WO2009001032A1 - Modulation of inflammation - Google Patents

Abstract

The invention relates to the use of a Trb-2 modulator in the preparation of a medicament for the treatment of an inflammatory disease or disorder, for example ACS.

Description

Modulation of Inflammation

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.

Elevated serum LDL levels have been implicated as a major risk factor in the development of atherosclerosis. Expression of inflammatory cytokines, chemokines by a variety of vascular cells, proliferation of vascular smooth muscle cells (Yang, CM., et al Br J Pharmacol. 132:1531-1541), migration of monocytes (Jing, Q. et al Circ Res. 87:52- 59.) and the differentiation of macrophages into foam cells (Zhao, M. et al. APMIS. 110:458-468) have ail been linked to LDL action. The above studies also demonstrate a fundamental role for Mitogen Activated Protein Kinase (MAPK) pathways in these processes.

Differentiation of monocyte/macrophages to foam cells is a hallmark of the development of atherosclerotic lesions/plaques. Rupture of the mature plaques of the vessel wall leads to one of the most common file threatening conditions in the western world, the Acute Coronary Syndromes, in part mediated by the pro-inflammatory activation of monocytes. A number of studies have demonstrated that MAPK pathways play a central role in both of these processes. In particular, the ERK and JNK pathways were reported to be important for LDL mediated monocyte/macrophage function (Ricci, R. et al. Science. 306:1558-1561 , Sumara, G. et al. Ce// MoI Life Sci. 62:2487-2494, Miller, Y.I. et al. Aiierioscler Thromb Vase Biol. 25:1213-1219). Whilst the involvement biological importance of these MAPK pathways is relatively well characterised, the molecular mechanisms which modulate the ability of monocyte/macrophages to respond to inflammatory stimuli in an LDL dependent fashion are poorly understood.

A novel protein family, human tribbles (trb) (Kiss-Toth, et al (2005) Biochem Soc Trans 33, 1405-1406, Kiss-Toth, et al (2006) Cellular Signalling 18, 202-214) has been identified as regulators of MAPKK activity (Kiss-Toth, et al(2004) J Biol Chem 279, 42703-42708)

The role of trb-2 in monocyte biology and the functional links between LDL, inflammatory activation of monocytes and trb-2 has been investigated in the present application. 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. MoI Immunol. 42:1275-1261), and in inflammatory conditions (Sung, H.Y. et al. Immunol Lett. 104:171-177). Functional studies on trb-2 have demonstrated that it is involved in the progression of mitosis in Xenopus embryos and that it is necessary for the normal development of the eye and the neouronal system (Saka, Y. et al. Dev Biol. 273:210-225). A recent publication by Keeshan et al. also suggests a role for trb-2 in cell division by showing that this gene is up-regulated in a subset of acute myeloid leukemias (AML) and that retroviral overexpresison of trb-2 induces AML in mice possibly via enhancing degradation of certain C/EBP protein forms (Keeshan, K. et al. Cancer Cell. 10:401-411). These results are in line with previous work in Drosophila, where Roth et al. have demonstrated that levels of the fly homologue of C/EBP (slbo) are critical for programmed mitosis and that slbo turnover is regulated by tribbles (Rorth, P. et al. MoI Cell. 6:23-30).

However, 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).

Previous research has attempted to interfere by LDL induced cellular events, such as macrophage proliferation, via the inhibition of p38 MAPK pathways (Senokuchi, T. et al. J Biol Chem. 280:6627-6633), for example with the use of statins. Accordingly, there remains a need to provide methods and composition to modulate LDL induced cellular events, in particular LDL induced IL-8 production.

The inventors have identified that 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.

BRIEF SUMMARY OF THE DISCLOSURE

In a first aspect 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. In one embodiment, the inflammatory disease or disorder is mediated by monocyte IL-8 production.

In a preferred embodiment, the inflammatory disease or disorder is a coronary disease, preferably acute coronary syndrome.

In a further aspect, the invention provides a trb-2 modulator for use as a medicament.

In a further aspect the invention provide a trb-2 modulator for use as a modulator of inflammation.

In a still further aspect the invention provides a trb-2 modulator for use as a modulator of monocyte response to an inflammatory stimulus.

In a further embodiment the invention provides a method of treating an inflammatory disease or disorder comprising administering to a subject in need thereof a trb-2 modulator. In one embodiment, the inflammatory disease or disorder is mediated by monocyte IL-8 production.

In a preferred embodiment, the inflammatory disease or disorder is a coronary disease, preferably acute coronary syndrome. Preferably, the modulator up regulates Trb-2 expression or activity.

In a preferred embodiment,- the Trb-2 modulator upregulates trb-2 binding to MEK1 or MKK7.

In a preferred embodiment, 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.

Still more preferably, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 3.

Alternatively, said modulator is an isolated nucleic acid molecule encoding a Trb-2 protein or a fragment thereof. Preferably, 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 of SEQ ID NO:1 or SEQ ID NO:3.

In a further aspect 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.

Preferably the assay is a cell based assay, more preferably, a monocytic cell based assay.

In a further aspect, 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. Preferably, the method is an in vitro method.

Preferably, the cell is a mammalian cell, more preferably, a human cell.

Preferably, the inflammatory stimulus comprises low-density lipoprotein (LDL).

Preferably, the modulation of cellular inflammatory response is a decrease.

Preferably, the compound up regulates Trb-2 expression or activity,

Preferably, the compound is small molecules that stimulates the binding activity of TRB- 2 to MEK1 or IVJKK7.

Preferably, 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.

Alternatively, the compound is an isolated nucleic acid molecule encoding an active Trb- 2 protein or a fragment thereof. Preferably, 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 of SEQ ID NO:1 or SEQ ID NO:3.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

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.

Figure 4. is a graphical representation showing that Trb-2 is an inhibitor of the activation of JNK and ERK pathways in THP-1 cells. (A) 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 β-actin and plotted on the right panels. (B) 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. (A) MEK1 , MKK4 and MKK7 are endogenously expressed in THP-1 cells and their expression levels can be downregulated by specific siRNAs. (B) THP-1 cells were transfected with siMAPKK or control siRNA, as indicated and stimulated by LPS. Production of IL-8 was detected by ELISA.

(C) PCA was performed in Raw264.7 cells using the Trb-2-V2 and MAPKK-V1 fusion protein expressing constructs. pEGFP-N2 and zip-V1/zip-V2 were used as positive controls for transfection efficiency (>30%) and PCA, respectively. A representative image for each PCA positive samples is shown. (D) Interaction between trb-2 and MAPKKs was also investigated in THP-1 cells, using the above constructs and analysied by FACS. Abbreviations: Z - zip, T - trb, M - MAPKK. Cotransfection of zip-v1/Trb2-V2 was used as a negative control and zip-v1/zip-v2 as a positive control. (E) FACS was used to show that the specificity of interaction between Trb-2 and MEK1. Three doses of untagged Trb-2 expression plasmid (relative to the amount of Trb2-V2) were cotransfected (1/10, 1/1 and 10/1) with the above PCA constructs and the mean .fluorescence intensity was calculated in the varying samples.

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.

(A) Responsiveness of primary monocytes from four healthy volunteers were assessed by stimulating cells with LPS or with the combination of LPS and AcLDL. IL-8 levels, in response to LPS alone were used as a unit for normalisation for each donor and values measured in samples with LPS-AcLDL costimulation were expressed relative to these. AcLDL treatment alone did not induce the production of IL-8 (not shown). (B) The relationship between the amount of IL-8 produced (LPS + AcLDL treatment) and trb-2 expresison was measured by linear regression. (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.

(A) AcLDL uptake by monocytes triggers reduction of Trb-2 expression, resulting in a hypersensitive state towards inflammatory stimuli, as exemplified by LPS produced IL-8 production. (B) The molecular basis of trbr2 regulatory function of MAPKK pathways in the expression of IL-8.

Figure 8 depicts the amino acid sequence of the trb-2 polypeptide, designated SEQ ID

NO:1.

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. 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.

The experimental evidence_, disclosed herein demonstrates that 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.

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.

The inventors have show that 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. Moreover, 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.

The results disclosed herein demonstrate that expression levels of the trb-2 gene product are regulated by AcLDL, sensitising monocytes towards inflammatory signals and, therefore, enabling them to produce elevated levels of a key inflammatory chemokine, IL-8. 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.

As used herein, 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. Preferably 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.

As used herein, the term "inflammatory agent", "inflammatory stimulus" or "inflammatory stimuli" refers to a compound capable of inducing an immune response. For example, that inflammatory stimulus may comprise low density lipoprotein (LDL), acLDL,, inflammatory cytokines, such as IL-1 , IL18, TNFα, lipopolysaccharide (LPS) or other TLR agonists, such as Lipoteichoic Acid (LTA) or CpG DNA.

As used herein, the term "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.

As used herein, the term "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.

Compounds or agents that have a stimulatory effect upon expression of a 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.

Compounds or agents that have inhibitory effect upon effect upon expression of a Trb-2 nucleic acid molecule or activity of a Trb-2 polypeptide (also referred to as "inhibitors") include small molecules that inhibit Trb-2 MEK1 or MKK7 binding activity, antisense trb- 2 nucleic acid molecules and anti-trb-2 antibodies.

As used herein, the term "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.

It is preferred that 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.

In order to be sufficiently selective and potent for inhibition, such 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.

Although 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. In addition, 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.

The term "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).

Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such 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. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.

In one embodiment, 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. In a further preferred embodiment of the invention the therapeutic molecule is an inhibitory RNA (sfRNA). siRNA 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). Typically 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.

In one embodiment, inhibition is associated with pretranscriptional silencing.

In one embodiment of the invention 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. Thus, 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).

In one embodiment of the invention the inhibitor is an antibody, or at least an effective binding part thereof, which binds to a trb-2 polypeptide according to the invention. The term "antibody" as used herein 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 (Ig) 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 λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), 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. 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, α, μ, σ, α, and γ (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. Thus, there are five 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). Further detail regarding antibody structure and their various functions can be found in, Using Antibodies: A laboratory manual, Cold Spring Harbour Laboratory Press.

The antibody may be a polyclonal or a monoclonal antibody that binds trb-2. As used herein, 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 immunσreacts.

Preferably, 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. In a further embodiment of the invention, said antibody is humanised by recombinant methods to combine the complimentarity determining regions of said antibody with both the constant

(C) regions and the framework regions from the variable (V) regions of a human antibody.

Preferably, 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. Alternatively, 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. The complimentarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V-region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.

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.

Preferably the cells are monocytic cells, i.e. monocytes or macrophages.

Alternatively, 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 β cell; epithelial cell (eg lung, gastric,) ; and a endothelial cell. Preferably, the cell is an endothelial cell. Preferably the cells are mammalian cells. More preferably the cells are human.

In one embodiment, 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 Alternatively, 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.

In one embodiment the administration of 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.

In one embodiment 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. Preferably 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 addition, 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.

In one embodiment, 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.

As used herein, 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.

Diseases and disorders that may be treated by modulating the expression or activity of a trb-2 nucleic acid molecule or polypeptide include inflammatory disorders, more preferably 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).

Acute coronary syndromes include Unstable Angina (UA), Non-ST Segment Elevation Myocardial Infarction (NSTEMI), and ST Segment Elevation Myocardial Infarction (STEMI).

Alternatively, the disorder is a vascular inflammatory disorder, for example atherosclerosis, heart disease, stroke, angina, thrombosis, myocardial infarction, ischemic heart disease, congestive heart failure. In one embodiment 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.

According to a yet further aspect of the invention there is provided a method of treatment of a mammal, preferably a human, comprising administering to said mammal a modulator according to the invention.

Accordingly, in vivo modulation provides both prophylactic and therapeutic methods.

The trb-2 modulators of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the trb-2 modulator and a pharmaceutically acceptable carrier. The term "pharmaceutically- acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.

When administered, the pharmaceutical 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.

The 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.

The 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.

The 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.

The 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).

As used herein, the term "trb-2 nucleic acid" refers to a nucleic acid sequence encoding a Trb-2 protein. As used herein, the terms "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.

In a preferred embodiment 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).

Alternatively 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).

In one embodiment the trb-2 nucleic acid molecule is an isolated nucleic acid molecule. With regards to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, 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.

Moreover, 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.

Further trb-2 nucleic acid molecules of the present invention are described below. In one embodiment, 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.

In one embodiment 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. In one embodiment the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 or SEQ ID NO:3. In yet another embodiment, 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.

In another embodiment, 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.

As used herein, the term "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 6χ sodium chloride/sodium citrate (SSC) at about 45⁰C, followed by one or more washes in 0.2χ SSC, 0.1% (w/v) SDS at 5O⁰C. Another example of stringent hybridization conditions are hybridization in 6χ SSC at about 45°C, followed by one or more washes in 0.2χ SSC, 0.1% (w/v) SDS at 55°C. A further example of stringent hybridization conditions are hybridization in 6^χ SSC at about 45°C, followed by one or more washes in 0.2χ SSC, 0.1% (w/v) SDS at 60⁰C. Preferably, stringent hybridization conditions are hybridization in 6x SSC at about 45°C, followed by one or more washes in 0.2χ SSC, 0.1% (w/v) SDS at 65⁰C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention) are 0.5 molar sodium phosphate, 7% (w/v) SDS at 65⁰C, followed by one or more washes at 0.2^χ SSC, 1% (w/v) SΘS at 65°C. Preferably, a trb-2 nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:2 or 4.

In one embodiment, 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.

In another embodiment, 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.

Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, 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. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein 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. In a preferred embodiment, 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, In yet another preferred embodiment, 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 (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) 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.

In one embodiment 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.

As used herein, 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).

In another embodiment 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. Preferably, 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. One example of 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.

In one embodiment the trb-2 nucleic acid molecule is a trb-2 encoding gene. As used herein, 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.

In one embodiment a trb-2 polypeptide is the polypeptide of SEQ ID NO:1. The sequence is approximately 343 amino acid residues in length.

In one embodiment a trb-2 polypeptide is the polypeptide of SEQ ID NO:3. The sequence is approximately 249 amino acid residues in length.

In one embodiment 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. As used herein, 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. For example, 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. Accordingly, 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.

In one embodiment 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.

In one embodiment the trb-2 protein is a chimeric or fusion protein. As used herein, a trb-2 "chimeric protein" or "fusion protein" comprises a trb-2 polypeptide operably linked to a non-trb-2 polypeptide. In a fusion protein 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.

A "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. For example, 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.

In one aspect 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.

Examples

LDL potentiates LPS induced IL-8 production and reduces Trb-2 expression In order to characterise molecular regulators of monocytes responses in inflammatory settings and the modulation of this process by LDL, the dynamics of acetylated LDL (AcLDL) uptake by THP-1 cells (human macrophage derived cells) was characterized. THP-1 and Raw 264.7 cells were purchased from ATCC and maintained in RPMI (Gibco) supplemented with 10% foetal calf serum (FCS), (L-glutamine) and penicillin- streptomycin.

Cells were incubated with Dil-labelled acLDL and 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 ,5x10⁵ THP-1 cells were treated with 5μg/ml Dil-labeled acLDL for 1 , 2, 4 and 24h and assayed by flow cytometry.

Whilst the uptake of LDL was readily detected after a short incubation (1-4 hrs), markedly elevated fluorescent signal was seen after 24 hrs of treatment. In order to assess the functional consequences of AcLDL uptake in modulating monocyte inflammatory responses, THP-1 cells were treated with AcLDL for 24 hrs, in the presence or absence of LPS. IL-8 production was measured by ELISA as a biologically relevant marker of inflammatory activation of monocytes. The results show that AcLDL treatment potentiates LPS induced IL-8 production in these cells (Fig. 1B). In order to demonstrates that the expression of IL-8 is regulated by MAPK pathways it has been demonstrated that blocking of JNK or MEK1 but not of p38 activity results in the inhibition of IL-8 production (Fig. 1C).

A transient regulation of trb-1 and trb-2 expression in THP-1 cells, in response to IL-1β stimulation (Figure 2A) was observed. As tribbles appear to be negative regulators of signalling, the observed downregulation of trb-2 by LPS suggested that this member of the tribbles family may be involved in regulating monocyte activation. In line with this, AcLDL treatment of THP-1 cells led to a marked decrease in trb-2 expression levels (Figure 2B). Detection of endogenous trb-2 protein in parallel samples by using polyclonal antisera, raised against the N-terminal region of trb-2 was also attempted. Detection of trb-2 was successful in cells transfected with a trb-2 expression plasmid. Reduced Trb-2 levels lead to elevated IL-8 production via the JNK and ERK MAPK pathways

To examine whether the observed down-regulation of trb-2 by AcLDL was involved in potentiating LPS induced IL-8 production, trb-2 levels were suppressed by transfecting siRNA against trb-2 (Fig. 3A). Transfections were performed using Nucleofector

(Amaxa) using program U-001 and Cell line Nucleofector Kit V solution (Amaxa). For most experiments, 1.0 * 10⁶ cells were used per nucleofection. siRNA SmartPool against human trb-2, MKK4, MKK7 and MEK1 were purchased from Dharmacon and used according to the manufacturer's recommendation.

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.

As MAPK pathways have previously been shown to play a major role in regulating the expression of IL-8 (5, 18-21) 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 20μM for MEK1 and JNK MAPK inhibitors and 0.2μM 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.

Altered trb-2 levels lead to modulated MAPK activation

In order to gain a mechanistic inside into the regulatory action of trb-2 in IL-8 expression, the effects of overexpressed trb-2 and sitrb-2 on ERK and JNK activation levels were investigated in LPS stimulated THP-1 cells (Fig. 4). Western blotting results showed that overexpressed trb-2 attenuates LPS dependent activation of ERK: in control transfected cells phospho-ERK (pERK) and phospho-JNK (pJNK) levels increased upon LPS treatment over the period of 60 minutes. However, trb-2 overexpression markedly decreased the activation of both MAPKs (Fig. 5A). Interestingly, both pJNK and pERK levels have risen in unstimulated, trb-2 overexpressing cells, compared to controls and LPS stimulation of these cells led to a time dependent decrease of pMAPK levels. In contrast, sitrb-2 treatment potentiated LPS dependent ERK and JNK activation (Fig. 5B). These observations confirm a negative regulatory role for trb-2 in control of ERK and JNK activation.

Trb-2 modulates IL-8 production via interaction with MAPKKs

The above experiments showed that reduced Trb-2 levels lead to elevated IL-8 production through enhanced activation of the JNK and ERK pathways. The interaction of trb-2 with MKK4/SEK-1 , MKK7 and MEK-1 , known activators of JNK or ERK, was therefore investigated. 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). 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.

The physically interactions of trb-2 with MAPKKs in monocytic cells was also investigated, using protein complementation assay (PCA) as described previously by Michnick and colleagues (Remy, I., and Michnick, 2004. Methods. 32:381-388).

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.

The results showed 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

The potential in vivo relevance of these findings was investigated by assessing the ability of human primary monocytes to produce IL-8 in response to LPS stimulation. As shown in Figure 6A, AcLDL increased the amount of IL-8 produced in response to LPS, similar to that seen in monocytic cell lines (Fig. 1 B). Furthermore, the level of IL-8 inversely correlated to the expression of trb-2 in primary monocytes (Fig. 6B). These data are in agreement with findings, described above in THP-1 cells, confirming that trb- 2 is a major regulator of monocytes responses in inflammatory settings.

Given that monocyte activation is a central event in the development of progression of coronary disease, the expression of tribbles in an inflammatory heart disease (Carter, A.M. 2OO5.D/a/b Vase Dis Res. 2:113-121 , Cirillo, M. 2006. J Thromb Haemost 4:2248- 2255, Meuwissen, M.. et al J CHn Pathol. 59:196-201 , Schieffer, B et al, Circulation. 110:3493-350, van Haeist, P.L et al, CHn Exp Immunol. 138:364-368), in Acute Coronary Syndromes, was investigated. Levels of gene expression were compared to samples obtained from patients with Chronic Stable Angina.

Total RNA was extracted from whole blood of patients with unstable angina and chronic stable angina using QIAamp RNA blood minikit (Qiagen) according to the manufacturer's description, htrb-1 , 2, 3, IL-1β, IL-ra and β-actin genes were analyzed by

Quantitative real time PCR using ABI prism 7900 sequence detection system (Applied

Biosystems). The sequences of all primers and probes used are listed in Table! To quantify transcript levels of the various genes, β-actin was used as a house keeping control, and each sample was normalized with respect to its β-actin transcript content.

Table 1

In line with previous findings, the balance of IL-1β/IL-1ra was altered in the Acute disease, highlighting the inflammatory nature of this condition. In addition, selective down-regulation of trb-2 was observed in these samples. This finding is compatible with the model, whereby the responsiveness of monocytes under inflammatory conditions is controlled through trb-2.

Mutation of a conserved serine (S239) in trb-2 abolishes MKK/trb-2 binding

The impact of elimination of putative phosphorylation sites on the ability of trib-2 to bind to MKKs and to inhibit their respective function was tested. Multiple alignments of tribbles orthologues identified six serine residues which are conserved in trb-1 , -2 and -3 across a range of mammalian species. In addition, a number of the residues were found to be present in Drosophila tribbles orthologues (Figure 12). An alanine scanning mutagenesis (S→A) was carried out to exchange the conserved serine residues in trb-2. The single amino acid mutations (S→A) on trbs-2-V2, were generated using Fusion Site-directed Mutation Cloning kits (New England Biolabs). The positions of the individual single residue mutations are illustrated in figure 12.

The resulting mutants were tested for their ability to bind to MEK1 or MKK4 in a PCA assay. Fluorescent microscopy showed the S239A trb2 mutant was no longer able to bind to the MKK proteins (Figure 13A). The present data confirms by western blotting that the wild type and S239A mutant trb-2 proteins are expressed at similar levels and thus the lack of binding is not due to poor expression of the mutant (Figure 13B).

Focussing on S239A mutant, the data shows that the mutants inability to bind to MKKs is generic (Figure 13C).

The data also demonstrates that 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. Moreover, the data demonstrates that an equal dose of the trb2 S239A exerted a significantly lower inhibitory effect than wt trb2 (figure 13E). 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.

For FACS analysis: HeLa cells were seeded onto 24-well plates. In total 400ng of plasmids were transfected per well (per sample). When two plasmids were co- transfected, 200ng of each plasmid were used. 24h after transfection, the cells were trysinized, fixed and resuspended. Flow cytomtric analysis (FACS) was performed on a FACSCalibre (Becton Dickson, USA) following the general guidelines from the manufacturer. Data were analysed using Cell Quest Pro software (Becton Dickinson, USA). The fluorescent intensity values were normalised by subtracting the background fluorescent value for the mock transfected cells from the FL1 (green) arithmetic mean fluorescence values for individual test samples. The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed

Claims

1. Use of a Trb-2 modulator in the preparation of a medicament for the treatment of an inflammatory disease or disorder.

2. Use according to claim 1 , wherein the inflammatory disease or disorder is mediated by monocyte IL-8 production.

3. Use according to claim 1 or 2, wherein the inflammatory disease or disorder is a coronary disease.

4. Use according to claim 3, wherein the coronary disease is acute coronary syndrome.

5. Use according to anyone of claims 1 to 4, wherein the Trb-2 modulator upregulates trb-2 binding to MEK1 or MKK7.

6. Use according to anyone of claims 1 to 4, wherein the modulator is an an active Trb-2 protein or a fragment thereof.

7. Use according to anyone of claims 1 to 4, wherein 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 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.

8. Use according to claim 7, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 3.

9. Use according to any one of claims 1 to 4, wherein said modulator is an isolated nucleic acid molecule encoding a Trb-2 protein or a fragment thereof.

10. Use according to claim 9, wherein 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.

11. Use according to claim 10, wherein the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4.

12. Use according to claim 10, wherein the isolated nucleic acid molecule encodes a polypeptide of SEQ ID NO:1 or SEQ ID NO:3.

13. A trb-2 modulator for use as a medicament.

14. A trb-2 modulator for use as a modulator of inflammation.

15. A trb-2 modulator for use as a modulator of monocyte response to an inflammatory stimulus.

16. A trb-2 modulator according to any one of claims 13, 14 or 15, wherein the modulator up regulates Trb-2 expression or activity.

17. A trb-2 modulator according to claim 16, wherein the Trb-2 modulator upregulates trb-2 binding to MEK1 or MKK7.

18. A trb-2 modulator according to claim 16, wherein the modulator is an an active Trb-2 protein or a fragment thereof.

19. A trb-2 modulator according to claim 16 wherein 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 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.

20. A trb-2 modulator according to claim 19, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 3.

21. A trb-2 modulator according to claim 16, wherein said modulator is an isolated nucleic acid molecule encoding a Trb-2 protein or a fragment thereof.

22. A trb-2 modulator according to claim 16, wherein 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.

23. A trb-2 modulator according to claim 21, wherein the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4.

24. A trb-2 modulator according to claim 21, wherein the isolated nucleic acid molecule encodes a polypeptide of SEQ ID NO:1 or SEQ ID NO:3.

25. 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.

26. A method according to claim 25, wherein the assay is a cell based assay.

27. A method according to claim 26, wherein the cell based assay is a monocyte based assay.

28. 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.

29. A method according to claim 28, wherein the method is an in vitro method.

30. A method according to claim 28 or 29, wherein the cell is a mammalian cell.

31. A method according to claim 30, wherein the mammalian cell is a human cell.

32. A method according to any one of claims 28 to 31 , wherein the inflammatory stimuli comprises low-density lipoprotein (LDL).

33. A method according to any one of claims 28 to 32, wherein said modulation of cellular inflammatory response is a decrease.

34. A method according to claim 33, wherein said compound up regulates Trb-2 expression or activity.

35. A method according to claim 34, wherein said compound is small molecules that stimulates the binding activity of TRB-2 to MEK1 or MKK7.

36. A method according to claim 34, wherein said compound is an active Trb-2 protein or a fragment thereof.

37. A method according to claim 34 wherein 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.

38. A method according to claim 37, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 3.

39. A method according to claim 34, wherein said compound is an isolated nucleic acid molecule encoding an active Trb-2 protein or a fragment thereof.

40. A method according to claim 39, wherein 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.

41. A method according to claim 40, wherein the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4.

42. A method according to claim 40, wherein the isolated nucleic acid molecule encodes a polypeptide of SEQ ID NO:1 or SEQ ID NO:3.

43. Use of a Trb-2 modulator in the preparation of a medicament as hereinbefore described with reference to the accompanying drawings.

44. A method of modulating monocyte response to an inflammatory stimulus as hereinbefore described with reference to the accompanying drawings.

45. A method of identifying a compound capable of modulating inflammation as hereinbefore described with reference to the accompanying drawings.