protein 3
  • CHUK conserved helix-loop-helix ubiquitous kinase
  • FIG1 interleukin- four induced gene 1 protein or a polynucleotide encoding the same
  • MHC2TA class II transactivator
  • HCST hematopoietic cell signal signal transducer
  • FceR2A Fc receptor, IgE, low affinity II, alpha polypeptide protem or a polynucleotide encoding the same
  • Standard techniques can routinely be utilized to determine these amounts.
  • such methods of the invention can routinely be performed using standard techniques for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample.
  • 35 involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or mRNA such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample.
  • a compound or an agent capable of detecting the polypeptide or mRNA such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample.
  • a control sample is determined to . contain none of a given molecule (e.g., T-bet mRNA or protem, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, or IgGl or IgE) and the molecule is determined to be present in the test- sample, the amount of the molecule in the test sample is greater than that present in the control sample.
  • the amount in the test sample can routinely be determined to be greater than, equal to, or less than that of the control sample.
  • the amount of a given molecule in test and qontrol samples will differ by at least 2-fold, and in certain instances, 2.5-fold, at least 3- fold, at ' least 4-fold, at least 5-fold, or at least 10-fold.
  • T-bet, STAT4 nucleic acid and amino acid sequences are well known to those of skill in the art.
  • GenBank accession number for human T-bet mRNA or protein, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, IgGl, IgE and gene x- xxxv are listed in the Table 1, 2 and 3.
  • Table 2 and 3 show the name of genes, accession number, functional description, and fold of changes induced by CpG and other stimuli. Gene expression was determined by DNA microarray analysis
  • Table 2 and 3 show the accession number and functional description of genes that are up- or . down-regulated by CpG, as well as their expression in response to IL-4, INF ⁇ , IL-12 or CpG plus , IL-12. .
  • the skilled person is aware of methods that are capable of being used to determine the difference in the amount relative to a test sample of any one, two, three, four, five of T-bet mRNA or protem, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, IgGl, IgE (germline transcript or protein) and gene x - xxxv.
  • One method comprises using a microarray for determining such difference in the amount relative to a test sample, wherein the microarray comprises one or more nucleic acid sequences immobilized onto a solid surface, said nucleic acid sequence or sequences exhibiting complementarity to one or more selected from the group consisting of: mRNA of (i) T-bet; (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; or (vi) STAT 4; or germlme transcript of (vii) IgG2a, (viii) IgGl or (ix) IgE, gene x - xxxv.
  • the microarray comprises one or more nucleic acid sequences immobilized onto a solid surface, said nucleic acid sequence or sequences exhibiting complementarity to one or more selected from the group consisting of: mRNA of (i) T-bet; (ii) IKKs; (iii) NF- ⁇ B; (i
  • the kit can, in addition, comprise a labeled compound or agent capable of detecting one or more of (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protem or polynucleotides encoding the same; (iii) NF- ⁇ B protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL-12 protem or a polynucleotide encoding the same; (vi) STAT 4 protem or a polynucleotide encoding the same; (vii) IgG2a protein or a germline transcript of the same, (viii) IgGl protem or a germline transcript of the same, (ix) IgE protein or a germline transcript of the same, gene x - xxxv in a biological sample and means for determining the amount of the polypeptide or
  • the method can comprise, for example: using (1) a first antibody (e.g., attached to a solid support) which binds to any one of IKKs, NF- ⁇ B, p38/phospho-p38, IL- 12, STAT4/phospho-STAT, T-bet, proteins (x) to (xxxv) and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a , detectable agent.
  • a first antibody e.g., attached to a solid support
  • a second antibody which binds to either the polypeptide or the first antibody and is conjugated to a , detectable agent.
  • the method can comprise, for example: using (1) an oligonucleotide, e.g., an oligonucleotide labeled for detection, which hybridizes to mRNA of one selected from the group consisting of: T-bet, IKKs, NF- ⁇ B, ⁇ 38, IL-12, STAT4, IgG2a, IgGl, IgE and gene x - xxxv nucleic acid sequence; or (2) a pair of primers useful for amplifying T-bet,
  • the method can also comprise, e.g., using a buffering agent, a preservative, or a protein-stabilizing agent.
  • the method can also comprise using components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).
  • detectable agent e.g., an enzyme or a substrate.
  • such methods can comprise using a nucleotide sequence, e.g., an oligonucleotide sequence, immobilized onto the surface of a solid support (e.g., a glass or porous solid support).
  • determination of the difference in the amount relative to a test sample of mRNA of T-bet, IKKs, NF- ⁇ B, p38, IL-12, or STAT4, or germline transcript of IgG2a, IgGl or IgE, or gene x - xxxv 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, for example, anchor PCR, RACE PCR or RT-PCR.
  • PCR polymerase chain reaction
  • Such methods can include the steps of collecting a cell sample, isolating mRNA from the cells of the sample, reverse transcribing the mRNA, contacting the sample with one or more primers which specifically hybridize to the selected sequence under conditions such that hybridization and amplification of the sequence (if present) occurs, and determining the amount of product that is present.
  • Alternative amplification methods can also routinely be utilized. Such methods can include, for example, self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA . 86: 1173-1177), Q-Beta Replicase'(Lizardi et al., 1988, Bio/Technology: 1197), or any other nucleic acid amplification method, followed by the detection/quantitation of the amplified molecules using techniques well known to those of skill in the art.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (preferably at least
  • SSC 0.1% SDS at 50-65'C (preferably 65'C).
  • Probes can comprise any readily detectable label moiety.
  • probes utilized herein 0 comprise a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor as a label moiety.
  • I mRNA sequence of (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12;
  • the amount of mRNA of (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL- 12; (vi) STAT 4; or germline transcript of (vii) IgG2a, (viii) IgGl, (ix) IgE, gene x-xxxv can be determined by hybridizing nucleic acid arrays, e.g., microarrays.
  • the expression of (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, (ix) IgE, gene x-xxxv, is measured or detected using a DNA microarray.
  • a DNA microarray or chip is a microscopic array of DNA fragments or synthetic oligonucleotides, disposed in a defined pattern on a solid support, wherein they are amenable to analysis by standard hybridization methods (see, e.g., Schena, 1996, BioEssays 18: 427).
  • Microarrays share certain preferred characteristics: The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably the microarrays are small, usually smaller than 5 cm, and they are made from materials that are stable ⁇ - under binding (e.g., nucleic acid hybridization) conditions.
  • Microarrays contain a surface to which sequences corresponding to gene products (e.g., mRNA, 0 cDNA, cRNA, or complements thereof), can be specifically hybridized or bound at a known position.
  • the binding sites of the microarray are polynucleotides, preferably DNA polynucleotides, that specifically hybridize to at least a portion of mRNA or cDNA of (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or germline transcript of (vii) IgG2a, (viii) IgGl, (ix) IgE, gene x-xxxv, or any combination of such
  • mRNA or cDNA molecules -produced by a subject mammal. That is, a given binding site or
  • the nucleotide sequence of each of the different polynucleotide bound to the surface is in the range of about 15 to about 100 nucleotides in length.
  • Polynucleotides can be synthesized using conventional methods, such as phosphoramidite- based synthesis methods.
  • the 0 binding site polynucleotide sequences can be derived from cDNA or genomic clones.
  • DNA microarrays can be probed using mRNA, extracted and, optionally, reverse transcribed and amplified from a sample.
  • Nucleic acid hybridization and wash conditions are optimally chosen so that the probe "specifically binds" or “specifically hybridizes” to a specific array site, i.e., the probe hybridizes, 5 duplexes or binds to a sequence array site- ith a complementary nucleic acid sequence but does not hybridize to a site with a non-complementary nucleic acid sequence.
  • one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base- pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than 0 a 5% mismatch.
  • a microarray of the invention farther comprises a binding site 5 designed to act as a negative control and/or a binding site designed to act as a positive control.
  • a positive control can relate to a constitutively expressed gene sequence, e.g., a ubiquitin sequence, HSC70, or GADPH.
  • a negative control can relate to a gene sequence not expressed in the test cell or tissue being assayed. *- .
  • hybridization conditions that can be utilized with ,DNA 0 microarrays are as follows: hybridization in 5 X SSC plus 0.2% SDS at 65°C for 4 hours followed by washes at 25°C in low stringency wash buffer (I X SSC plus 0.2% SDS) followed by 10 minutes at 25°C in high stringency wash buffer (0.1 X SSC plus 0.2% SDS) (Shena et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-19).
  • the use of a two-color fluorescence labeling and detection scheme to define alterations in gene expression has been described, e.g., in Shena et al., 1995, Science 270:467-470.
  • 5 cell states e.g., control and activated
  • experimental conditions e.g., hybridization conditions
  • cDNA from a single cell, and compare, for example, the absolute amount of a particular mRNA in sample cell.
  • the mRNA or cDNA are typically labeled with fluorescent dyes that emit at 0 different wavelengths.
  • fluorescent dyes include, but are not limited to, rhodamine, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, a- phthaldehyde and fluorescamine.
  • the fluorescence emissions at each site of a DNA array can be, preferably, detected by scanning confocal laser microscopy. In one embodiment, a separate scan, using, the appropriate excitation line, is carried out for each of two fluorophores used.
  • a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see, e.g., Shalon et al., 1996, Genome Research 6:639-645).
  • Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12 bit analog to digital board.
  • the scanned image is despeckled using a graphics 0 program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site.
  • a graphics 0 program e.g., Hijaak Graphics Suite
  • the level of hybridization to the site in the array corresponding to any particular gene will reflect the prevalence in the cell of mRNA transcribed from that gene.
  • detectable labeled e.g., with a fluorophore
  • the site on the array corresponding to a gene i.e., capable of specifically binding the product of the gene
  • the site on the array corresponding to a gene i.e., capable of specifically binding the product of the gene
  • a gene for which the encoded mRNA is prevalent will have a relatively strong signal.
  • Microarrays can be made in a number of ways well known to those of skill in the art.
  • the nucleic acid for the microarray can be generated by synthesis of synthetic.polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (e.g., Froehler et al., 1986, Nucleic Acid Res 14:5399-5407).
  • synthetic nucleic acids include non-natural bases, e.g., inosine.
  • nucleic acid analogues may be used as binding sites for' hybridization.
  • An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Eghohn et al, 1993, Nature 365:566-568; see also U.S. Patent No. 5,539, 083, Cook et al., entitled "Peptide nucleic acid combinatorial libraries and improved methods of synthesis,” issued July 23, 1996).
  • binding (hybridization) sites can also be used as binding sites for' hybridization.
  • the polynucleotide of the binding sites is RNA.
  • the nucleic acid or analogue is attached to a solid support to produce the binding site.
  • Solid supports may be made from glass, silicon, plastic (e.g., polypropylene, nylon, polyester), 15 polyacrylamide, nitrocellulose, cellulose acetate or other materials. In general, non-porous supports, and glass in particular, are preferred.
  • the solid support may also be treated in such a way as to enhance binding of oligonucleotides thereto, or to reduce nonspecific binding of unwanted substances thereto.
  • the glass support is treated with pplylysine or silane to facilitate attachment of oligonucleotides to the slide.
  • Methods of immobilizing DNA on the solid support may include direct touch, micropipetting (Yershov et al, Proc. Natl. Acad. Sci. USA, 1996, 93:4913-4918), or the use of controlled electric fields to direct a given oligonucleotide to a specific spot in the array (U.S. Patent No. 5,605, 662).
  • any type of array for example, dot blots on a nylon hybridization membrane (see Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold
  • DNA can typically be immobilized at a density of 50, 75, 100, up to 10,000 oligonucleotides per cm 2 and preferably at a density of about 1000 oligonucleotides per cm 2 .
  • nucleic acids can be attached to a surface by printing on glass plates (Schena et al., 1995, Science 270:467-470; DeRisi et al., 1996, Nature Genetics 14:457460; Shalon et al., 1996, Genome Res. 6:639- 645; and Schena et al., Proc. Natl. Acad! Sci. USA, 1996, 93(20):10614-19. )
  • As an alternative to immobilizing pre-fabricated oligonucleotides onto a solid support it is possible to synthesize oligonucleotides directly on the support (Maskos et al., 1993, Nucl. Acids Res.
  • microarrays e.g., by masking (l ⁇ 4askos and Southern, 1992, Nue. Acids Res. 20:1679- ' * ' • ' ⁇ 1684)', may also be used.
  • Standard techniques can also be utilized for determining the amount of the protein or proteins of interest (e.g., T-bet, the following signal molecules which can lead to the activation of KB binding site and/or STAT4-binding site containing promoters and/or T-bet promoter; IKKs, NF- ⁇ B, p38, IL-12, and STAT4, or IgG2a, IgGl or IgE protein, or gene x-xxxv protein) present in a sample.
  • T-bet the following signal molecules which can lead to the activation of KB binding site and/or STAT4-binding site containing promoters and/or T-bet promoter; IKKs, NF- ⁇ B, p38, IL-12, and STAT4, or IgG2a, IgGl or IgE protein, or gene x-xxxv protein
  • Such a determination of the amount of a protein present includes deterr ⁇ ining the total amount of a protem present, and also includes, especially with respect to determining the amount of a STAT protein present, determining the amount of a phosphorylated form of the protein present.
  • a preferred agent for detecting a protein of interest is an antibody capable of binding to a protein of interest, preferably an antibody with a detectable label.
  • such a determination can also be performed using standard techniques well known to those of skill in the art.
  • a determination can include, first, immunoprecipitation with an antibody that is specific for a phosphorylated amino acid residue, e.g., an anti-phosphotyrosine antibody, such that all exhibiting such a phosphorylated residue in a
  • the immunoprecipitated proteins can be contacted with a second antibody that is specific for the following protein of interest, e.g., (i) T-bet (ii) IKKs;
  • a phosphorylated protein of interest can be identified and quantitated using an antibody specific for the phosphorylated form of the particular protem itself, e.g., an antibody
  • protein from the sample to be analyzed can easily be isolated using techniques that are well known to those of skill in the art.
  • Protem isolation methods can, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, . "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, ' ' ⁇ * ⁇ ' ⁇ New York).
  • Preferred methods for the detection of the protein or proteins of interest involve their detection via interaction with a protein-specific antibody.
  • antibodies directed a protein of interest can be utilized as described herein.
  • Antibodies directed against (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, (ix) IgE, or (x)-(xxxv) are well known to those of skill in the art.
  • T-bet, STAT4 can be
  • antibodies can be generated utilizing standard techniques well known to those of skill in the art. See, e.g., Section 5. 3, below, for a more detailed discussion of such antibody generation techniques. Briefly,- such antibodies can be polyclonal, or more preferably, monoclonal.
  • An intact antibody, or a fragment thereof e.g., Fab or F(ab')2 can, for example, be used.
  • antibodies, or fragments of antibodies, specific for a protein of interest can be used to quantitatively or qualitatively detect the presence of the protein. This can be accomplished, for example, by immunofluorescence techniques. Antibodies (or fragments thereof) can, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ
  • In situ detection can be accomplished by removing a histological specimen (e.g., a biopsy specimen) from a patient, and applying thereto a labeled antibody thereto that is directed to a T-bet, STAT4 protein.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays for a protein of interest typically comprise incubating a biological sample, e.g., a biopsy or subject blood sample, of a detectable labeled antibody capable of identifying a protein of 30 interest, and detecting the bound antibody by any of a number of techniques well- known in the art.
  • a biological sample e.g., a biopsy or subject blood sample
  • detectable labeled antibody capable of identifying a protein of 30 interest
  • the term "labeled” can refer to direct labeling of the antibody via, e.g., coupling (i.e., physically linking) a detectable substance to the antibody, and can also refer to indirect labeling of the antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescence labeled secondary antibody.
  • the biological sample can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support can then be washed with suitable buffers followed by treatment with the detectable-labeled fingerprint gene-specific antibody.
  • the solid phase support can then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on solid support can then be detected by conventional means.
  • solid phase support or carrier any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, poly- ethylenej dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface can be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • T-bet ii) IKKs; (iii) NF- B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or (x)-(xxxv) specific antibody can be detectable labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunos ⁇ rbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A. et al., 1978, J. Clin. Pathol.
  • EIA enzyme immunoassay
  • the enzyme that is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • an appropriate substrate preferably a chromogenic substrate
  • enzymes which can be used to detectable label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta- 5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerolphosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6 ⁇ hosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • Detection can also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay R1A
  • the radioactive isotope e.g., 125 1, 131 1, 35 S or 3 H
  • fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, 2- phthaldehyde and fluorescarnine.
  • the antibody can also be detectable-labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid • (EDTA).
  • DTP A diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid •
  • the antibody also can be detectable labeled by coupling it to a chemilummescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemilummescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound can be used to label the antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic r-- protein increases the . efficiency of the chemilummescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Standard techniques can be utilized to determine the level of (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl; or (ix) IgE; or (x)-(xxxv) activity.
  • the activity of (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) ⁇ 38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl or (ix) IgE can be determined by detecting the binding of (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE , or (x)-(xxxv) to its cognate DNA binding element, via, for example, an electromobility shift assay ("EMSX"), detecting the expression of a gene whose expression is controlled by a promoter that is responsive to (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38;
  • T-bet-responsive promoter Genes whose expression is controlled by a T-bet-responsive promoter include, for example IFN- ⁇ , IL-12RJ3, et al, (see, Szabo SJ, et al, Cell 100(6), 655-69 (2000), and Afkarian M, et al., Nat Immunol. 3(6), 549-57 (2002)).
  • expression of such genes in the TLR9 expressing cell sample in the presence and absence of a test compound can routinely be determined using standard techniques, such as reporter assays.
  • EMSAs can also routinely be utilized to assess T-bet activity. Such techniques are well known to those of skill in the art. See, e.g., A ici et al., 1995, Cancer Research 55: 14452-4457.
  • T-bet ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or (x)-(xxxv) can also be assessed by detecting the production of cytokines, surface markers, Ig, in TLR9 expressing cells such as B cells and ' DC. Techniques known to those of skill in the art can be used for measuring these activities. For example, cytokine production can be assayed by ELISA. The effector function of T cells can be measured, for example, by a FACS analysis of surface markers.
  • the methods described herein for identifying reagents that modulate the activity of TLR9 comprise as a readout assaying whether a test compound has an effect on the expression and/or the activity of T-bet mRNA or protein, STAT4 mRNA or protein,IgGl, IgG2a, IgE, (x) ⁇ (xxxv) produced by TLR9 expressing cells, or produced by a cell that has the ability to respond to CpG.
  • the compounds can be further investigated.
  • the compounds identified via the present methods can be further tested in vivo in accepted animal models of inflammatory disorders. Further, the compounds identified can also be analyzed with respect to their specificity.
  • Reagents that modulate the activity of TLR9 identified via the present methods can be used in the manufacture of medicaments for the treatment of cancer, inflammatory diseases such as asthma
  • COPD chronic obstructive pulmonary disease
  • the invention provides assays for screening test compounds that bind to or modulate the activity of TLR9 or CpG recognizing receptor by determining the level relative to a test sample of T-bet mRNA or protein, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, or IgGl or IgE germline transcripts or protein, 0 gene x-xxxv mRNA or proteins in a tissue.
  • a test compound preferably binds to a TLR9 or CpG recognising receptor polypeptide or polynucleotide. More preferably, a test compound decreases or increases functional activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacological agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library 0 methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small 5 molecule libraries of compounds. See Lam, Anticancer Drug Des. 72, 145, 1997.
  • Test compounds can be screened for the ability to affect TLR9 or CpG activity by determining the level relative to a test sample of e.g., T-bet mRNA or protem or T-bet promoter activity measured by reporter assay, STAT4 mRNA or protein phosphorylation or STAT4 promoter activity measured by reporter assay, p38 mRNA or protein phosphorylation or enzyme activity, IL-12 or any one of (x) to (xxxv) mRNA or protein, IgG2a, or IgGl or IgE germline transcripts or protein in or secreted from a tissue using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • assay volumes typically range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released ' the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying . combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • Another high throughput screening method is described in Beutel et al, U.S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then " .
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • Test compounds can be tested for the ability to increase or decrease a biological effect of TLR9 by determining a level relative to a test sample of T-bet mRNA or protein or promoter activity, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, activation of IKK and NF- ⁇ B, IL-12 or any one of (x)-(xxxv) mRNA'Or protem, IgG2a, or IgGl or IgE germline transcripts or protein in a tissue.
  • Such biological effects can be determined for example using functional assays such as those described below. Functional assays can be carried out after contacting a cell membrane preparation, or an intact cell with a test compound.
  • a test compound which increases or decreases a functional activity of TLR9 polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent.
  • Polypeptides comprising amino acid sequences encoded by open reading frames of TLR9 are either expressed endogenously in appropriate reporter cells or are introduced recombinantly. Signal molecule or Igs activity can be monitored by measuring a cellular response (e.g., expression of a reporter gene or secretion of a neurotransmitter) triggered or modulated by the polypeptide's activity.
  • a cellular response e.g., expression of a reporter gene or secretion of a neurotransmitter
  • the activity of TLR9 modulation can be determined by, for example, (i) a reporter assay with cells expressing TLR9 endogenously or ectopically and reporter gene containing T-bet promoter element; (ii) a reporter assay with cells expressing TLR9 endogenously or ectopically and reporter gene containing STAT 4 binding elements and/or NF- ⁇ B binding sequences; (iii) a reporter assay with cells expressing TLR9 endogenously or ectopically and reporter gene containing T-box element or responsive to the T-bet protein, such as an IFN-gamma promoter; (iv) measurement of IL-12 protein amount by ELISA (v) comparing the degree of STAT 4 phosphorylation; (vi) determining the expression of IgG2a germline transcripts or the production of IgG2a protein in primary B cells or B cell lines; and the like, (vii) determining the inhibition of IL-4 alone or IL-4/CD40 ligatib
  • test compounds that increase or decrease a particular signal molecule gene
  • RNA or polypeptide product of particular signal molecule polynucleotide is identified.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator 0 - or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods 5 can be used.
  • the presence of polypeptide products of a polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • Such screening can be carried out, but not limited to, in an intact cell.
  • Any cell that expresses TLR9 receptor polynucleotide can be used in a cell-based assay system.
  • the TLR9 polynucleotide 0 can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the invention can comprise a reagent which alters .
  • the compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions can be administered to a patient alone, or in combination 0 with other agents, drugs or hormones.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical; sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patierit.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixtur of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, marrnitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, • or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 niM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated, condition. Such labeling would include amount, frequency, and method of administration.
  • T-bet ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or (x)-(xxxv) and polynucleotides encoding them can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme.
  • differences can be determined between the cDNA or genomic sequence encoding one of the signal molecules selected from the group consisting of (i) T-bet (ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, (ix) IgE and (x)-(xxxv) in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in hormal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as ⁇ probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radio-labeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers- et al. Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl.
  • the detection of a specific DNA sequence can be performed by methods such- as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
  • T-bet ii) IKKs; (iii) NF- ⁇ B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or (x)-(xxxv), can be modulated to treat allergic or inflammatory diseases such as asthma and COPD.
  • allergens typically elicit a specific IgE response and, although in most cases the allergens themselves have little or no intrinsic toxicity, they induce pathology when the IgE response in turn elicits an IgE-dependent or T cell-dependent hypersensitivity reaction.
  • allergens typically elicit a specific IgE response and, although in most cases the allergens themselves have little or no intrinsic toxicity, they induce pathology when the IgE response in turn elicits an IgE-dependent or T cell-dependent hypersensitivity reaction.
  • Hypersensitivity reactions can be local or systemic and typically occur within minutes after allergen exposure in individuals who have previously been sensitized to the respective allergen.
  • the hypersensitivity reaction of allergy develops when the allergen is recognized by IgE antibodies bound to specific receptors on the surface of effector cells, such as mast cells, basophils, or eosinophils, which causes the activation of the effector cells and the release of mediators that produce the acute signs and symptoms of the reactions.
  • Allergic diseases include asthma, allergic rhinitis (hay fever), atopic dermatitis, and anaphylaxis.
  • Asthma is thought to arise as a result of interactions between multiple genetic and environmental factors -and is characterized by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a narrowing of the airways, 2) airway hyperresponsiveness, and 3) airway inflammation.
  • Certain cells are critical to the inflammatory reaction of asthma and they include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE.- These effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to tissue destruction related to the disorder.
  • Other resident cells such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to its pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually makes asthma a chronic and disabling disorder requiring long-term management.
  • COPD chronic obstructive pulmonary (or airways) disease
  • Emphysema is characterized by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung.
  • Chronic bronchitis is defined ⁇ - clinically as the presence of chronic productive cough for three months in each of two successive years'. 1 '
  • airflow obstruction is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does also occur in non-smokers.
  • the inflammatory cell population comprises increased numbers of macrophages, neutrophils and CD 8+ lymphocytes.
  • Inhaled irritants such as cigarette smoke activate macrophages resident in the respiratory tract as well as epithelial cells leading to release of chemokines (e.g., interleukin-8) and other chemotactic
  • Signal molecules which can lead to the activation of KB binding site and/or STAT4-binding site containing promoters, or IgG2a, IgGl and IgE or a portion or biologically active variant thereof may be administered to a subject to prevent or treat an allergic or inflammatory disorder, such as asthma or COPD.
  • an agonist which is specific for Signal molecules which can lead to the activation of kB binding site and/or STAT4-binding site containing promoters, or IgG2a, IgGl. and IgE may be administered to a subject to regulate the intracellular signaling pathways involved in reorganization of the actin cytoskeleton and cell mobility, and thereby prevent or treat an allergic or inflammatory disorder, such as asthma or COPD.
  • an antagonist may be administered to a subject to prevent or treat inflammation of any type and, in particular, that which results from a particular disorder or conditions.
  • disorders and conditions associated with inflammation include, but are not limited to, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma.
  • an antibody specific for TLR9 may be used directly as an antagonist, or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which f. . express TLR9.
  • any of the therapeutic proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for
  • the invention further pertains to the use of novel agents identified by the screening assays 5- described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, . ribozyme, or a polypeptide-binding partner
  • an agent identified 0 as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above- described screening assays for treatments as described herein.
  • a reagent which affects TLR9 activity can be administered to a human cell, either in vitro or in vivo, to reduce TLR9 activity.
  • the reagent preferably binds to Ttlr9 is a polypeptide, the reagent 5 is preferably an antibody.
  • an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for 0 at least about 1 hour, and even more preferably for at least about 24 hours.
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung or liver.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing 5 with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmol of liposome delivered to about 106 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmol of liposome delivered to about ⁇ 106 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 106 cells.
  • a liposome is between about 100 and 500 nm, more preferably 0 between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes' having a cholesterol
  • a liposome comprises a compound capable of targeting the liposome to a tumor cell, such as a tumor cell ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • antibodies can be delivered to specific tissues in vivo using receptor- mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases extracellular matrix degradation relative to that which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED50 (the dose therapeutically effective in 50% of the ⁇ ' * ⁇ ,' ⁇ population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to 5- therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range 0 depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account 5 include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • 0 Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for. nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular 5 cells, conditions, locations, etc.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about ⁇ 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 ⁇ g to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to 0 about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the reagent is preferably an antisense oligonucleotide, a siRNA or a ribozyme.
  • Antisense oligonucleotides, siRNAs or ribozymes can be introduced into cells by a variety of methods, as described above or known in the art.
  • a reagent reduces expression of a TLR9 polynucleotide or activity of a TLR9 polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%
  • TLR9 polynucleotide or the activity of a TLR9 polypeptide
  • the effectiveness of the mechanism chosen to decrease the level of expression of a TLR9 polynucleotide or the activity of a TLR9 polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to TLR9- . specific mRNA, quantitative RT-PCR, immunologic detection of a TLR9 polypeptide, or measurement of TLR9 activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders 5 • described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • antibody is meant to, without limitation, refer to complete, intact antibodies, Fab fragments and F(ab)2 fragments thereof, and chimeric antibodies.
  • the term “modulates” means an increase or decrease in the amount or effect of a particular activity or protein.
  • the term "marker” refers to a biological molecule, e.g., a nucleic acid, peptide, etc. 0 or the status of said biological molecule (e.g. phosphorylation status) whose presence or concentration can be detected and correlated with the presence or absence of a reagent in an assay (e.g. a modulator, inhibitor or activator).
  • a biological molecule e.g., a nucleic acid, peptide, etc. 0 or the status of said biological molecule (e.g. phosphorylation status) whose presence or concentration can be detected and correlated with the presence or absence of a reagent in an assay (e.g. a modulator, inhibitor or activator).
  • modulator refers to an inhibitor or an activator.
  • Inhibitors can be any organic compound or an activator.
  • Inhibitors can be any organic compound
  • antagonists can be agonists, partial agonists, inverse agonist, or co-activators.
  • Inhibitors can be agonists, partial agonists, inverse agonist, or co-activators.
  • activators agonists, partial agonist, inverse agonists or co-activators
  • nucleic acids including but not limited to DNA, RNA, or DNA analog or RNA analog
  • proteins including but not limited to antibodies, peptides, peptidomimetics, carbohydrates, lipids and small molecules.
  • Small molecule refers to a composition, which has a molecular weight of less than 5 kD, or less than 4 kD, or more preferably less than 2,5 kD and most preferably less than 0,5 kD.
  • Small molecules can be organic (carbon-containing) or inorganic 0 molecules.
  • Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate TLR9 activity.
  • TLR9 antagonist examples include, but not limited to, antibodies (such as TLR9 antibody, IMG-305, IMGENEX), oligonucleotides, polypeptides, and small molecular weight compounds.
  • TLR9 agonist examples include, but not limited to, synthetic oligonucleotides (such as ODN2006), polypeptides, and small molecular weight compounds.
  • IL-12 receptor antagonist examples include, but not limited to, polypeptides, IL-12 neutralizing antibodies, soluble recombinant IL-12 receptors
  • IL-12 receptor agonist examples include, but not limited to, recombinant IL-12, IL-12 0 agonist antibodies, polypeptides which can activate IL-12 receptor.
  • T-bet could be regulated by CpG
  • mice splenocytes with 3 j ⁇ M CpG for 3 or 6 hours and measured the expression of mRNA by quantitative RT-PCR.
  • Cells treated with IFN- ⁇ were used as a positive control.
  • GpC active oligonucleotides with a reversed . CpG motif
  • the .TLR4 agonist E.coli LPS were used for determining the specificity of oligonucleotides and signaling pathways, respectively.
  • T-bet was slighfly elevated in CpG-treated splenocytes (3 -fold) at 3 hours, followed by a profound induction at 6 hours after stimulation, surpassing the induction produced by the IFN- ⁇ treatment ( Figure 1).
  • the effect of CpG on T-bet transcription was specific since the inactive GpC DNA failed to upregulate T-bet mRNA. LPS was incapable of inducing the transcription over the 6-hour time course, consistent with previous reports 8 .
  • TLR9 itself, or a yet unknown CpG-responding receptor, transmits the signal leading to the transactivation Of T-bet and phosphorylation of STATl
  • TLR9 _/" and MyDS ⁇ " ' " mice mice to define the signal.
  • FIG. 3A and 3B we show in Figure 3A and 3B that both CpG-induced T-bet mRNA and STATl phosphorylation were completely ablated in MyD88- or TLR9-deficient splenocytes, while the effect of IFN- ⁇ was unchanged.
  • TLR9 and MyD88 the induction of T-bet and ⁇ phosphorylation of STATl by CpG requires both TLR9 and MyD88.
  • T cell and B cell fractions were then isolated by sequential positive and negative selection and the T cell-and B cell-depleted splenocytes recovered (mainly NK cells and myeloid cells, designated as "non-T, non-B").
  • non-T, non-B T cell-and B cell-depleted splenocytes recovered.
  • T cells are not expected to be able to respond to CpG directly due to their lack of TLR9 expression, to preclude the possibility that CpG-induced signaling had not yet transmitted or failed to transmit to T cells, we measured IFN- ⁇ mRNA levels in the same samples.
  • Figure 4B a dramatic induction of IFN-gamma mRNA was seen in T cells with levels comparable to non-T, non-B splenocytes even at 6 hours post CpG stimulation. This shows that even in mixed cultures, CpG- cannot upregulate T-bet mRNA in-T cells in at least a period of 24 hours, despite being able to drive T cells to express IFN- ⁇ mRNA.
  • CpG-induced T-bet expression in B cells is IFN-gamma/STATl independent.
  • NF- B, p38, and IL-12 are required for CpG-induced T-bet expression.
  • IL-12 functions sequentially or additively with CpG
  • B cells were stimulated with CpG, IL-12, and IFN- gamma alone or in combination.
  • IL-12 synergistically enhances CpG- induced T-bet upregulation, in contrast to IFN- ⁇ which only appears to additively effect on T-bet expression.
  • IL-12 together with CpG stimulates a much greater induction of T-bet mRNA (130 times background) than that of CpG alone (12 times) or CpG plus IFN- ⁇ (20 times).
  • CpG inhibits IL-4/CD40-induced IgGl and IgE class switching via a direct action on B cells.
  • CpG can inhibit antigen-induced IgE production in mice and in PBMC from atopic patients, such an effect has not been reported in cultured B cells.
  • CpG can rapidly induce a drastic increase in T-bet mRNA in purified B cells and the report that ectopic expression of T-bet is sufficient to induce IgG2a switching in cultured B cells, and that T-bet- deficient B cells produce excess amounts of IgGl and IgE 15 .
  • splenic B cells purity>99% were cultured in a medium containing IL-4 (10 ng/ml) and anti-CD40 agonistic
  • I*- antibodies (3 microg/ml) in the presence or absence of CpG (3 microM) for 10-14 days, then cell number, purity and the secreted IgGl, IgG2a and IgE were assessed. Strikingly, IL-4 and CD40 ligation-induced IgE (36.1+6.3 ng/ml) was completely inhibited and IgGl production (246+30.9 ng/ml) showed an 87% reduction after CpG treatment (Table 4 and Figure 7A). Because neither the cell number nor the purity showed significant changes during the cell culture, this shows that CpG effectively inhibits IL-4/CD40-induced Th2-related IgE and IgGl production via a direct effect on B cells. This inhibition appeared to occur at the transcriptional level because
  • I ⁇ and I ⁇ l germline transcripts were completely diminished in B cells cultured with IL-4/CD40
  • IgE and IgGl class switching showed a positive correlation with T-bet mRNA ( Figure 7B and C).
  • CpG treatment or T-bet expression could inhibit IL-4-induced phosphorylation of STAT6.
  • B cells were pretreated with CpG for 20 hours, and then stimulated with IL-4 for 20 or 60 min. Activation of STAT6 was assessed by
  • T- bet promoter element or any one ot gene x-xxxv promoter elements.
  • TLR9 receptor Cells expressing endogenous or recombinant TLR9 receptor are transfected with a DNA construct having T-bet promoter element or any one of gene x-xxxv promoter elements, and reporter gene (such as luciferase gene).
  • the transfected cells are incubated at 37 ° C for 1 min to 3 days with a test compound.
  • a culture of the same type of cells that have not been transfected is incubated for 0 the same time without the test compound to provide a negative control.
  • the cells or culture medium are/is harvested to determine the reporter activity (such as luciferase activity by luminometry).
  • test compound that decreases or increases the reporteractivity relative to the activity obtained in the absence of the test compound is identified as a modulator (an inhibitor or activator 5 respectively) of TLR9. • '
  • Cells expressing endogenous or recombinant TLR9 receptor are transfected with a DNA construct
  • the transfected cells are incubated at 37 ° C for 1 min to 3 days with a test compound.
  • a culture of the same type of cells that have not been transfected or transfected with a reporter construct without KB or STAT4 binding elements is incubated for the same time without the test compound to provide a negative control.
  • the cells are harvested to determine the reporter activity by luminometry.
  • test compound that decreases or increases the luciferase activity relative to the activity obtained ' ' ⁇ * •' ⁇ in the" ' absence of the test compound is identified as a modulator (an inhibitor or activator respectively) of TLR9.
  • Cells expressing endogenous or recombinant TLR9 receptor are used. The cells are incubated at 37 ° C for 10 min to 7 days with a test compound. A culture of the same type of cells that have not 0 been tr nsfected is incubated for the same time without the test compound to provide a negative control.
  • the supematants of the reaction mixture are then collected.
  • the IL-12 concentration in the supematants is determined using a DuoSetTM ELISA Development Kit (GenzymeTechne, Minneapolis, USA) following the manufacturer's recommendations.
  • test compound that decreases or increases the amount of IL-12 relative to the amount obtained in the absence of the test compound is identified as a modulator (an inhibitor or activator respectively) of TLR9.
  • B cells expressing TLR9 receptor are used.
  • the B cells are incubated at 37 ° C for 0.1-21 days with a test compound.
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • the supematants of the reaction mixture are then collected.
  • the IgG2a concentration in the 5 supematants is determined using a DuoSetTM ELISA Development Kit (GenzymeTechne, Minneapolis, USA) following the manufacturer's recommendations.
  • ⁇ _. in the absence of the test compound is identified as a modulator (an inhibitor or activator respectively) of TLR9.
  • B cells expressing TLR9 receptor are used.
  • the B cells are incubated at 37 ° C for 3 to 21 days with IL-4 (0.01 ng/ml - 10 ⁇ g/ml, optimal concentration is 10 ng/ml) and CD40 ligation (CD40 ligand, or an agonistic antibody, a test compound.
  • IL-4 0.01 ng/ml - 10 ⁇ g/ml, optimal concentration is 10 ng/ml
  • CD40 ligation CD40 ligand, or an agonistic antibody
  • the supematants of the reaction mixture are then collected.
  • the IgGl and/or IgE concentration in the supematants is determined using a DuoSetTM ELISA Development Kit (GenzymeTechne, Minneapolis, USA) following the manufacturer's recommendations.
  • a test compound that decreases or increases the amount of IgGl and/or IgE relative to the amount obtained in the absence of the test compound is identified as a modulator (an inhibitor or activator respectively) of TLR9.
  • Table 2 and 3 show the accession number and functional description of genes that are up- or down-regulated by CpG, as well as their expression in response to IL-4, INF ⁇ , IL-12 or CpG plus IL-12.
  • Mouse splenic B cells were purified by negative selection and stimulated with indicated stimuli for 8 h and 24 h respectively.
  • Total RNA was purified and 5 ⁇ g of each was used for DNA microarray analysis (CodeLink Bioarray Mouse AmershamBiosciences). Image was visualized using Axon GenePixPro v4.0 and analyzed by CodeLinkTM software (AmershamBiosciences). The relative expression of mRNA was determined by the ratio of fluorescent intensity to the median.
  • Modlin RL Mammalian toll-like receptors. Ann Allergy Asthma Immunol. 88(6), 543-7 ⁇ * ⁇ - (2002).
  • T-bet is a STATl-induced regulator of IL-12R expression in naive CD4+ T cells. Nat Immunol. 3(6), 549-57 (2002).

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Abstract

The effect of TLR-9 modulation can be detected by determining e.g., the amount of T-bet mRNA or protein, STAT4 protein phosporylation, p38 activities, IL-12 mRNA or protein, inhibition of TH2-related IgG1 and IgE switching, present in a tissue. The present invention relates to method for identifying or evaluating reagents that modulate the activity of TLR9 using these members of the pathway such as T-bet, NF- B, IKK, STAT4, p38, IL-12 and Igs as markers. Reagents that modulate the activity of TLR9 identified by the present method are useful in the manufacture of medicaments for the treatment of a range of diseases including cancer, autoimmune diseases, inflammatory diseases such as asthma or COPD, immunological disorders and any other conditions involving aberrations of signal transduction.

Description

Methods for identification and validation of reagents that modulate the activity of TLR9 and methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of TLR9 related diseases
' *' FIELD OF THE INVENTION
5 The present invention relates to methods for identifying and evaluating reagents that modulate the activity of TLR9 (Toll-like receptor 9). The invention further relates to the use of reagents that modulate the activity of TLR9 in the manufacture of medicaments for the treatment of cancer, immunological diseases such as allergy, asthma, chronic obstructive pulmonary disease (COPD), autoimmune diseases, viral and parasitic infection, transplantation rejection, and conditions where 10 TLR9 signal transduction is disordered.
BACKGROUND OF THE INVENTION
In the mammalian immune system T cells can produce either TH1 or TH2 cytokines depending on the initial signal and environment. These initially secreted cytokines are thought to provide permissive signals that favor naϊve CD4+ helper T cells polarization towards the TH1 or TH2
15 subtype 1'2.
The expression of the master transcription factor T-bet (T box expressed in T cells) (T H1 gene regulator) or the transcription factors GATA3 and STAT6 (signal transducer and activator of transcription 6) (TH2 gene regulator) is enhanced in polarized T cells by IFN /STAT1, IL-12/STAT4 and IL-4/STAT6 respectively, and results in the differentiation to effector T cells3. 20 Differentiated TH.1 and TH2 have distinct functions and cytokine profiles.
Dysregulated TH1 or TH2 responses can cause diseases such as asthma which is characterized by Tκ2-associated pulmonary inflammation. Therefore, redirecting immune responses with therapeμtic reagents is an important strategy for disease treatments. This includes the redirection of TH2 mediated diseases which include allergies like asthma and TH1 mediated diseases which 25 include autoimmune disease like rheumatoid arthritis and multiple sclerosis.
The Toll-like receptor (TLR) family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition. Mammalian TLRs comprise a large family with extracellular leucine-rich repeats and a cytoplasmic Toll/interleukin-lR homology domain. Adapter protein MyD88 interacts with TLRs through TIR domain. Upon the activation by 30 mi.crobial components, MyD88 recruits IL-1 receptor associated kinase (IRAK) and TRAF6 to the receptors, and results in the activation of IKK-NF-κB and JNK-AP-1 signaling pathways. These twp MyD88-dependent pathways are common for all known TLRs and are well demonstrated by MyD88-defιcient mice4"6. So far 10 members have been identified.
Oligonucleotides containing CpG motifs induce the expression of TH1 cytokines7. Although TLR9 has been identified as the receptor for CpG oligonucleotides (WOO 1/32877), the CpG/TLR9 signaling pathway and the mechanism of action that leads to the unique protective effects against allergic inflammation are unknown. Since T cells do not express TLR9, it is not clear how CpG achieves its regulatory effects on T helper cells. Additionally, the inhibition of IgE production by CpG has been shown in vivo and in vitro in cultured peripheral blood mononuclear cells (PBMCs). The underlying mechanism still awaits to be discovered.
Since CpG oligonucleotides are useful modulators of various immune responses, there is considerable interest to understand the mechanisms of actions of CpG and the TLR9 signaling t . pathway. The signaling pathway needs to be understood in order to be able to provide assays for the identification of reagents that mimic the effect of CpG oligonucleotides and which are useful as therapeutics for diseases such as autoimmune diseases, allergic diseases including asthma and COPD. There is also a need to discover the TLR9 signaling pathway in order to identify novel therapeutic targets for the treatment of diseases such as autoimmune diseases, allergic diseases including asthma and COPD and to monitor and evaluate the efficacy of novel therapeutics.
SUMMARY OF THE INVENTION
The present invention relates to methods for identifying and evaluating reagents that modulate the activity of TLR9 using at least one of the following as a marker: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii)
NF-κB protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL-12 protein or a polynucleotide encoding the same; (vi) STAT4 protem or a polynucleotide encoding the same; (vii) IgG2a protein or a germline transcript of the same, (viii) IgGl protein or a germline transcript of the same, (ix) IgE protem or a germline transcript of the same, x) TCF2, protein or a polynucleotide encoding the same, xi) BATF protein or a polynucleotide encoding the same, xii) PLAUR protein or a polynucleotide encoding the same,
^ xiii) C/EBPδ protein or a polynucleotide encoding the same, xiv) ARHE (ras homolog gene faiftily) protein or a polynucleotide encoding the same, xv) NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same, xvi) GOTl (glutamate oxaloacetate transaminase
1) protein or a polynucleotide encoding the same, xvii) C/EBPβ protein or a polynucleotide encoding the same, xviii) TACSTD1 (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same, 'xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same, xx) SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same, xxi) ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same, xxii) UBEIC (ubiquitin-activating
•: -Λ,, enzyme E1C) protem or a polynucleotide encoding the same, xxiii) FNBP3 (formin binding 5 protem 3) protein or a polynucleotide encoding the same, xxiv) CHUK (conserved helix-loop- helix ubiquitous kinase) protein ΌΓ a polynucleotide encoding the same, xxv) SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same, xxvi) GNPNAT1 (glutamine repeat protein 1) protein or a polynucleotide encoding the same, xxvii) BAP29 (B-cell receptor- associated protein 29) protein or a polynucleotide encoding the same, xxviii) PML (promyelocytic
10 leukemia) protein or a polynucleotide encoding the same, xxix) MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same, xxx) FIG1 (interleukin-four induced gene 1) protein or a polynucleotide encoding the same, xxxi) MHC2TA (class II transactivator) protein or a polynucleotide encoding the same, xxxii) HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same, xxxiii)
15 FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same, xxxiv) Id2 protein or a polynucleotide encoding the same, xxxv) CD74 protein or a polynucleotide encoding the same. IKKs, NF-κB, p38, IL-12, and STAT4 are indicators for the activation of TLR9. Genes such as IgG2a, IgGl, IgE, PLAUR, ARHE, GOTl, TACSTD1, SCS, ESP15, UBElc, FNBP3, CHUK, SELL, GNPNAT1, BAP29, PML, MMKROX2R, FIG1, HCST,
20 FcεR2A, CD74 are the output signal molecules of NF- B, STAT4, T-bet, TCF2, C/EBPβ, C/EBPδ, NOP5/NOP58, BATF, LM04, Id2, and MHC2TA activation or repression. Any combination of two or more markers selected from the above can also be used.
The present invention also relates to the use of a reagent that alters the expression, amount, activity or phosphorylation, in a cell or tissue, of (i) T-bet protein or a polynucleotide encoding the
25 same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF-κB protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL- 12 protein or a polynucleotide encoding the same; (vi) STAT 4 protein or a polynucleotide encoding the same; (vii) IgG2a protein or a germline transcript of the same, (viii) IgGl protein or a germline transcript of the same and (ix) IgE protein or a germline transcript of the same; x)
30~ TCF2, protein or a polynucleotide encoding the same, xi) BATF protein or a polynucleotide encoding the same, xii) PLAUR protein or a polynucleotide encoding the same, xiii) C/EBPδ protein or a polynucleotide encoding the same, xiv) ARHE (ras homolog gene family) protein or a polynucleotide encoding the same, xv) NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same, xvi) GOTl (glutamate oxaloacetate transaminase 1) protein or
35 a polynucleotide encoding the same, xvii) C/EBPβ protem or a polynucleotide encoding the same, xviii) TACSTD1 (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same, xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same, xx) SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the "same, xxi) ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same, xxii) UBEIC (ubiquitin-activating enzyme E1C) protein or a polynucleotide encoding the same, xxiii) FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same, xxiv) CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same, xxv) SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same, xxvi) GNPNAT1 (glutamine repeat protein 1) protem or a polynucleotide encoding the same, xxvii) BAP29 (B-cell receptor-associated protein 29) protein or a polynucleotide encoding the same, xxviii) PML (promyelocytic leukemia) protein or a polynucleotide encoding the same, xxix) MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protem or a polynucleotide encoding the same, xxx) FIG1 (interleukin- four induced gene 1) protein or a polynucleotide encoding the same, xxxi) MHC2TA (class II transactivator) protein or a polynucleotide encoding the same, xxxii) HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same, xxxiii) FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same, xxxiv) Id2 protein or a polynucleotide encoding the same, xxxv) CD74 protein or a polynucleotide encoding the same by modulating TLR9 activity in the preparation of a medicament for the treatment of cancer, inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD), immunological disorders and conditions where TLR9 signal transduction is aberrant, the reagent not being a CpG oligonucleotide.
The present invention is based on the discovery that inhibition or activation of TLR9 activity can be detected by determining the amount, expression, activity or the phosphorylation of signal molecules which can lead to the activation of KB binding site and/or STAT4-binding site containing promoters or the T-bet, or gene vii-xxxv promoter in a tissue. The present invention is further based on the discovery that immune responses resulting from CpG-TLR9 interaction can be mimicked by increasing or diminishing the amount of mRNA or protein of T-bet or gene vii- xxxv, or the amount of mRNA or protein of the members, which can lead to the regulation of NF- KB and/or STAT4 activities, and/or output signal molecules of T-bet or gene vii-xxxv.
One embodiment of the invention provides a method for monitoring the effect of TLR-9 activation or inhibition by determining the difference in the level relative to a test sample of T-bet, IKKs, or NF-kB or gene x-xxxv mRNA QΓ protein, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a germline transcripts or protein, IgGl germline transcripts or protein, or IgE germline transcripts or protein in a tissue.
"Level" used herein includes, but not limited to, the amount of a protein, expression amount of mRNA, a gene activity, a protein activity, and the amount of phosphorylation.
Thus, in one aspect, the invention relates to methods for identifying and evaluating reagents that activate or inhibit TLR9-activtiy comprising determining the difference in the amount, expression, activity or phosphorylation relative to a test sample of at least one of the following: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF-κB- protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL-12 protein or a polynucleotide encoding the same; (vi) STAT 4 protein or a polynucleotide encoding the same; (vii) IgG2a protein or a germline transcript of the same, (viii) IgGl protein or,,a germline transcript of the same and (ix) IgE protein
I or a germline transcript of the same; x) TCF2, protem or a polynucleotide encoding the same, xi) BATF protein or a polynucleotide encoding the same, xii) PLAUR protein or a polynucleotide enco'ding the same, xiii) C/EBPδ protein or a polynucleotide encoding the same, xiv) ARHE (ras homolog gene family) protein or a polynucleotide encoding the same, xv) NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same, xvi) GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same, xvii) C/EBPβ protein or a polynucleotide encoding the same, xviii) TACSTD1 (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same, xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same, xx) SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same, xxi) ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same, xxii) UBE1C (ubiquitin-activating enzyme ElC) protein or a polynucleotide encoding the same, xxiii) FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same, xxiv) CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same, xxv) SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same, xxvi) GNPNAT1 (glutamine repeat protein 1) protein or a polynucleotide encoding the same, xxvii) BAP29 (B-cell receptor- associated protein 29) protein or a polynucleotide encoding the same, xxviii) PML (promyelocytic leukemia) protein or a polynucleotide encoding the same, xxix) MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same, xxx) FIG1 (interleukin-four induced gene 1) protem or a polynucleotide encoding the same, xxxi) MHC2TA (class II transactivator) protein or a polynucleotide encoding the same, xxxii) HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same, xxxiii) FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same, xxxiv) Id2 protein or a polynucleotide encoding the same, xxxv) CD74 protein or a polynucleotide encoding the same in a tissue .
In another embodiment, such method comprises determining the difference in the amount relative to a test sample of at least two, at least three, of each of (i) to (xxxv) as defined supra.
• 5 In one embodiment the difference in the amount relative to a test sample of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays.
In one embodiment the difference in the amount relative to a test sample of protein is determined, while in still other embodiment the difference in the amount relative to a test sample of mRNA and protem is determined. With respect to STAT4, when the difference in the amount relative to a
10 test sample of STAT4 is being determined, it is preferable that the amount of STAT4 protein be determined. In any such embodiment wherein a STAT protein amount is determined, the amount determined can be the total amount of the STAT protein present in a sample or, alternatively, can be the amount of phosphorylated STAT protein present in the sample. In certain embodiments, the difference in the amount relative to a test sample of mRNA is determined, and can, for example,
15 be determined via use of nucleic acid microarrays. In other embodiments, the difference in the amount relative to a test sample of protem is determined, while in still other embodiments, the difference in the amount relative to a test sample of mRNA and protein is determined.
In yet another aspect, the present invention relates to a method for identifying or evaluating reagents that modulate the activity of TLR9, said method comprises: (a) contacting cell expressing 0 TLR9 receptor or CpG recognizing receptor with a test compound; (b) determining the difference in the level relative to a test sample of at least one of the following: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF-κB protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL-12 protein or a polynucleotide encoding the same; (vi) STAT 4 protein
25 or a polynucleotide encoding the same; (vii) ϊgG2a protein or a germline transcript of the same, (viii) IgGl protein or a germlme transcript of the same and (ix) IgE protein or a germlme transcript of the same, x) TCF2, protein or a polynucleotide encoding the same, xi) BATF protein or a polynucleotide encoding the same, xii) PLAUR protem or a polynucleotide encoding the safhe,>,xiii) C/EBPδ protein or a polynucleotide encoding the same, xiv) ARHE (ras homolog gene
30 family) protem or a polynucleotide encoding the same, xv) NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same, xvi) GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide -encoding the same, xvii) C/EBPβ protein or a polynucleotide encoding the same, xviii) TACSTD1 (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same, xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same, xx) SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same, xxi) ESP 15 (epidermal growth factor receptor pathway -■ >. substrate 15) protein or a polynucleotide encoding the same, xxii) UBEl'C (ubiquitin-activating 5 enzyme E1C) protein or a polynucleotide encoding the same, xxiii) FNBP3 (formin binding protein 3) protein or a polynucldotide encoding the same, xxiv) CHUK (conserved helix-loop- helix ubiquitous kinase) protein or a polynucleotide encoding the same, xxv) SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same, xxvi) GNPNAT1 (glutamine repeat protem 1) protein or a polynucleotide encoding the same, xxvii) BAP29 (B-cell receptor-
10 associated protein 29) protein or a polynucleotide encoding the same, xxviii) PML (promyelocytic leukemia) protein or a polynucleotide encoding the same, xxix) MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein . or a polynucleotide encoding the same, xxx) FIG1 (interleukin-four induced gene 1) protein or a polynucleotide encoding the same, xxxi) MHC2TA (class II transactivator) protein or a polynucleotide encoding the same, xxxii) HCST
15 (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same, xxxiii) FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same, xxxiv) Id2 protein or a polynucleotide encoding the same, xxxv) CD74 protein or a polynucleotide encoding the same, present in (a); and (c) comparing the amount(s) in (a) to that/those present in a corresponding control cell expressing TLR9 receptor or CpG recognizing
20 receptor that has not been contacted with the test compound, so that if the amount of any one of (i) to (xxxv) is decreased or increased, relative to the amount in the control sample, a compound to be tested for an ability to modulate immune response is identified. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, at least four, at least five, or each of (i) to (xxxv) present in a cell expressing TLR9 receptor or CpG recognizing receptor
25 and comparing the amounts to those present in the control sample.
"Cell expressing TLR9 receptor or CpG recognizing receptor" includes, but not limited to, a B cell or a myeloid cell.
In certain embodiments, the difference in the amount relative to a test sample of mRNA is determined, in other embodiments, the difference in the amount relative to a test sample of protein, 30 or .protein modification is determined, while in still other embodiments, the difference in the amount relative to a test sample of mRNA and protein is determined. With respect to STAT4, when the amount of STAT4 is being determined, it is preferable that the . amount of STAT4 protem, or phosphorylation of STAT4 protein be determined. In any such embodiment wherein a STAT protein amount is determined, the amount determined can be the total amount of the STAT protein present in a sample or, alternatively, can be the amount of phosphorylated STAT protem present in the sample.
In a preferred embodiment of a method for identifying or evaluating reagents that modulate the activity of TLR9, said method comprises: (a) contacting cell samples expressing TLR9 or CpG- recognizing receptors with a test compound; (b) determining the amount of STAT4 mRNA or STAT4 protein or phosphorylated STAT4 present in the sample; and (c) comparing the amounts in (b) to those present in a corresponding control cell samples not bearing TLR9 or CpG-recognizing receptors that has not been contacted with the test compound, so that if the amount of STAT4 mRNA, or protem, or phospho-STAT4 is decreased or the amount of STAT4 mRNA, or protein, or phospho-STAT4 is increased relative to the amount in the control sample, a compound to be tested for an ability to modifying TLR9 or CpG recognizing receptors.
I The present invention further relates to a pharmaceutical composition. The composition comprises: (a) a reagent I that modulates the activity of a TLR9 polypeptide or polynucleotide; and b) a reagent II that modulates the activity of an IL-12 receptor polypeptide or polynucleotide, and c) a pharmaceutically acceptable carrier. Rreagent I can be a TLR 9 agonist or antagonist and the reagent II can be a IL-12 receptor agonist or antagonist. The composition can be used to treat the diseases such as an allergic disease when TLR9 agonist and IL-12 agonist are included in the composition. The composition can be used to treat an autoimmune disease when TLR9 antagonist and IL-12 antagonist are included in the composition.
The present invention further relates to a pharmaceutical composition. The composition comprisses: (a) a reagent I that modulates the activity of a TLR9 polypeptide or polynucleotide; and b) a reagent II that modulates the activity of a CD40 polypeptide or polynucleotide, and c) a pharmaceutically acceptable carrier. Rreagent I can be a TLR 9 agonist or antagonist and the reagent II can be a CD40 agonist or antagonist. The composition can be used to treat the diseases such as an allergic disease when TLR9 agonist and CD40 agonist are included in the composition. The composition can be used to treat an autoimmune disease when TLR9 antagonist and CD40 antagonist are included in the composition.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the induction of T-bet in mouse splenocytes. Spleen cells from 8-week old C57BL/6 mice were cultured at a density of 2.0xl06/ml in RPMIl 640/10%FCS with or without indicated stimuli. The concentration of IFN-γ, LPS, CpG and GpC are 50 ng/ml, 50 ng/ml, 3μM, and 3μM respectively. Cells were collected at 3 (A) or 6 (B) hours post stimulation and the mRNA levels of T-bet were measured by real time LightCycler PCR. Data are representative of three independent experiments (mean + SD).
Figure 2 shows Stimuli-induced STATlα/β phosphorylation in splenocytes. Spleen cells from 8-week old C57BL/6 mice were treated with indicated stimuli (using the concentrations shown in Figure 1) and cell lysates were made after incubation for 2, 5, and 18 hours. Phosphorylation of STAT1 was assessed by immunoblotting with antibodies against phospho-STATlα/β (pY701). Data are representative of three separate experiments.
Figure 3 shows T-bet transactivation and STAT1 phosphorylation in MyD88- or TLR9- deficient mice. Splenocytes from 129/C57BL/6/MyD88+ + and 129/C57BL/6/MyD88' ', 129/C57BL/6/TLR9+/" and 1129/C57BL/6/TLR9"'" mice were isolated and treated with indicated stimuli as described in Figure 1 and methods. Figure 3A shows T-bet induction with 3h (A), 6h
!
(B), 3h (C), and 6h (D) stimulation (ratio to the mRNA level in resting cells) and Figure 3E and 3F depict phosphorylation of Y701 in STATla/b. Anti-ERK2 antibodies were used as a control to detect total protein loading. Data are representative of 3 independent experiments (mean + SD) or a single experiment, representative of three.
Figure 4 shows the cells response to CpG in T-bet and IFN-γ expression. A and B, splenocytes were isolated from C57BL/6 mice and incubated in medium with or without CpG (3μM) for 6 hours. Thereafter, T cells were obtained by positive selection with anti-CD90 microbeads and B cells were purified by negative selection (B Cell Isolation Kit). "Non-T, non-B" represents the remaining cells after T and B cell depletion. The expression of T-bet (A) and IFN-γ (B) was assessed by LightCycler RT-PCR. Data are from one experiment, representative of three separate experiments. T-bet expression was measured in purified mouse splenic B cells treated with indicated stimuli for 6 hours (C) and in purified human peripheral B cells cultured in the presence or absence of ODN 2006 (CpG) or negative ODN 2006 (GpC) for 18 hours (D). 'Induction' indicates the ratio to non-stimulated cells. Data in (C) are from. one experiment, representative of four independent experiments.
Figure 5 shows the CpG-induced IFN-γ/STATl-independent T-bet expression in purified B ,_. cells. Splenic B cells (isolated by negative selection) and the remaining splenocytes (non-B) were treated with CpG for 6 hours, then the expression of T-bet was measured by LightCycler RT-PCR. Data are from single experiments, representative of three. (A). B cells were negatively purified from STAT1+ " and STATl"'" spleens and treated with indicated stimuli for 6 hours. Thereafter, T- bet mRNA was measured by LightCycler PCR (B). Data presented are the mean + SD of three independent experiments. Figure 6 shows the characterization of signal cascades involved in CpG-induced T-bet transactivation in purified B cells. Effect of cycloheximide (cycloheximide 3 μg/ml,) and inhibitors of kinase orNF-=κB (dexamethasone lOOnM, SB203580 10 μM, and wortmarmin 10 nM) on 'CpG-induced T-bet expression (B). Production of IL-12 from cells stimulated with LPS or CpG for 6 hours (C). Effects of neutralizing antibodies (D) and IL-12 deficiency (E) on CpG-induced T-bet expression. T-bet expression induced by the indicated stimuli (F). 'Induction' indicates ratio to non-stimulated cells. For (B, C, D, E), n=3; for (A) data is from a single experiment, representative of three (A).
Figure 7 shows the inhibition of IL-4 and CD40-ligation-induced IgGl, IgG2a and IgE production. B cells were isolated from splenocytes by negative selection and treated as described in Materials and Methods. (A) Ig production was determined by ELISA and cell purity was checked by FACS with PE-labeled anti-B220 antibody. Data represent four animals in each group. (B) Germline transcripts were measured by RT-PCR with primer sets corresponding to the sequences of the Iγl-Cγl, Iγ2a-Cγ2a and Iε-Cε exon-hinge region, respectively. The housekeeping gene GAPDH was used as a control. Shown is a single experiment, representative of three. (C) Samples from (B) were assessed for T-bet expression. (D) Effect of CpG treatment on IL-4- induced STAT6 phosphorylation. B cells were pretreated with CpG for 20 hours, and then stimulated with IL-4 for 20 or 60 min. Activation of STAT6 was assessed by western blotting with anti- phospho-STAT6. Anti-ERK2 antibodies were used as a control to detect total protein loading.
Figure 8 shows the expression of Id2 in resting B cells and B cells stimulated with CpG, IL-4, Poly( C) and LPS.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based on the discovery that CpG is engaged in the transcriptional regulation of transcription factors T-bet, TCF2, C/EBPβ, C/EBPδ, BATF, LM04, Id2, and MHC2TA. Furthermore, T-bet TCF2, C/EBPβ, C/EBPδ, BATF, LM04, Id2, and C2TA mRNA is specifically induced after binding of CpG to TLR9. LPS, the ligand of TLR4 is incapable of inducing T-bet expression. Namely, the inventor discovered that CpG can induce the expression of T-bet, TCF2, C/EBPβ, C/EBPδ, BATF, LM04, Id2, and MHC2TA which results in the inhibition of IL-4/CD- 40 'induced IgGl and IgE class switching by direct acting on B-cells.
The present invention is also based on the, discovery of the signalling pathway and the molecules therin that mediates the CpG-induced upregulation of T-bet mRNA. As a result of this finding the invention provides methods for identifying and evaluating reagents that modulate the activity of TLR9 using at least one of the following as markers: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF-κB protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL- ■ *,<. 12 protein or a polynucleotide encoding the same; (vi) STAT 4 protein or a polynucleotide 5 encoding the same; (vii) IgG2a protein or a germlme transcript of the same, (viii) IgGl protem or a germline transcript of the same and (ix) IgE protein or a germline transcript of the same, x) TCF2, protein or a polynucleotide encoding the same, xi) BATF protein or a polynucleotide encoding the same, xii) PLAUR protein or a polynucleotide encoding the same, xiii) C/EBPδ protem or a polynucleotide encoding the same, xiv) ARHE (ras homolog gene family) protem or a
10 polynucleotide encoding the same, xv) N0P5/N0P58 (nucleolar protein 5) protein or a polynucleotide encoding the same, xvi) GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same, xvii) C/EBPβ protein or a polynucleotide encoding the same, xviii) TACSTD1 (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same, xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same, xx)
15 SCS -(GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same, xxi) ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same, xxii) UBEIC (ubiquitin-activating enzyme E1C) protein or a polynucleotide encoding the same, xxiii) FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same, xxiv) CHUK (conserved helix-loop-helix ubiquitous kinase)
20 protem or a polynucleotide encoding the same, xxv) SELL (Selectin lymphocyte) protem or a polynucleotide encoding the same, xxvi) GNPNAT1 (glutamine repeat protein 1) protein or a polynucleotide encoding the same, xxvii) BAP29 (B-cell receptor-associated protein 29) protem or a polynucleotide encoding the same, xxviii) PML (promyelocytic leukemia) protem or a polynucleotide encoding the same, xxix) MMKROX2R (mouse mRNA for KROX-20 protein
25 containing zinc fingers) protein or a polynucleotide encoding the same, xxx) FIG1 (interleukin- four induced gene 1) protein or a polynucleotide encoding the same, xxxi) MHC2TA (class II transactivator) protein or a polynucleotide encoding the same,'xxxii) HCST (hematopoietic cell signal signal transducer) protein or a polynucleotide encoding the same, xxxiii) FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protem or a polynucleotide encoding the same,
30 xxxiv) Id2 protein or a polynucleotide encoding the same, xxxv) CD74 protein or a polynucleotide encoding the same.
Inhibition or activation of TLR9 activity can be detected by determining the amount of mRNA or protein of at least one of the following members of signal transduction pathway present within a tissue; T-bet, IKKs, NF-κB, p 8, IL-12, STAT4, Igs, and gene x - xxxv. Specifically, the
35 inhibition or activation of TLR9 activity can be detected by determining the amount, expression, or activity of at least one of the following: (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protein or polynucleotides encoding the same; (iii) NF-κB protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL- lr<, 12 "protein or a polynucleotide encoding the same; (vi) STAT 4 protein or a polynucleotide
5 encoding the same; (vii) IgG2a protein or a germline transcript of the same, (viii) IgGl protein or a germline transcript of the same and (ix) IgE protein or a germline transcript of the same, x)
TCF2, protein or a polynucleotide encoding the same, xi) BATF protein or a polynucleotide encoding the same, xii) PLAUR protein or a polynucleotide encoding the same, xiii) C/EBPδ protein or a polynucleotide encoding the same, xiv) ARHE (ras homolog gene family) protein or a
10 polynucleotide encoding the same, xv) NOP5/NOP58 (nucleolar protein 5) protein or a polynupleotide encoding the same, xvi) GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same, xvii) C/EBPβ protein or a polynucleotide encoding the same, r xviii) TACSTD1 (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same, xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same, xx)
15 SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same, xxi) ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same, xxii) UBEIC (ubiquitin-activating enzyme E1C) protein or a polynucleotide encoding the same, xxiii) FNBP3 (formin binding protein 3) protem or a polynucleotide encoding the same, xxiv) CHUK (conserved helix-loop-helix ubiquitous kinase)
20 protein or a polynucleotide encoding the same, xxv) SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same, xxvi) GNPNAT1 (glutamine repeat protein 1) protein or a polynucleotide encoding the same, xxvii) BAP29 (B-cell receptor-associated protem 29) protein or a polynucleotide encoding the same, xxviii) PML (promyelocytic leukemia) protein or a polynucleotide encoding the same, xxix) MMKROX2R (mouse mRNA for KROX-20 protein
25 containing zinc fingers) protein or a polynucleotide encoding the same, xxx) FIG1 (interleukin- four induced gene 1) protein or a polynucleotide encoding the same, xxxi) MHC2TA (class II transactivator) protein or a polynucleotide encoding the same,' xxxii) HCST (hematopoietic cell signal signal transducer) protein or a polynucleotide encoding the same, xxxiii) FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protem or a polynucleotide encoding the same,
30 xxxiv) Id2 protein or a polynucleotide encoding the same, xxxv) CD74 protein or a polynucleotide i- . encoding the same.
Standard techniques can routinely be utilized to determine these amounts. In general, such methods of the invention can routinely be performed using standard techniques for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample. This
35 involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or mRNA such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample. When comparing levels, such comparisons can be either quantitative or qualitative.
Thus, in qualitative instances, for example, in instances wherein a control sample is determined to. contain none of a given molecule (e.g., T-bet mRNA or protem, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, or IgGl or IgE) and the molecule is determined to be present in the test- sample, the amount of the molecule in the test sample is greater than that present in the control sample. In quantitative instances wherein both the control and test samples are determined to contain a given molecule, using standard techniques, the amount in the test sample can routinely be determined to be greater than, equal to, or less than that of the control sample. In general, the amount of a given molecule in test and qontrol samples will differ by at least 2-fold, and in certain instances, 2.5-fold, at least 3- fold, at'least 4-fold, at least 5-fold, or at least 10-fold.
T-bet, STAT4 nucleic acid and amino acid sequences are well known to those of skill in the art.
Representative examples of GenBank accession number for human T-bet mRNA or protein, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, IgGl, IgE and gene x- xxxv are listed in the Table 1, 2 and 3.
Table 2 and 3 show the name of genes, accession number, functional description, and fold of changes induced by CpG and other stimuli. Gene expression was determined by DNA microarray analysis
Table 1
Figure imgf000015_0001
Table 2
Figure imgf000016_0002
Figure imgf000016_0001
Table 2 continued
Figure imgf000017_0002
Figure imgf000017_0001
Table 2 continued
Figure imgf000018_0002
Figure imgf000018_0001
Table 3
Figure imgf000019_0002
Figure imgf000019_0001
Table 3 continued
Figure imgf000020_0002
Figure imgf000020_0001
Table 2 and 3 show the accession number and functional description of genes that are up- or . down-regulated by CpG, as well as their expression in response to IL-4, INFγ, IL-12 or CpG plus , IL-12. .
Further, additional forms, e.g., alleles or species homologues of such sequences can routinely be obtained and detected using the sequences described above in conjunction with standard cloning and hybridization techniques such as those find in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd. ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
Several methods known to the skilled artisan can be utilized for determining the difference in the amount 'relative to a test sample of T-bet mRNA or protein, STAT4 mRNA or protem phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, IgGl IgE and gene x - xxxv present within a sample. The skilled person is aware of methods that are capable of being used to determine the difference in the amount relative to a test sample of any one, two, three, four, five of T-bet mRNA or protem, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, IgGl, IgE (germline transcript or protein) and gene x - xxxv.
One method, for example, comprises using a microarray for determining such difference in the amount relative to a test sample, wherein the microarray comprises one or more nucleic acid sequences immobilized onto a solid surface, said nucleic acid sequence or sequences exhibiting complementarity to one or more selected from the group consisting of: mRNA of (i) T-bet; (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; or (vi) STAT 4; or germlme transcript of (vii) IgG2a, (viii) IgGl or (ix) IgE, gene x - xxxv. The kit can, in addition, comprise a labeled compound or agent capable of detecting one or more of (i) T-bet protein or a polynucleotide encoding the same; (ii) IKKs protem or polynucleotides encoding the same; (iii) NF-κB protein or a polynucleotide encoding the same; (iv) p38 protein or a polynucleotide encoding the same; (v) IL-12 protem or a polynucleotide encoding the same; (vi) STAT 4 protem or a polynucleotide encoding the same; (vii) IgG2a protein or a germline transcript of the same, (viii) IgGl protem or a germline transcript of the same, (ix) IgE protein or a germline transcript of the same, gene x - xxxv in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide).
For antibody-based methods, the method can comprise, for example: using (1) a first antibody (e.g., attached to a solid support) which binds to any one of IKKs, NF-κB, p38/phospho-p38, IL- 12, STAT4/phospho-STAT, T-bet, proteins (x) to (xxxv) and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a , detectable agent.
For oligonucleotide-based methods, the method can comprise, for example: using (1) an oligonucleotide, e.g., an oligonucleotide labeled for detection, which hybridizes to mRNA of one selected from the group consisting of: T-bet, IKKs, NF-κB, ρ38, IL-12, STAT4, IgG2a, IgGl, IgE and gene x - xxxv nucleic acid sequence; or (2) a pair of primers useful for amplifying T-bet,
IKKs, NF-κB, p38, IL-12, STAT4, IgG2a, IgGl, IgE and gene x-xxxv nucleic acid molecule. The method can also comprise, e.g., using a buffering agent, a preservative, or a protein-stabilizing agent. The method can also comprise using components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). t For microarray-based methods, such methods can comprise using a nucleotide sequence, e.g., an oligonucleotide sequence, immobilized onto the surface of a solid support (e.g., a glass or porous solid support).
Nucleic acid detection
In certain embodiments, determination of the difference in the amount relative to a test sample of mRNA of T-bet, IKKs, NF-κB, p38, IL-12, or STAT4, or germline transcript of IgG2a, IgGl or IgE, or gene x - xxxv, 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, for example, anchor PCR, RACE PCR or RT-PCR. Such methods can include the steps of collecting a cell sample, isolating mRNA from the cells of the sample, reverse transcribing the mRNA, contacting the sample with one or more primers which specifically hybridize to the selected sequence under conditions such that hybridization and amplification of the sequence (if present) occurs, and determining the amount of product that is present.
Alternative amplification methods can also routinely be utilized. Such methods can include, for example, self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA .86: 1173-1177), Q-Beta Replicase'(Lizardi et al., 1988, Bio/Technology: 1197), or any other nucleic acid amplification method, followed by the detection/quantitation of the amplified molecules using techniques well known to those of skill in the art. These schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (preferably at least
. 75%, more preferably at least" 85%, most preferably at least 951/o) identical to each other typically
' *■'■ remain hybridized to each other. Such stringent conditions are known to those skilled in the art and
5 can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-
6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in
6X sodium chloride/sodium citrate (SSQ at about 45'C, followed by one or more washes in 0.2 X
SSC, 0.1% SDS at 50-65'C (preferably 65'C).
Probes can comprise any readily detectable label moiety. For example, probes utilized herein 0 comprise a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor as a label moiety., I In alternate embodiments, mRNA sequence of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12;
(vi) STAT 4; or germline transcript of (vii) IgG2a, (viii) IgGl, (ix) IgE, gene x-xxxv can be detected "in situ" directly upon the sample, e.g., the biopsy sample. Techniques for such 5 procedures are well known to those of skill in the art. See, e.g., Nuovo, G.J., 1992, "PCR In Situ
Hybridization: Protocols and Applications," Raven Press, NY.
In other embodiments, the amount of mRNA of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL- 12; (vi) STAT 4; or germline transcript of (vii) IgG2a, (viii) IgGl, (ix) IgE, gene x-xxxv can be determined by hybridizing nucleic acid arrays, e.g., microarrays. In a specific embodiment of the 0 invention, the expression of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, (ix) IgE, gene x-xxxv, is measured or detected using a DNA microarray. A DNA microarray or chip is a microscopic array of DNA fragments or synthetic oligonucleotides, disposed in a defined pattern on a solid support, wherein they are amenable to analysis by standard hybridization methods (see, e.g., Schena, 1996, BioEssays 18: 427).
5 Microarrays share certain preferred characteristics: The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably the microarrays are small, usually smaller than 5 cm, and they are made from materials that are stable ι- under binding (e.g., nucleic acid hybridization) conditions.
Microarrays contain a surface to which sequences corresponding to gene products (e.g., mRNA, 0 cDNA, cRNA, or complements thereof), can be specifically hybridized or bound at a known position. For practicing the methods of the present invention, the binding sites of the microarray are polynucleotides, preferably DNA polynucleotides, that specifically hybridize to at least a portion of mRNA or cDNA of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or germline transcript of (vii) IgG2a, (viii) IgGl, (ix) IgE, gene x-xxxv, or any combination of such
, mRNA or cDNA molecules, -produced by a subject mammal. That is, a given binding site or
*' *' •'■ unique1' set of binding sites in the microarray will specifically bind the product (e.g., mRNA or
5 cDNA) of a single gene of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or germlme transcript of (vii) IgG2a, (Viii) IgGl, (ix) IgE and gene x-xxxv.
Preferably, the nucleotide sequence of each of the different polynucleotide bound to the surface is in the range of about 15 to about 100 nucleotides in length. Polynucleotides can be synthesized using conventional methods, such as phosphoramidite- based synthesis methods. Alternatively, the 0 binding site polynucleotide sequences can be derived from cDNA or genomic clones.
DNA microarrays can be probed using mRNA, extracted and, optionally, reverse transcribed and amplified from a sample.
Nucleic acid hybridization and wash conditions are optimally chosen so that the probe "specifically binds" or "specifically hybridizes" to a specific array site, i.e., the probe hybridizes, 5 duplexes or binds to a sequence array site- ith a complementary nucleic acid sequence but does not hybridize to a site with a non-complementary nucleic acid sequence. As used herein, one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base- pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than 0 a 5% mismatch. Preferably, the polynucleotides are perfectly complementary (no mismatches). It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls (see, e.g., Shalon et al., 1996, Genome Research 6:639645, and Chee et al., 1996, Science 274:610-614) or positive controls. Thus, in a preferred embodiment, a microarray of the invention farther comprises a binding site 5 designed to act as a negative control and/or a binding site designed to act as a positive control. For example, a positive control can relate to a constitutively expressed gene sequence, e.g., a ubiquitin sequence, HSC70, or GADPH. A negative control can relate to a gene sequence not expressed in the test cell or tissue being assayed. *- .
Exemplary, non-limiting examples of hybridization conditions that can be utilized with ,DNA 0 microarrays are as follows: hybridization in 5 X SSC plus 0.2% SDS at 65°C for 4 hours followed by washes at 25°C in low stringency wash buffer (I X SSC plus 0.2% SDS) followed by 10 minutes at 25°C in high stringency wash buffer (0.1 X SSC plus 0.2% SDS) (Shena et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-19). The use of a two-color fluorescence labeling and detection scheme to define alterations in gene expression has been described, e.g., in Shena et al., 1995, Science 270:467-470. An advantage of
. using mRNA, cRNA, or cDNA labeled with two different fluorophores is that a direct and
' *■' internally controlled comparison of the mRNA levels corresponding to each arrayed gene in two
5 cell states (e.g., control and activated) can be made, and variations due to minor differences in experimental conditions (e.g., hybridization conditions) will not affect subsequent analyses.
However, it will be recognized that it is also possible to use cDNA from a single cell, and compare, for example, the absolute amount of a particular mRNA in sample cell.
To facilitate detection the mRNA or cDNA are typically labeled with fluorescent dyes that emit at 0 different wavelengths. Examples of fluorescent dyes include, but are not limited to, rhodamine, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, a- phthaldehyde and fluorescamine. The fluorescence emissions at each site of a DNA array can be, preferably, detected by scanning confocal laser microscopy. In one embodiment, a separate scan, using, the appropriate excitation line, is carried out for each of two fluorophores used. 15 Alternatively, a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see, e.g., Shalon et al., 1996, Genome Research 6:639-645).
Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12 bit analog to digital board. In one embodiment the scanned image is despeckled using a graphics 0 program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site.
It will be appreciated that when mRNA or cRNA is hybridized to a microarray under suitable hybridization conditions, the level of hybridization to the site in the array corresponding to any particular gene will reflect the prevalence in the cell of mRNA transcribed from that gene. For 5 example, when detectable labeled (e.g., with a fluorophore) cRNA complementary to the total cellular mRNA is hybridized to a microarray, the site on the array corresponding to a gene (i.e., capable of specifically binding the product of the gene) that is not transcribed in the cell will have little or no signal (e.g., fluorescent signal), and a gene for which the encoded mRNA is prevalent will have a relatively strong signal.
30 Microarrays can be made in a number of ways well known to those of skill in the art.
With respect to the nucleic acids of the binding sites, the nucleic acid for the microarray can be generated by synthesis of synthetic.polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (e.g., Froehler et al., 1986, Nucleic Acid Res 14:5399-5407). In some embodiments, synthetic nucleic acids include non-natural bases, e.g., inosine. Additionally,
, it is possible to vary the charge on the phosphate backbone of the oligonucleotide, for example, by
'' '*■'■ thiolation or methylation, or even to use a peptide rather than a phosphate backbone. The making 5 of such modifications is within the skill of one trained in the art. Further, nucleic acid analogues may be used as binding sites for' hybridization. An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Eghohn et al, 1993, Nature 365:566-568; see also U.S. Patent No. 5,539, 083, Cook et al., entitled "Peptide nucleic acid combinatorial libraries and improved methods of synthesis," issued July 23, 1996). In addition, binding (hybridization) sites can also be
10 made from plasmid or phage clones of genes, cDNAs (e.g., expressed sequence tags), or inserts therefrom (Nguyen et al., 1995, Genomics 29:207-209). In yet another embodiment, the polynucleotide of the binding sites is RNA.
!
The nucleic acid or analogue is attached to a solid support to produce the binding site. Solid supports may be made from glass, silicon, plastic (e.g., polypropylene, nylon, polyester), 15 polyacrylamide, nitrocellulose, cellulose acetate or other materials. In general, non-porous supports, and glass in particular, are preferred. The solid support may also be treated in such a way as to enhance binding of oligonucleotides thereto, or to reduce nonspecific binding of unwanted substances thereto. Preferably, the glass support is treated with pplylysine or silane to facilitate attachment of oligonucleotides to the slide.
20. Methods of immobilizing DNA on the solid support may include direct touch, micropipetting (Yershov et al, Proc. Natl. Acad. Sci. USA, 1996, 93:4913-4918), or the use of controlled electric fields to direct a given oligonucleotide to a specific spot in the array (U.S. Patent No. 5,605, 662). In principal, any type of array, for example, dot blots on a nylon hybridization membrane (see Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold
25 Spring Harbor Laboratory, Cold Spring Harbor, New York), can used, although, as will be recognized by those of skill in the art, very small arrays are be preferred because hybridization volumes will be smaller. DNA can typically be immobilized at a density of 50, 75, 100, up to 10,000 oligonucleotides per cm2 and preferably at a density of about 1000 oligonucleotides per cm2.
30 In addition, nucleic acids can be attached to a surface by printing on glass plates (Schena et al., 1995, Science 270:467-470; DeRisi et al., 1996, Nature Genetics 14:457460; Shalon et al., 1996, Genome Res. 6:639- 645; and Schena et al., Proc. Natl. Acad! Sci. USA, 1996, 93(20):10614-19. ) As an alternative to immobilizing pre-fabricated oligonucleotides onto a solid support, it is possible to synthesize oligonucleotides directly on the support (Maskos et al., 1993, Nucl. Acids Res. 21: 2269-70; Fodor et al., 1991, Science 251: 767-73; Lipshutz et al., 1999, Nat. Genet., 21 (1 Suppl):20-4; McGall et al., Proc. Natl. Acad. Sci. USA 93: 13555-60, 1996). Other methods for . making microarrays, e.g., by masking (l\4askos and Southern, 1992, Nue. Acids Res. 20:1679- ' * ' •'■ 1684)', may also be used.
5 Protein detection
Standard techniques can also be utilized for determining the amount of the protein or proteins of interest (e.g., T-bet, the following signal molecules which can lead to the activation of KB binding site and/or STAT4-binding site containing promoters and/or T-bet promoter; IKKs, NF-κB, p38, IL-12, and STAT4, or IgG2a, IgGl or IgE protein, or gene x-xxxv protein) present in a sample. It 10 is to be' understood, that such a determination of the amount of a protein present includes deterrηining the total amount of a protem present, and also includes, especially with respect to determining the amount of a STAT protein present, determining the amount of a phosphorylated form of the protein present.
For example, standard techniques can be employed using, e.g., immunoassays such as, for 15 example, Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunocytochemistry, and the like to determine the amount of the protein or proteins of interest present in a sample. A preferred agent for detecting a protein of interest is an antibody capable of binding to a protein of interest, preferably an antibody with a detectable label.
20 With respect to determining the amount of a phosphorylated form of a protein of interest that is present in a sample, such a determination can also be performed using standard techniques well known to those of skill in the art. For example, such a determination can include, first, immunoprecipitation with an antibody that is specific for a phosphorylated amino acid residue, e.g., an anti-phosphotyrosine antibody, such that all exhibiting such a phosphorylated residue in a
25 sample will be immunoprecipitated. Second, the immunoprecipitated proteins can be contacted with a second antibody that is specific for the following protein of interest, e.g., (i) T-bet (ii) IKKs;
(iii) NF- B; (iv) p38; (v) IL-12; (vi) STAT 4; (vii) IgG2a, (viii) IgGl, (ix) IgE, or (x)-(xxxv). - ■ Alternatively, a phosphorylated protein of interest can be identified and quantitated using an antibody specific for the phosphorylated form of the particular protem itself, e.g., an antibody
30 specific for phosphorylated "STAT4 that does not recognize non- phosphorylated STAT4. Such antibodies exist, and are well known to those of skill in the art.
For such detection methods, protein from the sample to be analyzed can easily be isolated using techniques that are well known to those of skill in the art. Protem isolation methods can, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, . "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, '' ■*■'■ New York).
5 Preferred methods for the detection of the protein or proteins of interest involve their detection via interaction with a protein-specific antibody. For example, antibodies directed a protein of interest can be utilized as described herein. Antibodies directed against (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, (ix) IgE, or (x)-(xxxv) are well known to those of skill in the art. For example, antibodies directed against T-bet, STAT4 can be
10 obtained from such companies as Zymed Laboratories, Inc. (South San Francisco, CA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and Research Diagnostics, Inc., (Flanders, NJ). Altern&tively, such antibodies can be generated utilizing standard techniques well known to those of skill in the art. See, e.g., Section 5. 3, below, for a more detailed discussion of such antibody generation techniques. Briefly,- such antibodies can be polyclonal, or more preferably, monoclonal.
15 An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can, for example, be used.
For example, antibodies, or fragments of antibodies, specific for a protein of interest can be used to quantitatively or qualitatively detect the presence of the protein. This can be accomplished, for example, by immunofluorescence techniques. Antibodies (or fragments thereof) can, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ
20 detection of a protein of interest. In situ detection can be accomplished by removing a histological specimen (e.g., a biopsy specimen) from a patient, and applying thereto a labeled antibody thereto that is directed to a T-bet, STAT4 protein. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the protein of interest, but also its
25 distribution, its presence in lymphocytes within the sample. A wide variety of well-known histological methods (such as staining procedures) can be utilized in order to achieve such in situ detection.
Immunoassays for a protein of interest typically comprise incubating a biological sample, e.g., a biopsy or subject blood sample, of a detectable labeled antibody capable of identifying a protein of 30 interest, and detecting the bound antibody by any of a number of techniques well- known in the art. As discussed in more detail, below, the term "labeled" can refer to direct labeling of the antibody via, e.g., coupling (i.e., physically linking) a detectable substance to the antibody, and can also refer to indirect labeling of the antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescence labeled secondary antibody.
The biological sample can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support can then be washed with suitable buffers followed by treatment with the detectable-labeled fingerprint gene-specific antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on solid support can then be detected by conventional means.
By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, poly- ethylenej dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface can be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
One of the ways in which (i) T-bet (ii) IKKs; (iii) NF- B; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or (x)-(xxxv) specific antibody can be detectable labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosόrbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J.E., 1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, ENZYME EVIMUNOASSAY, CRC Press, Boca Raton, FL; Ishikawa, E. et al., (eds.), 1981, ENZYME EVIMLTNOASSAY, Kgaku Shoin, Tokyo). The enzyme that is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.- Enzymes which can be used to detectable label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta- 5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerolphosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6ρhosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection can also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect a protem of interest through the use of a radioimmunoassay (R1A) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine . Society, March, 1986, which is incorporated by reference herein). The radioactive isotope (e.g., 1251, 1311, 35S or 3H) can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
It is also possible to label the antibody with a fluorescent compound. When the fluorescence
I labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, 2- phthaldehyde and fluorescarnine.
The antibody can also be detectable-labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid • (EDTA).
The antibody also can be detectable labeled by coupling it to a chemilummescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
Examples of particularly useful chemilummescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound can be used to label the antibody of the present invention.
Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic r-- protein increases the . efficiency of the chemilummescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Standard techniques can be utilized to determine the level of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl; or (ix) IgE; or (x)-(xxxv) activity. For example, the activity of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) ρ38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl or (ix) IgE can be determined by detecting the binding of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE , or (x)-(xxxv) to its cognate DNA binding element, via, for example, an electromobility shift assay ("EMSX"), detecting the expression of a gene whose expression is controlled by a promoter that is responsive to (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or x-xxxv detecting the induction of a reporter gene that comprises a regulatory element that is responsive to (i) T-bet (ii) IKKs;. (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or x-xxxv, wherein the element is operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase.
Genes whose expression is controlled by a T-bet-responsive promoter include, for example IFN-γ, IL-12RJ3, et al, (see, Szabo SJ, et al, Cell 100(6), 655-69 (2000), and Afkarian M, et al., Nat Immunol. 3(6), 549-57 (2002)). Thus, expression of such genes in the TLR9 expressing cell sample in the presence and absence of a test compound can routinely be determined using standard techniques, such as reporter assays. EMSAs can also routinely be utilized to assess T-bet activity. Such techniques are well known to those of skill in the art. See, e.g., A ici et al., 1995, Cancer Research 55: 14452-4457.
The activity of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or (x)-(xxxv) can also be assessed by detecting the production of cytokines, surface markers, Ig, in TLR9 expressing cells such as B cells and' DC. Techniques known to those of skill in the art can be used for measuring these activities. For example, cytokine production can be assayed by ELISA. The effector function of T cells can be measured, for example, by a FACS analysis of surface markers.
As set forth above, the methods described herein for identifying reagents that modulate the activity of TLR9 comprise as a readout assaying whether a test compound has an effect on the expression and/or the activity of T-bet mRNA or protein, STAT4 mRNA or protein,IgGl, IgG2a, IgE, (x)~ (xxxv) produced by TLR9 expressing cells, or produced by a cell that has the ability to respond to CpG.
Upon identification of compounds to be tested for an ability to modify TLR9 activity, the compounds can be further investigated. In particular, for example, the compounds identified via the present methods can be further tested in vivo in accepted animal models of inflammatory disorders. Further, the compounds identified can also be analyzed with respect to their specificity. Reagents that modulate the activity of TLR9 identified via the present methods can be used in the manufacture of medicaments for the treatment of cancer, inflammatory diseases such as asthma
' and chronic obstructive pulmonary disease (COPD), immunological disorders and conditions where TLR9 signal transduction is aberrant.
5. Screening Methods
The invention provides assays for screening test compounds that bind to or modulate the activity of TLR9 or CpG recognizing receptor by determining the level relative to a test sample of T-bet mRNA or protein, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, IL-12 mRNA or protein, IgG2a, or IgGl or IgE germline transcripts or protein, 0 gene x-xxxv mRNA or proteins in a tissue. A test compound preferably binds to a TLR9 or CpG recognising receptor polypeptide or polynucleotide. More preferably, a test compound decreases or increases functional activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
Test Compounds
5 Test compounds can be pharmacological agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library 0 methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small 5 molecule libraries of compounds. See Lam, Anticancer Drug Des. 72, 145, 1997.
Methods for the synthesis of molecular libraries are well known in the art (see, for example, ._ DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S. A., 91, 11422, 1994; Zuckermann et α/., J Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., 0 Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 35^, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; ' Devlin, Science 249, 404-406," 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).
• High Throughput Screening
Test compounds can be screened for the ability to affect TLR9 or CpG activity by determining the level relative to a test sample of e.g., T-bet mRNA or protem or T-bet promoter activity measured by reporter assay, STAT4 mRNA or protein phosphorylation or STAT4 promoter activity measured by reporter assay, p38 mRNA or protein phosphorylation or enzyme activity, IL-12 or any one of (x) to (xxxv) mRNA or protein, IgG2a, or IgGl or IgE germline transcripts or protein in or secreted from a tissue using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format."
Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released' the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
Another example of a free format assay is described by Chelsky, "Strategies for Screening
Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual
Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995).
*"* Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying . combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change. Another high throughput screening method is described in Beutel et al, U.S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then". • placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse -sufficiently slowly, such that the assays can be performed without the test samples running together.
Functional Assays
Test compounds can be tested for the ability to increase or decrease a biological effect of TLR9 by determining a level relative to a test sample of T-bet mRNA or protein or promoter activity, STAT4 mRNA or protein phosphorylation, p38 mRNA or protein phosphorylation, activation of IKK and NF-κB, IL-12 or any one of (x)-(xxxv) mRNA'Or protem, IgG2a, or IgGl or IgE germline transcripts or protein in a tissue. Such biological effects can be determined for example using functional assays such as those described below. Functional assays can be carried out after contacting a cell membrane preparation, or an intact cell with a test compound. A test compound which increases or decreases a functional activity of TLR9 polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent.
Polypeptides comprising amino acid sequences encoded by open reading frames of TLR9 are either expressed endogenously in appropriate reporter cells or are introduced recombinantly. Signal molecule or Igs activity can be monitored by measuring a cellular response (e.g., expression of a reporter gene or secretion of a neurotransmitter) triggered or modulated by the polypeptide's activity.
More specifically, the activity of TLR9 modulation can be determined by, for example, (i) a reporter assay with cells expressing TLR9 endogenously or ectopically and reporter gene containing T-bet promoter element; (ii) a reporter assay with cells expressing TLR9 endogenously or ectopically and reporter gene containing STAT 4 binding elements and/or NF-κB binding sequences; (iii) a reporter assay with cells expressing TLR9 endogenously or ectopically and reporter gene containing T-box element or responsive to the T-bet protein, such as an IFN-gamma promoter; (iv) measurement of IL-12 protein amount by ELISA (v) comparing the degree of STAT 4 phosphorylation; (vi) determining the expression of IgG2a germline transcripts or the production of IgG2a protein in primary B cells or B cell lines; and the like, (vii) determining the inhibition of IL-4 alone or IL-4/CD40 ligatibn induced IgGl and/or IgE class switching or production; (viii) determining the expression of transcripts or proteins of one of gene x-xxxv in B cells. Gene Expression
In another embodiment, test compounds that increase or decrease a particular signal molecule gene
'- >. expression are identified. A cell expressing TLR9 receptor is contacted with a test compound, and the expression of an RNA or polypeptide product of particular signal molecule polynucleotide is
5 . determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator 0 - or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
The level of mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods 5 can be used. The presence of polypeptide products of a polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
Such screening can be carried out, but not limited to, in an intact cell. Any cell that expresses TLR9 receptor polynucleotide can be used in a cell-based assay system. The TLR9 polynucleotide 0 can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
Pharmaceutical Compositions
The invention also provides pharmaceutical compositions which can be administered to a patient 5 to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise a reagent which alters . The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination 0 with other agents, drugs or hormones.
In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical; sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patierit.
Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixtur of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, marrnitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, • or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 niM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co, Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated, condition. Such labeling would include amount, frequency, and method of administration.
Diagnostic Methods
The Human (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or (x)-(xxxv) and polynucleotides encoding them can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding one of the signal molecules selected from the group consisting of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, (ix) IgE and (x)-(xxxv) in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in hormal individuals, then the mutation is likely to be the causative agent of the disease. Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as ■ probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radio-labeled nucleotides or by automatic sequencing procedures using fluorescent tags.
Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers- et al. Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85, 4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such- as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
Altered levels of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE or any one of (x)-(xxxv) also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
Therapeutic Indications and Methods
The activity of (i) T-bet (ii) IKKs; (iii) NF-κB; (iv) p38; (v) IL-12; (vi) STAT 4; or (vii) IgG2a, (viii) IgGl, or (ix) IgE, or (x)-(xxxv), can be modulated to treat allergic or inflammatory diseases such as asthma and COPD.
Allergy is a complex process in which environmental antigens induce clinically adverse reactions. Asthma can be understood as an basically allergic disease of the lung and its tissues. The asthma inducing antigens, called allergens, typically elicit a specific IgE response and, although in most cases the allergens themselves have little or no intrinsic toxicity, they induce pathology when the IgE response in turn elicits an IgE-dependent or T cell-dependent hypersensitivity reaction. • Hypersensitivity reactions can be local or systemic and typically occur within minutes after allergen exposure in individuals who have previously been sensitized to the respective allergen. The hypersensitivity reaction of allergy develops when the allergen is recognized by IgE antibodies bound to specific receptors on the surface of effector cells, such as mast cells, basophils, or eosinophils, which causes the activation of the effector cells and the release of mediators that produce the acute signs and symptoms of the reactions. Allergic diseases include asthma, allergic rhinitis (hay fever), atopic dermatitis, and anaphylaxis.
Asthma is thought to arise as a result of interactions between multiple genetic and environmental factors -and is characterized by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a narrowing of the airways, 2) airway hyperresponsiveness, and 3) airway inflammation. Certain cells are critical to the inflammatory reaction of asthma and they include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE.- These effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to tissue destruction related to the disorder. Other resident cells, such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to its pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually makes asthma a chronic and disabling disorder requiring long-term management.
Chronic obstructive pulmonary (or airways) disease (COPD) is a condition defined physiologically as airflow obstruction that generally results from a mixture of emphysema and peripheral airway obstruction due to chronic bronchitis. Emphysema is characterized by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung. Chronic bronchitis is defined ι- clinically as the presence of chronic productive cough for three months in each of two successive years'.1' In COPD, airflow obstruction is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does also occur in non-smokers.
Chronic inflammation of the airways is a key pathological feature of COPD. The inflammatory cell population comprises increased numbers of macrophages, neutrophils and CD 8+ lymphocytes. Inhaled irritants such as cigarette smoke activate macrophages resident in the respiratory tract as well as epithelial cells leading to release of chemokines (e.g., interleukin-8) and other chemotactic
■ factors which act to increase the neutrophil/monocyte trafficking from the blood into lung tissue
*" and airways. Neutrophils and monocytes recruited into the airways can release a variety of potentially damaging mediators such as proteolytic enzymes and reactive oxygen species. Matrix degradation and emphysema, along with airway wall thickening, surfactant dysfunction and mucus hypersecretion are all potential sequelae of this inflammatory response that lead to impaired airflow and gas exchange.
In both asthma and COPD, although resident cells of the lungs play important parts in disease induction, the movement of inflammatory cells into respiratory tissues can be considered a prerequisite for the late-phase and chronic pathologies of these diseases. Members of the PP2C family of serine/threonine protem phosphotases have recently been shown to be important in the intracellular signaling pathways related to the reorganization of the actin cytoskeleton and cell mobility (Koh et al. Current Biology 12, 317-321, 2002). Therefore, in one embodiment, Signal molecules which can lead to the activation of KB binding site and/or STAT4-binding site containing promoters, or IgG2a, IgGl and IgE or a portion or biologically active variant thereof may be administered to a subject to prevent or treat an allergic or inflammatory disorder, such as asthma or COPD. In another embodiment, an agonist which is specific for Signal molecules which can lead to the activation of kB binding site and/or STAT4-binding site containing promoters, or IgG2a, IgGl. and IgE may be administered to a subject to regulate the intracellular signaling pathways involved in reorganization of the actin cytoskeleton and cell mobility, and thereby prevent or treat an allergic or inflammatory disorder, such as asthma or COPD.
In a further embodiment, . an antagonist may be administered to a subject to prevent or treat inflammation of any type and, in particular, that which results from a particular disorder or conditions. Such disorders and conditions associated with inflammation include, but are not limited to, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma. In one aspect, an antibody specific for TLR9 may be used directly as an antagonist, or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which f. . express TLR9.
In other embodiments, any of the therapeutic proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for
' adverse side effects.
•- ϊ,.
The invention further pertains to the use of novel agents identified by the screening assays 5- described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, . ribozyme, or a polypeptide-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified 0 as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above- described screening assays for treatments as described herein.
A reagent which affects TLR9 activity can be administered to a human cell, either in vitro or in vivo, to reduce TLR9 activity. The reagent preferably binds to Ttlr9 is a polypeptide, the reagent 5 is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for 0 at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung or liver.
A liposome useful in the present invention comprises a lipid composition that is capable of fusing 5 with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmol of liposome delivered to about 106 cells, more preferably about 1.0 μg of DNA per 16 nmol of liposome delivered to about ^ 106 cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 106 cells. Preferably, a liposome is between about 100 and 500 nm, more preferably 0 between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
Suitable liposomes for use in the present invention include those liposomes used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes' having a cholesterol
' backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a tumor cell, such as a tumor cell ligand exposed on the outer surface of the liposome.
Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151).. Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes. .
!
In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor- mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).
If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.
Determination of a Therapeutically Effective Dose
The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases extracellular matrix degradation relative to that which occurs in the absence of the therapeutically effective dose.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
Therapeutic efficacy and toxicity, e.g., ED50 (the dose therapeutically effective in 50% of the ■' * ■ ,'■ population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to 5- therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range 0 depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account 5 include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
0 Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for. nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular 5 cells, conditions, locations, etc.
Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about ^ 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 μg to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to 0 about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.
If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide, a siRNA or a ribozyme. Antisense oligonucleotides, siRNAs or ribozymes can be introduced into cells by a variety of methods, as described above or known in the art.
• Preferably, a reagent reduces expression of a TLR9 polynucleotide or activity of a TLR9 polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%
5. relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a TLR9 polynucleotide or the activity of a TLR9 polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to TLR9- . specific mRNA, quantitative RT-PCR, immunologic detection of a TLR9 polypeptide, or measurement of TLR9 activity.
0 In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders 5 • described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
0 The above disclosure generally describes the present invention, and all patents and patent applications cited in this disclosure are expressly incorporated herein. A more complete understanding can be obtained by reference to the following specific examples which are provided for purposes of illustration only and are not intended to limit the scope of the invention.
DEFINITIONS
5 As used herein, the term "antibody" is meant to, without limitation, refer to complete, intact antibodies, Fab fragments and F(ab)2 fragments thereof, and chimeric antibodies. r»
As usted herein, the term "modulates" means an increase or decrease in the amount or effect of a particular activity or protein.-
As used herein the term "marker" refers to a biological molecule, e.g., a nucleic acid, peptide, etc. 0 or the status of said biological molecule (e.g. phosphorylation status) whose presence or concentration can be detected and correlated with the presence or absence of a reagent in an assay (e.g. a modulator, inhibitor or activator).
As used herein the term modulator refers to an inhibitor or an activator. Inhibitors can be
•• y, antagonists, activators can be agonists, partial agonists, inverse agonist, or co-activators. Inhibitors
(like antagonists) and activators (agonists, partial agonist, inverse agonists or co-activators)
5. include but are not limited to nucleic acids, including but not limited to DNA, RNA, or DNA analog or RNA analog, proteins including but not limited to antibodies, peptides, peptidomimetics, carbohydrates, lipids and small molecules. Small molecule refers to a composition, which has a molecular weight of less than 5 kD, or less than 4 kD, or more preferably less than 2,5 kD and most preferably less than 0,5 kD. Small molecules can be organic (carbon-containing) or inorganic 0 molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate TLR9 activity.
The examples of TLR9 antagonist include, but not limited to, antibodies (such as TLR9 antibody, IMG-305, IMGENEX), oligonucleotides, polypeptides, and small molecular weight compounds.
5 The examples of TLR9 agonist include, but not limited to, synthetic oligonucleotides (such as ODN2006), polypeptides, and small molecular weight compounds.
The examples of IL-12 receptor antagonist include, but not limited to, polypeptides, IL-12 neutralizing antibodies, soluble recombinant IL-12 receptors
The examples of IL-12 receptor agonist include, but not limited to, recombinant IL-12, IL-12 0 agonist antibodies, polypeptides which can activate IL-12 receptor.
The invention is further illustrated by way of, the following examples which are intended to elucidate the invention. The foregoing examples are meant to illustrate the invention and are not to be construed to limit the invention in any way. Those skilled in the art will recognize modifications that are within the spirit and scope of the invention. It is intended that all references, 5 including each of the patents, applications, and printed publications, mentioned herein be herein,, incorporated by reference in their entirety. EXAMPLES
Example 1
CpG-DNA induced T-bet transcription.
To determine whether T-bet could be regulated by CpG, we treated mouse splenocytes with 3 jαM CpG for 3 or 6 hours and measured the expression of mRNA by quantitative RT-PCR. Cells treated with IFN-γ were used as a positive control. GpC (inactive oligonucleotides with a reversed . CpG motif) and the .TLR4 agonist E.coli LPS were used for determining the specificity of oligonucleotides and signaling pathways, respectively. We found that the mRNA level of T-bet was slighfly elevated in CpG-treated splenocytes (3 -fold) at 3 hours, followed by a profound induction at 6 hours after stimulation, surpassing the induction produced by the IFN-γ treatment (Figure 1). The effect of CpG on T-bet transcription was specific since the inactive GpC DNA failed to upregulate T-bet mRNA. LPS was incapable of inducing the transcription over the 6-hour time course, consistent with previous reports8.
Example 2
CpG-induced STATl phosphorylation.
Previously published data have shown that STATl activity is required for the induction of T-bet in CD4+ T and myeloid cells 8'9. To investigate the possibility that CpG-induced T-bet expression is also mediated by STATl, we assessed the phosphorylation of STATlα/β on tyrosine 701. As depicted in Figure 2, IFN-γ induced the phosphorylation of STATl quickly peaking at 2 hours, whereas CpG induced the phosphorylation of STATl to a maximum level at 5 hours where it was maintained over a long duration (up to 18 hours). Indeed, STATl phosphorylation induced by CpG (3 μM) was stronger and longer-lived than that by IFN-γ (50 ng/ml). Notably, although LPS can not induce T-bet expression (Figure 1), considerable phosphorylation of STATl was observed in cells treated with LPS (Figure 2 and refs. 10).
Example 3
CpQ-induced T-bet expression and STATl phosphorylation are both TLR9- and MyD88- dependent.
To clarify whether TLR9 itself, or a yet unknown CpG-responding receptor, transmits the signal leading to the transactivation Of T-bet and phosphorylation of STATl, we used TLR9_/" and MyDSδ"'" mice to define the signal. We show in Figure 3A and 3B that both CpG-induced T-bet mRNA and STATl phosphorylation were completely ablated in MyD88- or TLR9-deficient splenocytes, while the effect of IFN-γ was unchanged. Thus the induction of T-bet and ■ phosphorylation of STATl by CpG requires both TLR9 and MyD88. In contrast to CpG, LPS- induced STATl phosphorylation is MyD88-independent (Figure 3B). This shows that T-bet induction is peculiar to CpG/TLR9 signaling, while STATl phosphorylation can be induced by both TLR9 and TLR4 agonists.
Example 4
Identification of cells expressing T-bet in response to CpG
In order to further investigate the mechanism and functional consequences of CpG-induced up- regulation of T-bet, we sought to characterize cell types expressing T-bet in response to CpG. I Considering that some cellular effects of CpG are indirect (such as CpG-induced IFN-γ production in T cells and NK cells), we performed the first set of experiments using splenocytes. Cells were treated with CpG for 6 hours, the time point where T-bet expression reaches maximum, and for
24 hours, where secondary effects could be observed. The T cell and B cell fractions were then isolated by sequential positive and negative selection and the T cell-and B cell-depleted splenocytes recovered (mainly NK cells and myeloid cells, designated as "non-T, non-B"). When the mRNA levels of T-bet in these three populations were assessed by quantitative RT-PCR, a remarkable induction of T-bet was found in B cells and the non-T, non-B cell population (Figure 4A). Surprisingly, we could not observe increased T-bet expression in T cells at least over a time course of 24 hours. T cells are not expected to be able to respond to CpG directly due to their lack of TLR9 expression, to preclude the possibility that CpG-induced signaling had not yet transmitted or failed to transmit to T cells, we measured IFN-γ mRNA levels in the same samples. As shown in Figure 4B, a dramatic induction of IFN-gamma mRNA was seen in T cells with levels comparable to non-T, non-B splenocytes even at 6 hours post CpG stimulation. This shows that even in mixed cultures, CpG- cannot upregulate T-bet mRNA in-T cells in at least a period of 24 hours, despite being able to drive T cells to express IFN-γ mRNA.
The rapid induction of T-bet in B cells led us to examine whether CpG acts directly on B cells or ^ " through other cells, such as DCs, macrophages and monocytes. Splenic B cells were isolated (purity > 99%), and then treated with CpG for 6 hours. As depicted in Figure 4C, CpG, but not LPS, induced T-bet expression was clearly observed in highly purified B cells. We also examined T-bet induction in human B cells. Peripheral B cells were purified from three healthy donors and treated with ODN 2006 (2 JJ.M)", a well-characterized human B cell stimulatory CpG DNA11. As shown in Figure 4D, CpG-induced up-regulation of T-bet was clearly seen in the B cells from all three donors. These results show that CpG/TLR9 signaling could lead to a rapid induction of the key transcription factor T-bet directly in B cells, straightly linking innate and adaptive immunity ' through the B cell compartment.
Example 5
CpG-induced T-bet expression in B cells is IFN-gamma/STATl independent.
Since CpG can effectively induce STATl phosphorylation, (as shown in Figure 2), which we also confirmed in purified B cells (data not shown), we investigated the potential role for IFN-γ and STATl in CpG-induced T-bet upregulation. The production of IFN-γ is unlikely to have a major effect on CpG-induced T-bet expression since we saw no significant increase in IFN-γ transcript levels (Figure 4B) or in the amount of IFN-gamma protein in the culture medium as measured by
I
ELISA (below the detection limit 15 pg/ml (data not shown)) after CpG stimulation. If the phophorylation of STATl by CpG was not due to IFN-γ, but rather through type I IFNs which have been reported to be rapidly induced by CpG 12, we wondered whether STATl phosphorylation was required at all for the T-bet induction by CpG. In B cells from STATl''" mice, IFN-gamma induced T-bet expression is reduced severely (Figure 5B), consistent with previous reports for myeloid cells and T cells 8' 10. In contrast to IFN-γ, CpG-induced T-bet expression is not affected by STATl -deficiency, indicating that CpG and IFN-γ use differential signaling pathways in the regulation of T-bet transcription.
Example 6
NF- B, p38, and IL-12 are required for CpG-induced T-bet expression.
To further trace the downstream signal pathway induced by CpG/TLR9 that leads to T-bet upregulation, several pharmacological inhibitors were tested. When B cells were incubated with cycloheximide, an inhibitor of protein synthesis, CpG-induced •T-bet expression in B cells was completely diminished (Figure 6A), indicating a requirement for de novo protein synthesis. Further investigation was carried out with SB203580 and Wortmannin, specific inhibitors for p38 and PI-3 kinases respectively, as well as Dexamethasone, an effective inhibitor of NF-kB activity. Incubation of B cells with Dexamethasone or SB203580 resulted in a profound reduction in CpG- triggered T-bet expression (Figure 6B), while the effect of Wortmannin was not significant. Based on these data and the reports by Hacker13 and Choudhury14 that p38 plays an important role in CpG-induced IL-12 production in vitro and the anti-allergic effect of CpG in vivo, we investigated whether IL-12 might be involved in CpG-dependent T-bet upregulation in B cells. We first determined whether IL-12 was produced in CpG-treated B cells using B cell-depleted splenocytes and LPS treatment as a reference. Six hours post stimulation (where a clear induction in T-bet mRNA could be detected), secreted IL-12 was assayed by ELISA. As shown in Figure 6C, ' biologically significant amounts of IL-12 (1.6 ng/ml) were produced from CpG treated B cells, while the production from cells treated with LPS was much lower (200 pg/ml). IFN-γ could not be detected in the same culture medium (data not shown). When B cells were treated with a neutralizing antibody against IL-12, CpG-induced T-bet expression was markedly decreased, whereas an IFN-γ blocking antibody did not show significant effects, confirming that IL-12, but not IFN-γ is important for CpG-driven T-bet expression. To further address whether IL-12 functions sequentially or additively with CpG, B cells were stimulated with CpG, IL-12, and IFN- gamma alone or in combination. As shown in Figure 6E, IL-12 synergistically enhances CpG- induced T-bet upregulation, in contrast to IFN-γ which only appears to additively effect on T-bet expression. Thus IL-12 together with CpG stimulates a much greater induction of T-bet mRNA (130 times background) than that of CpG alone (12 times) or CpG plus IFN-γ (20 times). The discovery of the great synergistic effects of CpG and IL-12, suggests a novel strategies for the treatment of immune diseases such as allergy, asthma, by combining CpG or TLR9 agonists and IL-12 . On the other hand, a combination of TLR9 antagonists together with IL-12 antagonists would be effective in the treatment of diseases, such as autoimmunities. Such methods can not only effectively increase the efficacy of CpG or TLR9 modifying reagents, but also prevent the side effects caused by high concentration of CpG or TLR9 modifying reagents.
Example 7
CpG inhibits IL-4/CD40-induced IgGl and IgE class switching via a direct action on B cells.
Although CpG can inhibit antigen-induced IgE production in mice and in PBMC from atopic patients, such an effect has not been reported in cultured B cells. Based on our finding that CpG can rapidly induce a drastic increase in T-bet mRNA in purified B cells and the report that ectopic expression of T-bet is sufficient to induce IgG2a switching in cultured B cells, and that T-bet- deficient B cells produce excess amounts of IgGl and IgE 15, we thought that the induction of T- bet by CpG in B cells might play a role in Ig class-switching. To determine this, splenic B cells (purity>99%) were cultured in a medium containing IL-4 (10 ng/ml) and anti-CD40 agonistic
I*- antibodies (3 microg/ml) in the presence or absence of CpG (3 microM) for 10-14 days, then cell number, purity and the secreted IgGl, IgG2a and IgE were assessed. Strikingly, IL-4 and CD40 ligation-induced IgE (36.1+6.3 ng/ml) was completely inhibited and IgGl production (246+30.9 ng/ml) showed an 87% reduction after CpG treatment (Table 4 and Figure 7A). Because neither the cell number nor the purity showed significant changes during the cell culture, this shows that CpG effectively inhibits IL-4/CD40-induced Th2-related IgE and IgGl production via a direct effect on B cells. This inhibition appeared to occur at the transcriptional level because
I ε and Iγl germline transcripts were completely diminished in B cells cultured with IL-4/CD40
■ Ab and CpG (Figure 7B). To investigate the involvement of T-bet, mRNA was assessed by quantitative RT-PCR. As depicted in Figure 7C, no markable T-bet transcripts could be detected in cells treated with IL-4 and CD40 Ab. Conversely, a striking- up-regulation of T-bet mRNA was found in cells treated with CpG. Therefore the expression of T-bet correlated with the inhibition of
IgE and IgGl class switching. As expected, IgG2a class switching showed a positive correlation with T-bet mRNA (Figure 7B and C). Because activation of STAT6 plays a central role in IL-4- induced IgE and IgGl class switching, we wondered whether CpG treatment or T-bet expression could inhibit IL-4-induced phosphorylation of STAT6. B cells were pretreated with CpG for 20 hours, and then stimulated with IL-4 for 20 or 60 min. Activation of STAT6 was assessed by
Western blotting with an antibody recognizing phospho-STAT6. Figure 7D reveals that CpG i treatment did not interfere with IL-4-induced STAT6 phosphorylation, indicating that some other mechanism must be involved.
Our results to this point demonstrate that CpG or TLR9 modifing reagents can regulate both TH1- and TH2-related Ig class switching in a reciprocal, but B cell-intrinsic, T cell-independent way. The finding of CpG-mediated inhibition of IgE and IgGl has a consequence for the treatment of antibody-mediated allergic disorders, such as asthma. Moreover, different from the data described in WO03/022296, we found that CpG alone could induce the upregulation of IgG2a, a class of antibodies often found in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. This discovery provides the first direct evidence that bacterial infection can worsen autoimmune diseases and therefore the antagonists of TLR9 are expected to be able to protect patients from the susceptibility to bacterial infection as well as to ease up autoimmune responses in general. In addition, we also found CD40 signaling could enhance the CpG-induced upregulation of IgG2a and downregulation of IL-4-mediated IgE and IgGl switching. This discovery is particularly important, because it points out that modifying both TLR9 and CD40 will be more effective than regulating only TLR9. Table 4
Figure imgf000051_0001
N.D.: not detected
Example 8
Identification of test compounds that modulate TLR9 activity with the use of reporter assay 1: T- bet promoter element, or any one ot gene x-xxxv promoter elements.
Cells expressing endogenous or recombinant TLR9 receptor are transfected with a DNA construct having T-bet promoter element or any one of gene x-xxxv promoter elements, and reporter gene (such as luciferase gene). The transfected cells are incubated at 37°C for 1 min to 3 days with a test compound. A culture of the same type of cells that have not been transfected is incubated for 0 the same time without the test compound to provide a negative control. *
The cells or culture medium are/is harvested to determine the reporter activity (such as luciferase activity by luminometry).
A test compound that decreases or increases the reporteractivity relative to the activity obtained in the absence of the test compound is identified as a modulator (an inhibitor or activator 5 respectively) of TLR9. • '
Example 9
Identification of test compounds that modulate TLR9 activity with the use of reporter assay: STAT 4 BSNF-κB BS
Cells expressing endogenous or recombinant TLR9 receptor are transfected with a DNA construct
_ θ" having STAT 4 binding sites and/or NF-κB binding sites and a reportergene. The transfected cells are incubated at 37°C for 1 min to 3 days with a test compound. A culture of the same type of cells that have not been transfected or transfected with a reporter construct without KB or STAT4 binding elements is incubated for the same time without the test compound to provide a negative control. The cells are harvested to determine the reporter activity by luminometry.
A test compound that decreases or increases the luciferase activity relative to the activity obtained '' ■ *•'■ in the"' absence of the test compound is identified as a modulator (an inhibitor or activator respectively) of TLR9.
5 Example 10
Identification of test compounds that modulate TLR9 activity by measuring IL-12 protein amount by ELISA
Cells expressing endogenous or recombinant TLR9 receptor are used. The cells are incubated at 37°C for 10 min to 7 days with a test compound. A culture of the same type of cells that have not 0 been tr nsfected is incubated for the same time without the test compound to provide a negative control.
The supematants of the reaction mixture are then collected. The IL-12 concentration in the supematants is determined using a DuoSet™ ELISA Development Kit (GenzymeTechne, Minneapolis, USA) following the manufacturer's recommendations.
5 A test compound that decreases or increases the amount of IL-12 relative to the amount obtained in the absence of the test compound is identified as a modulator (an inhibitor or activator respectively) of TLR9.
Example 11
Identification of test compounds that modulate TLR9 activity by determining the IgG2a protein 0 production in primary B cells or B cell lines
B cells expressing TLR9 receptor are used. The B cells are incubated at 37°C for 0.1-21 days with a test compound. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
The supematants of the reaction mixture are then collected. The IgG2a concentration in the 5 supematants is determined using a DuoSet™ ELISA Development Kit (GenzymeTechne, Minneapolis, USA) following the manufacturer's recommendations.
A test compound that decreases or increases the amount of IgG2a relative to the amount obtained
<_. in the absence of the test compound is identified as a modulator (an inhibitor or activator respectively) of TLR9. Example 12
Identification of test compounds that modulate TLR9 activity by determining the inhibition oflL- 4/CD40 ligation-induced IgGl and IgE protein production in primary B cells or B cell lines
B cells expressing TLR9 receptor are used. The B cells are incubated at 37°C for 3 to 21 days with IL-4 (0.01 ng/ml - 10 μg/ml, optimal concentration is 10 ng/ml) and CD40 ligation (CD40 ligand, or an agonistic antibody, a test compound. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
The supematants of the reaction mixture are then collected. The IgGl and/or IgE concentration in the supematants is determined using a DuoSet™ ELISA Development Kit (GenzymeTechne, Minneapolis, USA) following the manufacturer's recommendations.
A test compound that decreases or increases the amount of IgGl and/or IgE relative to the amount obtained in the absence of the test compound is identified as a modulator (an inhibitor or activator respectively) of TLR9.
Example 13
Table 2 and 3 show the accession number and functional description of genes that are up- or down-regulated by CpG, as well as their expression in response to IL-4, INFγ, IL-12 or CpG plus IL-12. Mouse splenic B cells were purified by negative selection and stimulated with indicated stimuli for 8 h and 24 h respectively. Total RNA was purified and 5 μg of each was used for DNA microarray analysis (CodeLink Bioarray Mouse AmershamBiosciences). Image was visualized using Axon GenePixPro v4.0 and analyzed by CodeLinkTM software (AmershamBiosciences). The relative expression of mRNA was determined by the ratio of fluorescent intensity to the median.
References
1. Modlin RL. Mammalian toll-like receptors. Ann Allergy Asthma Immunol. 88(6), 543-7 < *■ - (2002).
2. ' Yang D, Biragyn A, Kwak LW, Oppenheim JJ. Mammalian defensins in immunity: more 5 than just microbicidal. Trends Immunol 23(6), 291-6 (2002)
3. Asnagli H and Murphy MK. Stability and commitment in T helper cell development. Current Opinion in Immunology. 13, 242-247 (2001).
4. Kline JN, Waldschmidt TJ, Businga TR, et al. Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol. 160, 2555-2559 (1998)
I 0 5. Sur S, Wild JS, Chhoudhury BK, et al. Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG oligodeoxynucleotides. J Immunol. 162, 6284-6293
" (1999)
6. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S. A Toll-like receptor recognizes bacterial DNA. Nature 5 2000 Dec 7;408(6813):740-5
7. Kaisho T and Akira S. Toll-like receptors as adjuvant receptors. Biochimica et Biophysica Acta 1589, 1-13 (2002)
8. Lighvani AA, Frucht DM, Jankovic D, Yamane H, Aliberti J, Hissong BD, Nguyen BV, Gadina M, Sher A, Paul WE, O'Shea JJ. T-bet is rapidly induced by interferon-gamma in 0 lymphoid and myeloid cells. Proc Natl Acad Sci U S A. 98(26), 15137-42 (2001).
9. Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Williams BR, Major J, Hamilton TA, Fenton MJ, Vogel SN. TLR4, but not TLR2, mediates IFN-beta-induced STATl alpha/beta-dependent gene expression in macrophages. Nat Immunol. 3(4), 392-8 (2002).
5 10: Afkarian M, Sedy JR, Yang J, Jacobson NG, Cereb N, Yang SY, Murphy TL, Murphy KM. T-bet is a STATl-induced regulator of IL-12R expression in naive CD4+ T cells. Nat Immunol. 3(6), 549-57 (2002).
11. Hartmann, G. and Krieg, A. M. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J Immunol. 164, 944-953 (2000). 12. Hoshino K, Kaisho T, Iwabe T, Takeuchi O, and Akira S. Differential involvement of IFN- β in Toll-like receptor-stimulated dendritic cell activation. Int. Immunol. 14, 1225-1231 (2002) "
13. Hacker H, Mischak H, Hacker G, et al. Cell type-specific activation of mitogen-activated 5. protein kinases by CpG-DNA controls interleukin-12 release from antigen-presenting cells. EMBOJ H, 6973-6982 (1999).
14. Choudhury BK, Wild JS, Alam R, Klinman DM, Boldogh I, Dharajiya N, Mileski WJ, Sur S. In Vivo Role of p38 Mitogen- Activated Protein Kinase in Mediating the Anti- inflammatory Effects of CpG Oligodeoxynucleotide in Murine Asthma. J Immunol. 0 169(10), 5955-5961(2002).
I
15. Peng SL, Szabo SJ, Glimcher LH. T-bet regulates IgG class switching and pathogenic autoantibody production. Proc Natl Acad Sci U S A. 99(8), 5545-50 (2002).

Claims

Claims:
1. A method of screening for agents which modulate TLR9 activity characterized in that at v ;,,_. •>■ least one marker selected from the group consisting of:
' i) T-bet protein, or a polynucleotide encoding the same;
5 ii) IKKs protein, or a polynucleotide encoding the same;
iii) NF-κB protein or a polynucleotide encoding the same;
iv) P38 protein or a polynucleotide encoding the same;
v) IL-12 protein, or a polynucleotide encoding the same;
vi) STAT4 protein, or a polynucleotide encoding the same;
0 " vii) IgG2a protem or a IgG2a germlme transcript;
viii) IgGl protein or a IgGl germline transcript;
ix) Ig E protein or a IgE germline transcript;
x) TCF2 protein or a polynucleotide encoding the same;
xi) BATF protein or a polynucleotide encoding the same;
5 xii) PLAUR protein or a polynucleotide encoding the same;
xiii) C/EBPδ protein or a polynucleotide encoding the same;
xiv) ARHE (ras homolog gene family) protein or a polynucleotide encoding the same;
xv) NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same;
xvi) GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide 0t- encoding the same;
xvii) C/EBPbeta protein or a polynucleotide encoding the same;
xviii) TACSTDl (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same; xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same;
xx) SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a ■ Λ> " polynucleotide encoding the same;
xxi) ESP 15 (epidermal growth factor receptor pathway substrate 15) protem or a 5 polynucleotide encoding the same;
xxii) UBEIC (ubiquitin-activating enzyme E1C) protein or a polynucleotide encoding the same;
xxiii) FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same;
xxiv) CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide 0 encoding the same;
• xxv) SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same;
xxvi) GNPNATl (glutamine repeat protein 1) protein or a polynucleotide encoding the same;
xxvii) BAP29 (B-cell receptor-associated protein 29) protein or a polynucleotide 5 encoding the same;
xxviii) PML (promyelocytic leukemia) protem or a polynucleotide encoding the same;
xxix) MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same;
xxx) FIG1 (interleukin-four induced gene 1) protein or a polynucleotide encoding the 0 same;
xxxi) MHC2TA (class II transactivator) protein or a polynucleotide encoding the same;
xxxii) HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding - ( the same;
xxxiii) FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a 5 polynucleotide encoding the same;
xxxiv) Id2 protein or a polynucleotide encoding the same; and xxxv) CD74 protein or a polynucleotide encoding the same
is detected.
2. The method of claim 1, comprising the step of determining the relative difference compared to "a control of the amount, expression, activity or phosphorylation of at least one marker selected from the group consisting of:
i) T-bet protein, or a polynucleotide encoding the same;
ii) IKKs protem, or a polynucleotide encoding the same;
iii) NF-κB protein or a polynucleotide encoding the same;
' iv) p38 protein or a polynucleotide encoding the same;
v) IL-12 protein, or a polynucleotide encoding the same;
vi) STAT4 protein, or a polynucleotide encoding the same;
vii) IgG2a protein or a IgG2a germline transcript;
viii) IgGl protein or a IgGl germline transcript;
ix) IgE protein or a IgE germline transcript;
x) TCF2 protein or a polynucleotide encoding the same;
xi) BATF protem or a polynucleotide encoding the same;
xii) PLAUR protein or a polynucleotide encoding the same;
xiii) C/EBPδ protein or a polynucleotide encoding the same;
xiv) ARHE (ras homolog gene family) protein or a polynucleotide encoding the same;
- xv) NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same;
xvi) GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same;
xvii) C/EBPβ protein or a polynucleotide encoding the same; xviii) TACSTDl (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same;
•: >. "■ xix) LM04 (LIM ONLY4) protein or a polynucleotide encoding the same;
xx) SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a 5 polynucleotide encoding the same;
xxi) ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same;
xxii) UBEIC (ubiquitin-activating enzyme EIC) protein or a polynucleotide encoding the same;
0 xxiii) FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same;
■ xxiv) CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same;
xxv) SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same;
xxvi) GNPNATl (glutamine repeat protein 1) protein or a polynucleotide encoding the 15 same;
xxvii) BAP29 (B-cell receptor-associated protein 29) protein or a polynucleotide encoding the same;
xxviii) PML (promyelocytic leukemia) protein or a polynucleotide encoding the same;
xxix) MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) 0 protein or a polynucleotide encoding the same; ..
xxx) FIG1 (interleukin-four induced gene 1) protein or a polynucleotide encoding the same;
. ^ xxxi) MHC2TA (class II transactivator) protein or a polynucleotide encoding the same;
xxxii) HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding
25 the same; xxxiii) FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same;
.. xxxiv) Id2 protem or a polynucleotide encoding the same; and
xxxv) CD74 protein or a polynucleotide encoding thfc same.
3. A method of screening for agents which modulate TLR9 activity, comprising the steps of:
a) contacting a test compound with a cell expressing TLR9, and
b) determining the relative difference of the amount, expression, activity, or phosphorylation compared to a control of at least one marker selected from the group consisting of:
I i. T-bet protein, or a polynucleotide encoding the same;
ii. IKKs protein, or a polynucleotide encoding the same;
iii. NF-kappaB protein or a polynucleotide encoding the same;
iv. p38 protein or a polynucleotide encoding the same;
v. IL-12 protein, or a polynucleotide encoding the same;
vi. STAT4 protein, or a polynucleotide encoding the same;
vii. IgG2a protein or a IgG2a germline transcript;
viii. IgGl protein or a IgGl germline transcript;
ix. IgE protein or a IgE germline transcript;
x. TCF2 protein or a polynucleotide encoding the same;
xi. BATF protein or a polynucleotide encoding the same;
■f xii. PLAUR protein or a polynucleotide encoding the same;
xiii. C/EBPδ protein or a polynucleotide encoding the same;
xiv. ARHE (ras hom log gene family) protein or a polynucleotide encoding the same; xv. NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same;
xvi. GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide »- j,,.. "■ encoding the same;
xvii. C/EBPbeta protein or a polynucleotide encoding the same;
5 xviii. TACSTDl (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same;
xix. LM04.(LIM ONLY4) protein or a polynucleotide encoding the same;
xx. SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same;
0 xxi. ESP15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same;
xxii. UBEIC (ubiquitin-activating enzyme EIC) protein or a polynucleotide encoding the same;
xxiii. FNBP3 (formin binding protem 3) protein or a polynucleotide encoding the same;
5 xxiv. CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same;
xxv. SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same;
xxvi. GNPNATl (glutamine repeat protein 1) protein or a polynucleotide encoding the same;
0 xxvii. BAP29 (B-cell receptor-associated protein 29) protein or a polynucleotide encoding the same;
xxviii. PML (promyelocytic leukemia) protein or a polynucleotide encoding the same;
xxix. MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same;
5 xxx. FIGl (interleukin-four induced gene 1) protein or a polynucleotide encoding the same; xxxi. MHC2TA (class II transactivator) protein or a polynucleotide encoding the same;
xxxii. HCST (hematopoietic cell signal transducer) protem or a polynucleotide encoding
< Λ - t, "■ the same;
xxxiii. FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protem or a
5 polynucleotide encoding the same;
xxxiv. Id2 protem or a polynucleotide encoding the same; and
xxxv. CD74 protein or a polynucleotide encoding the same.
4. A method of screening for agents which modulate TLR9 activity, comprising the steps of:
1 a) contacting a test compound with a cell expressing a CpG-recognizing receptor,
0 b) determining the relative difference compared to a control of the amount, expression, activity or phosphorylation of at least one marker selected from the group consisting of :
i. T-bet protein, or a polynucleotide encoding the same;
ii. IKKs protem, or a polynucleotide encoding the same;
5 iii. NF-kappaB protein or a polynucleotide encoding the same;
iv. p38 protein or a polynucleotide encoding the same;
v. IL-12 protein, or a polynucleotide encoding the same;
vi. STAT4 protein, or a polynucleotide encoding the same;
vii. IgG2a protem or a IgG2a germline transcript;
0 viii. IgGl protein or a IgGl germline transcript;
ix. IgE protein or a IgE germline transcript;
• x. TCF2 protein or a polynucleotide encoding the same;
xi. BATF protein or a polynucleotide encoding the same;
xii. PLAUR protein or a polynucleotide encoding the same; xiii. C/EBPdelta protein or a polynucleotide encoding the same;
xiv. ARHE (ras homolog gene family) protein or a polynucleotide encoding the same;
xv. NOP5/NOP58 (nucleolar protein 5) protem or a polynucleotide encoding the same;
xvi.. GOTl (glutamate * oxaloacetate transaminase 1) protein or a polynucleotide encoding the same;
xvii. C/EBPbeta protein or a polynucleotide encoding the same;
xviii. TACSTDl (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same;
xix. LM04 (LIM ONLY4) protein or a polynucleotide encoding the same;
xx. SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same;
xxi. ESP15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same;
xxii. UBEIC (ubiquitin-activating enzyme EIC) protein or a polynucleotide encoding the same;
xxiii. FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same;
xxiv. CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same;
xxv. SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same;
xxvi. GNPNATl (glutamine repeat protem 1) protein or a polynucleotide encoding the same;
xxvii. BAP29 (B-cell receptor-associated protein 29) protein or a polynucleotide encoding the same;
xxviii. PML (promyelocytic leukemia) protem or a polynucleotide encoding the same;
xxix. MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same; xxx. FIGl (interleukin-four induced gene 1) protein or a polynucleotide encoding the same;
•ι xxxi. MHC2TA (class II transactivator) protein or a polynucleotide encoding the same;
xxxii. HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same;
xxxiii. FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same;
xxxiv. Id2 protein or a polynucleotide encoding the same; and
'xxxv. CD74 protein or a polynucleotide encoding the same..
I .
5. A method of screening for agents which modulate TLR9 activity characterized in that at least two or more markers selected from the group consisting of:
i T-bet protein, or a polynucleotide encoding the same;
ii. IKKs protein, or a polynucleotide encoding the same;
iii. " NF-kappaB protein or a polynucleotide encoding the same;
iv. p38 protein or a polynucleotide encoding the same;
v. IL-12 protein, or a polynucleotide encoding the same;
vi. STAT4 protem, or a polynucleotide encoding the same;
vii. IgG2a protem or a IgG2a germline transcript;
viii. IgGl protein or a IgGl germline transcript; _ ix. IgE protein or a IgE germline transcript;
x. TCF2 protein or a polynucleotide encoding the same;
xi. BATF protein or a polynucleotide encoding the same;
xii. PLAUR protein or a polynucleotide encoding the same; xiii. C/EBP delta protein or a polynucleotide encoding the same;
xiv. ARHE (ras homolog gene family) protem or a polynucleotide encoding the same;
xv. NOP5/NOP58 (nucleolar protem 5) protein or a polynucleotide encoding the same;
xvi. GOTl (glutamate ' oxaloacetate transaminase 1) protem or a polynucleotide encoding the same;
xvii. C/EBPbeta protem or a polynucleotide encoding the same;
xviii. TACSTDl (Tumor-associated calcium signal transducer 1) protein' or a polynucleotide encoding the same;
! xix. LM04 (LIM ONLY4) protein or a polynucleotide encoding the same;
xx. SCS (GTP-specific succinyl-COA synthetase beta subunit) protem or a
. polynucleotide encoding the same;
xxi. ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same;
xxii. UBEIC (ubiquitin-activating enzyme EIC) protein or a polynucleotide encoding the same;
xxiii. FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same;
xxiv. CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same;
xxv. SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same;
xxvi. GNPNATl (glutamine repeat protem 1) protein or a polynucleotide encoding the same;
**■ - xxvii. BAP29 (B-cell receptor-associated protein 29) protein or a polynucleotide encoding the same;
xxviii. PML (promyelocytic leukemia) protein or a polynucleotide encoding the same;
xxix. MMKROX2R "(mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same; xxx. FIGl (interleukin-four induced gene 1) protein or a polynucleotide encoding the same;
,- v -<■ xxxi. MHC2TA (class II transactivator) protem or a polynucleotide encoding the same;
xxxii. HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding 5 the same;
xxxiii. FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same;
xxxiv. Id2 protein or a polynucleotide encoding the same; and
xxxv. CD74 protein or a polynucleotide encoding the same
0 are detected.
6. The method of claim 1, comprising the step of determining the relative difference compared to a control of the amount, expression, activity or phosphorylation of at least two or more markers selected from the group consisting of:
i T-bet protein, or a polynucleotide encoding the same;
5 ii. IKKs protein, or a polynucleotide encoding the same;
iii. NF-kappaB protem or a polynucleotide encoding the same;
iv. p38 protein or a polynucleotide encoding the same;
v. IL-12 protein, or a polynucleotide encoding the same;
vi. STAT4 protein, or a polynucleotide encoding the-same;
0 vii. IgG2a protein or a IgG2a germline transcript;
viii. IgGl protein or a IgGl germline transcript;
ix. IgE protein or a IgE germlme transcript;
x. TCF2 protein or a polynucleotide encoding the same;
xi. BATF protein or a polynucleotide encoding the same; xii. PLAUR protein or a polynucleotide encoding the same;
xiii. . C/EBPdelta protein or a polynucleotide encoding the same;
xiv. ARHE (ras homolog gene family) protein or a polynucleotide encoding the same;
xv. NOP5/NOP58 (nucleolar protein 5) protem or a polynucleotide encoding the same;
xvi. . GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same;
xvii. C/EBPbeta protein or a polynucleotide encoding the same;
, xviii. TACSTDl (Tumor-associated calcium signal transducer 1) protein or a ! polynucleotide encoding the same;
xix. LM04 (LIM ONLY4) protein or a polynucleotide encoding the same;
xx. SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same;
xxi. ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same;
xxii. UBEIC (ubiquitin-activating enzyme EIC) protein or a polynucleotide encoding the same;
xxiii. FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same;
xxiv. CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same;
xxv. SELL (Selectin lymphocyte) protem or a polynucleotide encoding the same;
xxvi. GNPNATl (glutamine repeat protein 1) protein or a polynucleotide encoding the **~ - same;
xxvii. BAP29 (B-cell receptor-associated protein 29) protein or a polynucleotide encoding the same;
xxviii. PML (promyelocytic leukemia) protein or a polynucleotide encoding the same; xxix. MMKROX2R (mouse mRNA for KROX-20 protem containing zinc fingers) protein or a polynucleotide encoding the same;
,- jϊ •<■ xxx. FIGl (interleukin-four induced gene 1) protein or a polynucleotide encoding the same;
5 xxxi. MHC2TA (class II transactivator) protein or a polynucleotide encoding the same;
xxxii. HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same;
xxxiii. FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same;
10 xxxiv. Id2 protein or a polynucleotide encoding the same; and
. xxxv. CD74 protem or a polynucleotide encoding the same.
7. A method of screening for agents which modulate TLR9 activity, comprising the steps of:
a) contacting a test compound with a cell expressing TLR9, and
b) determining the relative difference of the amount, expression, activity, or 15 , phosphorylation compared to a control of at least two or more markers selected from the group consisting of:
i. T-bet protein, or a polynucleotide encoding the same;
ii. IKKs protein, or a polynucleotide encoding the same;
iii. NF-kappaB protein or a polynucleotide encoding the same;
20 iv. p38 protein or a polynucleotide encoding the same;
v. IL-12 protein, or a polynucleotide encoding the same;
< , , vi. STAT4 protein, or a polynucleotide encoding the same;
vii. IgG2a protein or a IgG2a germline transcript;
viii. IgGl protein or-a IgGl germline transcript;
25 ix. IgE protein or a IgE germline transcript; x. TCF2 protein or a polynucleotide encoding the same;
xi. BATF protein or a polynucleotide encoding the same;
xii. PLAUR protein or a polynucleotide encoding the same;
xiii. C/EBPdelta protein or a polynucleotide encoding the same;
xiv. ARHE (ras homolog gene family) protein or a polynucleotide encoding the same;
xv. NOP5/NOP58 (nucleolar protein 5) protein or "a polynucleotide encoding the same;
xvi. GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same;
I xvii. C/EBPbeta protein or a polynucleotide encoding the same;
• xviii. TACSTDl (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same;
xix. LM04 (LIM ONLY4) protein or a polynucleotide encoding the same;
xx. SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same;
xxi. ESP15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same;
xxii. UBEIC (ubiquitin-activating enzyme EIC) protein or a polynucleotide encoding the same;
xxiii. FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same;
xxiv. CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same;
* , . xxv. SELL (Selectin lymphocyte) protem or a polynucleotide encoding the same;
xxvi. GNPNATl (glutamine repeat protein 1) protein or a polynucleotide encoding the same; xxvii. BAP29 (B-cell receptor-associated protein 29) protem or a polynucleotide encoding the same;
.1- xxviii. PML (promyelocytic leukemia) protein or a polynucleotide encoding the same;
' xxix. MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same;
xxx. FIGl (interleukin-four induced gene 1) protein or a polynucleotide encoding the same;
xxxi. MHC2TA (class II transactivator) protein or a polynucleotide encoding .the same;
'xxxii. HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same;
. xxxiii. FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protem or a polynucleotide encoding the same;
xxxiv. Id2 protein or a polynucleotide encoding the same; and
xxxv. CD74 protein or a polynucleotide encoding the same.
8. A method of screening for agents which modulate TLR9 activity, comprising the steps of:
a) contacting a test compound with a cell expressing a CpG-recognizing receptor,
b) determining the relative difference compared to a control of the amount, expression, activity or phosphorylation of at least two or more markers selected from the group consisting of:
i. T-bet protein, or a polynucleotide encoding the same;
ii. IKKs protein, or a polynucleotide encoding the same;
^ - iii. NF-kappaB protein or a polynucleotide encoding the same;
iv. p38 protein or a polynucleotide encoding the same;
v. IL-12 protein, or a polynucleotide encoding the same;
vi. STAT4 protein, or a polynucleotide encoding the same; vii. IgG2a protein or a IgG2a germline transcript;
viii. IgGl protein or a IgGl germline transcript;
ix. IgE protein or a IgE germline transcript;
x. TCF2 protein or a polynucleotide encoding the same;
xi. BATF protein or a polynucleotide encoding the same;
xii. PLAUR protein or a polynucleotide encoding the same;
xiii. C/EBPdelta protem or a polynucleotide encoding the same;
, xiv. ARHE (ras homolog gene family) protein or a polynucleotide encoding the same;
xv. NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same;
xvi. GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same;
. xvii. C/EBPbeta protein'or a polynucleotide encoding the same;
xviii. TACSTDl (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same;
xix. LM04 (LIM ONLY4) protein or a polynucleotide encoding the same;
xx. SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same;
xxi. ESP15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same;
xxii. UBEIC (ubiquitin-activating enzyme EIC) protein or a polynucleotide encoding the same; -. . xxiii. FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same;
xxiv. CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same;
xxv. SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same; xxvi. GNPNATl (glutamine repeat protem 1) protein or a polynucleotide encoding the same;
.. xxvii. BAP29 (B-cell receptor-associated protein 29) protein or a polynucleotide encoding the same;
xxviii. PML (promyelocytic leukemia) protein or a polynucleotide encoding the same;
xxix. MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same;
xxx. FIGl (interleukin-four induced gene 1) protein or a polynucleotide encoding the same;
xxxi. MHC2TA (class II transactivator) protein or a polynucleotide encoding the same;
. xxxii. HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same;
xxxiii. FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same;
xxxiv. Id2 protein or a polynucleotide encoding the same; and
xxxv. CD74 protein or a polynucleotide encoding the same.
9. The method of claim 3, 4, 7, or 8, wherein the cell is an activated B-cell.
10. The method of any of theclaims 1 to 9, wherein the difference is determined via use of nucleic acid microarrays.
11. A reagent that modulates the activity, amount or stability of a TLR9 polypeptide or polynucleotide, wherein said reagent is identified by the method of any of the claims 1 to 9 the reagent not being a CpG oligonucleotide.
12, A pharmaceutical composition, comprising:
a) the reagent of claim 11 , and
b) a pharmaceutically acceptable carrier.
13. Use of the reagent of claim 11 in the preparation of a medicament for modulating the activity of TLR9 in a disease.
14. Use of claim 13, wherein the disease is an allergic disease.
15. The use of a reagent that alters the expression, amount, activity or phosphorylation in a cell of
i T-bet protein or a polynucleotide encoding the same,
ii. IKKs protein, or a polynucleotide encoding the same;
iii. NF-kappaB protein or a polynucleotide encoding the same;
, iv. p38 protein or a polynucleotide encoding the same;
! v. IL-12 protein, or a polynucleotide encoding the same;
" vi. STAT4 protein, or a polynucleotide encoding the same;
vii. IgG2a protein or a IgG2a germline transcript;
viii. IgGl protein or a IgGl germline transcript;
ix. IgE protein or a IgE germline transcript;
x. . TCF2 protein or a polynucleotide encoding the same;
xi. BATF protein or a polynucleotide encoding the same;
xii. PLAUR protein or a polynucleotide encoding the same;
xiii. C/EBPδ protein or a polynucleotide encoding the same;
xiv. ARHE (ras homolog gene family) protein or a polynucleotide encoding the same;
xv. NOP5/NOP58 (nucleolar protein 5) protein or a polynucleotide encoding the same;
, xvi. GOTl (glutamate oxaloacetate transaminase 1) protein or a polynucleotide encoding the same;
xvii. C EBPbeta protein or a polynucleotide encoding the same; xviii. TACSTDl (Tumor-associated calcium signal transducer 1) protein or a polynucleotide encoding the same;
xix. LM04 (LIM ONLY4) protein or a polynucleotide encoding the same;
xx. SCS (GTP-specific succinyl-COA synthetase beta subunit) protein or a polynucleotide encoding the same;
xxi. ESP 15 (epidermal growth factor receptor pathway substrate 15) protein or a polynucleotide encoding the same;
xxii. UBEIC (ubiquitin-activating enzyme EIC) protem or a polynucleotide encoding the same;
I xxiii. FNBP3 (formin binding protein 3) protein or a polynucleotide encoding the same;
xxiv. CHUK (conserved helix-loop-helix ubiquitous kinase) protein or a polynucleotide encoding the same;
xxv. SELL (Selectin lymphocyte) protein or a polynucleotide encoding the same;
xxvi. GNPNATl (glutamine repeat protem 1) protein or a polynucleotide encoding the same;
xxvii. AP29 (B-cell receptor-associated protein 29) protein or a polynucleotide encoding the same;
xxviii. PML (promyelocytic leukemia) protein or a polynucleotide encoding the same;
xxix. MMKROX2R (mouse mRNA for KROX-20 protein containing zinc fingers) protein or a polynucleotide encoding the same;
xxx. FIGl (interleukin-four induced gene 1) protein or a polynucleotide encoding the same;
xxxi. MHC2TA (class II transactivator) protein or a polynucleotide encoding the same;
xxxii. HCST (hematopoietic cell signal transducer) protein or a polynucleotide encoding the same; xxxiii. FceR2A (Fc receptor, IgE, low affinity II, alpha polypeptide protein or a polynucleotide encoding the same;
,. ( ,,, xxxiv. Id2 protein or a polynucleotide encoding the same; and
• xxxv. CD74 protein or a polynucleotide encoding the same by modulating TLR9 activity 5 in the preparation of a medicament for the treatment of allergic diseases the reagent not being a CpG oligonucleotide.
16. A pharmaceutical composition comprising: (a) a reagent I that modulates the activity of a TLR9 polypeptide or polynucleotide; and b) a reagent II that modulates the activity of a IL-12 receptor polypeptide or polynucleotide, and c) a pharmaceutically acceptable carrier.
10 17. [ A pharmaceutical composition of claim 16, wherein said reagent I is a TLR 9 agonist and said reagent II is a IL-12 receptor agonist.
18. " A pharmaceutical composition of claim 16, wherein said reagent I is a TLR 9 antagonist and said reagent II is a IL-12 receptor antagonist.
19. Use of (a) a reagent I that modulates the activity of a TLR9 polypeptide or polynucleotide 15 and b) a reagent II that modulates the activity of a IL-12 receptor polypeptide or polynucleotide, in the preparation of a medicament for modulating the activity of TLR9 in a disease.
20. Use of claim 19, wherein the disease is an allergic disease and the reagent I is a TLR 9 agonist and the reagent II is a IL-12 receptor agonist.
20 21. Use of claim 19, wherein the disease is an autoimmune disease and the reagent I is a TLR 9 antagonist and the reagent II is a IL-12 receptor antagonist.
22. A pharmaceutical composition comprising: (a) a reagent I that modulates the activity of a TLR9 polypeptide or polynucleotide; and b) a reagent II that modulates the activity of a CD40 polypeptide or polynucleotide, and c) a pharmaceutically acceptable carrier.
25 23., A pharmaceutical composition of claim 22, wherein said reagent I is a TLR 9 agonist and said reagent II is a CD40 agonist.
24. A pharmaceutical composition of claim 22, wherein said reagent I is a TLR 9 antagonist and said reagent II is a CD40 antagonist.
25. Use of (a) a reagent I that modulates the activity of a TLR9 polypeptide or polynucleotide and b) a reagent II that modulates the activity of a CD40 polypeptide or polynucleotide, in the preparation of a medicament for modulating the activity of TLR9 in a disease.
26. Use of claim 25, wherein the disease is an allergic disease and the reagent I is a TLR 9 agonist and the reagent II is a CD40 agonist.
27. Use of claim 25, wherein the disease is an autoimmune disease and the reagent I is a TLR 9 antagonist and the reagent II is a CD40 antagonist.
PCT/EP2004/000641 2003-02-04 2004-01-27 Method of screening for agents which modulate the activity of tlr9 WO2004070344A2 (en)

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WO2006138478A2 (en) * 2005-06-17 2006-12-28 The Trustees Of The University Of Pennylvania Stimulation of toll-like receptors on t cells
WO2006138478A3 (en) * 2005-06-17 2009-04-30 Univ Pennsylvania Stimulation of toll-like receptors on t cells
WO2007127624A1 (en) * 2006-04-26 2007-11-08 The Uab Research Foundation Reducing cancer cell invasion using an inhibitor of toll like receptor signaling

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