AU2001240346B2

AU2001240346B2 - Methods of regulating cytokine signalling

Info

Publication number: AU2001240346B2
Application number: AU2001240346A
Authority: AU
Inventors: Manuel Baca; Louis Fabri; Douglas Hilton; Andrew Nash; Sandra Elaine Nicholson; Nicos A. Nicola; Jian-Guo Zhang
Original assignee: Walter and Eliza Hall Institute of Medical Research
Current assignee: Walter and Eliza Hall Institute of Medical Research
Priority date: 2000-03-09
Filing date: 2001-03-09
Publication date: 2006-09-14
Anticipated expiration: 2021-03-09
Also published as: AU2001240346C1; AU4034601A

Description

WO 01/66128 PCT/AU01/00263 -1- METHODS OF REGULATING CYTOKINE SIGNALLING FIELD OF THE INVENTION The present invention relates generally to a method for regulating cytokine signalling and agents useful for same. The method of the present invention is predicated in part on the identification of the molecular target of suppressor of cytokine signalling (SOCS) interaction in controlling cytokine signalling. The identification of the molecular target permits the development of assays to screen for a range of agonists and antagonists useful in modulating cytokine function. The present invention further provides, therefore, screening assays and more particularly high through-put screening assays for agonists and antagonists of SOCS-receptor interaction. Such agonists and antagonists are useful in the manufacture of medicaments for controlling cytokine signalling. Control of cytokine signalling is important for the treatment of a range of conditions including cancer, inflammatory conditions, immunological disorders and any other conditions involving aberrations of signal transduction.

BACKGROUND OF THE INVENTION Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

Cytokines control a wide variety of biological responses, thus the duration and intensity of their effects must be tightly regulated. Upon stimulation by cytokine, specific cell surface receptors oligomerise and cause activation of the JAK-STAT signalling pathway (Heim, 1999; Ihle et al., 1998; Leonard and O'Shea, 1998). The transient nature of this signalling cascade is partly a consequence of the action of negative regulatory molecules such as Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -2- SHP-1, PIAS-3 and the SOCS (suppressor of cytokine signalling) (alternate names SSI, JAB, CIS) proteins, each of which inhibit the JAK-STAT signalling pathway and ensure the appropriate level of response to a particular cytokine stimulus is maintained (Endo et al.. 1997; Gisselbrecht, 1999; Hilton, 1999; Naka et al., 1997; Starr et al., 1997; Yoshimura, 1998).

The SOCS family of proteins comprises a number of members, including SOCS-1 through SOCS-7 and CIS, and the expression of several of these is known to be induced by cyvtokines (Hilton, 1999; Hilton et al., 1998; Yoshimura, 1998). SOCS-7 and CIS contain two regions of homology a central SH2 domain and a C-terminal 40 amino acid motif known as the SOCS box. While the SOCS box acts to recruit elongins BC, a protein complex implicated in the proteasomal degradation pathway (Kamura et al., 1998; Zhang et al., 1999), the SH2 domains of the SOCS proteins are responsible for specific binding to activated (phosphorylated) signalling molecules and may also play a role in the mechanism of signal suppression. For instance, CIS binds via its SH2 domain to phosphorylated erythropoietin (EPO) and interleukin-3 (IL-3) receptors at the same sites used for binding, thus preventing docking and activation of this transcription factor (Matsumoto et al., 1997; Yoshimura et al., 1995). By contrast, the SH2 domain of SOCS-1 binds to activated JAKs, and together with the action of an additional protein interaction motif upstream of the SH2 domain, results in inhibition of the kinase catalytic activity (Nicholson et al., 1999; Sasaki et al., 1999).

A number of studies have identified cytokines which can induce the expression of SOCS-3 mRNA, including CNTF (Bjorbaek et al., 1999), LIF (Bousquet et al., 1999), IL-6 (Starr et al., 1997), IL-11 (Auernhammer and Melmed, 1999a), leptin (Bjorbaek et al., 1998), IL-2 (Cohney et al., 1999), IL-10 (Donnelly et al., 1999; Ito et al., 1999), prolactin (Pezet et al., 1999), growth hormone (Adams et al., 1998) and insulin. Overexpression of SOCS-3 results in the inhibition of signalling by each of these cytokines, and under these conditions SOCS-3 has been shown to physically associate with either JAK kinase (Sasaki et al., 1999), or the receptors for growth hormone (Hansen et al., 1999), IL-2R (Cohney et al., 1999), and insulin receptors. However, given that overexpression can lead to elevated Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 protein levels at which non-specific interactions can result, it is difficult to assess whether all of these observations have a genuine biological relevance. Alternatively, gene knockout studies have shown that SOCS-3' mice die embryonically from a disease possibly associated with excessive foetal erythropoiesis (Marine et al., 1999).

Recently, it was proposed that the mechanism by which SOCS-3 inhibits signalling is identical to that of SOCS-1. As had been demonstrated for SOCS-1 (Yasukawa et al., 1999), SOCS-3 was also shown to associate with JAK2 in intact cells, and to a synthetic phosphopeptide encompassing the activation loop from JAK2 (Sasaki et al., 1999). The region of SOCS-3 immediately N-terminal to the SH2 domain has also been shown to be important for biological activity (Narazaki et al., 1998; Nicholson et al., 1999; Sasaki et al., 1999), and based on sequence similarity to SOCS-1, was also proposed to function as a kinase active site inhibitor. However, despite these similarities, there is evidence to suggest that the mechanism of signalling suppression used by SOCS-3 differs from that of SOCS- 1. Unlike SOCS-1, SOCS-3 does not inhibit the catalytic activity of JAK1 or JAK2 in an in vitro kinase reaction (Nicholson et al., 1999). Furthermore, the kinetics of IL-6 signal suppression, as measured by inhibition of STAT3 phosphorylation, is considerably slower for SOCS-3 as compared to SOCS-1 (Suzuki et al., 1998). While forced expression of SOCS-1 in M1 cells results in rapid and total inhibition of STAT3 phosphorylation within 30 minutes, inhibition by SOCS-3 slowly increases over the course of several hours (Suzuki et al., 1998). These differences between SOCS-1 and SOCS-3 have been explained on the basis of a weaker affinity of SOCS-3 for JAK kinase (Masuhara et al., 1997; Sasaki et al., 1999). However, another possible explanation is that the primary binding target for SOCS-3 is not the JAK kinase, but other molecules within the signalling cascade such as the phosphorylated cytokine receptors or STAT proteins.

In work leading up to the present invention, the inventors sought to identify the molecular target of SOCS-3 and quantify the affinity of this interaction. Based on peptide binding data, the subject inventors have demonstrated that a single high affinity binding site exists for SOCS-3 on the gpl30 receptor, centred around phosphotyrosine-757 (pY757). The letter before a single letter abbreviation for an amino acid for tyrosine) means Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -4the amino acid is phosphorylated. The numerical value after the amino acid letter is the residue number. Binding is phosphorylation dependent, and suppression of signalling by SOCS-3, but not SOCS-1, is impaired if this residue is mutated to phenylalanine. Furthermore, SOCS-3 binds to a gpl30-derived pY757 phosphopeptide with an affinity that is approximately 10 4 -fold higher than binding to a phosphopeptide derived from the activation loop in JAK2, previously reported to be the biologically relevant docking site for SOCS-3 (Sasaki et al., 1999).

Taken together, these data show that suppression of gp 130-mediated signalling by SOCS-3 involves recruitment to the phosphorylated receptor in a manner that is distinct to the mechanism of inhibition used by SOCS-1. These data also have similar implications in relation to other cytokine receptors such as erythropoietin (EPO) receptor and leptin receptor which have phosphotyrosine regions homologous to those recognized by SOCS-3 on Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1, <400>2, etc. A sequence listing is provided after the claims.

One aspect of the present invention relates to a method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS molecule and a region within a cytoplasmic domain of a receptor for said cytokine or related molecule.

Another aspect of the present invention contemplates a method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS-3 or related molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule.

A further aspect of the present invention provides a method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS-3 or related molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule wherein said region comprises a phosphorylated tyrosine.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -6- Still another aspect of the present invention contemplates a method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS-3 or related molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule wherein the receptor is selected from gp130, leptin receptor, EPO receptor or any other receptor having a region homologous to the amino acid sequence in the cytoplasmic domain of gpl30 to which SOCS-3 binds.

Yet another aspect of the present invention contemplates a method of inhibiting signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of an agonist of interaction between a SOCS molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule.

Still yet another aspect of the present invention provides a method of facilitating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of an antagonist of interaction between a SOCS molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule.

Even still another aspect of the present invention contemplates a method of identifying an agent capable of modulating signalling of a cytokine or related molecule, said method comprising screening for agents which are capable of interfering or otherwise antagonizing or promoting or otherwise agonizing interaction between a SOCS molecule and a cytoplasmic domain of a receptor for said cytokine or related molecule.

Even yet another aspect of the present invention contemplates a method for identifying a modulator of interaction between a SOCS molecule or part, fragment, or derivative or homologue or analogue thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:- Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -7immobilizing one of said SOCS molecules or receptor or their parts, fragments, derivatives, homologues or analogues to a solid support via binding partners wherein one of said binding partners is attached or otherwise anchored to said solid support and another of said binding partners is linked to said SOCS or receptor molecules; contacting said immobilized SOCS or receptor molecule with the other of receptor or SOCS molecule in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.

Another aspect of the present invention provides a method for identifying a modulator of interaction between a SOCS molecule or part, fragment, or derivative, or homologue or analogue thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:immobilizing said receptor or its parts, fragments, derivatives, homologues or analogues to a solid support via binding partners wherein one of said binding partners is attached or otherwise anchored to said solid support and another of said binding partners is linked to said receptor molecule; contacting said immobilized receptor molecule with said SOCS molecule or its parts, fragments, derivatives, homologues or analogues in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -8- A further aspect of the present invention provides a method for identifying a modulator of interaction between a SOCS molecule or part, fragment, or derivative, or homologue or analogue thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:immobilizing one of said SOCS molecules or receptor or their parts, fragments, derivatives, homologues or analogues to a solid support via binding partners comprising biotin and streptavidin wherein one of said biotin or streptavidin is attached or otherwise anchored to said solid support and the another of said biotin or streptavidin is linked to said SOCS or receptor molecule; contacting said immobilized SOCS or receptor molecule with the other of receptor or SOCS molecule in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.

Still another aspect of the present invention provides a method for identifying a modulator of interaction between a SOCS molecule or part, fragment, or derivative, or homologue or analogue thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:immobilizing said receptor or its parts, fragments, derivatives, homologues or analogues to a solid support via binding partners comprising biotin and streptavidin wherein streptavidin is attached or otherwise anchored to said solid support and said biotin is linked to said receptor molecule; contacting said immobilized receptor molecule with said SOCS molecule or its parts, fragments, derivatives, homologues or analogues in the presence of a potential agonist or antagonist; and Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -9measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.

Yet another aspect of the present invention contemplates a composition comprising a modulator of interaction between a SOCS molecule and a cytoplasmic domain of a receptor molecule, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a photographic representation showing that gpl30-derived phosphopeptide interacts specifically with SOCS-3. Biotinylated phosphopeptides were synthesized corresponding to the regions of murine gpl30 surrounding each cytoplasmic tyrosine residue (a pY812 peptide was not synthesized, as this tyrosine is not conserved in human or to regions surrounding the known tyrosine phosphorylation sites in STAT-1 and STAT-3. Peptides shown in were immobilized on streptavidin-agarose resin and incubated with recombinant SOCS-3. After washing and elution of bound protein, samples were analyzed by SDS-PAGE. Only the gpl30 pY757 peptide showed any significant binding of SOCS-3 as detected by SDS-PAGE analysis of the resin eluates.

Figure 2 is a graphical representation showing functional characterization of SOCS-3 protein. Biosensor analysis of SOCS-3 binding to the gpl30-derived phosphopeptide biotin-STASTVEpYSTVVHSG. [SEQ ID NO:4] Sensorgrams correspond to a 2-fold serial dilution series of SOCS-3 (8.65-1110 nM) passed over a chip bearing the immobilized peptide. Determination of binding constants by Scatchard-type analysis.

Data shown in was used to calculate the association constant for SOCS-3 binding to the immobilized gpl30 peptide. Plateau response values at equilibrium (RU) for the four highest concentrations of SOCS-3 used were plotted against the ratio of response to SOCS- 3 concentration The association binding constant (KA 2.38 x 107 M 1 was calculated from the slope of the plot of RU/c versus RU. The dissociation constant I/KA 42 nM.

Figure 3 is a graphical representation showing a comparison of SOCS-3 affinity for phosphorylated versus non-phosphorylated gpl30 peptide, and JAK-derived peptides. (A) Solution binding of phosphorylated and non-phosphorylated forms of gpl30 (750-764) peptide to SOCS-3 was measured in a competitive binding assay. The IC50's for inhibition of SOCS-3 binding to immobilized ligand were 110 4.6 nM for the phosphorylated peptide and 2.1 0.2 mM for the unphosphorylated peptide. Solution binding of JAKderived peptides to SOCS-3 measured in the competitive binding assay. These phosphopeptides represent the activation loop sequences in JAK1, 2 and 3 and contain a Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 11 phosphotyrosine residue corresponding to the autophosphorylation site. The amino acid sequences of these peptides were JAKI: IETDKE(pY)YTVKDDRD (SEQ ID NO:1), JAK2: LPQDKE(pY)YKVKEPGE (SEQ ID NO:2), JAK3: LPLGKD(pY)YVVREPGQ (SEQ ID NO:3). The IC50 values for these peptides were: JAK1, 230 6.6 mM; JAK2, 1200 50 mM; JAK3, 140 4.8 mM. The IC50 value for the gpl30 (750-764) phosphopeptide, run in the same experiment, was 110 4.6 nM.

Figure 4 is a photographic representation showing that SOCS-3, but not SOCS-1, inhibition of gpl30 signalling is mediated through gpl30 tyrosine 757. 293T cells were transiently transfected with cDNAs expressing SOCS-1 and either EPO/gpl30 or EPO/gp130Y757F in the presence of the APRE-luc and Sra-p-gal reporter genes. Cells were incubated in the presence or absence of 10 U/ml hEPO overnight and cell extracts prepared. Luciferase activity from triplicate samples was determined and normalized against f-galactosidase activity. SOCS-1 protein levels in 293T cells expressing EPOigpl30 were determined by Western blot with anti-Flag antibody. (C) SOCS-1 protein levels in 293T cells expressing EPO/gpl30Y757F. 293T cells were transiently transfected with cDNAs expressing SOCS-3 and either EPO/gpl30 or EPO/gpl30Y757F in the presence of the APRE-luc and Sra-p-gal reporter genes. (E) SOCS-3 protein levels in 293T cells expressing EPO/gpl30 were determined by Western blot with anti-Flag antibody. SOCS-3 protein levels in 293T cells expressing EPO/gpl30Y757F.

Figure 5 is a representation showing a comparison of murine and human gpl30, human leptin and human EPO phosphopeptide binding sites.

Figure 6 is a graphical representation of human SOCS-3 binding to the phosphopeptides murine gpl30 (pY757) human EPO (pY601) and human leptin (pY985).

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 12- DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated in part on the identification of the molecular target of a SOCS molecule. In particular, the molecular target is determined to be a region on the cytoplasmic domain of a receptor for the cytokine. The identification of this molecular target permits the development of assays to identify agonists and antagonists of cytokines or related molecule-mediated signal transduction. Such agonists and antagonists are useful in the manufacture of medicaments for the treatment of conditions involving aberrations in signal transduction.

Accordingly, one aspect of the present invention relates to a method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS molecule and a region within a cytoplasmic domain of a receptor for said cytokine or related molecule.

The present invention is particularly directed to the SOCS-3 molecule. This is done, however, with the understanding that the present invention extends to any SOCS molecule which regulates cytokine signalling via interaction with the cytoplasmic domain of a cytokine receptor. Most preferably, however, the SOCS molecule is SOCS-3.

Reference herein to a "SOCS-3" molecule includes all mutants and derivatives including parts and fragments thereof as well as homologues of SOCS-3. It also extends to analogues of SOCS-3 or analogues of parts or fragments of SOCS-3 which may be useful in assays for ligands. A "homologue" of SOCS-3 includes the equivalent or similar molecule from another species or a molecule which has similar physiological, biochemical, immunological or binding kinetic properties to SOCS-3. Identification of the species from which a particular SOCS-3 molecule is isolated is shown by the singles letters for human and for murine mouse).

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 13 Reference herein to a "cytokine" is used in its broadest sense and includes molecules related to cytokines at the functional, biological, immunological or biochemical levels. A functionally related molecule may, for example, interact with the same receptor as a cytokine. Examples of related molecules include growth factors, growth hormones, leptin and chemokines.

In one particularly preferred embodiment, the cytokine receptor is gp130 or a functionally or structurally related receptor. However, the present invention extends to the cytoplasmic domain of any cytokine receptor or related molecule including the EPO receptor and the leptin receptor. Reference herein to a "receptor' includes both receptors specific for a particular cytokine as well as non-specific receptors capable of interaction with the cytokine or related molecule.

In an even more particularly preferred embodiment, the region on gpl30 to which the SOCS-3 binds or otherwise interacts is a region spanning Y757 or other mammalian homologous regions in other receptors.

Accordingly, another aspect of the present invention contemplates a method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS-3 or related molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule.

Preferably the modulator inhibits SOCS-3 interaction with the cytoplasmic domain of a receptor.

Preferably the modulator inhibits SOCS-3 interaction with or proximal to a region comprising a phosphorylated tyrosine.

Accordingly, in a particularly preferred embodiment there is provided a method for regulating signalling of a cytokine or related molecule in an animal subject, said method Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -14comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS-3 or related molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule wherein said region comprises a phosphorylated tyrosine.

In a particularly preferred embodiment, the phosphorylated tyrosine is at position 757 pY757) or its functional equivalent in other receptors.

Examples of regions within particular receptors comprising a tyrosine which is capable of phosphorylation and which is proximal to where SOCS-3 or a related molecule binds include the following:- Y757 STASTVEYSTVVHSG [SEQ ID NO:4] Y757 NTSSTVQYSTVVHSG [SEQ ID leptin receptor Y985 QRQPFVKYATLISNS [SEQ ID NO:6] EPO receptor Y401 ASAASFEYTILDPSS [SEQ ID NO:7].

The terms "Y757", "Y985" and "Y401" relate to the residue number of the phosphorylated tvrosine residue.

Accordingly, another aspect of the present invention contemplates a method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS-3 or related molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule wherein the receptor is selected from gpl30, leptin receptor, EPO receptor or any other receptor having a region homologous to the amino acid sequence in the cytoplasmic domain of gpl30 to which SOCS-3 binds.

Examples of other receptors having homologous regions to those in gpl30 which interact with SOCS-3 include the IL-6 family of receptors such as those which interact with Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 leukemia inhibitory factor (LIF), CNTF, interleukins such as IL-6 and IL-11, OSM and cardiotrophin-1 (CT-1).

Reference to these specific receptors and target amino acid sequences includes reference to mutants and derivatives including fragments and parts thereof as well as homologues and analogues thereof.

The preferred animal subject of the present invention is a human, however, the present invention extends to primates, livestock animals sheep, pigs, cows, goats, horses), laboratory test animals mice, rats, guinea pigs, hamsters), companion animals (e.g.

dogs, cats) or captive wild animals.

The modulator of interaction between a SOCS molecule SOCS-3) and a receptor may be an agonist or antagonist. In essence, a molecule which promotes or otherwise facilitates SOCS binding is an agonist of SOCS but such a molecule acts to inhibit cytokine- or related molecule-mediated signal transduction. Conversely, an antagonist of SOCS interaction facilitates cytokine- or related molecule-mediated signal transduction.

Accordingly, another aspect of the present invention contemplates a method of inhibiting signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of an agonist of interaction between a SOCS molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule.

In an alternative embodiment, the present invention provides a method of facilitating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of an antagonist of interaction between a SOCS molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -16- Preferably, the SOCS molecule is SOCS-3 or a related molecule. More preferably, the SOCS molecule is SOCS-3. Preferably, the site of interaction between the SOCS molecule and the receptor comprises or is proximal to a site comprising a phosphorylated tyrosine.

In a particular preferred embodiment, the receptor and site of interaction is selected from:- Y757 STASTVEYSTVVHSG [SEQ ID NO:4] Y757 NTSSTVQYSTVVHSG [SEQ ID leptin receptor Y985 QRQPFVKYATLISNS [SEQ ID NO:6] EPO receptor Y401 ASAASFEYTILDPSS [SEQ ID NO:7], or their mammalian equivalents or homologues thereof or their mutants and derivatives including parts and fragments thereof.

Reference to equivalents, homologues, mutants and derivatives including parts and fragments thereof preferably includes regions carrying the SH2 domain. The present invention is particularly directed to the use of a phosphopeptide comprising an SH2 domain or equivalent region in the detection of antagonists and agonists in the interaction between this region and the cytoplasmic domain of a cytokine or related molecule receptor.

The agent capable of agonizing or antagonizing interaction between the SOCS molecule and the cytoplasmic domain of the cytokine receptor may be a proteinaceous or nonproteinaceous molecule. A proteinaceous molecule includes a peptide, polypeptide or protein or a complex thereof with, for example, a lipid, phospholipid or carbohydrate. A proteinaceous molecule may also be modified by the attachment of one or more nonproteinaceous sections or portions A non-proteinaceous molecule includes any organic chemical. Conveniently, the agent is identified following screening of a chemical library.

Chemical libraries are well known to those skilled in the art and may be derived from natural product sources such as but not limited to coral, plants and plant parts including bark, roots, flowers, leaves and stems, micro-organisms, marine macro-organisms and insects. Alternatively, chemical libraries may be collections of synthetic organic compounds or be produced by combinatorial chemical approaches. Alternatively, non- Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AUO 1/00263 17proteinaceous molecules may be produced through rational design, or through other conventional chemical approaches. A non-proteinaceous molecule may also be modified by the attachment of one or more proteinaceous sections or portions.

Any number of screening procedures may be adopted to identify the agonists and antagonist. In one example, a cytokine receptor is linked to a reporter molecule such that upon interaction between a SOCS molecule and the receptor, the reporter molecule provides an identifiable signal. An "identifiable signal" may be presence of a signal or absence of a signal. The amount or extent of signalling is then measured, quantitatively or qualitatively in the presence of potential agonists or antagonists. Any number of variations may be adopted to screen for agonists and antagonists. Variations of two hybrid screening and phage labelling may also be employed. Cell based assays and molecular assays may also be employed.

Accordingly, another aspect of the present invention contemplates a method of identifying an agent capable of modulating signalling of a cytokine or related molecule, said method comprising screening for agents which are capable of interfering or otherwise antagonizing or promoting or otherwise agonizing interaction between a SOCS molecule and a cytoplasmic domain of a receptor for said cytokine or related molecule.

The SOCS molecule and/or receptor molecule may be parts, fragments, derivatives, homologues or chemical analogues of all or a portion of the molecule. A portion of SOCS could include the SH2 domain or its equivalent.

More particularly, and in one preferred method, agonists and antagonists of SOCS interaction with a receptor are identified using biosensor technology. In one embodiment, phosphopeptides derived from a receptor or its mutants, derivatives, homologues or analogues are biotinylated such as in or at their N-terminal region. The biotinylated phosphopeptides are then immobilized to a solid support such as the surface of a chip via a streptavidin coating. A SOCS molecule capable of interacting with all and some of the immobilized phosphopeptides or binding portions or fragments or derivatives or Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -18 homologues or analogues of a SOCS molecule are then brought into contact with immobilized phosphopeptides. Such interactions may be in the presence or absence or a range of potential agonists or antagonists. Sensorgrams are then compiled to identify or analyse binding signals such as electrical or optical signals. Biotin/streptavidin represents one convenient means of immobilizing binding peptide. However, any of a host of other capturing pairs or binding partners may be used provided that these do not interfere with binding of the SOCS molecule or agonist/antagonist.

Accordingly, another aspect of the present invention contemplates a method for identifying a modulator of interaction between a SOCS molecule or part, fragment, or derivative or homologue or analogue thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:immobilizing one of said SOCS molecules or receptor or their parts, fragments, derivatives, homologues or analogues to a solid support optionally via binding partners wherein one of said binding partners is attached or otherwise anchored to said solid support and another of said binding partners is linked to said SOCS or receptor molecules; contacting said immobilized SOCS or receptor molecule with the other of receptor or SOCS molecule in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.

Preferably, the SOCS molecules in SOCS-3 or a related molecule. Most preferably, the SOCS molecule in SOCS-3. Although the SOCS molecule or receptor may be first immobilized to the solid support, it is particularly convenient for the receptor or a part, fragment, derivative, homologue or analogue to be anchored. Preferably, anchoring is via Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -19binding partners although the present invention extends to direct binding of the SOCS molecule or receptor molecule or portions thereof to the solid support.

According to these preferred embodiments, there is provided a method for identifying a modulator of interaction between a SOCS molecule or part, fragment, or derivative, or homologue or analogue thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:immobilizing said receptor or its parts, fragments, derivatives, homologues or analogues to a solid support optionally via binding partners wherein one of said binding partners is attached or otherwise anchored to said solid support and another of said binding partners is linked to said receptor molecule; contacting said immobilized receptor molecule with said SOCS molecule or its parts, fragments, derivatives, homologues or analogues in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.

The solid support is preferably in the form of a chip such as a biochip.

Any anchoring means may be employed to anchor the molecules to the solid support.

Generally, binding pairs are employed such as but not limited to biotin/streptavidin, DNA/DNA binding protein, antibody/antigen, FLAG/anti-FLAG antibodies, protein/protein binding molecule and complementary nucleic acid molecules.

The signal emission may be in any convenient means. Generally, interaction or loss of interaction between a SOCS molecule and a receptor or fragments, parts, derivatives, homologues or analogues thereof causes or otherwise facilitates production of an electrical Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 or optical signal via a suitable biological recognition system and electrochemical transducer. Electrochemical transducers include potentiometric, amperometric, optical and other physicochemical transducers. Potentiometric devices measure the accumulation of charge density at the surface of an electrode; amperometric sensors monitor currents generated when electrons are exchanged between a biological system and an electrode; an optical biosensor correlates changes in concentration, mass, or number to direct changes in the characteristics of light; other physicochemical sensors monitor biological interactions through changes in enthalpy, ionic conductance and mass. An "electrode" may also include a chip such as a biochip.

The streptavidin coated biosensor chips from pharmacia are particularly useful in the practice of the present invention.

According to this preferred embodiment there is provided a method for identifying a modulator of interaction between a SOCS molecule or part, fragment, or derivative, or homologue or analogue thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:immobilizing one of said SOCS molecules or receptor or their parts, fragments, derivatives, homologues or analogues to a solid support optionally via binding partners comprising biotin and streptavidin wherein one of said biotin or streptavidin is attached or otherwise anchored to said solid support and the another of said biotin or streptavidin is linked to said SOCS or receptor molecule; contacting said immobilized SOCS or receptor molecule with the other of receptor or SOCS molecule in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AUI01/00263 -21 In a preferred embodiment, anchoring of the receptor to the solid support is via biotin and streptavidin.

More particularly, there is provided a method for identifying a modulator of interaction between a SOCS molecule or part, fragment, or derivative, or homologue or analogue thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:immobilizing said receptor or its parts, fragments, derivatives, homologues or analogues to a solid support optionally via binding partners comprising biotin and streptavidin wherein streptavidin is attached or otherwise anchored to said solid support and said biotin is linked to said receptor molecule; contacting said immobilized receptor molecule with said SOCS molecule or its parts, fragments, derivatives, homologues or analogues in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.

Another useful biosensor employs Surface Plasmon Resonance (SPR) developed by Quantech Ltd. SPR is a quantum optical-electrical assay and is based on coupling or transferring energy carried by photons of light to electrons in a metal. The wavelength of light at which coupling energy transfer) occurs is characteristic of the particular metal and the environment of the metal surface which is illuminated. When there is a match or resonance between the energy of the light photons and the electrons at the metal surface, a transfer of energy occurs. The coupling, can be observed by measuring the amount of light reflected by the metal surface. All the light at most wavelengths is reflected except at the resonant wavelength, where almost all the light is absorbed. The measuring device is, in effect a reflectance spectrophotometer.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -22 The coupling of light into a metal surface results in the creation of a plasmon, a group of excited electrons which behave like a single electrical entity. The plasmon, in turn, generates an electrical field which extends about 100 nanometers above and below the metal surface. The characteristic of this phenomenon which makes SPR an analytical tool is that any change in the chemical composition of the environment within the range of the plasmon field causes a change in the wavelength of light which is absorbed rather than reflected and the magnitude of the shift is quantitatively related to the magnitude of the chemical change.

There are many other protocols for identifying binding or absence of binding or a reduction and these are encompassed by the present invention. Other such protocols include electrophoretic and chromatographic detection means.

As described above, chemical analogues of a SOCS molecule and/or a receptor are usefully employed in screening assays for ligands agonists or antagonists) due to enhanced chemical stability and/or coupling and/or signalling due to the chemical modifications. All such chemical modification to the SOCS molecules or receptor molecules including to their parts, fragments, portions, derivatives or homologue, are contemplated by the present invention. Reference herein to a "SOCS" molecule or a "receptor" molecule includes analogues and in particular chemical analogues including chemical modifications. Chemical modifications include modifications to side chains of peptides, polypeptides and proteins.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -23 The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4chloromercuribenzoate, 4 -chloromercuriphenylsulphonic acid, phenylmercury chloride, 2chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with Nbromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand. may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 WO 0166128PCT/AU01/00263 24 TABLE 1 Non-conventional Code Non-conventional Code amino acid amino acid ca-aminobutyric acid a-amino-oa-methylbutyrate amino cyc lopropanecarboxylate aminoisobutyrice acid aminonorbomylcarboxylate cyclohexvlalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamine D-giutamic acid D-histidine D-isoleucine D-leucine D-lysine D-methionine D-omithine D-phenylalanine D-proline D-serine D-threonine D-tryptophan Abu Mgabu Cpro Aib Norb Chexa Cpen Dal Darg Dasp Dcvs Dgln Dglu Dhis Dule Dleu Dlys Dmet Domn Dphe Dpro Dser Dthr Dtrp L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-Nmethylhistidine L-N-methylisolleucine L-N-methylleucine L-N-methvllysine L-N-methylmethionine L-N-methylnorleucine L-N-methvlnorvaline L-N-methvlornithine L-N-mnethylphenylalanine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyro sine L-N-methylvaline L-N-methylethylglycine L-N-mecthyl-t-butylglycinc L-norleucine L-norvaline Nmiala Nmarg Nmasn Nmasp Nmcys Nm gin Nmglu Nmihis Nmile Nmleu Nmlys Nmmnet Nmnle Nmnva Nmorn Nmphe Nmpro Nmscr Nmthr Nmtrp Nmtyr Nmnval Nmetg Nmtbug NMe Nva Substitute Sheet (Rule 26) ROIAU WO 01/66128 WO 0166128PCT/AUO1 /00263 25 D-tyrosine D-valine D-ca-methylalanine D-a-methylarginine D-ca-methylasparagine D-a.-methylaspartate D-a-methylcysteine D-ca-methylglutamine D-u-methylhistidine D-ca-methylisoleucine D-c-methylleucine D-c-methyllysine D-a-methylmethionine D-c-rethylomithine D-a-methylphenylalanine D -a-methylpro line D-oa-methylserine D-ca-methylthrzonine D-c-methyltryptophan D-a-methvltyro~sine D-oa-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine D-N-methylglutamine D-N-methylglutamate D-N-methylhisti dine D-N-methylisoleucine Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmnhis Dmile Dmleu Dmlys Dmmet Dmom Dmnphe Dmnpro Dmnser Dmthr Dmtrp Dmnty Dmnval Dnala Dnmarg Dnasri Dnmasp Dnmcys Dnmgln Dnmglu Dnm~his Drnile cu-methvI-aminoisobuty-rate ca-methy1l-y- aminobutyrate cu-methvlcyclohexylalanine ca-methvlcvlcopentylalanine ou-methyl-au-napthylalanine ca-methylp enicillamine N-(4-aminobutyl)glycine N-(2 -arnino ethyl) glycine N-(3 -aminopropyl)glycine N-amino-oa-methylbutyrate a-napthylalanine N-benzyiglycine N-(2-carbamylethyl)glycine N-(carbamylrnethyl)glycine N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutvlglycine N-cycloheptylglycine N-cyciohexylglycine N-cyclodecylglycine N-cylcododec yl glycine N-cyclooetylglycine N-cyclopropylgiycine N-cycloundocylglycine N-(2,2-diphenyl ethyl) glycine N-(3 ,3-diphcnylpropyl)glycine N-(3-g-uanidinopropyl)glycine 1 -hydroxyethyl)glycine N-(hydroxyethyl))glycine N-(imidazo lylethyl))glycine Maib Mgabu Mchexa Mcpen Manap Mpen NglU Nacg Nom Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe Narg Nthr Nser Nhis Substitute Sheet (Rule 26) ROIAU WO 01/66128 WO 0166128PCT/AUO1/00263 D-N-methylleucine D-N-methyllysine N-methylcvclohexylalanine D-N-methylomithine N-methylgIlycine N-methylaminoi sobutyrate 1 -methylpropyl)glycine N-(2 -methvlpropyl)glycine D-N-methvltryptophan D-N-methyltyro sine D-N-,,-methx'lvaline y-aminobutyric acid L-t-butylglycine L-ethylglycine L-homophenylalanine L-a-methylargirnne L-a-methylaspartate L-u-methylcysteine L-a-methylglutamine L-c-methylhistidine L-ca-methylisoleucine L-c-methylleucine L-ca-methylmeffiionine L-c-methylnorvaline L-a-methylphenylalanine L-ca-methylserine L-ca-methvltryptophan L-c-methylvaline N-(N-(2,2-diphenylethyl) carbamvlmethyl)glycine Dnmleu Damlys Nm chex a Dnmom Nala Nmaib Nile Nleu DnmtT-p Dnmtyr Dnmval Gabu Tbug Etg Hphe Marg Masp Meys Mgln Mhis Mile Mleu Mmet Mnva Mphc Mser Mtrp Mval Nnbhmn N-(3-indolylyethyl)glycine N-methyl--y-aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenyl al anine D-N-methylpro line D-N-methylserine D-N-methylthreonine 1 -methylethyl)glycine N-methyl a-napthylalanine N-methylp enicillamine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillamine L-a-methylalanine L-a-methylasparagine L-a-methyl-t-butylglycine L-m ethyl ethylglycine L-ou-niethiylglutarnate L-ca-methylhomophenylalanine N-(2-methylthioethyl)glycine L-a-methyllysine L-ca-methyinorleucine L-oa-methylomithine L-c-methylproline L-a-methylth-reonine L-ca-methyltyrosine L-N-methylhomophenylalanine ,3 -diphenyipropyl) carbarnylmethyl)glycine Nhtrp Nmgabu Dnmmet Nmcpen Dnmphe Dnmpro Dnmser Drmithr Nval Ninanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Mew~ Mglu Mhphe Nmet Mlys Mnle Mom Mpro Mthr Mtyr Nmhphe Nnbhe Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -27l-carboxy-1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane Crosslinkers can be used, for example, to stabilize 3D conformations, using homobifunctional crosslinkers such as the bifunctional imido esters having (CH2), spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Ca and N a-methylamino acids, introduction of double bonds between Ca and Cg atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

Still a further aspect of the present invention is directed to an agent useful for modulating signalling of a cytokine or a related molecule, said agent capable of interfering or otherwise antagonizing or promoting or otherwise agonizing interaction between a SOCS molecule and a cytoplasmic domain of a receptor for said cytokine or related molecule.

Preferably, the SOCS molecule is SOCS-3 or related molecule. Most preferably, the SOCS molecule is SOCS-3. The preferred receptors comprise a phosphorylated tyrosine in a region comprising or proximal to the site of interaction with the SOCS molecule.

The present invention, therefore, contemplates a composition comprising a modulator of interaction between a SOCS molecule and a cytoplasmic domain of a receptor molecule, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.

The modulator may be an agonist or an antagonist of the SOCS/receptor interaction and may be useful in modulating cytokine- or related molecule-mediated signal transduction.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AUO 1/00263 -28- This may be useful in a range of conditions including cancer, inflammatory disorders and autoimmune and other immunological disorders. The composition may be regarded as a pharmaceutical composition and/or an agent.

Composition forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The carrier can be a solvent or dilution medium comprising, for example, water, cthanol, polvol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of superfactants. The preventions of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the active ingredient and optionally other active ingredients as required, followed by filtered sterilization or other appropriate means of sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying and the freeze-drying technique which yield a powder of active ingredient plus any additionally desired ingredient.

When the modulator is suitably protected, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet or administered via breast milk. For oral therapeutic Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -29administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 1% by weight of modulator. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.

The amount of modulator in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosgae unit form contains between about 0.1 itg and 200 mg of modulator. Alternative dosage amounts include from about 1 gg to about 1000 mg and from about 10 jg to about 500 mg. These dosages may be per individual or per kg body weight. Administration may be per hour, day, week, month or year.

The tablets, troches, pills, capsules, creams and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 substances is well known in the art and except insofar as any conventional media or agent is incompatible with the modulator, their use in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of encoding a modulator, when the modulator is a proteinaceous molecule. The vector may, for example, be a viral vector. In this regard, a range of gene therapies are contemplated by the present invention including isolating certain cells, genetically manipulating and returning the cell to the same subject or to a genetically related or similar subject.

The present invention further contemplates antibodies and other immunological reagents directed to the modulators identified by the subject screening assays.

The present invention is further directed to a use of a SOCS molecule and/or its receptor in the manufacture of an assay to screen for agonists or antagonists of SOCS/receptor interaction.

The present invention is further described by the following non-limiting Examples.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -31 EXAMPLE 1 SOCS-3 protein expression Murine SOCS-3 cDNA was PCR-amplified and cloned into the NdeI/BamHI sites of pET15b (Novagen). The hexahistidine tagged protein was expressed in BL21 DE3 pLysS E. coli (Stratagene) and purified from cells lysed with 7 M guanidinium hydrochloride by immobilized metal affinity chromatography. Purified protein was refolded by dialysis into PBS containing 2 mM DTT, 2 mM EDTA and 0.02% v/v Tween 20. Refolded material was purified to homogeneity by ion exchange chromatography on a Mono S column (Pharmacia). Recombinant SOCS-3 was characterized by SDS-PAGE analysis, and MALDI mass spectrometry. The concentration of a stock solution of SOCS-3 used for quantitative binding analyses was determined by amino acid analysis.

EXAMPLE 2 Peptide synthesis All synthetic peptides were synthesized with C-terminal amides using a Rink amide MBHA resin support and Fmoc amino acids activated with HBTU. Phosphotyrosinecontaining peptides were prepared using Fmoc-O-benzyl-L-phosphotyrosine which was activated with HATU, and coupled for 1 hour. Deprotection and cleavage was effected using 95% trifluoroacetic acid containing 5% triisopropylsilane, and crude peptides were purified by reverse phase HPLC. Purified peptides were characterized by MALDI mass spectrometry. Biotinylated peptides were prepared by coupling o-biotin to the amino terminus of resin-bound peptides prior to cleavage and deprotection.

EXAMPLE 3 Qualitative analysis of SOCS-3 phosphopeptide interactions Biotinylated phosphopeptides were prepared corresponding to fragments of murine surrounding each cytoplasmic tyrosine residue (Figure 1A), with the exception of tyrosine 859 which is not conserved in the human receptor. Biotinylated phosphopeptides were also Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -32prepared corresponding to the known tyrosine phosphorylation sites in murine STAT-1 (Y701) and STAT-3 (Y705). Biotinylated peptides were immobilized onto streptavidinagarose resin (Pierce), and 20 pL of these resins were incubated for 1 hour at room temperature with 1 mL of 50 pg/mL SOCS-3 in 10 mM Tris pH 7.5 containing 0.1% v/v Tween-20. Following removal of the protein solution, resin was washed twice with 1 mL PBS/0.1% v/v Tween-20, and bound protein eluted with 25 pL of SDS sample buffer containing 10 mM DTT. Samples were analyzed by SDS-PAGE on 12% w/v polyacrylamide gels.

EXAMPLE 4 Biosensor Analyses Phosphopeptides containing an N-terminal biotin moiety were immobilized onto a streptavidin-coated Biosensor chip (Pharmacia) at a density of -200 Resonance Units (Rus). Binding of recombinant SOCS-3 was measured by flowing solutions of protein diluted into PBS, 0.1% v/v Tween 20 over the chip at a flow rate of 15 gL/min. For kinetic analysis of SOCS-3 binding, samples of protein were diluted and immediately analysed to minimize losses of protein due to surface absorption. Following each binding measurement, residual SOCS-3 was stripped from the immobilized ligand by washing with 6 M guanidinium hydrochloride (pH followed by PBS, 0.1% v/v Tween 20. Binding profiles were analyzed using the BIAevaluation software version 3.0 (Pharmacia). To correct for non-specific binding, sensorgrams obtained for binding of SOCS-3 to the noncognate gpl30( 6 74-688) phosphopeptide were subtracted from those for binding to the gpl30(750-764) phosphopeptide. The dissociation constant Kd was calculated from a Scatchard type analysis of the equilibrium response measurements obtained at different concentrations of SOCS-3.

A competitive binding assay for measuring the affinities of SOCS-3 ligands in solution was developed using the Biosensor instrument. Briefly, the phosphorylated gpl30 (750- 764) peptide, containing an N-terminal biotin moiety, was immobilized onto a streptavidin Biosensor chip. A dilution series of peptide ligand was incubated with a fixed, Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -33 subsaturating amount of SOCS-3 (-100 nM) in a PBS, 0.1% v/v Tween 20 buffer containing 0.5 mg/mL BSA. Samples were left overnight at 4'C prior to analysis on the Biosensor so that the rate of protein loss due to absorption was minimal through the course of the experiment. The response level of bound protein was recorded at a fixed time point within the sensorgram, and divided by the corresponding level of SOCS-3 bound to the peptide-chip in the absence of competing ligand. These fractional binding values were fitted to the equation f 1/(1+(c/ICso) m where c the concentration of soluble SOCS-3 ligand, m the curvature constant and IC 5 0 values correspond to the concentration of ligand required to displace 50% of the bound SOCS-3.

EXAMPLE Luciferase assays, Immunoprecipitation and Western Analysis Generation of the expression vectors for SOCS-1 and SOCS-3, luciferase assays and Western blot analysis have been described previously (Nicholson et al., 1999). Briefly, 293T cells were transiently transfected with SOCS expression plasmids, a LIF-responsive reporter construct (APRE-luc; Endo et al., 1997) and a 0-galactosidase reporter construct under a constitutive promoter (Sra-p-gal; Ogilvy et al., 1998). Luciferase activity from triplicate samples was determined and normalized against p-galactosidase activity.

EXAMPLE 6 Identification ofpY757 in gpl30 as a potential SOCS-3 interaction site SOCS-3 expression (like that of SOCS-1) is induced by the IL-6 family of cytokines and can inhibit their biological actions (Masuhara et al., 1997; Minamoto et al., 1997; Nicholson et al., 1999; Starr et al., 1997). These cytokines utilize common features of the JAK-STAT signalling pathway each shares gpl30 as a common co-receptor, while JAKs 1, 2 and STAT-1 and -3 are responsible for transmitting the downstream signals. Thus a common mechanism of inhibition for each of the IL-6 family cytokines might require that SOCS-3 interact with either gpl30, the JAK kinases, or STAT proteins. Association between SOCS-3 and the JH1 (kinase) domain of JAK2 has been previously demonstrated Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -34in qualitative "pull-down" experiments, moreover, the site of interaction has been identified as the autophosphorylation site within the activation loop (Sasaki et al., 1999).

To investigate whether SOCS-3 might interact with tyrosine phosphorylated sites within or relevant STAT proteins, the inventors prepared phosphopeptides corresponding to regions surrounding each of the gpl30 cytoplasmic tyrosine residues, in addition to peptides based on the known tyrosine phosphorylation sites within STAT-1 and STAT-3 (Figure 1A). These peptides contained an N-terminal biotin moiety and were immobilized onto streptavidin-agarose resin. Recombinant SOCS-3 was incubated with peptide resin, and after washing, any bound protein was eluted with SDS sample buffer. SDS-PAGE analysis showed that only one peptide was able to efficiently capture SOCS-3 (Figure 1B).

This peptide represented a fragment of gpl30 corresponding to amino acids 750-764 and was centred around the phosphorylated tyrosine residue 757 (pY757).

EXAMPLE 7 gpl30 contains a high affinity, phosphorylation-dependent SOCS-3 binding site centred around pY757 The binding affinity of SOCS-3 to the phosphorylated gpl30(750-764) peptide was measured using surface plasmon resonance (SPR). The biotinylated form of this peptide was immobilized onto a streptavidin biosensor chip and binding of recombinant SOCS-3 was analyzed by SPR on a Biosensor instrument. As a non-specific control, the phosphorylated gpl30(674-688) peptide was also immobilized in a separate channel on the same chip. Concentration-dependent binding of SOCS-3 to the immobilized gpl30(750- 764) phosphopeptide was observed (Figure 2A), while no specific binding was detected to the gpl30(674-688) phosphopeptide. The dissociation constant for peptide binding was calculated from a Scatchard analysis of the equilibrium response values at different concentrations of SOCS-3 (Figure 2B). The affinity of this interaction (Kd 42 nM) is high relative to that for the other peptides and characteristic of the binding constants observed for other SH2 domains binding to high affinity phosphopeptide ligands.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 To ascertain whether SOCS-3 binding was dependent upon the phosphorylation state of the peptide, a competitive binding assay was developed using the Biosensor instrument (Karlsonn, 1994). A subsaturating amount of SOCS-3 was incubated with different dilutions of a soluble competing peptide, and this mixture was then passed over the biosensor chip containing the immobilized gpl30(750-764) phosphopeptide. IC5o values were determined from plots of soluble peptide concentration versus fractional SOCS-3 bound. By this method, the relative binding affinities for phosphorylated and nonphosphorylated gp130(750-764) peptides were determined (Figure 3A). While an ICso value of 110 nM was obtained for the phosphorylated peptide, the non-phosphorylated analogue bound with an ICso of 2.0 mM. Thus, binding of SOCS-3 to the gp130(750-764) peptide is phosphorylation-dependent, with an 18,000-fold difference in affinity mediated by the phosphate moiety. The inventors also used this competition assay to reassess the potential solution binding of the phosphopeptides listed in Figure 1A. With the exception of the biotinylated gpl30(750-764) phosphopeptide, none of these peptides showed measurable binding to SOCS-3 at concentrations up to 100 gM.

EXAMPLE 8 SOCS-3 binds selectively to gpl30, but only weakly to JAK kinase peptides Earlier reports (Sasaki et al., 1999) have demonstrated that endogenous SOCS-3 is able to associate with full length JAK2. This interaction was mapped to the JH1 (kinase) domain of JAK2, and preparation and assaying of a series of phosphopeptides corresponding to known tyrosine phosphorylation sites within this domain identified residue pY1007 as the site of SOCS-3 interaction. This corresponds to the activation loop within the kinase domain of JAK2. The inventors, therefore, sought to make a quantitative comparison between the affinity of SOCS-3 for the gpl30 (750-764) phosphopeptide versus phosphopeptides derived from the activation loops of JAKs 1-3. The affinities of the JAKderived phosphopeptides for SOCS-3 were measured in the competitive binding assay, and the binding constants were compared to that for the gpl30 (750-764) phosphopeptide (Figure 3B). The tightest binding JAK phosphopeptide was that from JAK-3 with an value of 110 pM, however, this affinity is 1,000-fold weaker than for gp 130(750-764). The Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -36- JAK2-derived peptide bound SOCS-3 with even lower affinity, some 10,000-fold weaker than gpl30(750-764). The observed affinities of the JAK-derived peptides for SOCS-3 seem inordinately weak for an SH2 domain binding to a phosphopeptide ligand derived from a biologically-relevant target. Based on this binding data, it seems far more likely that pY757 on gpl30 is the physiological site of SOCS-3 interaction, and not the activation loop on the JAK kinases.

EXAMPLE 9 pY757 is important for inhibition ofgpl30 signalling by SOCS-3 The peptide studies indicated that tyrosine 757 was likely to be the preferred binding site for the SOCS-3 SH2 domain and consequently have an important role in the specificity and mechanism of SOCS-3 action. It was therefore important to confirm these findings using a biological assay. A chimeric receptor was created that contained the erythropoietin receptor (EPOR) extracellular domain and the gpl30 cytoplasmic domain (EPOR/gp130).

A second receptor was created containing the EPO receptor extracellular domain and the cytoplasmic domain in which tyrosine 757 had been mutated to phenylalanine (EPOR/gpl30Y757F). 293T cells were transiently transfected with cDNAs expressing the receptor constructs and either SOCS-3 or SOCS-1 in the presence of the APRE-luc and Sra-p-gal reporter genes. Cells were then incubated in the presence of 10U/ml EPO overnight and luciferase activity determined. Mutation of Y757 in the gpl30 cytoplasmic domain has been reported to result in a hyperactive receptor complex, presumably from the loss of a negative regulator such as SHP-2. These results are consistent with these observations as EPO activation of the EPOR/gpl30Y757F receptor resulted in a four-fold higher level of luciferase activity than activation of the EPO/gpl30 receptor. When high levels of SOCS DNA were transfected (170 ng), SOCS-3 inhibited both EPOR/gpl30 and EPOR/gp130Y757F equally. However, SOCS-3 was able to inhibit the wild-type cytoplasmic domain (EPOR/gpl30) at much lower concentrations of DNA than the mutated gpl30 cytoplasmic domain (EPOR/gp130Y757F) (50% inhibition at 4 ng compared with 54 ng SOCS-3 DNA). This difference was not observed when SOCS-1 DNA was used and strongly suggests that SOCS-3 acts by binding to gpl30 tyrosine 757.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AU01/00263 -37- When SOCS-3 is highly over-expressed, ie when 170 ng DNA is transfccted, it is likely that the levels of SOCS-3 protein are saturating and SOCS-3 also inhibits signalling by binding to members of the JAK family in a non-specific manner.

EXAMPLE SOCS-3 recognizes an extended epitope on SH2 domains recognize tyrosine phosphorylated polypeptides as ligands. While a central phosphotyrosine residue is the primary binding determinant, other amino acids within the immediate vicinity of the phosphotyrosine residue also contribute to the specificity of the association. For prototypical SH2 domains, such as that from src, the sidechains of the 1st (pY+1) and 3rd (pY+3) amino acid residues downstream from the phosphotyrosine residue contribute to the overall protein-ligand association by interacting with specific residues within the SH2 domain. More generally, 2 or 3 of the amino acids between the pY+1 and pY+5 residues contribute to SH2 binding, while residues upstream from pY do not participate in binding interactions.

To better understand the structural basis of SOCS-3 ligand binding specificity, a series of gpl30(750-764) phosphopeptide analogues were synthesized. These analogues were either truncated or contained single alanine substitutions relative to the gpl30 (750-764) parent phosphopeptide. The affinities of these peptides for SOCS-3 was measured in the competitive binding assay, and compared to that of the gpl30 (750-764) phosphopeptide (Table Based on the data from this series of peptides, the side chains of at least 5 nonphosphotyrosine residues appear to be interacting with SOCS-3, these being the pY-1 and 2 residues, in addition to pY+3, +4 and While it is possible that the pY+1 and +2 sidechains also participate in interactions with SOCS-3, this is not apparent from the alanine substitution data. Thus, compared to most other SH2 domains SOCS-3 recognizes an extended polypeptide epitope and this interaction includes ligand residues that are Nand C-terminal to phosphotyrosine.

Substitute Sheet (Rule 26) RO/AU WO 01/66128 WO 0166128PCT/AUOI /00263 38 TABLE 2 IC 50 values for gp 130(750-764) phosphopeptide and analogues Peptidle 1C 50 (nM) ICso analogue!

C

50 wild-type STASTVE(pY)STVVHSG [SEQ ID NO:8] 110 STASTVE(pY)ATVVHSG [SEQ ID NO:9J 72 0.65 STASTVE(pY)SAVVHSG [SEQ ID NO: 10] 110 STASTVE(pY)STAVHSG [SEQ ID NO: 11] 3400 31 STASTVE(pY)STVA-SG [SEQ ID NO.-121 1100 STASTVE(pY)STVVASG [SEQ ID NO: 13J] 770 STASTVE( Y)STVVHSG [SEQ ID NO: 14] 2.0 x 106 18,000 STASTVE(pY)STVVHS [SEQ ID NO: 15J 61 0.55 STASTVE(pY)STVVH [SEQ ID NO: 16] 77 0.70 STASTVE(pY)STVV [SEQ ID NO: 17] 1100 AcTVE(pY)STVVHSG [SEQ ID NO:18S] 120 1.1 AcVE(pY)STVVHSG [SEQ ID NO: 19] 110 AcE(pY)STVVHSG [SEQ ID NO:20J 970 8.8 Ac(pY7JSTVVHSG [SEQ ID NO:21] 9600 87 AcAE(pY)STVVHSG [SEQ ID NO:22] 580 5.3 AeVA(pY)STVVHSG [SEQ ID NO:23] 550 Substitute Sheet (Rule 26) RO/AU WO 01/66128 PCT/AUO1/00263 -39- EXAMPLE 11 Effects of binding of SOCS-3 to gpl30, EPO and leptin SOCS-3 regulates signalling by binding to the intracellular region of the receptor subunit gpl30 when tyrosine (757) is phosphorylated. Docking of SOCS to the receptor inhibits the binding of other signalling proteins to the receptor, preventing signal transduction.

The present example shows that there are critical residues within the sequence of the phospho-tyrosine peptide of gpl30 that SOCS-3 specifically binds. These residues along with the phospho-tyrosine can be used to identify key signalling regions on other cytokine receptors. Examples of these can be found for both leptin receptor and erythropoietin (EPO) receptor (Figure Specific binding of the EPO and Leptin receptor phospho-tyrosine peptides to SOCS-3 can be demonstrated using the biosensor as described in Example 4. Briefly, biotinylated (Y757), EPO and leptin peptides (Figure 5) are immobilized to a streptavidin chip on the biosensor. A gpl30 phospho-tyrosine peptide (Y681; FNSKDQM(pY)SDGNFTD) is also coupled to the sensorchip to serve as a control. Simultaneous binding of SOCS-3 (100 nM) to mgp 130, EPO and leptin phospho-tyrosine peptides and control peptide can be monitored on a Biacore 2000. Subtraction of the control sensorgram from the other channels demonstrates specificity of the signal.

The binding of SOCS-3 to these peptides is shown (Figure These interactions form the basis for a high throughput molecular screen designed to identify antagonists of the interaction of SOCS-3 with both the EPO and leptin receptors.

Substitute Sheet (Rule 26) RO/AU WO 0 1 /66128 WO 0166128PCT/AU01 /00263 40

BIBLIOGRAPHY

Adams et al. (1999) JBiol Chiei 273:1285-1287 Auernhamnier, C.J. and Melmed, S. (1999) Endocrinology 140: 1559-1566 Bjorbaek et al. (1999) Endocrinology 140: 2035-2043 Bjorbaek et al. (1999) JBiol Chem 274: 30059-30065 Bjorbaek et al. (1998) i~fol Cell 1: 619-625 Bousquet et (1999) J Clin Invest 104: 1277-1285 Cohney et (1999) M1o! Cell Biol 19: 4980-4988 Donnelly et ai. (1999) Jlnteiferon Cvtokine Res 19: 563-573 Endo et al. (1997) Nature 387: 92 1-924 Gisselbrecht, S. (1999) Fur Cytokine Netiv 10: 463-470 Hansen et al. (1999) Mol Endocrinol 13: 18 32-1843 Heim, M.H. (1999) JRecept Signal TransductRes 19: 75-120 Hilton, D.J. (1999) Cell Mol Life Sci 55: 1568-1577 Hilton et al. (1998) Proc NatlAcad Sci USA 95: 114-119 IhIe (1998) Ann N YAcad Sci 865: 1-9 Ito et al. (1999) Blood 93: 145 6-1463 Kamura et (1998) Genes Dev 12: 3872-3 881 Leonard, W.J. and O'Shea, J.J. (1998) Annu Revlrnniunol 16: 293-322 Marine et al. (1999) Cell 98: 617-627 Masuhara et (1997) Biochern Biophys Res Commun 239: 439-446 Matsumoto et al. (1997) Blood 89: 3148-3154 Minamoto et al. (1997) Biochem Biophys Res Comnmun 237: 79-83 Naka et al. (1997) Nature 387: 924-929 Narazaki et al. (t998) Proc NatlAcad Sci USA 95: 13130-13134 Nicholson, S.F. and Hilton, D.J. (1998) JLeukoc Biol 63: 665-668 Nicholson et (1999) EMBO J18: 375-385 Ogilvy et a. (199 8) Blood 91: 419-43 0 Pezet et al. (1999) JBiol Chem 274: 24497-24502 Sasaki et (1999) Genes Cells 4: 339-351 Substitute Sheet (Rule 26) RO/AU WO 01/66128 WO 0166128PCTIAUOI/00263 41 Starr et (1997) Mature 387: 917-921 Suzuki et al. (1998) Oncogene 17: 2271-2278 Yasukawa et a. (1999) EMBO J 18: 1309-1320 Yashimura, A. (1998) Leukemiia 12: 1851-1857 Yoshimura et al. (1995) EMBO J 14: 2816-2826 Zhang et a. (1999) Proc Nat! Acad Sci USA 96: 2071-2076 Substitute Sheet (Rule 26) RO/AU

Claims

29- 8-06:12!02PM:DAVIES COLLISON CAVE 18/ P.1APERUEH RS CimaM2WtM1%2G4330QaLcC*.29/gt VO 0 -42- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said Sanimal an effective amount of a modulator of interaction between a SOCS-3 molecule and 0 a region within a cytoplasmic domain of a gpl30 receptor for said cytokine or related e, molecule. 0 o 2. A method according to Claim 1 wherein the cytokine receptor is for the IL-6 family of receptors. 3. A method according to Claim 2 wherein the cytokine receptor is for CNTF, an interleukin, LIF, OSM or CT-1. 4. A method according to any one of Claims 1 to 3 wherein the SOCS molecule interacts with a region of the receptor comprising or proximal to a phosphorylated tyrosine. A method according to any one of Claims 1 to 4 wherein the animal is a mammal. 6. A method according to Claim 5 wherein the mammal is a human. 7. A method according to any one of Claims 1 to 6 wherein the SOCS molecule interacts with the amino acid sequence set forth in SEQ ID NO:4. 8. A method according to any one of Claims 1 to 6 wherein the SOCS molecule interacts with the amino acid sequence set forth in SEQ ID 9. A method according to any one of Claims I to 6 wherein the SOCS molecule interacts with the amino acid sequence set forth in SEQ ID NO:6. COMS ID No: SBMI-04597096 Received by IP Australia: Time 12:35 Date 2006-08-29 29- 8-06;12:02PM:DAVIES COLLISON CAVE 19/ o ;Z| P IQ75KVW-im CimlwfllAurW3l diw.d-rA-M -43- A method according to any one of Claims 1 to 6 wherein the SOCS molecule interacts with the amino acid sequence set forth in SEQ ID NO:7. 11. A method for regulating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of a modulator of interaction between a SOCS-3 or related molecule and a region within the cytoplasmic domain of gpl30 or any other receptor having a region homologous to the amino acid sequence in the cytoplasmic domain of to which SOCS-3 binds. 12. A method according to Claim 11 wherein the amino acid sequence set forth in SEQ ID NO:4. 13. A method according to Claim 11 wherein the amino acid sequence set forth in SEQ ID 14. A method according to Claim 11 wherein the amino acid sequence set forth in SEQ ID NO:6. A method according to Claim 11 wherein the amino acid sequence set forth in SEQ ID NO:7. SOCS molecule interacts with the SOCS molecule interacts with the SOCS molecule interacts with the SOCS molecule interacts with the 16. A method of inhibiting signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of an agonist of interaction between a SOCS-3 molecule and a region within the cytoplasmic domain of a gpl130 receptor for said cytokine or related molecule. 17. A method of facilitating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site within said animal an effective amount of an antagonist of interaction between a SOCS-3 molecule COMS ID No: SBMI-04597096 Received by IP Australia: Time 12:35 Date 2006-08-29 29- 8-06:12:02PM;DAVIES COLLISON CAVE 20/ 0-44- I Z4 and a region within the cytoplasmic domain of a gp30 receptor for said cytokine or Crelated molecule. 18. A method of identifying an agent capable of modulating signalling of a cytokine or related molecule, said method comprising screening for agents which are capable of o interfering or otherwise antagonizing or promoting or otherwise agonizing interaction c,1 between a SOCS-3 molecule and a cytoplasmic domain of a gpl30 receptor for said ocytokine or related molecule. 19. A method for identifying a modulator of interaction between a SOCS-3 molecule or part, fragment, or derivative or homologue or analogue thereof and a gpl30 receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:- immobilizing one of said SOCS molecules or receptor or their parts, fragments, derivatives, homologues or analogues to a solid support optionally via binding partners wherein one of said binding partners is attached or otherwise anchored to said solid support and another of said binding partners is linked to said SOCS or receptor molecules; contacting said immobilized SOCS or receptor molecule with the other of gpl3O receptor or SOCS-3 molecule in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues. A method for identifying a modulator of interaction between a SOCS-3 molecule or part, fragment, or derivative, or homologue or analogue thereof and a gpl30 receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:- immobilizing said receptor or its parts, fragments, derivatives, homologues or analogues to a solid support optionally via binding partners wherein one of said binding partners is attached or otherwise anchored to said solid support and another of said binding partners is linked to said receptor molecule; COMS ID No: SBMI-04597096 Received by IP Australia: Time 12:35 Date 2006-08-29 29- 8-06;12:02PM;DAVIES COLLISON CAVE 21/ Va 0 0 ci ci a', P.XCIEOcft aMOMONlVW 633MII d AI11-W=Mh contacting said immobilized receptor molecule with said SOCS molecule or its parts, fragments, derivatives, homologues or analogues in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues. 21. A method according to any one of Claims 16 to 20 wherein the cytokine receptor is for the IL-6 family of receptors. 22. A method according to Claim 21 wherein the cytokine receptor is for CNTF, an interleukin, LIF, OSM or CT-1. 23. A method according to any one of Claims 16 to 22 wherein the SOCS-3 molecule interacts with a region of the receptor comprising or proximal to a phosphorylated tyrosine. 24. A method according to any one of Claims 16 to 23 wherein the interacts with the amino acid sequence set forth in SEQ ID NO:4. A method according to any one of Claims 16 to 23 wherein the interacts with the amino acid sequence set forth in SEQ ID 26. A method according to any one of Claims 16 to 23 wherein the interacts with the amino acid sequence set forth in SEQ ID NO:6. 27. A method according to any one of Claims 16 to 23 wherein the interacts with the amino acid sequence set forth in SEQ ID NO:7. SOCS molecule SOCS molecule SOCS molecule SOCS molecule 28. A method according to Claim 19 or 20 wherein the solid support is a biochip. COMS ID No: SBMI-04597096 Received by IP Australia: Time 12:35 Date 2006-08-29 29- 8-06;12:02PM;DAVIES COLLISON CAVE 22/ rMFUKWutrMSI flhCni2Ix'oIAON433Gclmooc.jIMSGw 0 0 -46- 29. A method according to Claim 19 or 20 wherein the binding partners are biotin and streptavidin. ci A method for identifying a modulator of interaction between a SOCS-3 molecule or part, fragment, or derivative, or homologue or analogue thereof and a gpl30 receptor for a 0 cytokine or related molecule or part, fragment derivative or homologue or analogue ci\ thereof, said method comprising:- Simmobilizing one of said SOCS molecules or receptor or their parts, fragments, o derivatives, homologues or analogues to a solid support optionally via binding partners comprising biotin and streptavidin wherein one of said biotin or streptavidin is attached or otherwise anchored to said solid support and the another of said biotin or streptavidin is linked to said SOCS or receptor molecule; contacting said immobilized SOCS or receptor molecule with the other of receptor or SOCS molecule in the presence of a potential agonist or antagonist; and measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues.
31. A method of identifying a modulator of interaction between a SOCS-3 molecule or part, fragment, or derivative, or homologue or analogue thereof and a gpl30 receptor for a cytokine or related molecule or part, fragment derivative or homologue or analogue thereof, said method comprising:- immobilizing said receptor or its parts, fragments, derivatives, homologues or analogues to a solid support optionally via binding partners comprising biotin and streptavidin wherein streptavidin is attached or otherwise anchored to said solid support and said biotin is linked to said receptor molecule; contacting said immobilized receptor molecule with said SOCS molecule or its parts, fragments, derivatives, homologues or analogues in the presence of a potential agonist or antagonist; and COMS ID No: SBMI-04597096 Received by IP Australia: Time 12:35 Date 2006-08-29 29- 8-06:12:02PM:DAVIES COLLISON CAVE *23/ 0 0-47- ;Z measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS and receptor or their parts, fragments, derivatives, homologues or analogues. 32, A method according to Claim 30 or 31 wherein the cytokine receptor is for the IL-6 ofamily of receptors.
33. A method according to Claim 30 or 31 wherein the cytokine receptor is for CNTF, an interleukin, LIE, OSM or CT-1.
34. A method according to any one of Claims 30 to 33 wherein the SOCS molecule interacts with a region of the receptor comprising or proximal to a phosphorylated tyrosine. A method according to any one of Claims 30 to 34 wherein the SOCS molecule interacts with the amino acid sequence set forth in SEQ ID NO:4.
36. A method according to any one of Claims 30 to 34 wherein the SOCS molecule interacts with the amino acid sequence set forth in SEQ ID
37. A method according to any one of Claims 30 to 34 wherein the SOCS molecule interacts with the amino acid sequence set forth in SEQ ID NO:6.
38. A method according to any one of Claims 30 to 34 wherein the SOCS molecule interacts with the amino acid sequence set forth in SEQ ID NO:7.
39. A method according to any one of Claims 30 to 33 wherein the solid support is a biochip. A method of facilitating signalling of a cytokine or related molecule in an animal subject, said method comprising administering to said animal or to a site in this said animal an effective amount of an antagonist of interaction between a SOCS-3 molecule and a COMS ID No: SBMI-04597096 Received by IP Australia: Time 12:35 Date 2006-08-29 29- 9-06:12:02PM:DAVIES COLLISON CAVE 24/ r91*P..Ia a*n~dm&-,v~ -48- ,1 region in the cytoplasmic domain of a gp130 receptor for said cytokine or related molecule.
41. An agent when used for modulating signalling of a cytokine or a related molecule, said agent capable of interfering or otherwise antagonizing or promoting or otherwise oagonizing interaction between a SOCS-3 molecule and a cytoplasmic domain of a creceptor for said cytokine or related molecule.
42. A composition comprising a modulator of interaction between a SOCS-3 molecule and a cytoplasmic domain of a gpl30 receptor molecule, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.
43. A method according to Claim 40 or an agent when used according to Claim 41 or a composition according to Claim 42 wherein the modulator is an agonist of SOCS-3/gpl receptor interaction.
44. A method according to Claim 40 or an agent when used according to Claim 41 or a composition according to Claim 42 wherein the modulator is an antagonist of SOCS- 3/gpl3O receptor interaction. A method according to Claim 40 or an agent when used according to Claim 41 or a composition according to Claim 42 when used in the treatment of cancer, an inflammatory condition or an autoimmune or other immunological disorder.
46. Use of a SOCS-3 molecule and/or gp 130 receptor or part thereof in the manufacture of an assay to screen for agonists or antagonists of SOCS-3/gp130 receptor interaction.
47. Use according to Claim 46 wherein the part of the SOCS molecule comprises an SH2 domain. COMS ID No: SBMI-04597096 Received by IP Australia: Time 12:35 Date 2006-08-29 29- 8-06;12:02PM:DAVIES COLLISON CAVE 25/ F:?PERUIFtnu CloI2006%AoWG453Bclmac-df2910M 0 -49-
48. A method according to any one of Claims 1 to 40 or 44 to 45, an agent when used Saccording to Claim 41, a compositions according to Claim 42 or a use according to any one of Claims 46 to 47 substantially as hereinbefore described with reference to the Figures and/or Examples. O DATED this 29th day of August, 2006 CNK THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCH Sby DAVIES COLLISON CAVE "i Patent Attorneys for the Applicant(s) CI COMS ID No: SBMI-04597096 Received by IP Australia: Time 12:35 Date 2006-08-29