WO2002100416A1 - Socs-5 molecules, screening therefore and therapeutic uses thereof - Google Patents

Abstract

The present invention relates generally to molecules which are capable of interacting with members of the family of suppressors of cytokine signalling (SOCS) proteins or with a growth factor associated therewith and/or a growth factor receptor. The molecules range from intracellular proteinaceous targets for which the SOCS is a ligand to chemicals including proteinaceous entities identified inter alia by screening of natural products, chemical libraries and/or through rational drug design. The identification of intracellular targets of a SOCS protein and/or the identification of other interactors of the SOCS molecule permits the development of a range of therapeutic and diagnostic applications. The present invention particularly relates to SOCS-5 and its involvement in various physiological processes such as those mediated by growth factor signalling and in particular epidermal growth factor (EGF)-mediated signalling. The present invention provides, therefore, ligands of SOCS-5, EGF and/or EGF receptor. Furthermore, ligands for target proteins linked to a SOCS box can be used to direct target proteins to degradation in vivo.

Description

THERAPEUTIC AND DIAGNOSTIC MOLECULES

FIELD OF THE INVENTION

The present invention relates generally to molecules which are capable of interacting with members of the family of suppressors of cyto ine signalling (SOCS) proteins or with a growth factor associated therewith and/or a growth factor receptor. The molecules range from intracellular proteinaceous targets for which the SOCS is a ligand to chemicals including proteinaceous entities identified inter alia by screening of natural products, chemical libraries and/or through rational drug design. The identification of intracellular targets of a SOCS protein and/or the identification of other interactors of the SOCS molecule permits the development of a range of therapeutic and diagnostic applications. The present invention particularly relates to SOCS-5 and its involvement in various physiological processes such as those mediated by growth factor signalling and in particular epidermal growth factor (EGF)-mediated signalling. The present invention provides, therefore, ligands of SOCS-5, EGF and/or EGF receptor. Furthermore, ligands for target proteins linked to a SOCS box can be used to direct target proteins to degeneration in vivo.

BACKGROUND OF THE INVENTION

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

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 any country.

Cells continually monitor their environment in order to modulate physiological and biochemical processes which in turn affects future behaviour. Frequently, a cell's initial interaction with its surroundings occurs via receptors expressed on the plasma membrane. Activiation of these receptors, whether through binding endogenous ligands (such as cytokines) or exogenous ligands (such as antigens), triggers a biochemical cascade from the membrane through the cytoplasm to the nucleus.

Of the endogenous ligands, cytokines represent an important and versatile group. However, of particular importance are molecules which regulate cytokine function. An example of this class of molecules are members of the family of suppressors of cytokine signalling (SOCS).

SOCS proteins contain a C-terminal homology domain which is termed the "SOCS box" [Starr et al, Nature (London) 387: 917-921, 1997]. The first member of this family was called CIS (cytokme-inducible SH2-containing protein) [Yoshimura et al, EMBO J. 14: 2816-2826, 1995] and was shown to inhibit erythropoietin and interleukin-3 receptor signalling. SOCS-1 was cloned from a retroviral expression library as a cDNA whose constitutive expression inhibited interleukin-6-induced differentiation of Ml [Starr et al, 1997, supra] cells and it was simultaneously cloned as a protein that interacted with activated JAK kinases (JAK-binding protein, JAB) [Endo et al, Nature (London) 387: 921-924, 1997] and as a protein with antigenic similarity to STATs (STAT-inducible STAT inliibitor, SSI) [Naka et al, Nature (London) 387: 924-929, 1997]. The sequence similarity of SOCS-1 and CIS led to the identification of six additional members of tins family (SOCS-2-7) each with an SH2 domain and a C-terminal SOCS box [Starr et al, 1997, supra; Hilton et al, Proc.Natl Acad. Sci. USA 95: 114-119, 1998; Masuhara et al, Biochem. Biophys. Res. Commun. 239: 429-446, 1997; Minamoto et al, Biohcm. Biophys. Res. Commun. 237: 79-83, 1997]. An additional twelve proteins have been described that contain a C-terminal SOCS box but instead of an SH2 domain they contain different protein-protein interaction domains including WD40, ankyrin repeats, SPRY or small GTPase domains [Hilton et al, 1998, supra].

Following binding to their receptors, many cytokines activate receptor-associated cytoplasmic kinases called JAKs which in turn phosphorylate the receptor cytoplasmic domain and associated signal transducers and activators of transcription (STATs). Phosphorylated STAT dimers translocate to the nucleus and activate transcription of specific genes including those of CIS and some of the SOCS. SOCS proteins then recognize activated signalling molecules (including JAKs and cytokine receptors) through their SH2 and N-terminal domains and inhibit their activity [Narazaka et al, Proc. Natl. Acad. Sci. USA 95: 13130-13134, 1998]).

SOCS-5 is one member of the suppressor of cytokine signalling (SOCS) family of proteins and comprises the characteristic central SH2 domain and C-tenninal SOCS box. The physiological function of SOCS-5 is not clearly understood. A biochemical analysis of SOCS-5 has revealed that it is a ubiquitously expressed cytosolic protein. Like other proteins containing a SOCS box, SOCS-5 binds to elongins B and C. This suggests that SOCS-5 may act as an E3 ubiquitin ligase, targeting proteins which interact through its SH2 domain for ubiquitination and proteasomal degradation.

In work leading up to the present invention, the inventors sought to identify molecules which interact with SOCS-5 and in particular but not limited to the SH2 domain. Such molecules and in particular endogenous proteinaceous molecules and complexes of endogenous proteinaceous molecules enable elucidation of signalling pathways involving SOCS-5 and related SOCS molecules. The inventors have surprisingly identified that SOCS-5 interacts with inter alia the epidermal growth factor (EGF) receptor and hence, is involved in EGF-mediated signalling.

Epideπnal growth factor (EGF) or the closely related transforming growth factor α (TGF- α) mediate a wide variety of biological responses, including proliferation and differentiation of epidermal, neural and fibroblast cells. The EGF-receptor (EGF-R) family consists of four family members, EGF-R (Erb-Bl), Erb-B2 (HER2/Neu), Erb-B3 (HER3) and Erb-B4 (HER4). Following ligand binding, the EGF-R -can signal either as a homodimeric protein or as a heterodimer with ErbB-2 or ErbB-4. Signalling is initiated by activation of the intrinsic tyrosine kinase domain present in each receptor subunit, although in the case of Erb-B3, the kinase domain is inactive with the heterodimeric-binding partner initiating signalling. There at least eight known ligands for the EGF-R family, these include; EGF, TGF-α, amphiregulin, β-cellulin, neu differentiation factors, epiregulin, and heparin binding-EGF-like growth factor. Differential activation of signalling pathways has been observed with different ligand and receptor subunit combinations, contributing to the diversity of EGF-R signalling.

In accordance with the present invention, it is proposed that the SOCS-5 and related SOCS molecules are involved in suppression of EGF-mediated signalling. This indicates that SOCS-5 and related SOCS molecules have a role in mediating a range of biological responses including modulation of the proliferation and differentiation of epidermal, neural and fibroblast cells. The present invention provides, therefore, ligands of SOCS-5, EGF and/or EGF-R. It is further proposed that since EGF-R and Erb-B2 are frequently over- expressed in human tumors, SOCS-5 agonists or mimetics are useful in inhibiting EGF- R/Erb-b2 signalling and, hence, tumor growth.

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 NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:l), <400>2 (SEQ ID NO:2), etc. A sequence listing is provided after the claims.

The present invention is predicated in part on the surprising observation that SOCS-5 associates with the EGF-receptor family including Erb-B2 in a phosphotyrosine- independent manner. It is proposed, in accordance with the present invention, that SOCS-5 and related SOCS molecules modulate and in particular suppress mitogenic responses and other signalling mediated via EGF and its isoforms or homologs. The identification of SOCS-5's involvement in EGF-mediated signalling indicates that this molecule is involved in biological activities such as controlling EGF-mediated proliferation and/or differentiation of epidermal, neural and/or fibroblast cells. The identification of the involvement of SOCS-5 in EGF-mediated signalling provides a target for the identification of chemical and proteinaceous molecules capable of modulating SOCS-5 activity and, more particularly, their use as therapeutic agents in the treatment of conditions associated with EGF-mediated signalling such as but not limited to conditions involving aberrant proliferation and/or differentiation of epidermal, neural or fibroblast cells. The present invention further provides effector molecules for EGF such as those which antagonize or agonize EGF-R/Erb-B2 signalling. This is important, for example, in the treatment of tumors which frequently have over-expressed levels of EGF-R and Erb-B2.

The tenn "aberrant" includes both over proliferation and/or differentiation or poor proliferation and/or differentiation. Accordingly, one aspect of the present invention contemplates a method for modulating EGF-mediated/EGF-related molecule-mediated signalling in an animal cell or in an animal comprising the cell wherein the method comprises administering to the cell or the animal comprising the cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and/or an EGF-receptor or EGF-like receptor or a component in an EGF-receptor or EGF-like molecule receptor complex.

The effector molecule may be a SOCS-ligand or an EGF-ligand. The preferred SOCS molecule is SOCS-5. Reference to EGF receptor or EGF-R includes the EGF-R family such as but not limited to Erb-B2.

Another aspect of the present invention contemplates, therefore, a method of modulating the effects of EGF-mediated or EGF-like molecule-mediated signalling in an animal cell, by introducing to the animal cell or animal comprising the cell an antagonist of SOCS-5 for a time and under conditions sufficient to inhibit, reduce or otherwise suppress SOCS-5 inhibition of EGF-mediated or EGF-like molecule mediated signalling or sufficient to induce, enhance or otherwise facilitates EGF-mediated or EGF-like molecule-mediated signalling.

Without limiting the present invention to any one theory or mode of action, it is proposed that the preferred ligands of SOCS-5 interact with the N-terminal region and/or the SH2 domain of SOCS-5.

The present invention further contemplates a method for ameliorating the symptoms of cancer or tumor growth in a subject or inhibiting the growth or spread of a cancer or tumor in a subject, the method comprising administering to the subject an agonist of SOCS-5 for a time and under conditions sufficient to inhibit EGF-R/Erb-B2 signalling on the cancer/tumor cell.

The present invention further provides a means of targeting unwanted or aberrant proteins in a cell for degeneration by introducing directly or via genetic therapy to a cell a ligand of the protein fused, associated or otherwise linked to the SOCS-5 SOCS box. The resulting complex recruits the E3 ligase complex to ubiquitinate the target protein resulting in its degradation.

A further aspect of the present invention contemplates a composition and in particular a pharmaceutical composition comprising an effector molecule of SOCS-5 or a related SOCS molecule or EGF and one or more pharmaceutically acceptable carriers and/or diluents.

Yet another aspect of the present invention further provides genetically modified animals in which one or both alleles of SOCS-5 axe mutated alone or in combination with other mutations in one or both alleles for other genes such as encoding another SOCS molecule.

Still another aspect of the present invention provides a method of producing a genetically modified non-human animal, the method comprising introducing into embryonic stem cells of an animal a genetic construct comprising a SOCS-5 nucleotide sequence carrying a single or multiple nucleotide substitution, addition and/or deletion or inversion or insertion or any other mutation or genetic event which inactivates the gene or otherwise prevents expression of the gene to an active product wherein there is sufficient SOCS-5 nucleotide sequences to promote homologous recombination with a SOCS-5 gene within the genome of the embryonic stem cells selecting for said homologous recombination and selecting embryonic stem cells which carry a mutated SOCS-5 gene and then generating a genetically modified animal from the embryonic stem cell. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photographic representation showing association of SOCS-5 with the EGF-R complex. BaF/ERX cells stably expressing either SOCS-4 or SOCS-5 were incubated with or without 50 ng/ml EGF for 15 minutes on ice and lysed. EGF-R proteins were then immunoprecipitated using mAb 528 (Sato et al, Mol. Biol. Med. 1(5): 511-529, 1983) and associated FLAG-tagged SOCS proteins detected by Western analysis using anti-FLAG antibody (A). Westerns were then re-probed using anti-EGF-R antibody (LICR mAb 806) (C). Lysates were analyzed by Western blot with anti-phosphotyrosine antibodies (B). Levels of SOCS-4 and SOCS-5 protein were determined by immunoprecipitation using anti-FLAG antibody conjugated to agarose (M2-beads) and Western blot with rat anti- FLAG antibodies (D).

Figure 2 is a representation showing Top panel: FACS analysis of BaF/ERX lines showing EGF-receptor expression levels. Ba/F3 cells, which have not been transfected with EGF-R cDNA, are shown in the histogram to the left of Panel A. EGF-R levels are shown to the right of panel A and in subsequent panels. Bottom panel: SOCS-5 expression levels. Equal cell numbers of BaF/ERX puromycin resistant lines and BaF/ERX cells expressing SOCS-5 were lysed and analyzed by immunoprecipitation and Western blot using anti-FLAG antibody. A, B, C, D, E: independent puromycin resistant lines. F, G, H, I, J: independent SOCS-5 clones.

Figure 3 is a graphical representation showing the effect of SOCS-5 expression on EGF- stimulated proliferation. (A) The number of cells obtained per well of the parental BaF/ERX line, two puromycin resistant lines and three independent SOCS-5 clones are shown in response to a three day incubation of increasing concentrations of EGF. (B) The number of cells obtained per well is shown in response to increasing concentrations of IL- 3.

Figure 4 is a graphical representation showing EGF-R in vitro kinase assay. BaF/ERX puromycin resistant lines and SOCS-5 clones were incubated with 50 ng/ml EGF for 15 minutes on ice and lysed. EGF-R proteins were immunoprecipitated with mAb 528 and subjected to an in vitro kinase assay. Kinase activity is shown as cpm incorporated into a substrate peptide. A,B,C,D,E: independent puromycin resistant lines. F, G, H, I, J: independent SOCS-5 clones.

Figure 5 is a graphical representation showing EGF-R tyrosine phosphorylation in BaF/ERX cells expressing SOCS-5. Panels A & B: BaF/ERX cells which are either puromycin resistant clones or which stably express SOCS-5 were stimulated with EGF for the times indicated, lysed and the EGF-R immunoprecipitated using monoclonal antibody 528 (mAb; Sato et al, 1983, supra). Phosphorylated receptor protein was detected by Western blot with anti-phosphotyrosine antibody (4G10; Upstate Biotechnology). The results are shown for two independent puromycin resistant clones and two independent SOCS-5 clones.

Figure 6 is a graphical representation showing proteins associated with the EGF-R protein complex. BaF/ERX cells or BaF/ERX cells stably expressing SOCS-5 were incubated with or without 50 ng/ml EGF for 15 minutes on ice, lysed and the EGF-R immunoprecipitated using mAb 528. Associated proteins were then detected by Western blot with appropriate antibodies. (A) Anti-FLAG antibody (WEHI). (B) Anti-Statl antibody (Transduction Laboratories, Lexington, KY). (C) anti-cbl antibody (C40320; Transduction Laboratories). (D) anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Lake Placid, NY). (E) anti-EGF-R antibody (LICR Mab 806).

Figure 7 is a photographic representation showing (A) Stat5 tyrosine phosphorylation in BaF/ERX cells expressing SOCS-5. Upper panel: Untransfected BaF/ERX cells and BaF/ERX cells stably expressing SOCS-5 were incubated with 50 ng/ml EGF for the times indicated, lysed and tyrosine phosphorylated proteins immunoprecipitated using anti- phosphotyrosine antibodies. Tyrosine phosphorylated Stat5A/B proteins were detected using anti-phospho-Stat5A/B antibody. Lower panel: cell extracts were analyzed by SDS- PAGE and Western blot with anti-Stat5A/B antibodies (S21520). (B) p44/42 MAPK phosphorylation in BaF/ERX cells expressing SOCS-5. Upper panel: Untransfected BaF/ERX cells and BaF/ERX cells stably expressing SOCS-5 were incubated with 50 ng/ml EGF for the times indicated, lysed and analyzed by SDS-PAGE and Western blot using anti-phospho-MAPK antibody (#9106; Cell Signalling Technology, Beverly, MA). Lower panel: cell extracts were analyzed by Western blot with anti-MAPK antibody.

Figure 8 is a photographic representation of Northern blot analysis of mRNA expression of mouse SOCS-5 (4.5-kb transcript) and SOCS-4 in BaF/ERX cells following incubation with EGF (Panels A and C, respectively). The integrity of the RNA was confirmed by hybridization with GAPDH (1.4-kb transcript) (Panels B and D).

Figures 9A and 9B are photographic representations showing that the SOCS-5 SOCS box mediates interaction with elongin B/C complex.

Figures 10A and 10B are photographic representations showing that the SOCS-5 SOCS box mediates interaction with elongin B/C complex.

Figures 11 A and 11B are photographic representations showing that the SOCS-5 SOCS box mediates interaction with elongin B/C complex.

Figures 12A to 12C are graphical representations showing that the presence of SOCS-5 results in reduced EGF-R expressed on the surface of cells following EGF stimulation.

Figures 13A to 13D are photographic representations showing SOCS-5 association with EGF-R complex in the absence of EGF stimulation.

Figures 14A to 14F are photographic representations showing that SOCS-5 associates with both EGF-R and Erb-B2/Neu. The following are abbreviations used in the specification:

ABBREVIATIONS

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification that SOCS-5 is involved in the EGF-mediated and EGF-like molecule-mediated signalling pathway and in the regulation of biological responses mediated directly or indirectly by EGF- or EGF-related molecules. It is proposed, in accordance with the present invention, that SOCS-5, induced by cytokines, associates with EGF-receptor or a EGF-like molecule receptor thereby suppressing its function as a mediator of EGF-mediated signalling. An EGF-receptor or EGF-R includes reference to the EGF-R family which encompasses inter alia Erb-B2. This may contribute to aberrations in the proliferation and/or differentiation of epidermal, neural or fibroblast cells. EGF-mediated signalling mediation is important, therefore, in the treatment of inflammation, cancer, obesity and a range of neurodegenerative disorders. Cancer is a particularly useful target given the over-expression of EGF-R and Erb-B2 on many cancer and tumor cells. The present invention provides a target, therefore, to identify therapeutic molecules capable of modulating the expression or activity of SOCS-5 and related SOCS molecules for use in manipulating EGF- and EGF-like molecule-mediated signalling pathways. Preferred therapeutic molecules are agonists of SOCS-5. An "agonist" includes a SOCS-5 mimetic.

Accordingly, one aspect of the present invention contemplates a method for modulating EGF-mediated/EGF-related molecule-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and or an EGF-receptor or EGF-like receptor or a component in an EGF-receptor or EGF-like molecule receptor complex.

The term "modulating" means that the effector molecule may be an agonist or antagonist of SOCS interaction with its ligand. An agonist including a mimetic would promote suppression of signalling mediated by the SOCS molecule. Such a molecule is useful in the treatment of cancer where EGF-R and Erb-B2 are over-expressed on the cancer cells. An antagonist would inhibit the suppression of signalling. Consequently, in conditions where EGF- or EGF-like molecule-mediated signalling is required to be down-regulated then an agonist is preferred. In conditions where an excess of EGF- or EGF-like molecule- mediated signalling is required, then an antagonist is preferred.

Reference herein to "EGF" or "epidermal growth factor" includes EGF or its functionally equivalent homologs and isoforms. It also includes all members of the EGF-R family involved in SOCS signalling such as, in the case of SOCS-5, Erb-B2.

Accordingly, another aspect of the present invention contemplates a method for modulating EGF-mediated and/or EGF-like molecule-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and EGF-receptor or EGF-like molecule receptor or a component in an EGF-receptor or EGF-like molecule receptor complex.

An EGF-like molecule includes TGF-α, amphiregulin, β-cellulin, Neu differentiation factors, epiregulin and heparin-EGF-like growth factor.

The present invention is particularly directed to SOCS-5 but extends to any SOCS molecule capable of interacting with and suppressing the function of EGF- and EGF-like molecules-mediated pathways. Examples of other SOCS molecule contemplated herein include SOCS-4.

Accordingly, another aspect of the present invention provides a method for modulating EGF-mediated and/or EGF-like molecule-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between SOCS-5 molecule and an EGF-receptor or EGF-like molecule receptor or a component in an EGF-receptor or EGF-like molecule receptor complex. A "component" in the EGF or EGF-like molecule mediated signalling pathways includes Erb-Bl, Erb-B2 (HER2 Neu), Erb-B3 (HER) and Erb-B4 (HERA).

The effector molecules of the present invention may be proteinaceous or may be chemical molecules identified from, for example, screening a chemical library or following natural product screening. The tenn "natural product screening" includes screening environmental and biological locations such as coral, river beds, plants, microorganisms, rock formations, antartic or artic regions or sea water or sea beds for chemical molecules which are capable of interacting with the SOCS molecule and in particular SOCS-5 thereby modulating its function, hi a particular embodiment, the molecule is capable of interacting with the SH2 domain and/or N-terminal domain of SOCS-5.

Reference to modulating EGF-mediated signalling includes modulating EGF-R levels. This may be accomplished by any number of ways such as involving receptor protein synthesis, recycling of the receptor protein, internalization of a receptor complex or degradation of receptor complex following internalization.

Accordingly, another aspect of the present invention contemplates a method of modulating the effects of EGF-mediated or EGF-like molecule-mediated signalling in an animal cell, said method comprising introducing to said animal cell or animal comprising said cell an antagonist of SOCS-5 for a time and under conditions sufficient to inhibit, reduce or otherwise suppress SOCS-5 inhibition of EGF-mediated or EGF-like molecule mediated signalling or sufficient to induce, enhance or otherwise facilitates of EGF-mediated or EGF-like molecule-mediated signalling.

Accordingly, another aspect of the present invention is directed to a SOCS-5 antagonist wherein said antagonist inhibits SOCS-5-mediated suppression of EGF or EGF-like molecule-mediated signalling pathways.

hi another embodiment, the effector molecule is an agonist. Accordingly, yet another aspect of the present invention contemplates a method of modulating the effects of EGF-mediated or EGF-like molecule-mediated signalling in an animal cell, said method comprising introducing to said animal cell or animal comprising said cell an agonist of SOCS-5 for a time and under conditions sufficient to promote, induce or otherwise facilitate SOCS-5 inhibition of EGF-mediated or EGF-like molecule mediated signalling or sufficient to inhibit, reduce or otherwise suppress EGF-mediated or EGF-like molecule-mediated signalling.

An agonist is particularly useful in the treatment of cancer or other conditions involving over-expression of EGF-R or Erb-B2.

Accordingly, another aspect of the present invention is directed to a SOCS-5 agonist wherein said agonist facilitates or enhances SOCS-5 suppression of EGF or EGF-like molecule-mediated pathways.

An agonist is also referred to herein as a SOCS-5 mimetic.

An antagonist or agonist generally have some effect or act as a ligand of the N-tenninal domain of SOCS-5 or the SH2 domain or the SOCS-5 SOCS box region.

Reference to an "animal" includes a human, primate, livestock ammal (e.g. sheep, pig, horse, cow donkey), laboratory test animal (e.g. mouse, rat, guinea pig, rabbit, hamster) or companion animals (e.g. dog, cat). Preferably, for therapeutic applications, the target animal is a human. For test applications or for assessing therapeutic protocols, the target may be a laboratory test animal such as a mouse or rat.

As stated above, the effector molecule may be found following natural product screening or screening of chemical libraries or may be generated by preparing derivatives or analogs of SOCS (e.g. SOCS-5) itself or a SOCS ligand (e.g. EGF or EGF-like molecule). The terms "derivatives" or its singular form "derivative" in relation to a SOCS molecule and in particular SOCS-5 includes parts, mutants, fragments and analogs as well as hybrid or fusion molecules and glycosylation variants of the SOCS. Particularly useful derivatives comprise single or multiple amino acid substitutions, deletions and/or additions to the N- terminal domain of SOCS-5 or the SH2 domain and/or the SOCS box amino acid sequence.

Derivatives may act as antagonists or agonists. The present invention further extends to homologs of SOCS-5 which include the functionally or structurally related molecules from different animal species. The present invention also encompasses analogs and mimetics. Mimetics include a class of molecule generally but not necessarily having a non-amino acid structure and which functionally are capable of acting in an analogous or antagonistic manner to the protein for which it is a mimic, in this case, SOCS-5. Mimetics may comprise a carbohydrate, aromatic ring, lipid or other complex chemical structure or may also be proteinaceous in composition.

The present invention further extends to a range of deletion mutants of SOCS and in particular SOCS-5 carrying deletions in the SH2 domain and/or the SOCS box and/or the N-terminal region of SOCS-5. Molecules are also contemplated by the present invention which encompasses only the carboxy terminal region or amino terminal region or fused to another peptide, polypeptide or protein.

As stated above, the present invention contemplates agonists and antagonists of SOCS-5. Antibodies are one example of an antagonist although these are more useful in diagnostic applications or in the purification of SOCS peptides.

In one embodiment, the present invention provides antagonists of SOCS-5. Such antagonists may be used, for example, in the treatment or prophylaxis of cytokine mediated dysfunction such as autoimmunity, immune suppression or hyperactive immunity or other condition including but not limited to dysfunctions in the haemopoietic, endocrine, hepatic and neural systems. Dysfunctions mediated by other signal transducing elements such as hormones or endogenous or exogenous molecules, antigens, microbes and microbial products, viruses or components thereof, ions, hormones and parasites are also contemplated by the present invention. They may also be useful in promoting degradation or inhibiting degradation. They may also be useful in modulating proliferation of neural cells, epidermal cells and fibroblast cells.

Analogs of SOCS-5, especially those acting as antagonists contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.

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 ; 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 ρyridoxal-5 -phosphate followed by reduction with NaBH .

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 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide 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-3- hydroxy-5-phenylpentanoic acid, 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 below:-

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib D-valine Dval α-memyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnnigln N-(3-guanidinopropyl) glycine Narg

D-N-methylglutamate Dmnglu N-(l-hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-mefhylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nht D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l-methylpropyl) glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dmnval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methyltlireonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diρhenylρropyl) Nnbhe carbamylmethyl)glycine carbamyhnethyl)glycine 1 -carboxy- l-(2,2-diphenyl- Nmbc ethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n 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 C_α and N_α-methylamino acids, introduction of double bonds between C_α and C_β atoms of amino acids and the formation of cyclic peptides or analogs 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.

These types of modifications may also be important to stabilize SOCS molecules if administered to an individual or if used as a diagnostic reagent.

Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

The present invention also provides agonists of SOCS-5. Such molecules are useful to inhibit EGF-R and/or Erb-B2 signalling. They may be identified or comprise molecules as described above for antagonists. Agonists are particularly considered to be useful in the treatment or amelioration of the symptoms of cancer or tumor growth. Such cancers or tumors are those which over-express EGF-R or Erb-B2.

The present invention provides, therefore, methods for identifying SOCS-5, EGF-receptor or EGF-like molecule receptor or components in an EGF-receptor or EGF-like molecule receptor complex or EGF ligands. In one embodiment, the present invention contemplates a method for screening for a ligand of SOCS-5 or EGF-receptor or EGF-like molecule receptor or component in an EGF- receptor or EGF-like molecule receptor complex, said method comprising contacting a potential ligand of SOCS-5, or EGF-receptor or EGF-like molecule receptor or component in an EGF-receptor or EGF-like molecule receptor complex with a cell which is capable of expressing a nucleic acid molecule encoding a component in the EGF-mediated or EGF- like molecule mediated signalling together with a reporter molecule capable of directly or indirectly providing an identifiable signal and screening for an increase or decrease in said signal in the presence of SOCS-5.

Clearly, there are many ways of screening for SOCS-5 ligands including agonists and antagonists. For example, plasmids or vectors are generated encoding FLAG-tagged SOCS-5 or FLAG-tagged SOCS-5 containing a mutated SH2 domain (e.g. S5mSH2, see Example 13) or other mutated region. These plasmids may be introduced alone into a cell or co-introduced with a vector encoding an EGF-R. Cells are then incubated with or without EGF and with or without a potential agonist or antagonist of SOCS-5 and the levels of EGF-mediated signalling can then be readily determined. For example, the EGF- R may be fused to a reporter molecule or a physiological function of EGF-mediated signalling monitored.

Another aspect of the present invention contemplates a method of modulating levels of SOCS-5 in a cell, said method comprising contacting a cell containing a SOCS-5 gene with an effective amount of an inhibitor of expression of the SOCS-5 gene for a time and under conditions sufficient to modulate levels of said SOCS-5 protein.

Still a further aspect of the present invention contemplates a method of modulating signal transduction in a cell containing a SOCS-5 gene comprising contacting said cell with an effective amount of an inhibitor of SOCS-5 gene expression for a time sufficient to modulate levels of SOCS-5 protein with the cell. Yet a further aspect of the present invention contemplates a method of modulating EGF- or EGF-like mediated signalling, said method comprising administering to a subject a modulating effective amount of a molecule for a time and under conditions sufficient to decrease or increase the biological activity of SOCS-5 or down-regulating or up-regulating the suppressing effects of SOCS-5.

Still a further aspect of the present invention contemplates a composition and in particular a pharmaceutical composition comprising an effector molecule of SOCS-5 or a related SOCS molecule and one or more pharmaceutically acceptable carriers and/or diluents.

The present invention, therefore, contemplates a composition and in particular a pharmaceutical composition comprising an effector molecule as defined above and one or more pharmaceutically acceptable carriers and/or diluents. These components are referred to as the "active ingredients".

The pharmaceutical 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, ethanol, polyol (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 microoganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmersol and the like. In many cases, it will be preferable to include isotinic 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, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they 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. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, baccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. 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 active compound 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 dosage unit form contains between about 0.1 μg and 2000 mg of active compound. Alternative dosage amounts include from about 1 μg to about 1000 mg and from about 10 μg to about 500 mg.

The present invention also extends to forms suitable for topical application such as creams, lotions and gels as well as a range of "paints" which are applied to skin and through which the active ingredients are absorbed. In addition, the complex or components thereof may be associated with penetration or the TAT protein of HIV.

Pharmaceutically acceptable barriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and except insofar as any conventional media or agent is incompatible with the active ingredient, their use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable barrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts range from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

Dosages may also be expressed per body weight of the recipient. For example, from about 10 ng to about 1000 mg/kg body weight, from about 100 ng to about 500 mg/kg body weight and for about 1 μg to above 250 mg/kg body weight may be administered.

The pharmaceutical 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 modulating levels of polypeptides involved in the complex. The vector, may, for example, be a viral vector. Genetic molecule-based compositions may also include agonists and antagonists of SOCS-5 expression including antisense molecules, ribozymes and co-suppression molecules.

The present invention further provides an animal model useful for assessing potential agonists and antagonists of SOCS-5 function.

Accordingly, the present invention further provides genetically modified animals in which one or both alleles of SOCS-5 are mutated alone or in combination with other mutations in one or both alleles for other genes such as encoding another SOCS molecule. Preferably, the genetically modified animals are laboratory test animals such as murine species (e.g. mice, rats), rabbits, guinea pigs or hamsters, livestock animals such as sheep, pigs, horses or cows or non-human mammals such as primates. Conveniently, and preferably, the genetically modified animal is a murine species such as a mouse or rat.

Genetically modified animals with one or both alleles of SOCS-5 mutated in combination with other mutations in one or both alleles for other genes are referred to herein as "composite" genetically modified animals.

The genetic modification is generally in the form of a mutation such as a single or multiple nucleotide substitution, deletion and/or addition or inversion or insertion. Generally, such a genetically modified animal is referred to as a "knock-out" animal.

Genetically modified animals and in particular knock-out murine animals may be prepared by any number of means. In one method, a targeting DNA construct is prepared comprising a nucleotide sequence which is sufficiently homologous to a target sequence such a SOCS-5 to permit homologous recombination. The SOCS-5 targeting sequence may be isogenic or non-isogenic to the target SOCS-5 sequence. The targeting DNA construct generally comprises a selectable marker within the targeting sequence such that by homologous recombination, the target SOCS-5 gene is disrupted by an insertional mutation. The targeting DNA construct is generally introduced into an embryonic stem cell or embryonic stem cell line.

As an alternative to using a selectable marker, a mutation may be introduced which induces a phenotypic change which may then be selected or detected.

Accordingly, another aspect of the present invention provides a method of producing a genetically modified non-human animal, said method comprising introducing into embryonic stem cells of an animal a genetic construct comprising a SOCS-5 nucleotide sequence carrying a single or multiple nucleotide substitution, addition and/or deletion or inversion or insertion or any other mutation or genetic event which inactivates the gene or otherwise prevents expression of the gene to an active product wherein there is sufficient SOCS-5 nucleotide sequences to promote homologous recombination with a SOCS-5 gene within the genome of said embryonic stem cells selecting for said homologous recombination and selecting embryonic stem cells which carry a mutated SOCS-5 gene and then generating a genetically modified animal from said embryonic stem cell.

Preferably, the genetically modified animal is a murine species such as a mouse or rat.

The SOCS-5 nucleotide sequence may be isogenic or non-isogenic to the SOCS-5 gene in the embryonic stem cell.

The term "isogenic" means that the SOCS-5 nucleotide sequence in the construct is derived from the same animal strain from which the embryonic stem cell has been derived or a strain exhibiting the same genotype.

The present invention further contemplates non-homologous-mediated integration of the target DNA sequence.

A range of selectable markers may be employed and reference may be made to U.S. Patent No. 5,789,215 for general methodologies. Breeding protocols may also be adopted to introduce mutations or other genetic modifications into SOCS-5. In one approach, an EMS or other mutagenized mouse is crossed with a non-mutagenized mouse to produce a Gl generation. The Gl generation may then be crossed with an index strain to produce GIFI kindreds which are then screened phenotypically for mutation in SOCS-5. Mutations in SOCS-5 may be dominant or recessive and mutations may be detected directly on SOCS-5 or by its effect on another gene or on its effect in alleviating the effects of a first mutation on another gene.

All genetically modified animals in accordance with the present invention, including knock-out mice, carry mutations or other genetic events in one or both SOCS-5 alleles alone or in combination with mutations in other genes or genetic region.

The present invention further contemplates antibodies to SOCS-5.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milstein, European Journal of Immunology 6: 511-519, 1976).

Another aspect of the present invention contemplates a method for detecting SOCS-5 in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for SOCS-5 or its derivatives or homologs for a time and under conditions sufficient for an antibody-SOCS-5 complex to form, and then detecting said complex. The presence of SOCS-5 may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non- competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen- labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control ample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is one which might contain SOCS-5 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fennentation fluid and supernatant fluid such as from a cell culture.

i a typical forward sandwich assay, a first antibody having specificity for the SOCS-5 or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about 37°C including 25°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.

Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By "reporter molecule", as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, -galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, winch yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorecein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.

As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody- hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. The ability to identify SOCS-5 immunologically permits diagnostic assays for SOCS-5 alone or in a complex with other molecules such as molecules involved in growth factor or hormone signalling.

Diagnostic assays may also involve immobilizing SOCS-5 and screening for molecules which interact with SOCS-5. Alternatively, particular ligands may be immobilized to screen for SOCS-5. The ability to quantitate SOCS-5 or its ligands or complexes thereof provides significant diagnostic and therapeutic value.

SOCS-5 quantitation may also be conducted at the genetic level where mRNA transcript levels may be determined for the SOCS-5 gene. Such genetic assays may also be conducted at the DNA level especially to identify polymorphisms of SOCS-5.

In one embodiment, differential hybridization or differential priming is useful in distinguishing between polymorphisms of the SOCS-5 gene. Single nucleotide polymorphism (SNP) technology may also be employed.

The ability to identify SOCS-5 immunologically permits diagnostic assays for SOCS-5 alone or in a complex with other molecules such as molecules involved in growth factor signalling.

The present invention further provides a means of targeting particular proteins for degradation within the cell. This aspect is conveniently accomplished by generating fusion molecules comprising a ligand for the target protein which is fused or otherwise associated with the SOCS-5 SOCS box or a functional equivalent thereof.

The target protein-ligand-SOCS-5 SOCS box then recruits the E3 ligase complex which ubiquitinates the target protein, resulting in degradation of the protein.

Accordingly, another aspect of the present invention provides a method for the selective degradation of a target protein in a cell, said method comprising introducing to said cell an agent comprising a ligand for said target protein fused or otherwise associated with a SOCS-5 SOCS box or introducing a genetic molecule capable of encoding said agent, wherein upon binding of the target protein to the agent, the resulting complex recruits the E3 ubiquitin ligase which ubiquitinates the target protein resulting in its degradation.

Target proteins include cancer-associated proteins, pathogen-associated proteins and aberrant proteins.

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

EXAMPLE 1 SOCS-5

SOCS molecules are described in International Patent Application No. PCT/AU97/00729 [WO 98/20023]. The nucleotide and amino acid sequences of human SOCS-5 are shown in SEQ ID NOS:l and 2, respectively. The nucleotide and amino acid sequences of murine SOCS-5 are shown in SEQ ID NOS:3 and 4, respectively.

EXAMPLE 2 Generating anti-SOCS-5 monoclonal antibodies

Mice are immunized with a GST-SOCS-5 N-terminal peptide. Splenocytes from immunized mice are then fused with myeloma cells and hydridomas selected. Hybridoma supernatant fluids are then screened by ELISA to detect monoclonal antibodies (mAbs) which recognized GST SOCS-5 N-terminal peptide but not GST alone.

The mAbs identified are then characterized by isotype, immunoprecipitation, Western blot and epitope to which it binds (defined by amino acid residue sequence).

EXAMPLE 3

Generation of knock-out mice

The SOCS-5 gene is disrupted in mouse embryonic stem (ES) cells by homologous recombination of a targeting vector carrying a selectable marker. ES cell clones are identified carrying the targeted allele. SOCS-5 knock-out mice generated using these ES cells are shown to be genotypically SOCS-5^"/".

EXAMPLE 4 Model system of EGF-R signalling

The role of SOCS-4 and SOCS-5 in the regulation of tyrosine kinase receptor pathways was investigated using a model system of EGF-R signalling. Ba/F3 cells expressing the EGF-receptor show an increased survival response to EGF but will not proliferate in response to EGF. A mitogenically responsive line was derived by selection of Ba F3 cells expressing the EGF-receptor in EGF. The resulting cell line (BaF/ERX) grows continuously in EGF and is dependent on EGF for normal transit of the cell cycle. One of the advantages of the BaF/ERX line is that the EGF-R (Erb-Bl) is the only EGF-R family member present, allowing analysis of tins receptor subunit in the absence of heterodimeric binding partners.

EXAMPLE 5

SOCS-5 associates with the EGF-R complex

BaF/ERX cells were electroporated with expression plasmids for FLAG epitope-tagged SOCS-4 and SOCS-5, and multiple independent clones obtained for each. To assess the inherent variability in the EGF response, multiple independent lines were also derived which were transfected with the plasmid conferring puromycin resistance alone. All lines were derived and maintained by growth in IL-3. The ability of SOCS-4 and SOCS-5 to associate with the EGF-R complex was then examined by immunoprecipitation of EGF-R proteins and Western blot analysis for the presence of FLAG-tagged SOCS-4 and SOCS-5. Despite equivalent levels of protein expression, SOCS-5, but not SOCS-4 was observed to constitutively associate with the EGF-R complex. This appeared to occur in a phosphotyrosine independent manner. The results are shown in Figure 1.

EXAMPLE 6 SOCS-5 inhibits the EGF mitogenic response

Having determined that SOCS-5 was present in the EGF-R complex, the inventors examined the effect of SOCS-5 expression on EGF proliferation. Expression of the SOCS- 5 protein was confirmed in each case by immunoprecipitation and Western blot analysis with anti-FLAG antibodies. Maintenance of EGF-R expression levels was confirmed by FACS analysis using anti-EGF-R antibodies. Six independent SOCS-5 expressing clones were examined, all of which had a dramatically inhibited growth response to EGF. In contrast, all puromycin resistant lines and the parental BaF/ERX line, demonstrated a consistent proliferative response to EGF, increasing in cell number over the three day assay period. To confirm that this effect was specific to EGF signalling, cell lines were analyzed for their proliferative response to LL-3. Results are shown in Figures 2 and 3. All six SOCS-5 clones and the puromycin resistant lines demonstrated a similar response to IL-3.

EXAMPLE 7 SOCS-5 does not inhibit EGF-R kinase activity

SOCS-1 and SOCS-3 have been shown to act through inhibition of JAK protein tyrosine kinase activity. An in vitro kinase assay was performed to address whether SOCS-5 inhibition of EGF-R signalling was mediated through direct inhibition of the EGF-R tyrosine kinase domain. Puromycm resistant lines and SOCS-5 expressing lines were incubated in the presence and absence of EGF, lysed and the EGF-R proteins immunoprecipitated. Immunoprecipitates were then incubated in the presence of γ-ATP and a substrate peptide. A high background activity is observed due to antibody aggregation of the receptor. No significant difference in EGF-R kinase activity was observed in the SOCS-5 expressing lines in comparison with the puromycin lines, suggesting that SOCS-5 inhibition of EGF proliferation is not mediated through a direct inhibition of EGF-R kinase activity. Consistent with this, no differences were observed in levels of EGF-R tyrosine phosphorylation (Figure 4).

EXAMPLE 8

SOCS-5 associates with the EGF-R in an EGF-independent manner in an over-expression system

SOCS-3 has been shown to bind to tyrosine residues within the IL-6 signalling subunit gpl30, the Leptin receptor and the erythropoietin receptor, binding to the same docking site as the SHP2 tyrosine phosphatase. CIS is also recruited to receptor phosphotyrosines, and is thought to act by blocking recruitment of Stat5 molecules to the receptor complex. The inventors, therefore, examined the recruitment of the signalling molecules, Statl and cbl to the EGF-R complex in cells expressing SOCS-5. EGF-R proteins were immunoprecipitated using specific antibodies and analyzed by Western Blot for the presence of FLAG-tagged SOCS-5, Statl and cbl. Phosphorylation of the EGF-R and EGF-R expression levels was confirmed by Western blot with anti-phosphotyrosine and anti-EGF-R antibodies, respectively. Again, SOCS-5 was observed to associate in a constitutive fashion with the EGF-R complex. In contrast, both Statl and cbl were recruited to the complex in an EGF-dependent manner, consistent with previous reports. As the majority of EGF-R signalling molecules are recruited to the EGF-R following activation of the tyrosine kinase domain and phosphorylation of receptor tyrosine residues, this suggests that SOCS-5 may interact with the EGF via an alternative mechanism. It remains possible that low levels of receptor phosphorylation occurs in the absence of EGF and are involved in the SOCS-5 interaction. The results are shown in Figures 5 and 6.

EXAMPLE 9 SOCS-5 does not inhibit EGF-mediated activation of STATS or MAPK pathways

The inventors also assessed the effect of SOCS-5 expression on activation of several signalling pathways initiated by activation of the EGF-R. There was no significant difference observed in EGF-stimulated Stat5 tyrosine phosphorylation or in EGF- stimulated MAPK activation in cells expressing SOCS-5 compared with parental BaF/ERX cells. This suggests that SOCS-5 does not inhibit EGF mitogenic signalling through inhibition of the Stat5 or Ras/MAPK pathways. The results are shown in Figure 7.

EXAMPLE 10 SOCS-5 can induce SOCS-4 and SOCS-5 mRNA expression

To date, the majority of the SOCS proteins are induced in response to cytokine stimulation, acting as part of a negative feedback loop. The inventors sought to determine whether EGF induced the expression of SOCS-5. This was achieved by performing a Northern analysis to determine whether EGF treatment induced SOCS-4 or SOCS-5 mRNA expression in BaF/ERX cells (Figure 8). A weak induction of both SOCS-4 and SOCS-5 mRNA was observed following a two hour incubation of the cells in EGF, suggesting that both SOCS- 4 and SOCS-5 may be transcriptionally regulated by EGF.

EXAMPLE 11

SOCS-4 and SOCS-5 SOCS boxes interact with the

Elongin B and Elongin C complex

Figures 9, 10 and 11 demonstrate that the SOCS-5 and SOCS-4 "SOCS boxes" interact with the elongin B/C complex. Furthermore, mutation of the conserved SOCS box residues, shown to be important in the VHL SOCS box for binding to elongin C, also results in disruption of the SOCS-5 and SOCS-4 elongin B/C complex. These datasuggest that the SOCS-4 and SOCS-5 SOCS boxes are important for their physiological function.

293T cells were transiently transfected with expression plasmids for either FLAG-tagged SOCS-5 (F-S5), FLAG-tagged SOCS-5 in which the SOCS box is deleted (F-S5ΔSB) or vector alone (vector). Cells were then lysed and FLAG-tagged proteins immunoprecipitated using M2-beads. Immunoprecipitated proteins were eluted using FLAG peptide, concentrated and separated by SDS-PAGE electrophoresis on a 12% v/v gel. Association of the elongin B/C complex was detected by Western blotting with a rabbit polyclonal antibody that detects both elongin B and elongin C (Figure 9A). Reprobing the blot with rat anti-FLAG antibody revealed equivalent amounts of both SOCS-5 proteins (Figure 9B).

293 T cells were transiently transfected with expression plasmids for either FLAG-tagged SOCS-5 (SOCS-5), FLAG-tagged SOCS-5 in which the SOCS box is deleted (SOCS- 5ΔSB), FLAG-tagged SOCS-5 containing a mutated SOCS box (SOCS-5mutSB) or vector alone (vector). The residues mutated in the SOCS-5 SOCS box are lysine 484 to proline and cysteine 488 to phenylalanine. Residues mutated in the SOCS-4 SOCS box are lysine 386 to proline and cysteine 390 to phenylalanine. Newly synthesized proteins were metabolically labelled by growing cells in medium containing ³⁵S-methionine/cysteine for either 30, 60 or 180 minutes. FLAG-tagged proteins were immunoprecipitated with M2- beads, eluted using sample buffer and separated by SDS-PAGE. Proteins were then transferrred electrophoretically to PVDF membranes and SOCS proteins visualized by exposure to film (Figure 10A). The presence of elongin C was detected by probing the membranes with anti-elongin C antibody (Figure 10B).

293T cells were transiently transfected with expression plasmids for either FLAG-tagged SOCS-4 (F-S4), FLAG-tagged SOCS-4 in which the SOCS box is deleted (F-S4ΔSB), FLAG-tagged SOCS-4 containing a mutated SOCS-box (F-S4mutSB) or vector alone (vector). Cells were then lysed and FLAG-tagged proteins immunoprecipitated using M2- beads. Immunoprecipitated proteins were eluted using FLAG peptide, concentrated and separated by SDS-PAGE electrophoresis on a 12% v/v gel. Association of Elongin B was detected by Western blotting with a rabbit polyclonal antibody (Figure 11 A). Reprobing the blot with rat anti-FLAG antibody revealed equivalent amounts of SOCS-4 proteins (Figure 1 IB).

EXAMPLE 12 SOCS-5 modulates EGF-R levels and mutation of the SOCS-5 SH2 domain results in loss of effect

The data presented in Figures 12A to 12C demonstrate that the presence of SOCS-5 results in reduced EGF-R expressed on the surface of cells, following EGF stimulation. This appears to be mediated at least in part by the SH2-domain, as a single point mutation in the SH2 domain can abrogate this effect.

Puromycin resistant BaF/ERX clones expressing SOCS-5 (5.4.2, 5.8.5, 5A.9.2), SOCS-5 containing a mutated SH2 domain (S5mSH2.4.4.) or the puromycin resistant gene alone (Purol) and the parental BaF/ERX cell line were washed twice and resuspended in 50 ng/ml mEGF in DME with 10% w/v FCS at 37°C. 1 x 10⁶ aliquots were removed at 0 and 4 hrs for determination of available EGF receptors on the cell surface. Cells were incubated with ¹²⁵I mEGF in the presence and absence of excess unlabelled EGF (50 μg/ml) on iceTor 60 minutes. Binding of ¹²⁵I mEGF was detected using a gamma counter and corrected for non-specific binding. Each cell line was assayed in duplicate. The results are expressed as a percentage of binding at 0 hrs.

Puromycin resistant BaF/ERX clones expressing SOCS-5 (5.8.5), SOCS-5 containing a mutated SH2 domain (S5mSH2.1.2, S5mSH2.8.2) or the puromycin resistant gene alone (Purol) and the parental BaF/ERX cell line were washed twice and resuspended in 50 ng/ml mEGF in DME with 10% w/v FCS at 37°C. 1 x 10⁶ aliquots were removed at 0 and 4 hrs for determination of available EGF receptors on the cell surface as described in Figure 12 A.

FACS Analysis: 5x10 cells were also removed at 0 and 4 hrs for analysis with the anti- EGF-R antibody, 528. MAb 528 recognizes an epitope within the ligand-binding domain of the EGF receptor. It, therefore, competes with EGF for binding to the receptor and would be expected to primarily detect unoccupied EGF-R on the cell surface. Cells were incubated with primary antibody (528) for 30 min on ice, washed and incubated with secondary antibody (anti-mouse Ig-FITC) 30 min on ice. Dead cells were excluded from the analysis on the basis of propidium iodide uptake.

Protein expression levels of SOCS-5 proteins containing a mutated SH2 domain. Equal cell numbers of BaF/ERX clones expressing SOCS-5 (5.8.5), SOCS-5 containing a mutated SH2 domain (S5mSH2.1.2, S5mSH2.8.2, S5mSH2.1.2) or the puromycin resistant gene alone (Purol) were lysed and the FLAG-tagged SOCS-5 proteins immunoprecipated using M2 beads. Precipitated proteins were separated by SDS-PAGE and detected by western blotting with rat anti-FLAG antibody. The results indicate that the mutated proteins are expressed at equivalent levels to the SOCS-5 protein. EXAMPLE 13

Mutation of the SOCS-5 SH2 domain can eliminate the interaction of SOCS-5 and the EGF-R

293 T cells were transiently transfected with expression plasmids for FLAG-tagged SOCS- 5 (SOCS-5) or FLAG-tagged SOCS-5 containing a mutated SH2 domain (S5mSH2) and either vector alone (-) or an expression plasmid for the EGF-R (+). Cells were incubated with (+) or without (-) 50 ng/ml EGF for 15 min on ice and lysed. Following lysis, receptor proteins were immunoprecipitated using anti-EGF-R antibody (528) and protein complexes separated by SDS-PAGE under reducing conditions. Association of the FLAG-tagged SOCS-5 proteins with the receptor complex was detected by Western blot with rat anti- FLAG antibody (Figure 13 A). The blot in (A) was then stripped and re-probed with anti- EGF-R antibody (806) to show relative amounts of EGF-R protein precipitated (Figure 13B). Cell lysates were also Western blotted with anti-phosphotyrosine antibody (4G10) to confirm phosphorylation of the receptor following EGF stimulation (Figure 13C). Expression levels of the SOCS-5 proteins were confirmed by immunoprecipitation using M2 beads and Western blot with rat anti-FLAG antibody.

These data are consistent with SOCS-5 associating with the EGF-R complex in the absence of EGF stimulation. This association is then lost following mutation of the SOCS-5 SH2 domain.

EXAMPLE 14 SOCS-5 appears to associate with both the EGF-R and Erb-B2/Neu

293T cells were transiently transfected with expression plasmids for SOCS-5 (S5) alone or SOCS-5 in combination with expression plasmids for either the EGF-R or Erb-B2. Control transfections were performed with empty vector (pEFBOS). Cells were lysed and proteins immunoprecipitated using specific antibodies to FLAG-tagged SOCS-5 (M2 beads), EGF- R (528) or Erb-B2 (Ab5). Protein complexes were then separated using SDS-PAGE and FLAG-tagged proteins detected using rat anti-FLAG antibody. SOCS-5 was shown to co- immunoprecipitate with both EGF-R proteins and Erb-B2 proteins (Figure 14A). Membranes were stripped and re-probed with the appropriate anti-receptor antibodies to determine the amount of receptor protein immunoprecipitated in each lane. Both endogenous EGF-R and Erb-B2 were detected in the 293T cells and the levels of both were enhanced following transfection of expression plasmids for EGF-R and Erb-B2 (Figures 14C and 14D). Western blotting of cell lysates using anti-phosphotyrosine antibody detected tyrosine phosphorylation of the over-expressed EGF-R (Figurel4D) and Western blotting of cell lysates using the anti-EGF-R and anti-Erb-B2 antibodies, respectively, again confirmed increased levels following transfection (Figures 14E and 14F).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

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Hilton, D.J., Richardson, R.T., Alexander, W.S., Viney, E.M., Willson, T.A., Sprigg, N.S., Starr, R., Nicholson, S.E., Metcalf, D.& Nicola, N.A. (1998) Proc. Natl. Acad. Sci. USA 95: 114-119.

Kohler and Milstein (1976) European Journal of Immunology 6: 511-519.

Kohler and Milstein, (1975) Nature 256: 495-499.

Masuhara, M., Sakamoto, H., Matsumoto, A., Suzuki, R., Yasukawa, H., Mitsui, K., Wakioka, T., Tanimura, S., Sasaki, A., Misawa, H., Yokouchi, M., Ohtsuba, M. & Yoshimura, A. {1997) Biochem. Biophys. Res. Commun. 239: 429-446.

Minamoto, S., Ikegame, K., Ueno, K., Narazaki, M., Naka, T., Yamamoto, H., Matsumoto, T., Saito, M., Hosoe, S. & Kishimoto, T. (1997) Biochem. Biophys. Res. Commun. 237: 79-83.

Naka, T., Narazaki, M., Hirata, M., Matsumoto, T., Minamoto, S., Aono, A., Nishimoto, N., Kajita, T., Taga, T., Yoshizaki, K., Akira, S. & Kishimoto, T. (1997) Nature (London) 387: 924-929.

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Claims

1. A method for modulating EGF-mediated/EGF-related molecule-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and or an EGF-receptor or EGF-like receptor or a component in an EGF-receptor or EGF-like molecule receptor complex.

2. The method of Claim 1 wherein the SOCS is SOCS-5.

3. The method of Claim 1 or 2 wherein the EGF-like molecule is selected from TGF-α, amphiregulin, β-cellulin, epiregulin and heparin-EGF-like growth factor.

4. The method of Claim 1 wherein the component in an EGF-receptor or EGF- like molecule receptor is Erb-Bl, Erb-B2 (HER2/Neu), Erb-B3 (HER) and Erb-B4 (HER4).

5. The method of Claim 4 wherein the component in an EGF-receptor or EGF- like molecule receptor is Erb-B2.

6. The method of Claim 1 wherein the effector molecule is a proteinaceous molecule.

7. The method of Claim 1 wherein the effector molecule is a non-proteinaceous chemical molecule.

8. The method of Claim 6 or 7 wherein the effector molecules are identified following chemical library screening or natural product screening.

9. The method of Claim 2 wherein the effector molecule interacts with an SH2 domain of SOCS-5 and/or the N-terminal domain of SOCS-5.

10. The method of Claim 2 wherein the effector molecule interacts with the SOCS- 5 SOCS box.

11. The method of Claim 6 or 7 or 8 or 9 wherein the effector molecule is an antagonist of SOCS-5.

12. The method of Claim 6 or 7 or 8 or 9 wherein the effector molecule is an agonist of SOCS-5.

13. A method for screening for a ligand of SOCS-5 or EGF-receptor or EGF-like molecule receptor or component in an EGF-receptor or EGF-like molecule receptor complex , said method comprising contacting a potential ligand of SOCS-5 or EGF- receptor or EGF-like molecule receptor or component in an EGF-receptor or EGF-like molecule receptor complex with a cell which is capable of expressing a nucleic acid molecule encoding a component in the EGF-mediated or EGF-like molecule mediated signalling together with a reporter molecule capable of directly or indirectly providing an identifiable signal and screening for an increase or decrease in said signal in the presence of SOCS-5.

14. The method of Claim 13 wherein the ligand is a SOCS-5 antagonist.

15. The method of Claim 13 wherein the ligand is a SOCS-5 agonist.

16. The method of Claim 13 wherein the ligand is an EGF-receptor or EGF-like molecule receptor or component in an EGF-receptor or EGF-like molecule receptor complex antagonist.

17. The method of Claim 13 wherein the ligand is an EGF-receptor or EGF-like molecule receptor or component in an EGF-receptor or EGF-like molecule receptor complex agonist.

18. An isolated ligand of SOCS-5.

19. The ligand of Claim 18 wherein the ligand is a SOCS-5 antagonist.

20. The ligand of Claim 18 wherein the ligand is a SOCS-5 agonist.

21. The ligand of Claim 18 wherein the ligand is an EGF-receptor or EGF-like molecule receptor or component in an EGF-receptor or EGF-like molecule receptor complex antagonist.

22. The ligand of Claim 18 wherein the ligand is an EGF-receptor or EGF-like molecule receptor or component in an EGF-receptor or EGF-like molecule receptor complex agonist.

23. The ligand of Claim 18 wherein the ligand is an antibody to SOCS-5.

24. The ligand of Claim 23 wherein the antibody is a monoclonal antibody.

25. A composition comprising the ligand of Claim 18 or 19 or 20 or 21 or 22 or 23 or 24 together with one or more pharmaceutically acceptable carriers and/or diluents.

26. A method of modulating levels of SOCS-5 in a cell, said method comprising contacting a cell containing a SOCS-5 gene with an effective amount of an inhibitor of expression of the SOCS-5 gene for a time and under conditions sufficient to modulate levels of said SOCS-5 protein.

27. A method of modulating levels of SOCS-5 in a cell, said method comprising contacting a cell containing a SOCS-5 gene with an effective amount of an activator of expression of the SOCS-5 gene for a time and under conditions sufficient to modulate levels of said SOCS-5 protein.

28. A method of modulating signal transduction in a cell containing a SOCS-5 gene comprising contacting said cell with an effective amount of an inhibitor of SOCS-5 gene expression for a time sufficient to modulate levels of SOCS-5 protein with the cell.

29. A method of modulating signal transduction in a cell containing a SOCS-5 gene comprising contacting said cell with an effective amount of an activator of SOCS-5 gene expression for a time sufficient to modulate levels of SOCS-5 protein with the cell.

30. A method of modulating EGF- or EGF-like mediated signalling, said method comprising administering to a subject a modulating effective amount of a molecule for a time and under conditions sufficient to decrease or increase the biological activity of SOCS-5 or down-regulating or up-regulating the suppressing effects of SOCS-5.

31. A method of producing a genetically modified non-human animal, said method comprising introducing into embryonic stem cells of an animal a genetic construct comprising a SOCS-5 nucleotide sequence carrying a single or multiple nucleotide substitution, addition and/or deletion or inversion or insertion or any other mutation or genetic event which inactivates the gene or otherwise prevents expression of the gene to an active product wherein there is sufficient SOCS-5 nucleotide sequences to promote homologous recombination with a SOCS-5 gene within the genome of said embryonic stem cells selecting for said homologous recombination and selecting embryonic stem cells which carry a mutated SOCS-5 gene and then generating a genetically modified animal from said embryonic stem cell.

32. The method of Claim 31 wherein the genetically modified animal is a rat.

33. The method of Claim 31 wherein the genetically modified animal is a mouse.

34. The method of Claim 31 wherein the SOCS-5 nucleotide sequence is isogenic to the SOCS-5 in the embryonic stem cell.

35. The method of Claim 31 wherein the SOCS-5 nucleotide sequence is non- isogenic to the SOCS-5 gene in the embryonic stem cell.

36. A method of producing a genetically modified non-human animal, said method comprising introducing into embryonic stem cells of an animal a genetic construct comprising a SOCS-5 nucleotide sequence and one or more other SOCS-family member nucleotide sequences said nucleotide sequences carrying single or multiple nucleotide substitutions, additions and/or deletions or inversions or insertions or any other mutations or genetic events which inactivate the gene or otherwise prevent expression of the gene to an active product wherein there is sufficient SOCS-5 nucleotide sequence and other SOCS- family member nucleotide sequences to promote homologous recombinations with a SOCS-5 gene and said other SOCS-family member gene within the genome of said embryonic stem cells selecting for said homologous recombinations and selecting embryonic stem cells which carry a mutations in a SOCS-5 gene and one or more other SOCS-family member genes and then generating a genetically modified animal from said embryonic stem cell.

37. A method for detecting SOCS-5 in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for SOCS- 5 or its derivatives or homologs for a time and under conditions sufficient for an antibody- SOCS-5 complex to form, and then detecting said complex.

38. A method for the selective degradation of a target protein in a cell, said method comprising introducing to said cell an agent comprising a ligand for said target protein fused or otherwise associated with a SOCS-5 SOCS box or introducing a genetic molecule capable of encoding said agent, wherein upon binding of the target protein to the agent, the resulting complex recruits the E3 ubiquitin ligase which ubiquitinates the target protein resulting in its degradation.

39. A method for screening for a ligand of SOCS-5 which antagonizes or agonizes SOCS-5 interaction with EGF-R, said method comprising contacting a cell which produces SOCS-5 and EGF-R with a potential ligand and screening for enhanced or reduced EGR- mediated signalling.

40. A method for screening for a ligand of SOCS-5 which antagonizes or agonizes SOCS-5 interaction with EGF-R, said method comprising contacting an EGF-R phosphopeptide with potential ligands and screening for enhanced or reduced binding between SOCS-5 and the EGF-R phosphopeptide.