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WO2009001032A1 - Modulation de l'inflammation - Google Patents

Modulation de l'inflammation Download PDF

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Publication number
WO2009001032A1
WO2009001032A1 PCT/GB2008/002047 GB2008002047W WO2009001032A1 WO 2009001032 A1 WO2009001032 A1 WO 2009001032A1 GB 2008002047 W GB2008002047 W GB 2008002047W WO 2009001032 A1 WO2009001032 A1 WO 2009001032A1
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Prior art keywords
trb
seq
nucleic acid
polypeptide
modulator
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Endre Kiss-Toth
David Crossman
Sheila Francis
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University of Sheffield
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University of Sheffield
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to methods of modulating inflammation. More particularly, the invention relates to methods of modulating low density lipoprotein (LDL) induced inflammation and methods of treatment of inflammatory ' diseases and disorders.
  • LDL low density lipoprotein
  • Elevated serum LDL levels have been implicated as a major risk factor in the development of atherosclerosis.
  • the above studies also demonstrate a fundamental role for Mitogen Activated Protein Kinase (MAPK) pathways in these processes.
  • MAPK Mitogen Activated Protein Kinase
  • Trb-2 was described as a gene, which is up-regulated by mitogens in dog thyroid cells (Wilkin, F et al. Eur J Biochem. 248:660-668, Wilkin, F. et al. J Biol Chem. 271:28451- 28457.). Most recent data on this gene and its protein product was also mainly correlative, describing differential expression in prostate cancer (Bisoffi, M., et al J Urol. 172:1145-1150), autoimmune uveitis (Zhang, Y. et al.
  • LDL induced IL-8 production has also been shown recently to play an important role in cell spreading and wound closure, both of which are fundamental processes in physiological wound healing (Dobreva, I. et al. J Biol Chem. 281 :199-205).
  • trb-2 plays a central role in the modulation of IL-8 expression, via an inhibitory effect upon the ERK and JNK pathways.
  • a regulatory role for LDL in trb-2 expression has also been identified.
  • the invention provides the use of a Trb-2 modulator in the preparation of a medicament for the treatment of an inflammatory disease or disorder.
  • the inflammatory disease or disorder is mediated by monocyte IL-8 production.
  • the inflammatory disease or disorder is a coronary disease, preferably acute coronary syndrome.
  • the invention provides a trb-2 modulator for use as a medicament.
  • the invention provide a trb-2 modulator for use as a modulator of inflammation.
  • the invention provides a trb-2 modulator for use as a modulator of monocyte response to an inflammatory stimulus.
  • the invention provides a method of treating an inflammatory disease or disorder comprising administering to a subject in need thereof a trb-2 modulator.
  • the inflammatory disease or disorder is mediated by monocyte IL-8 production.
  • the inflammatory disease or disorder is a coronary disease, preferably acute coronary syndrome.
  • the modulator up regulates Trb-2 expression or activity.
  • the Trb-2 modulator upregulates trb-2 binding to MEK1 or MKK7.
  • the modulator is an active Trb-2 protein or a fragment thereof. More preferably, the modulator is an isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide consisting of the amino acid sequence of
  • SEQ ID NO:1 or 3 wherein the fragment comprises at least 30 contiguous amino acids of SEQ ID NO:1 or 3; b) a naturally Qccurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3, c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4; and d) - a polypeptide comprising an amino acid sequence which is at least 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3.
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 3.
  • said modulator is an isolated nucleic acid molecule encoding a Trb-2 protein or a fragment thereof.
  • the modulator is an isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% homologous to the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 200 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; and c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3. More preferably, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4. Alternatively, the isolated nucleic acid molecule encodes a polypeptide comprising an
  • the invention provides a method of identifying a compound capable of modulating inflammation comprising assaying the ability of the compound to modulate the nucleic acid expression or polypeptide activity of trb-2, thereby identifying a compound capable of modulating inflammation.
  • the assay is a cell based assay, more preferably, a monocytic cell based assay.
  • the invention provides a method of modulating monocyte response to an inflammatory stimulus comprising contacting a monocytic cell with a compound that modulates the expression or activity of Trb-2.
  • the method is an in vitro method.
  • the cell is a mammalian cell, more preferably, a human cell.
  • the inflammatory stimulus comprises low-density lipoprotein (LDL).
  • LDL low-density lipoprotein
  • the modulation of cellular inflammatory response is a decrease.
  • the compound up regulates Trb-2 expression or activity
  • the compound is small molecules that stimulates the binding activity of TRB- 2 to MEK1 or IVJKK7.
  • the compound is an active Trb-2 protein or a fragment thereof. More preferably, the compound is an isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide consisting of the amino acid sequence of
  • SEQ ID NO:1 or 3 wherein the fragment comprises at least 30 contiguous amino acids of SEQ ID NO:1 or 3; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3, c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4; and d) a polypeptide comprising an amino acid sequence which is at least 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3. More preferably, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 3.
  • the compound is an isolated nucleic acid molecule encoding an active Trb- 2 protein or a fragment thereof.
  • the compound is an isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% homologous to the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 200 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; and c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3. More preferably, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4. Alternatively, the isolated nucleic acid molecule encodes a polypeptide
  • Figure 1 is a graphical representation showing that AcLDL potentiates LPS induced IL-8 production.
  • A The dynamics of AcLDL uptake by THP-1 cells was investigated by dil labelled AcLDL and analysed by FACS. 24hrs incubation was used in all subsequent experiments, unless specifically stated otherwise.
  • B The impact of AcLDL and LPS alone and in combination was studied on IL-8 protein production in THP-1 cells. IL-8 levels were quantified by ELISA.
  • C the involvement of MAPK pathways in LPS induced IL-8 production was measured by the use of inhibitors of specific MAPK pathways.
  • Figure 2. is a graphical representation showing that AcLDL and LPS modulates tribbles expression in monocytes.
  • A Expression levels of tribbles-1 , -2 and -3 were assessed in response to LPS by qRT-PCR.
  • B Similarly, and tribbles-2 expression was measured in response to AcLDL treatment.
  • Figure 3 is a graphical representation showing that downregulated trb-2 expression results in enhanced IL-8 production via ERK and JNK MAPK pathways.
  • A The efficiency of trb-2 knockdown by siRNA was assessed using qRT-PCR.
  • B The impact of reduced trb-2 levels on LPS induced IL-8 production was measured by ELISA.
  • C The specific MAPK pathways involved in the tribbles-2 mediated IL-8 production was characterised by using MAPK inhibitors as above. In order to determine the relative involvement of the various MAPK pathways, the IL-8 levels in samples without the MAPK inhibitors (as measured on 2B) were taken as 100% and values obtained from the MEK1 and JNK inhibitor treated samples were expressed as a percentage of their respective controls.
  • FIG. 4 is a graphical representation showing that Trb-2 is an inhibitor of the activation of JNK and ERK pathways in THP-1 cells.
  • THP-1 cells were stimulated by LPS in control transfected or trb-2 overexpressing cells and the level of EK and JNK activation was assayed by Western blotting. A representative experiment is shown on the left. The images were digitised and the intensity of the signal quantified from at least three independent experiments. pMAPK levels were normalised to ⁇ -actin and plotted on the right panels.
  • THP-1 cells were transfected with control or trb-2 specific siRNA and EK and JNK activation was assessed as on (4A).
  • Figure 5. is a graphical representation showing that Trb-2 interacts with MEK1 and MKK7 but not with MKK4.
  • MEK1 , MKK4 and MKK7 are endogenously expressed in THP-1 cells and their expression levels can be downregulated by specific siRNAs.
  • THP-1 cells were transfected with siMAPKK or control siRNA, as indicated and stimulated by LPS. Production of IL-8 was detected by ELISA.
  • Figure 6 is a graphical representation showing that Trb-2 levels are inversely correlated to the IL-8 produced in response to LPS in primary monocytes and are downregulated in Acute Coronary Syndromes.
  • (C) Expression of members of the tribbles family were compared between samples obtained from individuals with Stable Chronic Angina and Acute Coronary Syndromes (ACS) by qRT-PCR (N 15 in each group). Since ACS is accompanied by a proinflammatory phenotype, the levels of IL-1 D and IL-1ra and the ration of the pro- and anti-inflammatory cytokines were used as positive controls.
  • Figure 7 is a schematic representation of the role of trb-2 in monocytes biology in inflammatory settings.
  • Figure 8 depicts the amino acid sequence of the trb-2 polypeptide, designated SEQ ID NO:
  • Figure 9 depicts the nucleotide sequence of the nucleic acid molecule that encodes the trb-2 polypeptide, designated SEQ ID NO:2.
  • Figure 10 depicts the amino acid sequence of the kinase domain oftrb-2, designated SEQ ID NO:3. The kinase domain is located at amino acid residues 61 to 309 of SEQ ID NO:1.
  • Figure 11 depicts the depicts the nucleotide sequence of the nucleic acid molecule that encodes the trb-2 kinase domain, designated SEQ ID NO:4, and is located at nucleic acid residues 1406 to 2164 of SEQ ID NO:2.
  • Figure 12 depicts a multiple alignment of Tribbles orthologues.
  • Figure 13a is a representation of the results of fluorescent microscopy and depicts both wild type and S239A trb-2 binding to MKK proteins.
  • Figure 13b is a western blot depicting levels of expression of wild type and S239A trb-2.
  • Figure 13c depicts the results of FACS analysis and depicts both wild type and S239A trb-2 binding to MKK proteins.
  • Figure 13d depicts the results of a wild type and S239A Trb-2-v2 luciferase assay.
  • Figure 13e depicts the results of a titration luciferase assay and demonstrates the inhibitory effect of wild type and S239A trb-2 on and demonstrates the S239A mutant trb-2 is able to block AP-1 activation less efficiently than wild type, in line with the loss of trb-2/MAPK interaction, as shown on fig 13c DETAILED DESCRIPTION
  • the present invention is based on the surprising finding that Tribbles-2 (Trb-2), a modulator of mitogen activated protein kinase (MAPK), 1 and 7, is a molecular regulator of monocyte response to inflammatory stimuli.
  • Trb-2 Tribbles-2
  • MPK mitogen activated protein kinase
  • the inventors have surprisingly demonstrated that treatment of monocytes with LDL, potentiates lipopolysaccaride (LPS) induced IL-8 production via down regulation of trb-2 expression.
  • trb-2 expression levels are key in modulating IL-8 production by monocytes.
  • the inventors have found that treatment of monocytes with LDL increases the amount of IL-8 produced in response to LPS (figure 1b).
  • the inventors have also demonstrated that treatment of monocytes with LDL leads to a marked decrease in the expression of trb-2 (Figure 2b), highlighting a role for trb-2 in modulating monocyte IL-8 production in response to an inflammatory stimuli.
  • the role of trb-2 as a modulator monocyte inflammatory response was confirmed by suppressing monocyte trb-2 levels, by transfecting with siRNA against trb-2 (figure 3a). Monocytes were then exposed to inflammatory stimuli, LPS, and an inflammatory response induced. As illustrated in figure 3b, the results show that siRNA treated cells produced significantly higher levels of IL-8 compared to control cells.
  • Inflammatory activation of monocytes is a key event in inflammatory disease progression.
  • Monocyte activation is a central event in the development and progression of coronary disease.
  • the inventors have shown that trb-2 expression plays a role in an inflammatory heart disease, Acute Coronary Syndrome (ACS). Levels of trb-2 expression were compared in patients with ACS and in patients with Chronic Stable Angina. Consistent with the in vitro findings reported here, selective down regulation of trb-2 was observed in patients with ACS (figure 6 a, b and c). These results confirm that the responsiveness of monocytes to inflammatory stimuli is controlled through the action of trb-2.
  • ACS Acute Coronary Syndrome
  • the inventors propose a model for the role of trb-2 in monocyte response to inflammatory stimuli. As illustrated in figure 7a, LDL uptake by monocytes triggers a reduction in trb-2 expression. A reduction in trb-2 induces a hypersensitive state towards inflammatory stimuli, such as LPS induced production of IL-8.
  • trb-2 decreases the activation of JNK and ERK in response to LPS (figure 5a), thereby confirming a negative regulatory role for trb-2 in the control of ERK and JNK activation.
  • the binding of trb-2 to MKK7 and MKK4 (figure 5c) confirms the mechanism by which trb-2 activates ERK and JNK, as illustrated in figure 7b.
  • Trb-2 is a cytoplasmic protein (22), suggesting that this protein may have a distinct biological function from the other tribbles family members trb-1 and trb-3, which are expressed in the nucleus.
  • a highly selective regulation of trb-2 expression in a human disease, ACS (Fig. 6C) is observed here for the first time. This selective regulation of expression and unique intracellular distribution identifies trb-2 as a target for future drug research, which aims to inhibit inflammatory activation of monocytes.
  • the polypeptide sequence of Trb-2 is designated SEQ ID NO:1 and illustrated in figure 8.
  • the trb-2 polypeptide sequence is encoded by an isolated nucleic acid molecule, designated SEQ ID NO:2, illustrated in figure 9.
  • the polypeptide sequence of the trb-2 kinase-like domain is designated SEQ ID NO:3 and is illustrated in figure 10.
  • the kinase domain is located at amino acid residues 61 to 309 of SEQ ID NO:1.
  • the kinase domain is encoded by the nucleotide sequence of SEQ ID NO:4, illustrated in figure 1.1 , and located at nucleic acid residues 1406 to 12164 of SEQ ID NO:8.
  • the kinase like domain alone is capable of binding MKK7 and MEK1.
  • the modulation of trb-2 nucleic acid expression or polypeptide activity can be used to inflammatory response, more particularly, inflammatory response, for example monocyte IL-8 production.
  • the term "inflammatory response” refers to the activation of inflammatory cells in response to an inflammatory agent of stimulus. The activation of cells leads to a series of events involving pro-inflammatory cytokines, adhesion molecules and reactive oxygen species.
  • the term "inflammatory cell” refers to a cell type seen in an inflammatory response, such as a neutrophil, monocyte or macrophage.
  • the inflammatory response is the production of a pro-inflammatory cytokine by a monocytic cell. More preferably, the inflammatory response is the production of an interleukin by a monocytic cell, more preferably IL-8.
  • inflammatory agent refers to a compound capable of inducing an immune response.
  • that inflammatory stimulus may comprise low density lipoprotein (LDL), acLDL,, inflammatory cytokines, such as IL-1 , IL18, TNF ⁇ , lipopolysaccharide (LPS) or other TLR agonists, such as Lipoteichoic Acid (LTA) or CpG DNA.
  • LDL low density lipoprotein
  • acLDL acLDL
  • inflammatory cytokines such as IL-1 , IL18, TNF ⁇
  • LPS lipopolysaccharide
  • LTA Lipoteichoic Acid
  • modulate refers to the alteration, i.e. the up regulation or down regulation, of gene expression, the level of RNA molecules or of activity of one or more proteins, protein fragments or protein subunits. Modulation is such that the aforementioned expression, level, or activity is greater than or less than that observed in the absence of the modulation. Modulation can be a reduction, inhibition or down regulation of the aforementioned expression, level, or activity. Alternatively, modulation can be an increase, stimulation or up-regulation.
  • Modulation of inflammatory response can be achieved by contacting a cell with or exposing a cell to a Trb-2 modulator.
  • Trb-2 modulator refers to a compound or agent that has a stimulatory or inhibitory effect on, for example, expression of a Trb-2 nucleic acid molecule or activity of a Trb-2 polypeptide.
  • Trb-2 nucleic acid molecule or activity of a Trb-2 polypeptide include, small molecules that stimulate the MEK1 or MKK7 binding activity of TRB-2, an active Trb-2 protein or a fragment thereof, or a nucleic acid molecule encoding a Trb-2 protein or a fragment thereof that has been introduced into the cell.
  • Trb-2 nucleic acid molecule or activity of a Trb-2 polypeptide include small molecules that inhibit Trb-2 MEK1 or MKK7 binding activity, antisense trb- 2 nucleic acid molecules and anti-trb-2 antibodies.
  • antisense oligonucleotide or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • the antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.
  • the antisense oligonucleotide may be constructed and arranged so as to bind selectively with the target, i.e. trb-2 under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.
  • antisense oligonucleotides should comprise at least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and more preferably, at least 15 consecutive bases which are complementary to the target. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites.
  • 3'-untranslated regions may be targeted. The 3'- untranslated regions are known to contain pis acting sequences which act as binding sites for proteins involved in stabilising mRNA molecules.
  • antisense oligonucleotides is to be construed as materials manufactured either in vitro using conventional oligonucleotide synthesising methods which are well known in the art or oligonucleotides synthesised recombinantly using expression vector constructs.
  • the present invention includes pharmaceutical preparations containing natural and/or modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding proteins the modulation of which results in beneficial therapeutic effects, together with pharmaceutically acceptable carriers (eg polymers, liposomes/cationic lipids).
  • pharmaceutically acceptable carriers eg polymers, liposomes/cationic lipids.
  • Antisense oligonucleotides may be administered as part of a pharmaceutical composition.
  • a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art (eg liposomes).
  • the compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • inhibition is associated with post transcriptional silencing, using a modulator such as siRNA to mediate cleavage of a target nucleic acid molecule (e.g. RNA) or to inhibit translation via a process known as RNA interference.
  • a modulator such as siRNA to mediate cleavage of a target nucleic acid molecule (e.g. RNA) or to inhibit translation via a process known as RNA interference.
  • the therapeutic molecule is an inhibitory RNA (sfRNA).
  • sfRNA molecules are RNA molecules that function to bind to specific cellular target molecules, thereby inducing the specific degradation of the targeted mRNA. As a consequence, synthesis of specific proteins can be greatly diminished. This therefore. allows the specific elimination of expression of certain genes.
  • Systems for both transient and permanent expression of siRNA have been developed which may be incorporated into the said Ad or Ad vector (Brummelkamp, Bernards et al. 2002).
  • si RNA's are small double stranded RNA molecules that vary in length from between 10-100 base pairs in length although large siRNA's e.g. 100-1000 bp can be utilised. Preferably the siRNA's are about 20 base pairs in length. Preferably siRNA molecules are RNA molecules that function to bind to trb-2 molecules.
  • inhibition is associated with pretranscriptional silencing.
  • the inhibitor is a ribozyme.
  • Ribozymes are catalytic RNA molecules having ribonuclease activity. They are capable of cleaving a single- stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes for example hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave Trb-2 mRNA transcripts to thereby inhibit translation of Trb-2 mRNA.
  • a ribozyme having specificity for an Trb-2-encoding nucleic acid can be designed based upon the nucleotide sequence of an Trb-2 encoding nucleic acid molecules disclosed herein (e.g., SEQ ID NO:2, SEQ ID NO:4).
  • the inhibitor is an antibody, or at least an effective binding part thereof, which binds to a trb-2 polypeptide according to the invention.
  • antibody refers to immunoglobulin molecules and immunologically active portions thereof, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as trb-2.
  • a molecule which specifically binds to trb-2 is a molecule which binds trb-2, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains trb-2.
  • Immunoglobulins are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (K or ⁇ ), and one pair of heavy (H) chains ( ⁇ , ⁇ , ⁇ , ⁇ and ⁇ ), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are non-variable or constant.
  • the carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant" (C) region.
  • C constant
  • the amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable" (V) region.
  • the H chains of Ig molecules are of several classes, ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ (of which there are several sub-classes).
  • An assembled Ig molecule consisting of one or more units of two identical H and L chains, derives its name from the H chain that it possesses.
  • Ig isotypes IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the differences in the H chains, i.e., IgGI , lgG2, lgG3 and lgG4).
  • IgGI IgGI , lgG2, lgG3 and lgG4
  • the antibody may be a polyclonal or a monoclonal antibody that binds trb-2.
  • the term "monoclonal antibody” refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of trb-2.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular trb-2 protein with which it immun ⁇ reacts.
  • the antibody is humanised.
  • a humanised monoclonal antibody to a trb-2 polypeptide is produced as a fusion polypeptide in an expression vector suitably adapted for transfection or transformation of prokaryotic or eukaryotic cells.
  • said antibody is humanised by recombinant methods to combine the complimentarity determining regions of said antibody with both the constant
  • said antibody is provided with a marker including a conventional label or tag, for example a radioactive and/or fluorescent and/or epitope label or tag.
  • said antibody is a chimeric antibody.
  • Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are .combined with human antibody C-regions.
  • Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V-regions. The C-regions from the human antibody are also used.
  • CDRs complimentarity determining regions
  • Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation.
  • Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not illicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is desirable when using therapeutic antibodies in the treatment of diseases.
  • Humanised antibodies are designed to have less "foreign" antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.
  • the modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject).
  • In vitro modulation provides methods for treating cells in culture.
  • the cells are monocytic cells, i.e. monocytes or macrophages.
  • said cell is selected from the group consisting of: a nerve cell; a mesenchymal cell; a muscle cell (cardiomyocyte); a liver cell; a kidney cell; a blood cell (eg erythrocyte, CD4+ lymphocyte, CD8+ lymphocyte; panceatic ⁇ cell; epithelial cell (eg lung, gastric,) ; and a endothelial cell.
  • the cell is an endothelial cell.
  • the cells are mammalian cells. More preferably the cells are human.
  • the modulatory method involves administering compound or agent that has a stimulatory effect upon expression of a Trb-2 nucleic acid molecule or activity of a Trb-2 polypeptide
  • the modulatory method involves administering a compound or agent that has an inhibitory effect upon expression of a Trb-2 nucleic acid molecule or activity of a Trb-2 polypeptide.
  • a trb-2 stimulator up regulates the expression and / or activity of trb-2 and upregulates the binding of trb-2 to MEK1 and / or MKK7.
  • the administration of a trb-2 stimulator down regulates IL-8 production in response to an inflammatory stimuli.
  • In vitro modulatory methods including cell culture may be carried out in any suitable vessel.
  • the vessel is selected from the group consisting of: a petri-dish; cell culture bottle or flask; multiwell plate.
  • “Vessel” is construed as any means suitable to contain a cell culture.
  • In vivo modulation provides methods of treating a subject having a disease or disorder, or at risk of having a disease or disorder, associated the expression or activity of a trb-2 nucleic acid or polypeptide.
  • in vivo modulation provides methods of treating subject having a disease or disorder, or at risk of having a disease or disorder, that may be treated by modulating the expression or activity of a trb-2 nucleic acid molecule or polypeptide.
  • the modulatory method involves administering a trb-2 modulator.
  • the modulatory method may be a method to stimulate trb-2 expression or activity. It is beneficial to stimulate trb-2 expression or activity of trb-2 activity in diseases or disorders in which trb-2 is abnormally down regulated, or in diseases or disorders in which increased trb-2 activity is likely to have a beneficial effect.
  • the modulatory method may be a method to inhibit trb-2 expression or activity. It is beneficial to inhibit trb-2 activity in diseases or disorders in which trb-2 is abnormally up regulated or in diseases or disorders in which reduced trb-2 activity is likely to have a beneficial effect.
  • the term "monocyte response to an inflammatory stimulus” refers to the production of proinflammatory cytokines by monocytic cells in response to an inflammatory agent, i.e. the production of interleukine, such as 1L-8.
  • IL8 is a chemo- attractant of neutrophils. Upon release IL-8 binds to its receptor and induces a signal transduction pathway that results in a biological response, for example the release of the primary granule constituents of neutrophils.
  • inflammatory disorders include inflammatory disorders, more preferably coronary inflammatory disorders such as acute coronary syndromes.
  • coronary inflammatory disorders such as acute coronary syndromes.
  • the term "acute coronary syndromes" or "ACS" refers to a group of coronary disorders that result from ischemic insult to the heart. ACS can result from an accumulation of lipid, for example LDL or oxidised or modified LDL, together with macrophages and other inflammatory cells, which results in leads to plaque growth and ultimately plaque instability. Rupture of the mature plaques results in thrombosis, causing occlusion of the coronary arterial lumen and presentation of an acute coronary syndrome (ACS).
  • ACS acute coronary syndrome
  • Acute coronary syndromes include Unstable Angina (UA), Non-ST Segment Elevation Myocardial Infarction (NSTEMI), and ST Segment Elevation Myocardial Infarction (STEMI).
  • UUA Unstable Angina
  • NSTEMI Non-ST Segment Elevation Myocardial Infarction
  • STMI ST Segment Elevation Myocardial Infarction
  • the disorder is a vascular inflammatory disorder, for example atherosclerosis, heart disease, stroke, angina, thrombosis, myocardial infarction, ischemic heart disease, congestive heart failure.
  • the disorder is rheumatoid arthritis.
  • a disorder in which increased trb-2 activity is likely to have a beneficial effect is for example any disorder where it would be beneficial to upregulate immune response, for example, where it would be beneficial to upregulate local immune response.
  • a method of treatment of a mammal comprising administering to said mammal a modulator according to the invention.
  • in vivo modulation provides both prophylactic and therapeutic methods.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the trb-2 modulator and a pharmaceutically acceptable carrier.
  • pharmaceutically- acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be topical, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal, intranasal, intracerebral or epidural.
  • compositions of the invention are administered in effective amounts.
  • An "effective amount” is the amount of a composition that alone, or together with further doses, produces the desired response.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of the active ingredient for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the response can, for example, be measured by measuring the physiological effects of the composition, such as decrease of disease symptoms etc.
  • Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
  • Trb-2 modulators that are nucleic acid molecules of can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057).
  • trb-2 nucleic acid refers to a nucleic acid sequence encoding a Trb-2 protein.
  • nucleic acid molecule and “nucleic acid” include DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • a trb-2 nucleic acid is the nucleic acid molecule of SEQ ID NO:2.
  • the sequence is approximately 4221 nucleotides in length and encodes a 343 amino acid polypeptide designated Trb-2 (SEQ ID NO:1).
  • the trb-2 nucleic acid is the nucleic acid molecule of SEQ ID NO:4.
  • the sequence is approximately 759 nucleotides in length and encodes a 249 amino acid kinase domain (SEQ ID NO:3).
  • the trb-2 nucleic acid molecule is an isolated nucleic acid molecule.
  • isolated includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5'- and/or 3'-ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the trb-2 nucleic acid molecule comprises a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, or a portions or fragment thereof.
  • the nucleic acid molecule comprises a fragment of the nucleic acid molecule of SEQ ID NO:2 or SEQ ID NO:4, for example a fragment of 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 consecutive nucleotides of SEQ ID NO: 2 or 4.
  • the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 or SEQ ID NO:3.
  • the trb-2 nucleic acid molecule encodes fragments of SEQ ID NO:1 or 3, preferably the fragments are biologically active fragments, i.e. having MEK1 and / or MKK7 binding activity.
  • the trb-2 nucleic acid molecule has a nucleic acid sequence that is the complement of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:4, or portions or fragments thereof. In other embodiments, the trb-2 nucleic acid molecule has a nucleic acid sequence that is sufficiently complementary to the nucleotide sequence shown in of SEQ ID NO:2 or SEQ ID NO:4 such that it can hybridize to the nucleotide sequence shown in any of SEQ ID NO:2 or 4, thereby forming stable duplexes.
  • hybridizes under stringent conditions describes conditions for hybridization and washing.
  • Stringent conditions are known to those skilled in the art and can be found in available references (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6). Aqueous and non-aqueous methods are described in that reference and either can be used.
  • a preferred example of stringent hybridization conditions are hybridization in 6 ⁇ sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by one or more washes in 0.2 ⁇ SSC, 0.1% (w/v) SDS at 5O 0 C.
  • SSC sodium chloride/sodium citrate
  • stringent hybridization conditions are hybridization in 6 ⁇ SSC at about 45°C, followed by one or more washes in 0.2 ⁇ SSC, 0.1% (w/v) SDS at 55°C.
  • a further example of stringent hybridization conditions are hybridization in 6 ⁇ SSC at about 45°C, followed by one or more washes in 0.2 ⁇ SSC, 0.1% (w/v) SDS at 60 0 C.
  • stringent hybridization conditions are hybridization in 6x SSC at about 45°C, followed by one or more washes in 0.2 ⁇ SSC, 0.1% (w/v) SDS at 65 0 C.
  • Particularly preferred stringency conditions are 0.5 molar sodium phosphate, 7% (w/v) SDS at 65 0 C, followed by one or more washes at 0.2 ⁇ SSC, 1% (w/v) S ⁇ S at 65°C.
  • a trb-2 nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:2 or 4.
  • the trb-2 nucleic acid molecule has a nucleic acid sequence that is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length of the nucleotide sequence shown in SEQ ID NO:2 or 4, or portions or fragments thereof.
  • the trb-2 nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide that is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length the polypeptide of SEQ ID NO: 1 or 3, or portions or fragments thereof.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least .50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence.
  • the amino acid residues or -nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. MoI. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6,
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using VNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the trb-2 nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4.
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 2 or 4, or substitution, deletion or insertion of non- critical residues in non-critical regions of the protein.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the Trb-2 nucleic acid molecules of the invention can be isolated based on their homology to the nucleic acid molecules of the invention using the nucleotide sequences described in SEQ ID NO:2 or 4, or a portion thereof, as a hybridization probe under stringent hybridization conditions.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • any of the nucleic acid molecule described previously comprises specific changes in the nucleotide sequence so as to optimize expression, activity or functional life of the Trb-2 polypeptides.
  • the nucleic acids described previously are subjected to genetic manipulation and disruption techniques.
  • Various genetic manipulation and disruption techniques are known in the art including, but not limited to, DNA Shuffling (US 6,132,970, Punnonen J et al, Science & Medicine, 7(2): 38-47, (2000), US 6,132,970), serial mutagenesis and screening.
  • mutagenesis is error-prone PCR, whereby mutations are deliberately introduced during PCR through the use of error-prone DNA polymerases and reaction conditions as described in US 2003152944, using for example commercially available kits such as The GeneMorph ® Il kit (Stratagene ® , US). Randomized DNA sequences are cloned into expression vectors and the resulting mutant libraries screened for altered or improved protein activity.
  • the trb-2 nucleic acid molecule is a trb-2 encoding gene.
  • the term “gene” refers to nucleic acid molecules which include an open reading frame encoding protein, and can further include non-coding regulatory sequences and introns.
  • a trb-2 polypeptide is the polypeptide of SEQ ID NO:1.
  • the sequence is approximately 343 amino acid residues in length.
  • a trb-2 polypeptide is the polypeptide of SEQ ID NO:3.
  • the sequence is approximately 249 amino acid residues in length.
  • the trb-2 polypeptide molecule is an isolated polypeptide.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the trb-2 protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of trb-2 protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • Biologically active portions of an trb-2 protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the trb-2 protein (e.g., the amino acid sequence shown in SEQ ID NO:1 or 3), which include fewer amino acids than the full length trb-2 proteins, and exhibit at least one activity of an trb-2 protein.
  • a biologically active portion of a trb-2 protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a "biologically active portion" of protein includes fragment of protein that participate in an interaction between molecules and non-molecules.
  • Biologically active portions of protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the protein, e.g., the amino acid sequences shown in SEQ ID NO: 2 or 4, which include fewer amino acids than the full length protein, and exhibit at least one activity of the encoded protein.
  • a biologically active portion of protein can be a polypeptide that is, for example, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acids in length of SEQ ID NO: 1 , or 3.
  • Biologically active portions of protein can be used as targets for developing agents that modulateTrb-2-mediated activities, e.g., biological activities described herein.
  • a trb-2 protein has the amino acid sequence shown of SEQ ID NO:1 or 3.
  • Other useful trb-2 proteins are substantially identical to SEQ ID NO:1 or 3 and retain the functional activity of the protein of SEQ ID NO:1 or 3 yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • such trb-2 proteins and polypeptides posses at least one biological activity described herein such as, (1) the ability to bind MEK1 and/or MKK7.
  • a useful isolated trb-2 protein is a protein which includes an amino acid sequence at least about 45%, preferably 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:1 or 3 and retains the functional activity of the trb-2 proteins of SEQ ID NO:1 or 3.
  • the trb-2 protein has the amino acid sequence shown of SEQ ID NO:1 or 3, comprising conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • the trb-2 protein is a chimeric or fusion protein.
  • a trb-2 "chimeric protein" or “fusion protein” comprises a trb-2 polypeptide operably linked to a non-trb-2 polypeptide.
  • the trb-2 polypeptide can correspond to all or a portion of a trb-2 protein, preferably at least one biologically active portion of an trb- 2 protein.
  • "Operably linked” as used herein, refers to a combination of the polypeptide linked together in a functional relationship with one another, for example, fused in-frame to each other.
  • Variants of trb-2 protein which may function as either trb-2 agonists or as trb-2 antagonists can be identified by screening combinatorial libraries of mutants, of the trb-2 protein for trb-2 protein agonist or antagonist activity.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of (e.g., the sequence of SEQ ID NO:2 or 4) without removing or, more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change.
  • amino acid residues that are conserved among the polypeptides of the present invention e.g., those present in the conserved kinase domain of trb-2 are particularly non-amenable to alteration.
  • the invention provides screening assays for identifying modulators of trb-2 nucleic acid expression or polypeptide activity.
  • Assays can be cell free assays or cell based assays. The assays determine the ability of a test compound to modulate (stimulate or inhibit) the expression or activity of trb-2.
  • LDL potentiates LPS induced IL-8 production and reduces Trb-2 expression
  • AcLDL acetylated LDL
  • acLDL LDL uptake was measured by FACS (Fig.1A). Specifically, monocyte uptake of acLDL was evaluated by flow cytometry with lipoproteins labelled with the fluorescent probe DiI (Invitrogen). 1 ,5x10 5 THP-1 cells were treated with 5 ⁇ g/ml Dil-labeled acLDL for 1 , 2, 4 and 24h and assayed by flow cytometry.
  • trb-2 levels were suppressed by transfecting siRNA against trb-2 (Fig. 3A). Transfections were performed using Nucleofector
  • IL-8 production was induced in siRNA transfected cells by LPS treatment, as above.
  • the results on Figure 3B show that indeed, sitrb-2 treated THP-1 cells produce significantly higher levels of IL-8, compared to cells transfected with control siRNA.
  • THP-1 cells were transfected with sitrb-2 or control siRNA, and treated with MEK1 or JNK inhibitors (Fig. 3C). In both cases, the inhibitors attenuated IL-8 levels to the same extent, indicating that trb-2 modulates IL-8 production via these pathways.
  • MEK1 inhibitor (PD98059), p38 MAPK inhibitor (SB203580) and JNK MAPK inhibitor (SP600125) were purchased from Calbiochem and used 20 ⁇ M for MEK1 and JNK MAPK inhibitors and 0.2 ⁇ M for p38 MAPK inhibitor. The cells were treated for 1h with inhibitors before the LPS treatment.
  • Anti-MKK7, Anti-MKK4 and Anti-MEK-1 antibodies were purchased from Cell Signalling Technology and used according to the manufacturers recommendation.
  • Anti trb-1 polyclonal antibody was developed in collaboration with Millipore/Upstate.
  • Trb-2 modulates IL-8 production via interaction with MAPKKs
  • Trb-2 levels lead to elevated IL-8 production through enhanced activation of the JNK and ERK pathways.
  • MKK4/SEK-1 , MKK7 and MEK-1 known activators of JNK or ERK.
  • the results show that all 3 MAPKKs are expressed in THP-1 cells and that their expression can be inhibited by specific siRNA treatment (Fig. 5A).
  • siRNA treatment As expected, down-regulation of kinase levels by siRNA led to impaired IL-8 production, in response to LPS (Fig. 5B), indicating that all three proteins contribute to the activation of IL-8 expression.
  • PCA protein complementation assay
  • RAW 267.4 and THP-1 cells were used to examine the interactions between Trb-2 and MEK1 , MKK4 and MKK7.
  • the PCA plasmids Trb-2-V2 and MEK1 (MKK4/7)-V1 were generated by tagging Trb-2 and MKKs with half of Venus mutant YFP (V1 and V2) as described before (Remy, I., and Michnick, 2004. Methods. 32:381-388).
  • the interaction between trb-2 and MKKs were examined by both fluorescent microscopy and FACS.
  • Trb-2 binding to MKK7 and MEK1 but not to MKK4 was detected in both by fluorescent microscopy (Fig. 5C) and by FACS analysis (Fig. 5D).
  • the specificity of the interactions was assessed by FACS (Fig. 5E). Whilst co-expression of one fusion protein with the complementary fragment of YFP alone did not result in a significant fluorescence (treatments 2 and 3, Fig. 5E), the fluorescence seen in samples co- expressing MAPKK and trb fusion proteins (sample 5, Fig. 5E) decreased in a dose dependent fashion, when an increasing amount of trb-2 expression plasmid (not expressing the fusion YFP partner) was co-transfected (samples 6-8 on Fig. 5E).
  • Trb-2 controls IL-8 production in primary monocytes and its expression is selectively down-regulated in Acute Coronary Syndromes
  • trb-2 inhibits MKK mediated upregulation of the activity of AP1-luciferase reporter in HELA cells(figure 13D).
  • the reporter is activated by co- transfecting a MEKK1 expression plasmid.
  • the reporter activation is then locked by adding an increasing dose of trb-2 expression construct, wild type and S239A mutant.
  • the data demonstrates that an equal dose of the trb2 S239A exerted a significantly lower inhibitory effect than wt trb2 (figure 13E).
  • trb2 For luciferase assay to study the function of trb2-V2, HeLa cells were seeded onto a 96- well plate one day before transfection.
  • the doses used for transfection were the following (for a repeat of 4 wells): AP1-Luc 500ng, TK-rLuc, 100ng, MEKK1 10ng, the range of the amount of wt/S239trb2-V2 used was: 1 ng, 5ng, 10ng, 50ng, 100ng and 200ng.
  • pcDNA3.2+ was used (also ranged from 1ng to 200ng) to maintain the total amount of the plasmids the same for every testing samples.

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Abstract

La présente invention concerne l'utilisation d'un modulateur de Trb-2 dans la préparation d'un médicament pour le traitement d'une maladie ou d'un trouble inflammatoire, par exemple l'ACS.
PCT/GB2008/002047 2007-06-22 2008-06-13 Modulation de l'inflammation Ceased WO2009001032A1 (fr)

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WO2002053743A2 (fr) * 2001-01-08 2002-07-11 Interleukin Genetics, Inc. Voies de signalisation du gene mammifere tribbles, procede et reactifs associes

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WO2002053743A2 (fr) * 2001-01-08 2002-07-11 Interleukin Genetics, Inc. Voies de signalisation du gene mammifere tribbles, procede et reactifs associes

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Title
EDER ET AL: "LDL uptake by monocytes in response to inflammation is MAPK dependent but independent of tribbles protein expression", IMMUNOLOGY LETTERS, ELSEVIER BV, NL, vol. 116, no. 2, 24 January 2008 (2008-01-24), pages 178 - 183, XP022513153, ISSN: 0165-2478 *
GUAN, H. (REPRINT) ET AL: "Low-density lipoprotein controls IL-8 production in monocytes via tribbles -2", HEART, (JUL 2008) VOL. 94, SUPP. [2], PP. A44-A44. ISSN: 1355-6037. PB - B M J PUBLISHING GROUP, BRITISH MED ASSOC HOUSE, TAVISTOCK SQUARE, LONDON WC1H 9JR, ENGLAND., July 2008 (2008-07-01), XP009106447 *
WEI SHU-CHEN ET AL: "Tribbles 2 (Trb2) is a negative regulator of Nf-kappa B signaling", GASTROENTEROLOGY, vol. 132, no. 4, Suppl. 2, April 2007 (2007-04-01), & DIGESTIVE DISEASE WEEK MEETING/108TH ANNUAL MEETING OF THE AMERICAN-GASTROENTEROLOGICAL-ASSOCIATION; WASHINGTON, DC, USA; MAY 19 24, 2007, pages A706, XP009106423, ISSN: 0016-5085 *
WEI SHU-CHEN ET AL: "Tribbles 2 (Trib2), a novel regulator in toll-like receptor 5 signaling, is decreased in inflamed intestinal mucosa", GASTROENTEROLOGY, vol. 134, no. 4, Suppl. 1, April 2008 (2008-04-01), & DIGESTIVE DISEASE WEEK MEETING/109TH ANNUAL MEETING OF THE AMERICAN-GASTROENTEROLOGICAL-ASSOCIATION; SAN DIEGO, CA, USA; MAY 17 22, 2008, pages A515, XP009106424, ISSN: 0016-5085 *

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