WO2004015417A1

WO2004015417A1 - A method and agents useful for same

Info

Publication number: WO2004015417A1
Application number: PCT/AU2003/001027
Authority: WO
Inventors: Christopher John Greenhalgh; Louis Jerry Fabri; Anne Louise Thaus; Jian-Guo Zhang; Phillip Owen Morgan; Warren S Alexander; Manuel Baca; Andrew Donald Nash; Douglas J Hilton; Ian Street
Original assignee: The Walter And Eliza Hall Institute Of Medical Research
Priority date: 2002-08-13
Filing date: 2003-08-13
Publication date: 2004-02-19
Also published as: AU2002950745A0

Abstract

The present invention relates generally to a method for regulating cytokine signaling 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 signaling (SOCS) interaction in controlling cytokine signaling. 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 signaling. Control of cytokine signaling is important for the treatment of a range of conditions including cancer, inflammatory conditions, immunological disorders, growth disorders and any other conditions involving aberrations of signal transduction.

Description

A METHOD AND AGENTS USEFUL FOR SAME

FIELD OF THE INVENTION

The present invention relates generally to a method for regulating cytokine signaling 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 signaling (SOCS) interaction in controlling cytokine signaling. 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 signaling. Control of cytokine signaling is important for the treatment of a range of conditions including cancer, inflammatory conditions, immunological disorders, growth disorders and any other conditions involving aberrations of signal transduction. In addition, the present invention provides mutant growth hormone receptors that exhibit reduced sensitivity to SOCS-2 modulation of signalling .

BACKGROUND OF THE INVENTION

Bibliographic details of references provided in the subject specification are listed at the end of the specification.

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.

The suppressor of cytokine signaling (SOCS) proteins are a family of eight SH2 domain containing proteins, comprising cytokine-inducible SH2 domain-containing protein (CIS) and SOCS-1-7. Studies in many laboratories have implicated SOCS proteins in the attenuation of cytokine action through inhibition of the Janus Kinase (JAK)/Signal Transducer and Activators of Transcription (STAT) signal transduction pathway. SOCS proteins operate as part of a classical negative feedback loop, in which activation of cytokine signaling leads to their expression. Once produced, SOCS proteins bind to key components of the signaling apparatus to deactivate and possibly target them for degradation via a conserved C-terminal motif, called the SOCS Box, that recruits ubiquitin ligases (reviewed in Krebs and Hilton, J. Cell Sci. 113(16): 2813-2819, 2000; Yasukawa et al, Annu. Rev. Immunol. 18: 143-164, 2000; Greenhalgh and Hilton, J. Leukoc. Biol. 70(3): 348-356, 2001).

While in vitro studies have suggested that SOCS proteins may be promiscuous in their activity, gene deletion studies in mice have highlighted their importance in a limited number of signaling pathways. SOCS-1 is a key regulator of IFNγ signaling, T-cell homeostasis and lactation (Marine et al., Cell 98(5): 609-616, 1999; Alexander et al, Cell 98(5): 597-608; Lindeman et al, Genes Dev. 15(13): 1631-1636, 2001), while SOCS-3 is thought to play crucial roles in erythropoiesis and placental function (Marine et al, Cell 98(5): 617-627, 1999; Roberts et al, Proc. Natl. Acad. Sci. USA 98(16): 9324-9329, 2001). CIS deficient mice are reported to have no phenotype, although CIS transgenic mice display growth retardation and defects in mammary development defects which are accompanied by reductions in STAT5 phosphorylation (Matsumoto et al, Mol. Cell Biol. 19(9): 6396-6407, 1999) and show similarities to the phenotypes observed in STAT5a and STAT5b deficient mice (Teglund et al, Cell 93(5): 841-850, 1998; Udy et al, Proc. Natl. Acad. Sci. USA 94(14): 7239-7244, 1997; Liu et al, Genes Dev. 11(2): 179-186, 1997).

SOCS-2 deficient animals exhibit accelerated post-natal growth resulting in a 30-50% increase in body weight by 12 weeks of age, significant increases in bone and body lengths, thickening of the skin due to collagen deposition and increases in internal organ size (Metcalf et al, Nature 405(6790): 1069-1073, 2000). This phenotype has striking similarities to those of insulin-like growth factor (IGF)-I and growth hormone (GH) transgenic mice (Palmiter et al, Science 222(4625): 809-814, 1983; Mathews et al, Endocrinology 123(6): 2827-2833, 1988). Further investigation of the SOCS-2^"7" phenotype identified significant increases of IGF-I mRNA in some tissues and lower levels of major urinary protein (MUP) the expression of which is regulated by intermittent GH secretion (Metcalf et al, 2000, supra). Recently, STAT5 phosphorylation in response to GH has been shown to be modestly prolonged in SOCS-2^"7" primary hepatocytes compared with to those from wild type mice, and much of the acceleration of growth in SOCS-2^"7" mice requires the presence of STAT5b, a key mediator of GH action (Greenhalgh et al, Molecular Endocrinology 16(6): 1394-1406, 2002).

There is a need to determine the mechanism of action of SOCS-2 and then to develop through rationale drug design, antagonists and agonists of SOCS-2 activity.

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.

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2 deficient mice, which grow to one and half times the size of their littermates, suggests that SOCS may attenuate growth hormone signaling. In vitro studies advocate that while SOCS-2 can inhibit growth hormone action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, the inventors generated and analyzed transgenic mice that over-express SOCS-2. These mice are not growth deficient and are, in fact, significantly larger than wild-type mice. The over-expressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while peptide binding studies with recombinant SOCS-2 defined the phosphorylated tyrosine residues, particularly residues such as Tyrosines 595, 534 and 487 on the human GH receptor, or their equivalent residues in cytokine receptors from other animal species, as the sites of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.

Accordingly, one aspect of the present invention relates to a method for regulating signaling 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.

Preferably, the SOCS molecule is SOCS-2 and the modulator of SOCS-2 activity modulates interaction between SOCS-2, and in particular, interaction between the SH2 domain of SOCS-2, and a region within the cytoplasmic domain of the receptor for GH. This region comprises at least one phosphorylated tyrosine. Most preferably, the at least one phosphorylated tyrosine is selected from tyrosine 595, tyrosine 534 and tyrosine 487 on the human GH receptor (referred to herein as pY595, pY534 and pY487 or ptyr595, ptyr534 and ptyr487, respectively), or their equivalent residues in cytokine receptors from other animal species. Notwithstanding that the SH2 domain of SOCS-2 is a particularly useful target for antagonist or agonist screening, the present invention does extend to any region motif or conformational pocket on SOCS-2 as the target for antagonist or agonist screening.

In an even more preferred embodiment, a modulator of SOCS-2 / GH interactivity interacts with two or more phosphorylated tyrosine residues, such as pY595, pY534 and pY487, on the human GH receptor, or their equivalent residues in GH receptors from other animal species.

In another preferred embodiment, a modulator of SOCS-2 / GH interactivity binds to the SH2 domain of SOCS-2 to modulate interaction between SOCS-2 and phosphorylated tyrosine residues, such as pY595, pY534 and pY487 on the GH receptor, or the equivalent residues on the GH receptor in a non-human animal species.

The modulator may be an agonist or antagonist of SOCS-2 interaction with the cytokine receptor.

The present invention further contemplates a method of identifying an agent capable of modulating signaling 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-2 molecule and the cytoplasmic domain of the GH receptor or related molecule.

Yet another aspect of the present invention contemplates a method for identifying a modulator of interaction between a SOCS-2 molecule or a part, fragment, derivative, or homolog or analog thereof comprising the SH2 domain of SOCS-2 and a receptor for GH or related molecule or a part, fragment, derivative or homolog or analog thereof comprising one or more phosphorylated tyrosines selected from tyrosine 595, tyrosine 534 and tyrosine 487 of the human GH receptor, or equivalent residue thereof in a non-human species, said method comprising:-

immobilizing one of said SOCS-2 molecule or the receptor or their parts, fragments, derivatives, homologs or analogs to a solid support via binding partners wherein one of said binding partners is attached or otherwise anchored to said solid support and the other of said binding partners is linked to said SOCS-2 or receptor molecule or their parts, fragments, derivatives, homologs or analogs;

contacting said immobilized SOCS-2 or receptor molecule or their parts, fragments, derivatives, homologs or analogs with the other of receptor or SOCS-2 molecule or their parts, fragments, derivatives, homologs or analogs 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-2 and receptor or their parts, fragments, derivatives, homologs or analogs.

A further aspect of the present invention provides a method for identifying a modulator of interaction between a SOCS-2 molecule or a part, fragment, derivative, or homolog or analog thereof comprising the SH2 domain of SOCS-2 and a receptor for GH or related molecule or part, fragment, derivative or homolog or analog thereof comprising one or more phosphorylated tyrosines selected from tyrosine 595, tyrosine 534 and tyrosine 487 of the human GH receptor, or equivalent residue thereof in the GH receptor from a non- human species, said method comprising:-

immobilizing one of said SOCS-2 molecule or receptor or their parts, fragments, derivatives, homologs or analogs 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-2 or receptor molecule or their parts, fragments, derivatives, homologs or analogs;

Yet another aspect of the present invention contemplates a composition comprising a modulator of interaction between a SOCS-2 molecule and a cytoplasmic domain of the GH receptor molecule, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.

The abbreviations used in the subject specification are listed in Table 1 :

TABLE 1

Abbreviations

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graphical/photomicrographic representation showing that SOCS-2 can act as a dual effector on GH signaling. 293T cells were transfected with pig GH receptor and CIS, SOCS-1, SOCS-3, or a range of SOCS-2 concentrations (ng) were stimulated with rhGH and the luciferase activity from a LHRE-luciferase reporter was measured. Data are corrected for transfection efficiency by co-transfection of a β-galactosidase expressing plasmid and luciferase activity in the absense of GH then expressed as a percentage of wild-type activity which is assigned a value of 100%. A sample of lysate from each group was Western blotted and probed with antibodies against FLAG. Data are the mean of three independent experiments.

Figure 2 are photomicrographic/graphical representations showing generation and analysis of SOCS-2 transgenic mice. (A) Expression of SOCS-2 protein was examined in a range of tissues from the F9 and F33 transgenic lines by immunoprecipitation of FLAG-tagged proteins, Western transfer then probing with specific antibodies to detect FLAG tagged proteins. Forty μg of protein from organ lysates were blotted for Hsp70 levels to confirm loadings. Asterisks denote non-specific bands. Note: FLAG-SOCS-2 is detectable in F33 line muscle samples but has not reproduced well in this figure. (B) Growth curves for male wild-type and transgenic mice were constructed using data from 15-36 mice at each point. a and b indicates significant differences (PO.05) between wild-type and F9 (closed circles) or F33 transgenic mice, respectively. (C) Differences in organ weights of 12 week old male F9 (open bars) and F33 (closed bars) transgenic mice compared to wild-type mice are represented as a percentage increase over the mean of wild-type mouse organ weight. Mes LN, sem ves and nose-anus refer to mesenteric lymph node, seminal vesicle and nose-anus body length, respectively. Asterisks denote significant differences from wild-type mice (PO.05).

Figure 3 is a photomicrographic representation showing that SOCS-2 interacts with endogenous GH receptors. Organ lysates were made from C57B1/6, SOCS-2 F33 transgenic mice and SOCS-2 F33 transgenic mice injected with 100 μg recombinant porcine GH for 40 min prior to sacrifice. Anti-FLAG immunoprecipitations and Western blots before being probed with antibodies against mouse GH receptor, followed by antibodies to detect Flag.

Figure 4 are representations showing that SOCS-2 interacts with Tyr 595 of the human GH receptor. (A) Diagram of the location of tyrosine residues phosphorylated in response to GH and the sequence of the synthesized phosphopeptides. (B) Immobilized phosphopeptides were incubated with recombinant SOCS-2 SH2 domain protein fused to the NusA protein, washed, separated on SDS-PAGE and Coomassie stained. The specificity of the SOCS-2 SH2 domain interaction with was tested with non- phosphorylated Tyr595 and NusA with phosphorylated-Tyr595.

Figure 5 is a schematic representation showing the refinement of the SOCS-2 protein purification scheme. This improved protein purification subsequently improves reproducibility of the alpha screen assay making it suitable for a high throughput screen.

Figure 6 is a representation of an SDS-PAGE gel analysis of thrombin cleaved SOCS-2. NusA.SOCS-2(6xHis) was bound to Talon (Registered Trademark) resin (5ml) and following buffer exchange the NusA portion was cleaved using thrombin. Cleaved SOCS- 2 remains bound to the column whereas cleaved NusA is present in the unbound elution fraction. SOCS-2 is recovered using 150mM imidazole in PBS. Overall yield of soluble SOCS-2 is approximately 2mg per liter.

Figure 7, panels A and B, are graphical representations of Biacore based analysis of the binding of SOCS-2 to immobilised GHR phosphopeptide Y595 and inhibition using soluble phosphopeptide. Panel A is a graphical representation showing dose response analysis-doubling dilutions of SOCS-2 between low dose 200nM and a high dose of 250μM. Affinity (K_D) was calculated to be 250nM using an algorithm derivation of association (ka) and dissociation rates (kd). Panel B is a graphical representation illustrating competition of SOCS-2 (1.45μM) binding to immobilised GHR phosphopeptide Y595 using soluble GHR phosphopeptide Y595 at a concentration range of 0.5 to lOμM.

Figure 8 is a schematic representation of the prototype SOCS-2 screen format using alpha screen readout. The SOCS-2 alpha screen is based on the interaction of SOCS-2 (immobilised on donor beads) and GHR phosphopeptide comprising the residues of interest such as Y595, Y534 and/or Y487 (immobilised onto acceptor beads). Laser excitation of the solution at 680nm results in the liberation of singlet oxygen from the donor beads (SOCS-2). Complex formation results in activation of the acceptor beads (eg. GHR Y595) reacting with singlet oxygen thereby generating a measurable fluoresce emission at 580 to 620nm.

Figure 9 is a tabular representation showing the preliminary analysis of SOCS-2 stability in the SOCS-2 prototype screen. SOCS-2 was assessed at various concentrations (as indicated) with GHR phosphopeptide Y595. Alpha screen analysis was performed at days 2, 9 and 13 to examine stability of the signal. Data is expressed as a value of signal to noise ratio.

Figure 10 is a graphical representation depicting the results of a Biacore analysis, measuring the ability of GHR phosphopeptides (specifically Y332, Y487, Y534 and Y595) to inhibit SOCS-2 binding to immobilized peptides.

Figure 11 is a graphical representation of the signal emanating from each of the mutant constructs when not stimulated by growth hormone (clear bars), or stimulated with growth hormone stimulation (black bars). The asterisks denote significant differences (PO.05). 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 a cytokine. The identification of this molecular target permits the development of assays to identify agonists and antagonists of cytokine 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.

The present invention is particularly directed to the SOCS-2 molecule. This is done, however, with the understanding that the present invention extends to any SOCS molecule which regulates GH signaling via interaction with the cytoplasmic domain of the GH receptor. Most preferably, however, the SOCS molecule is SOCS-2.

Before describing the present invention in detail, it is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulations of agents, manufacturing methods, methodologies, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent, as well as two or more agents. In describing and claiming the present invention, the following terminology is used in accordance with the definitions set forth below.

Reference herein to a "SOCS-2" molecule includes the molecule which suppresses cytokine signalling and is referred to in Hilton et al. (Hilton et al., Proc. Natl. Acad. Ssi. USA 95:114-119, 1998) as SOCS-2 and which is further described in the patent family represented by PCT/AU97/00729 (published as WO98/20023), and all mutants and derivatives including parts and fragments thereof as well as homologs of SOCS-2. It also extends to analogs of SOCS-2 or analogs of parts or fragments of SOCS-2 which may be useful in assays for ligands. A "homolog" of SOCS-2 includes the equivalent or similar molecule from another species or a molecule which has similar physiological, biochemical, immunological or binding kinetic properties to SOCS-2. Identification of the species from which a particular SOCS-2 molecule is isolated is shown by the singles letters "h" for human and "m" for murine (e.g. mouse).

It is a particularly preferred embodiment of the present invention that a mutant, derivative, part, fragment or homolog of SOCS-2 comprises the SH -domain of SOCS-2.

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 is GH and the cytokine receptor is the GH receptor or structurally related receptor. The GH receptor may be from a human or other animal species. 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 especially preferred embodiment, the cytokine is GH and the cytokine receptor is the GH receptor.

In still another preferred embodiment, the region to which SOCS-2 binds or otherwise interacts is a region of the cytoplasmic domain of the GH receptor comprising at least one phosphotyrosine.

In an even more particularly preferred embodiment, the region to which SOCS-2 binds or otherwise interacts is a region spanning tyrosine (Y) 595, Y534 or Y487 of the GH receptor or other homologous regions in other animal cytokine receptors. Reference herein to Y595, Y534 and Y487 in the human GH receptor is to be understood to also contemplate their equivalent residues in GH receptors from other non-human animal species.

Accordingly, another aspect of the present invention contemplates a method for regulating signaling 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-2 or related molecule and a region within the cytoplasmic domain of a receptor for said cytokine or related molecule which corresponds to or is homologous with regions comprising or proximal to Y595, Y534 and/or Y487 of the human GH receptor, or equivalent residues in GH receptors from other animal species.

Preferably the modulator inhibits SOCS-2 interaction with the cytoplasmic domain of the GH receptor. More particularly, the modulator inhibits the SH2 domain of SOCS-2 interacting with the cytoplasmic domain of the GH receptor. As indicated above, however, the present invention extends to any region, motif or conformational pocket on SOCS-2 as the target for antagonist or agonist screening.

Accordingly, in a particularly preferred embodiment there is provided a method for regulating signaling of GH or a GH-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-2 or related molecule and a region within the cytoplasmic domain of the GH receptor or related molecule wherein said region comprises at least one phosphorylated tyrosine.

In a particularly preferred embodiment, the region comprises pY595, pY534 and/or pY487 of the human GH receptor or their functional equivalents in other receptors.

Accordingly, another aspect of the present invention contemplates a method for regulating signaling of a GH 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-2 or related molecule and a region comprising or proximal to pY595, pY534 and/or pY487 within the cytoplasmic domain of the human GH receptor or the equivalent residues in other animal cytokine receptors.

Preferably, the modulator interacts with at least one phosphorylated tyrosine, and more preferably interacts with two or more phosphorylated tyrosine residues.

In another preferred embodiment, the modulator binds to the SH2 domain of SOCS-2 to modulate interaction between SOCS-2 and at least one phosphorylated tyrosine residue on the human GH receptor, such as pY595, pY534 and pY487 or the equivalent residue(s) thereof in a GH receptor from another animal species.

Reference to the GH receptor includes reference to mutants and derivatives including fragments and parts thereof as well as homologs and analogs thereof.

The preferred animal subject of the present invention is a human, however, the present invention extends to all vertebrates, including primates, livestock animals (e.g. sheep, pigs, cows, goats, horses), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters), companion animals (e.g. dogs, cats), or captive wild animals. The modulator of interaction between a SOCS molecule (e.g. SOCS-2) and a receptor may be an agonist or antagonist. In essence, a molecule which promotes or otherwise facilitates SOCS-2 binding is an agonist of SOCS-2 but such a molecule acts to inhibit cytokine- or related molecule-mediated signal transduction. Conversely, an antagonist of SOCS-2 interaction facilitates cytokine- or related molecule-mediated signal transduction.

Accordingly, another aspect of the present invention contemplates a method of inhibiting signaling of GH 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-2 molecule and a region within the cytoplasmic domain of a receptor for GH or related molecule.

In an alternative embodiment, the present invention provides a method of facilitating signaling of a GH 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-2 molecule and a region within the cytoplasmic domain of a receptor for GH or related molecule.

Again, the preferred region within the GH receptor cytoplasmic domain comprises at least one phosphorylated tyrosine. In an especially preferred embodiment, the at least one phosphorylated tyrosine is pY595, pY534 and/or pY487 in the human GH receptor, or the equivalent residue(s) thereof in the GH receptor from another animal species.

Reference to equivalents, homologs, mutants and derivatives including parts and fragments thereof preferably includes regions carrying the SH2 domain of SOCS-2. The present invention is particularly directed to the use of a SOCS-2 peptide comprising the SH2 domain or equivalent region in the detection of antagonists and agonists in the interaction between this region and the cytoplasmic domain of the GH receptor.

The agent capable of agonizing or antagonizing interaction between the SOCS-2 molecule and the cytoplasmic domain of the cytokine receptor may be a proteinaceous or non- proteinaceous 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 non- proteinaceous 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- proteinaceous 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-2 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 signaling 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 signaling 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-2 molecule and a cytoplasmic domain of a receptor for said cytokine or related molecule. The SOCS-2 molecule and/or receptor molecule may be parts, fragments, derivatives, homologs or chemical analogs of all or a portion of the molecule. A portion of SOCS-2 will preferably include the SH2 domain or its equivalent. The preferred cytokine in this context is GH and the preferred receptor is the GH receptor.

More particularly, and in one preferred method, agonists and antagonists of SOCS-2 interaction with a receptor are identified using biosensor technology. In one embodiment, phosphopeptides derived from a receptor or its mutants, derivatives, homologs or analogs 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-2 molecule capable of interacting with all and some of the immobilized phosphopeptides or binding portions or fragments or derivatives or homologs or analogs of a SOCS-2 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-2 molecule or agonist/antagonist.

Accordingly, another aspect of the present invention contemplates a method for identifying a modulator of interaction between a SOCS-2 molecule or part, fragment, or derivative, or homolog or analog thereof and a receptor for a cytokine or related molecule or part, fragment derivative or homolog or analog thereof, said method comprising:-

immobilizing one of said SOCS-2 molecules or the receptor or their parts, fragments, derivatives, homologs or analogs 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-2 or receptor molecule; contacting said immobilized SOCS-2 or receptor molecule with the other of receptor or SOCS-2 molecule in the presence of a potential agonist or antagonist; and

Preferably, the cytokine is GH, the parts, fragments, or derivatives, or homologs or analogs of SOCS-2 comprises the SH2 domain of SOCS-2, the receptor is the GH receptor and the parts, fragments, or derivatives, or homologs or analogs of the GH receptor comprise one or more phosphorylated tyrosines selected from tyrosine 595, tyrosine 534 and tyrosine 487 of the human GH receptor, or the equivalent residue(s) thereof in a GH receptor from another animal species.

Although the SOCS-2 molecule or receptor may be first immobilized to the solid support, it is particularly convenient for the receptor or a part, fragment, derivative, homolog or analog to be anchored. Preferably, anchoring is via binding partners although the present invention extends to direct binding of the SOCS-2 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-2 molecule or part, fragment, or derivative, or homolog or analog thereof and a receptor for a GH or related molecule or part, fragment derivative or homolog or analog thereof, said method comprising:-

immobilizing said receptor or its parts, fragments, derivatives, homologs or analogs 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-2 molecule or its parts, fragments, derivatives, homologs or analogs in the presence of a potential agonist or antagonist; and

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

Preferably, the parts, fragments, or derivatives, or homologs or analogs of SOCS-2 comprises the SH2 domain of SOCS-2, the receptor is the GH receptor and the parts, fragments, or derivatives, or homologs or analogs of the GH receptor comprise one or more phosphorylated tyrosines selected from tyrosine 595, tyrosine 534 and tyrosine 487 of the human GH receptor, or the equivalent residues thereof from a cytokine receptor in another animal species

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-2 molecule and a receptor or fragments, parts, derivatives, homologs or analogs thereof causes or otherwise facilitates production of an electrical 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-2 molecule or part, fragment, or derivative, or homolog or analog thereof and a receptor for a GH or related molecule or part, fragment derivative or homolog or analog thereof, said method comprising :-

immobilizing one of said SOCS-2 molecules or receptor or their parts, fragments, derivatives, homologs or analogs 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-2 or receptor molecule;

contacting said immobilized SOCS-2 or receptor molecule with the other of receptor or SOCS-2 molecule in the presence of a potential agonist or antagonist; and

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-2 molecule or part, fragment, or derivative, or homolog or analog thereof and a receptor for a GH or related molecule or part, fragment derivative or homolog or analog thereof, said method comprising:-

immobilizing said receptor or its parts, fragments, derivatives, homologs or analogs 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-2 molecule or its parts, fragments, derivatives, homologs or analogs in the presence of a potential agonist or antagonist; and

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 (i.e. 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.

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 analogs of a SOCS-2 molecule and/or a receptor are usefully employed in screening assays for ligands (e.g. agonists or antagonists) due to enhanced chemical stability and/or coupling and/or signaling due to the chemical modifications. All such chemical modification to the SOCS-2 molecules or receptor molecules including to their parts, fragments, portions, derivatives or homolog, are contemplated by the present invention. Reference herein to a "SOCS-2" molecule or a "receptor" molecule includes analogs and in particular chemical analogs 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 NaBHj; 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 NaBHj.

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 in Table 2. 25

TABLE 2

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 26

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-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 Dnmgln N-(3 -guanidinopropyl)gly cine Narg

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

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis 27 -

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nht

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethiomne 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-( 1 -methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval 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-methylthreonine 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-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine - 28 -

l-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=T 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 Cβ 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.

Still a further aspect of the present invention is directed to an agent useful for modulating signaling 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-2 or a related molecule. Most preferably, the SOCS molecule is SOCS-2. The preferred receptors comprise at least one phosphorylated tyrosine in a region comprising or proximal to the site of interaction with the SOCS molecule. Most preferably, the receptor is the GH receptor.

In an especially preferred embodiment, the agent binds to either the SH2 domain of SOCS- 2 or to at least one phosphorylated tyrosine of the GH receptor selected from tyrosine 595, tyrosine 534 and tyrosine 487, in the human GH receptor, or equivalent thereof in another species, to modulate interaction between SOCS-2 and the GH receptor. - 29 -

The present invention, therefore, contemplates a composition comprising a modulator of interaction between a SOCS-2 molecule and a cytoplasmic domain of a receptor molecule, preferably the GH receptor, 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-2/receptor interaction and may be useful in modulating cytokine- or related molecule-mediated signal transduction. This may be useful in a range of conditions including cancer, inflammatory disorders, growth 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, 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 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 - 30 -

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 administration, 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 μg and 200 mg of modulator. Alternative dosage amounts include from about 1 μg to about 1000 mg and from about 10 μg 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 - 31 -

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 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-2 molecule and/or the GH receptor in the manufacture of an assay to screen for agonists or antagonists of SOCS- 2/GH receptor interaction.

The present invention further contemplates mutant growth hormone receptors wherein the mutant growth hormone receptor has altered sensitivity to SOCS-2 mediated modulation. Preferably, the mutant growth hormone receptors contemplated by the present invention have decreased or eliminated SOCS-2 mediated modulation of signalling and/or decreased binding affinity with SOCS-2. - 32 -

The present invention specifically contemplates mutant growth hormone receptors comprising an amino acid substitution at one or more phosphorylated tyrosine residues, which are preferably, but not limited to residues such as, Y487, Y534 and Y595, in the human GH receptor, or their equivalents thereof in the GH receptor from another animal species.

In a further preferred embodiment, the mutant growth hormone receptor comprises two or more amino acid substitutions at phosphorylated tyrosine residues, such as Y487, Y534 and Y595, and more preferably comprises a double amino acid substitution at residues Y487 and Y595 in the human GH receptor, or equivalent substitution in the GH receptor of another animal species.

In addition, the present invention contemplates a eukaryotic cell which expresses a mutant growth hormone receptor as hereinbefore defined, and an animal subject comprising one or more of said cells.

The genetically modified animal may be a mammalian, avian, reptilian or fish species. Preferably, the genetically modified animal is a livestock animal, such as a pig, sheep, cattle or goat.

The animal models of the present invention may be in the form of the animals or may be, for example, in the form of embryos for transplantation. The embryos are preferably maintained in a frozen state and may optionally be sold with instructions for use.

The genetically modified animals may express the mutant growth hormone receptor and a genetically modified animal according to this aspect is referred to as a "knock-in" animal.

Accordingly, the present invention further contemplates a nucleic acid molecule, which encodes a mutant growth hormone receptor, as described supra. In addition, vectors comprising nucleic acid molecules that encode the genetic sequence of a mutant growth - 33 -

hormone receptor are also contemplated by the present invention. Specifically, these vectors include but are not limited to:

(i) expression vectors wherein the vector comprises the subject nucleic acid operably connected to a promoter; and/or

(ii) vectors designed for gene replacement wherein the vector comprises the subject nucleic acid sequence and optionally other nucleic acid sequences that promote homologous recombination between the vector-borne nucleic acid sequence and the genomic homolog of the subject nucleic acid sequence.

Still yet another aspect of the present invention is directed to the use of a targeting vector as defined above in the manufacture of a genetically modified animal comprising a modified or mutant growth hormone receptor.

Even still another aspect of the present invention is directed to the use of a targeting vector as defined above in the manufacture of a genetically modified mouse comprising a modified or mutant growth hormone receptor.

Preferably, the vector is DNA. A selectable marker in the targeting vector allows for selection of targeted cells that have stably incorporated the targeting DNA. This is especially useful when employing relatively low efficiency transformation techniques such as electroporation, calcium phosphate precipitation and liposome fusion where typically fewer than 1 in 1000 cells will have stably incorporated the exogenous DNA. Using high efficiency methods, such as microinjection into nuclei, typically from 5-25% of the cells will have incorporated the targeting DNA; and it is, therefore, feasible to screen the targeted cells directly without the necessity of first selecting for stable integration of a selectable marker. Either isogenic or non-isogenic DNA may be employed. - 34 -

Examples of selectable markers include genes conferring resistance to compounds such as antibiotics, genes conferring the ability to grow on selected substrates, genes encoding proteins that produce detectable signals such as luminescence. A wide variety of such markers are known and available, including, for example, antibiotic resistance genes such as the neomycin resistance gene (neό) and the hygromycin resistance gene (hyg). Selectable markers also include genes conferring the ability to grow on certain media substrates such as the tk gene (thymidine kinase) or the hprt gene (hypoxanthine phosphoribosyltransferase) which confer the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine); and the bacterial gpt gene (guanine/xanthine phosphoribosyltransferase) which allows growth on MAX medium (mycophenolic acid, adenine and xanthine). Other selectable markers for use in mammalian cells and plasmids carrying a variety of selectable markers are described in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbour, New York, USA, 1990.

The preferred location of the marker gene in the targeting construct will depend on the aim of the gene targeting. For example, if the aim is to disrupt target gene expression, then the selectable marker can be cloned into targeting DNA corresponding to coding sequence in the target DNA. Alternatively, if the aim is to express an altered product from the target gene or to super-express a SOCS-2 gene, then the selectable marker can be placed outside of the coding region, for example, in a nearby intron.

The selectable marker may depend on its own promoter for expression and the marker gene may be derived from a very different organism than the organism being targeted (e.g. prokaryotic marker genes used in targeting mammalian cells). However, it is preferable to replace the original promoter with transcriptional machinery known to function in the recipient cells. A large number of transcriptional initiation regions are available for such purposes including, for example, metallothionein promoters, thymidine kinase promoters, β-actin promoters, immunoglobulin promoters, SN40 promoters and human cytomegalovirus promoters. A widely used example is the pSN2-«eø plasmid which has the bacterial neomycin phosphotransferase gene under control of the SN40 early promoter and confers in mammalian cells resistance to G418 (an antibiotic related to neomycin). A - 35 -

number of other variations may be employed to enhance expression of the selectable markers in animal cells, such as the addition of a poly(A) sequence and the addition of synthetic translation initiation sequences. Both constitutive and inducible promoters may be used.

The DNA is preferably modified by homologous recombination. The target DNA can be in any organelle of the animal cell including the nucleus and mitochondria and can be an intact gene, an exon or intron, a regulatory sequence or any region between genes.

Homologous DNA is a DNA sequence that is at least 70% identical with a reference DNA sequence. An indication that two sequences are homologous is that they will hybridize with each other under stringent conditions (Sambrook et al, 1990, supra).

The present invention further contemplates co-suppression (i.e. sense suppression) and antisense suppression to down-regulate expression of SOCS-2. This would generally occur in a target test animal such as to generate a disease model.

In addition to providing a diagnostic capability as described above, the isolated nucleic acid molecules of the present invention may also provide a therapeutic capability by being used to correct or complement an abnormality detected in a subject. To deliver the appropriate sequence to a recipient cell or tissue of a subject, an isolated nucleic acid molecule of the present invention may be cloned into a suitable genetic construct such as a suitable vector.

A "vector" is a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e. a vector that exists as an extra-chromosomal entity, 36 -

the replication of which is independent of chromosomal replication. Examples include a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may also contain a means for assuring self- replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.

Vectors suitable for gene therapy applications are well known in the art. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which it is to be introduced. The vector may also include an additional genetic construct comprising a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those skilled in the art and include the nptll gene that confers resistance to the antibiotics kanamycin, and G418 (Geneticin®) and the hph gene which confer resistance to the antibiotic hygromycin B.

Accordingly, in a related embodiment, the present invention provides a genetic construct comprising a promoter or functional equivalent thereof operably linked to a nucleotide sequence encoding SOCS-2.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers), which alter gene expression in response to developmental and/or external stimuli, or in a tissue- specific manner. A promoter is usually, but not necessarily, positioned upstream (5') of a gene region, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. As is known in the art, some variation in this distance can be accommodated - 37 -

without loss of promoter function.

The selection of an appropriate promoter sequence to regulate expression of a transcription factor encoded by an isolated nucleic acid molecule of the present invention is an important consideration. Examples of suitable promoters include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in eukaryotic animal cells and, especially, human cells. The promoter may regulate the expression of the nucleic acid molecule differentially with respect to the cell, tissue or organ in which expression occurs, or with respect to the developmental stage at which expression occurs.

Preferably, the promoter is capable of regulating expression of a nucleic acid molecule in a eukaryotic cell, tissue or organ, at least during the period of time over which the regulated gene is expressed therein, and more preferably also immediately preceding the commencement of detectable expression of the regulated gene in said cell, tissue or organ.

Particularly preferred promoters for use with the nucleic acid molecules of the present invention include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, CaMV 35S promoter, SCSV promoter, SCBV promoter and the like. Those skilled in the art will readily be aware of additional promoter sequences other than those specifically described.

In the present context, the terms "in operable connection with" or "operably linked" or similar shall be taken to indicate that expression of the nucleic acid molecule is under the control of the promoter sequence, with which it is spatially connected, in a cell, tissue, organ or whole organism.

The genetic construct of the present invention may also comprise a 3' non-translated sequence. A 3' non-translated sequence refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is - 38 -

characterized by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon.

Accordingly, a genetic construct comprising a nucleic acid molecule of the present invention, operably linked to a promoter, may be cloned into a suitable vector for delivery to a cell or tissue in which regulation is faulty, malfunctioning or non-existent, in order to rectify and/or provide the appropriate regulation. Vectors comprising appropriate genetic constructs may be delivered into target eukaryotic cells by a number of different means well known to those skilled in the art of molecular biology. A particularly useful method introducing genetic material into animal hosts is nuclear transfer. In this method the construct is injected into male pronuclei of fertilized eggs which are then implanted into pseudo-pregnant females. In addition, nuclear transfer may also be used to introduce new genetic material into other cell types, which include, but are not limited to embryonic stem (ES) cells and fibroblasts.

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

- 39 -

EXAMPLE 1

Generation of transgenic mice

SOCS-2 transgenic mice were generated by ligating the mouse SOCS-2 coding region into the pUbiFLAG vector in which expression of the gene of interest is driven by the human ubiquitin C promoter (Kile et al, Mol. Cell Biol. 21(18): 6189-6197, 2001). The construct was linearized, purified on agarose gels and injected into the male pronuclei of C57BL/6 fertilized eggs, which were then implanted into pseudopregnant females. Genomic DNA was extracted from tail samples taken from offspring at weaning, digested with Xbal, Southern blotted and hybridized with probes derived from the coding region of SOCS-2. Mice carrying transgenes were bred to establish whether they transmitted them to progeny, then Northern blotting was performed as previously described (Alexander et al, Blood 87(6): 2162-2170, 1996) on RNA from a range of tissues to evaluate expression.

EXAMPLE 2

Growth analysis

Growth curves, organ weights and bone measurements were performed as previously described (Metcalf et al, 2000, supra).

EXAMPLE 3 Analysis of protein expression/interaction in SOCS-2 transgenic mice

Protein extracts and immunprecipitation experiments from tissues were prepared and performed essentially as described in (Kile et al, 2001, supra), except that tissues were lysed in muscle lysis buffer (0.5% v/v NP40, 50 mM Tris pH 8.0, 1 mM EDTA, 150 mM NaCl, 10% v/v glycerol, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, and protease inhibitors (Boehringer Manheim)) and 40 μg of total protein from each organ lysate was blotted on Western blots and probed to determine HSP70 levels using the antibody sc-24 (Santa Cruz). Antibodies against mouse GH receptor were provided by the University of California. - 40 -

EXAMPLE 4

Transfection assays

Transfections were performed in 293 T cells plated at 2 x 10⁵ cells/ml in 2 ml of DME with 10%) fetal calf serum (FCS) using Fugene transfection reagents (Roche). Briefly, 100 ng each of GH receptor plasmid, LHRE-luc reporter plasmid (Goffin et al, J Biol Chem 1996 Jul 12; 271(28) pp 16573-9), β-galactosidase plasmid, and 0-100 ng pEFSOCS-2Flag plasmid (Nicholson et al, EMBO J. 18(2): 375-385, 1999) made up to 100 ng with pEFBOS plasmid were transfected into a 6-well plate 24 h after cells were plated out. Twenty four hours later, cells were washed once with mouse-tonicity phosphate buffered saline (MT- PBS) and the culture medium was replaced with DME containing 1% w/v bovine serum albumin (BSA). Cells were left to equilibrate for 1 h before 500 ng/ml of E. coli derived recombinant human (rh)GH (Prospec) was added to appropriate groups. Cells were incubated for a further 16 h before being lysed and assayed for luciferase and β- galactosidase activity as described (Nicholson et al, 1999, supra).

EXAMPLE S

Histolopathological examination

The heart, lungs, brain, skin, muscle, spleen, thymus, mesenteric lymph node, liver, kidneys, bladder, seminal vesicles, uterus and testicles were collected from 12 week old animals, weighed, and then fixed in 10% v/v saline-buffered formalin before being sectioned, haematoxylin/eosin stained and analyzed as previously described (Metcalf et al, 2000, supra).

EXAMPLE 6 Generation of recombinant SOCS-2 proteins

DNA encoding the murine SOCS-2 SH-2 domain (amino acids 37-159) was amplified from the ρEF-SOCS-2 plasmid by PCR and ligated into the pET-43.1 NusA fusion protein expression plasmid (Novagen). DNA encoding a hexameric histidine amino acid (hexa- - 41 -

HIS) sequence was engineered into the 3' primer to aid in purification. The vector was transformed into BL21(DE3) cells, and overnight 37°C cultures were diluted 1:10 in 400 ml of media and grown at 30°C until OD₆₀o was 0.6 units before being induced with 0.1 mM IPTG. Cells were harvested 2 hours post-induction and stored at -20°C. Cell pellets were lysed in 10 ml of lysis buffer per 50 ml of culture (MT-PBS containing 0.2 mg/ml lysozyme, 1% Triton X-100, 1 mM PMSF and 30 ug/ml DNase I) for 60 min at 4°C. The total cell lysate was centrifuged for 10 min at 27,000 g at 4°C to remove cell debris. The supernatant was then loaded onto a Ni-NTA column (Qiagen) equilibrated in buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0), washed with buffer containinglO mM β- mercaptoethanol and 10 mM imidazole eluted with 200 mM imidazole. Fractions were collected and then EDTA and β -mercaptoethanol were added to achieve final concentrations of 2 mM and 40 mM. Fractions that contained His-tagged proteins were pooled, applied to a size-exclusion chromatography column (Sephadex 200) preequilibrated with MT-PBS containing 10 mM β-mercaptoethanol. Samples were subsequently analyzed by SDS-PAGE and Western blotting using anti-His antibodies (pentaHIS, Qiagen).

EXAMPLE 7 Peptide synthesis

Synthetic peptides with a C-terminal amide were synthesized using Fmoc chemistry. Amino acids activated with O-benzotriazol-l-yl-N^N'jN^N'-tetramethyluronium hexaflurophosphate. Peptides were biotinylated by coupling d-biotin to the Ν-terminus of resin bound peptides before cleavage and deprotection. Peptide products were purified by RP-HPLC and analyzed by MALDI mass spectrometry.

EXAMPLE 8 Recombinant SOCS-2 protein/GH receptor peptide interaction

Biotinylated phosphopeptides were immobilized on streptavadin-agarose resin (Pierce, Australia) using standard procedures, and 20 μl of resin was incubated with 25 μg ΝusA- - 42 -

SOCS-2-SH2 in 1 ml Tris pH 7.5, 0.1% w/v Tween 20 for 2 h at 4°C. Beads were then washed twice with cold PBS/0.1% v/v Tween 20 and boiled in 25 μl of 2 x reducing loading buffer (125 mM Tris-HCl pH 6.8, 20% v/v glycerol, 4% w/v SDS and 5% v/v β- mercaptoethanol). SDS-PAGE was performed on samples as in Greenhalgh, 2002, supra.

EXAMPLE 9 SOCS-2 can enhance and suppress GH signaling

The effects of SOCS expression on GH-induced transcription were analyzed by transient transfections of 293 T cells with a GH-responsive STAT5 -dependent luciferase reporter, porcine GH receptor, and a β-galactosidase containing plasmid in the presence or absence of plasmids encoding CIS, SOCS-1, SOCS-2 or SOCS-3. In the absense of exogenous SOCS proteins, GH stimulation resulted in a 16-fold increase of reporter activity. The expression of increasing levels of transfected SOCS-2 initially caused repression of reporter activity to 40% of that seen in the absence of SOCS, but at higher concentrations of SOCS-2 a recovery from inhibition and a significant enhancement of reporter activity was observed (Figure 1). Expression of SOCS-1 and SOCS-3 prevented any significant reporter activity and CIS was also a potent inhibitor of GH-induced activity.

EXAMPLE 10

Transgenic expression of SOCS-2 does not inhibit growth, but enhances it

To determine whether high levels of SOCS-2 could also potentiate GH signaling in vivo, SOCS-2 transgenic mice were generated. Three independent lines of mice that transmitted the SOCS-2 transgene were produced and two of these (Line F33 and Line F9) were analyzed further. Expression of SOCS-2 from the transgene in these lines was confirmed by immunoprecipitation of SOCS-2 protein via the FLAG epitope from lysates of all organs examined (Figure 2A).

Given the accelerated growth in mice deficient for SOCS-2, it was interesting to observe that SOCS-2 transgenic mice suffered from no apparent growth retardation throughout the - 43 -

post-natal period but, rather, displayed enhanced growth. Male mice were significantly heavier from 3 weeks of age resulting in a 13-15% increase in body weight (Figure 2B). This trend was confirmed in male organ weights with both lines demonstrating an overall increase in the size of most organs and tissues, with some being significant enlarged, particularly the carcass (Figure 2C). The enhancement of growth was also reflected in a small (2-6%), but significant, increase in tibia, femur and radius lengths in F9 males, but only the radius was longer in F33 males. Significant, changes were also found in the growth rates, body and organ weights of female transgenic mice, although they were of a lesser magnitude.

EXAMPLE 11 Histological and Hematopoietic analysis

Histological examination of 3 month old mice from both transgenic lines compared to wild-type mice revealed no consistent abnormalities or defects in any organ or tissue examined. The skin of male transgenic mice had a tendency to be thicker, although this was difficult to accurately quantitate. No differences were detectable in white blood cell, hematocrit or platelet numbers between groups, and normal numbers of progenitor cell- derived colonies were generated in cultures of marrow cells from transgenic mice when stimulated with a range of cytokines (data not shown). Despite data suggesting that the onset of blast crisis in patients with chronic myeloid leukemia CML is accompanied by the development of high levels of SOCS-2 mRNA (Schultheis et al, Blood 99(5): 1766-1775, 2002), indication that elevated SOCS-2 levels led either to leukemia development or to maturation arrest in hematopoeitic cells of any lineage was found.

EXAMPLE 12 SOCS-2 interacts with the GH receptor in vivo

Due to the unexpected effects of SOCS-2 over-expression on mouse growth, the inventors wanted to confirm that the SOCS-2 transgene product was functional. Based on the hypothesis that SOCS-2 may regulate or bind to the GH receptor, immunoprecipitations of .44 -

the SOCS-2 protein were performed from a number of tissues from wild-type and transgenic mice, before and after GH injection. These lysates were then electrophoresed and examined by Western blotting. As expected FLAG-tagged SOCS-2 was detected in transgenic but not wild type mice and this was observed to interact with the growth hormone receptor, especially after growth hormone injection (Figure 3). A similar experiment was also performed where an animal was injected with IGF-I and the Western blot was probed with antibodies against the IGF-I receptor, but no interaction was detected.

EXAMPLE 13 SOCS-2 interacts with Tyrosine 595 of the human GH receptor

Previous studies have shown that SOCS-2 can interact with the phosphorylated GH receptor in vitro (Ram and Waxman, J Biol. Chem. 274(5): 35553-35561, 1999; Hansen et al, Mol. Endocrinol, 13(11): 1832-1843, 1999), while the inventors have shown here an in vivo association. To further define the nature of this interaction, we designed biotinylated phopho-tyrosine (pTyr) peptides to each of the seven tyrosine residues phosphorylated on the intracellular region of human GH receptor (Figure 4a). These peptides were bound to Streptavidin-Sepharose before being incubated with a recombinant fusion protein of NusA and the SH2 domain of SOCS-2. Significant phosphopeptide/fusion protein interaction was detected with the pTyr595 peptide (Figure 4b), and the specificity of the interaction was confirmed by incubating NusASOCS-2 SH2 protein with either phosphorylated or non-phosphorylated Tyr595 peptide, and NusA recombinant protein with pTyr595 peptide.

EXAMPLE 14

Refined SOCS-2 protein purification method

In a large number of preliminary experiments that assessed bacterial expression of SOCS-2

(full-length and SH2 domain) using a variety of C- and N-terminal tags or fusions, difficulties were encountered with both the level of expression and precipitation of the purified protein. A fusion of the mouse SOCS-2 SH2 domain and the E.coli NusA protein - 45 -

was generated, expressed and purified in an attempt to overcome these issues. This fusion protein remained in solution at useful working concentrations for sufficient time to allow the identification of substrate peptides (see below), but protein instability remained a significant problem and precluded further activities including the development of high throughput screening (HTS) strategies. Further refinement of the SOCS-2 purification strategy has overcome instability issues to yield active SOCS-2 protein at approximately 2mg/L bacterial culture with greater than 60% purity (see Figures 5 and 6). This has facilitated significant progress in the development of a HTS (see below).

EXAMPLE 15

Growth hormone receptor mutational analysis protocols

Luciferase assays

293T cells were transfected with Lipofectamine 2000 reagents according to the manufacturer's instructions. For SOCS-2 domain analysis, 4 x 10⁵ cells were transfected with 200 ng each of growth hormone (GH) receptor plasmid (Hansen et al, J Biol Chem 271(21): 12669-12673, 1996), LHRE-luc reporter plasmid (Goffin et al, J Biol Chem 271(28): 16573-16579, 1996), β-galactosidase plasmid (Ogilvy et al., Blood 91: 419-430, 1998), and 0-200 ng of the pEFSOCS-2Flag plasmid (Nicholson et al, supra) made up to 200 ng with pEFBOS plasmid were transfected into a 24 well plate 6 h after cells were plated out. Twenty four hours later, cells were washed once with mouse-tonicity phosphate buffered saline (MT-PBS) and the culture medium was replaced with DME containing 1% bovine serum albumin (BSA). Cells were left to equilibrate for 1 h before 500 ng/ml of recombinant pig (rp)GH was added to the test groups. Cells were incubated for a further 16 hours before being lysed and assayed for luciferase and β-galactosidase activity as described (Greenhalgh et al, J. Biol. Chem. 270(43): 40181-40184, 2002).

GH receptor constructs for mutational analysis were generated by PCR of the GH receptor coding region from the pMet-Ig7 GHR plasmid (sourced from Nils Billestrup - described in Hansen et al, J Biol. Chem. 271(21): 12669-12673, 1996) and inserting it into the pEFBOS vector (Mizushima et al., Nucl. Acids Res., Vol 18(17): 5322, 1990). The Y487F, - 46 -

Y595F, Y534F and Y487 595F receptor mutants were generated by using overlapping PCR strategies to introduce a tyrosine to phenylalanine mutations into these sites. All constructs were sequenced in their entirety before use. Luciferase assays were performed as described above except that only 2 ng of each GH receptor plasmid was used.

Elongin B/C interaction

200 ng of either pEFFlag, pEFSOCSlFlag or pEFSOCS2Flag (Nicholson et al, supra) were transfected into 293T cells as described above. After 48 h the cells were washed and lyzed before being precipitated with antibodies against the FLAG epitope. After SDS- PAGE separation and Western transfer, blots were probed with antibodies against Elongin B and C (Krebs et al., Molecular and Cellular Biology 22(13): 4567-4578, 2002) then reprobed with antibodies directed against the FLAG epitope.

EXAMPLE 16 SOCS-2 actions depend on a number of domains

SOCS proteins have been defined as consisting of a central Src homology-2 (SH2) domain, an N-terminal domain of varying length and a C-terminal motif termed the SOCS-Box. While each of the three domains for SOCS-1 and SOCS-3 has been ascribed a function, little is known as to what role each of these plays in the SOCS-2 protein. Transient transfection assays expressing mutated SOCS-2 molecules were used to elucidate their roles. SOCS-2 that had a point mutation in a conserved arginine (R73K) within the SH2 domain showed only slightly impaired inhibitory/enhancement effects, while additional mutation of sites adjacent of this residue (R73K, D74E, S75C) led to a more complete cessation of activity. SOCS-2 lacking the N-terminus (37aa missing from the N-terminus) failed to have any significant effects on GH-mediated STAT5 reporter activity, while SOCS-2 with the N-terminus of SOCS-1 substituted onto the molecule has some inhibitory capacity at higher concentrations of transfected construct. A SOCS-2 construct lacking a SOCS-Box displayed no inhibitory effects at all, but surprisingly caused an enhancement of signalling even at low concentrations. Given the importance of this motifs effects on signalling we investigated whether SOCS-2 also bound elonginB/C complexes as has been - 47 -

shown for other SOCS molecules, and found an association between SOCS-2 and ElonginB/C in immunoprecipitation studies.

EXAMPLE 17 SOCS-2 interacts with 3 residues on the GH receptor

We have previously shown that SOCS-2 interacts with endogenous GH receptor from a number of different mouse tissues and that this interaction is mediated through at least one tyrosine in a phosphorylation dependant manner (Greenhalgh et al., supra). Modification of recombinant SOCS-2 protein purification strategies led to a significant improvement in stabilization of protein activity that allowed a more thorough examination of SOCS-2 GH receptor interactions.

Biacore analysis using recombinant SOCS-2 SH2 domain protein passed over phosphorylated peptides derived from the GH receptor confirmed that Tyr595, and indicated that Tyr487 and Tyr534, are also sites of interaction. The affinities of these interactions were tested by calculating the disassociation and association constants (K_d and K_a) and IC₅₀ values for each. The affinity of the SOCS-2 to immobilised Y595 peptide was calculated to be 250 nM (Figure 7a). The SOCS-2- Y595 interaction was examined further in soluble competition experiments where it was found that incubation of SOCS-2 protein with soluble Y595 for 2h prior to biosensor analysis displaced SOCS-2 binding to the immobilised form of Y595. Based on this data the IC50 of this competition was calculated to be 1.83 μM (Figure 7b), suggesting an equimolar competition.

A prototype high-throughput screen based on the interaction of SOCS-2 to the GHR phospho-peptide Y595 and alpha screen technology has now been developed (Figure 8). Initial data using this screening format suggested that the SOCS-2 protein was stable and the interaction could be tested over a two-week period (Figure 9). Data is expressed as a value of signal to noise. More recently, the time frame has been extended to 4 weeks, a period more suitable for high throughput screening. Further work is now progressing to examine soluble competition of this interaction for the Y487 and Y534 peptides. 48

Biacore analysis

All Biacore analyses were performed as per manufacturers instructions. Briefly, biotinylated phosphopeptides (1-lOOμg/ml) were bound to streptavidin-coated Biosensor chips (SA5, Pharmacia) at a density of 200-450 response units (RU). SOCS-2 binding studies were measured in buffer A (20mM Hepes, 0.15M NaCl, 3mM EDTA, 0.02% Tween 20) at a flow rate of 10 μl/min. Chips were washed with 0.1M sodium Hydroxide between binding measurements to remove residual SOCS-2 protein, then washed with buffer A. Binding profiles were analysed using BIAENALUATION software Ver. 3.1 (Pharmacia). Non-specific binding by SOCS-2 was corrected for by subtracting sensograms of pY332 from those of SOCS-2 interacting peptides. Dissociation constant (KD) was calculated for each SOCS-2 interacting peptide using a separate ka and kd determination algorithim.

The ability of GHR phosphopeptides to inhibit the SOCS-2 was measured by displacement of SOCS-2 binding to immobilized phosphopeptides by varying concentrations of soluble peptide incubated with SOCS-2. SOCS-2 (at a defined concentration: 0.5 micromolar) was incubated with phosphopeptides Y595, Y487, and Y534 in O.lmg/ml BSA for 3 hours prior to biosensor analysis. The mixture was run simultaneously over 4 biosensor channels containing one of the following phosphopeptides. (Y332, Y487, Y534, and Y595) (Figure 10). The level of bound protein was recorded at a fixed time point within the sensorgram and divided by the corresponding level of SOCS-2 bound to the peptide chip in the absence of competitive ligand.

EXAMPLE 18

SOCS-2 actions are dependent upon specific sites on the GH receptor

To biologically validate the relevance of the candidate GH receptor interaction sites we generated GH receptor constructs for transfection studies that lacked one or more of the tyrosine residues identified by the Biacore analysis. Increasing titrations of SOCS-2 have previously demonstrated a biphasic response in terms of GH-stimulated STAT5 reporter - 49 -

activity derived from wild-type GH receptors (Greenhalgh et ah, supra). Analysis of the Y487F mutant revealed that this construct was still inhibitable by SOCS-2, while there appeared to be some weakening of the inhibitory effects of SOCS-2 in the Y595F mutant. However, deletion of both the 487 and 595 residues (Y487, 595F) removed the inhibitory effects of SOCS-2 and also impacted heavily of the 'recovery' or 'enhancement' effects of SOCS-2 . Analysis of the Y534F mutant revealed that the 534 tyrosine did not play a role in controlling the negative or positive effects of SOCS-2, although the degree of inhibition in the Y534F mutant was stronger than observed in the wild-type construct.

EXAMPLE 19

Growth hormone receptor mutation analysis

SOCS-2 actions are dependant upon specific sites on the GH receptor. To biologically validate the relevance of the candidate GH receptor interaction sites we generated GH receptor constructs that lacked one or more of the tyrosine residues identified by the Biacrore analysis and used these in transfection studies. The activity of these constructs was examined by their ability to stimulate the STAT5 -responsive luciferase reporter with and without GH stimulation (Figure 11). While the Y534F receptor signalling was not different to that of the wild-type, significant increases were observed in the Y595F and Y487F constructs. Interestingly, the receptor lacking both 487 and 595 residues (Y487,595F) had a more than three a fold increase in reporter activity compared to wild- type receptors. Increasing titrations of SOCS-2 have previously demonstrated a biphasic response in terms of GH-stimulated STAT5 reporter activity derived from wild-type GH receptors and it was expected that removal of SOCS-2 binding sites would ameliorate these effects. Analysis of the Y487F mutant construct revealed that SOCS-2 could still inhibit by this receptor although the degree of recovery may have been lessened, while there appeared to be some weakening of the inhibitory effects of SOCS-2 in the Y595F mutant. However, deletion of both the 487 and 595 residues removed all the inhibitory effects of SOCS-2 and also impacted heavily of the recovery effects of SOCS-2. Analysis of the Y534F mutant revealed that 534 tyrosine did not play a role in controlling the negative or - 50 -

positive effects of SOCS-2, although the degree of inhibition in the Y534F mutant was slightly stronger than observed in the wild-type construct.

EXAMPLE 20 Possible model of SOCS-2 activity

Given that it is necessary to delete both the Y487 and Y595 residues to delete the negative effects of SOCS-2 one can suppose that the regulation of GH signalling is more complicated than that of other SOCS proteins regulatory roles in cytokine signalling. Little is known about the role that these residues play, although it been reported that both of these residues are the major sites of interaction by SHP-2 (Stofega et al., Mol. Endo. 14(9): 1338-1350, 2000). We have confirmed these observations using recombinant SHP-2 protein in our Biacore analysis. The role of SHP-2 in GH signalling is unclear, but there is some data to indicate that it may play a negative (Stofega et al., supra) or a positive role (Kim et al, J. Biol. Chem. 273(4): 2344-2354, 1998) in GH signalling. Interestingly, the data that says SHP-2 is a negative regulator could be reinterpreted as SOCS-2 causing the observed effects (Stofega et al, supra). The fact that SOCS-2 and SHP-2 bind to the same sites on the GH receptor set the scene for potential for interplay between these molecules, or competition between for binding to these sites to illicit their effects. This story has a number of similarities to that observed between SOCS-3 and SHP-2 on the gpl30 receptor. It is anticipated that further characterisation of these interactions will decipher the interplay between these molecules.

Analysis of the SOCS-2 molecule has implicated all three domains as having important functions. While the N-terminus is required for SOCS-2's negative effects it is unclear how this works given that we have shown that the SH2 domain is the only domain required for binding to the GH receptor peptides. Interestingly, the SOCS-Box was essential for the negative effects of SOCS-2 to be observed. Deletion of the SOCS-Box from other SOCS molecules has had no effect on signalling reporter output in overexpression systems studied to date (Yasukawa et al, EMBO J 18: 1309-1320, 1999; Narazaki et al, Proc. Natl. Acad. Sci. USA 95: 13130-13134, 1998; Nicholson et al, supra). This implies that - 51 -

the SOCS-Box plays a crucial role in the regulation and supports the hypothesis that the

SOCS-Box is a linker to protein destruction/degredation. This is also supported by our finding that SOCS-2 interacts with Elongins B and C which form part of the E3 ubiquitin ligase complex that adds ubiquitin to targets.

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.

- 52 -

BIBLIOGRAPHY

Alexander et al., Blood 87(6): 2162-70, 1996;

Alexander et al, Cell 98(5): 597-608, 1999;

Goffin et al, JBiol Chem 271(28): 16573-16579, 1996;

Greenhalgh and Hilton, JLeukoc Biol 70(3): 348-56, 2001;

Greenhalgh et al, J. Biol. Chem. 270(43): 40181-40184, 2002;

Greenhalgh et al, Molecular Endocrinology 16(6): 1394-1406, 2002;

Η.msβn et al, Mol Endocrinol 13(11): 1832-1843, 1999;

Hansen et al, JBiol. Chem. 271(21): 12669-12673, 1996;

Kile et al, Mol Cell Biol 21(18): 6189-6197, 2001;

Kim et al., J Biol Chem 273(4): 2344-2354, 1998;

Krebs and Hilton, J Cell Sci 113(Pt 16): 2813-2819, 2000;

Krebs et al., Molecular and Cellular Biology 22(13): 4567-4578, 2002;

Lindeman et al, Genes Dev 15(13): 1631-1636, 2001;

Liu et al, Genes Dev 11(2): 179-86, 1997;

Marine et al, Cell 98(5): 609-616, 1999;

Marine et al., Cell 98(5): 617-627, 1999

Matthews et al, Endocrinology 123(6): 2827-33, 1988;

Matsumoto et al, Mol Cell Biol 19(9): 6396-407, 1999;

Metcalf et al, Nature 405(6790): 1069-1073, 2000;

Mizushima et al, Nucl Acids Res., Vol 18(17): 5322, 1990;

Narazaki et al, Proc. Natl. Acad. Sci. USA 95: 13130-13134, 1998;

Nicholson et al, EMBOJ 18(2): 375-385, 1999;

Ogilvy et al, Blood ^'91: 419-430, 1998;

Palmiter et al, Science 222(4625): 809-814, 1983;

Ram and Waxman, JBiol Chem 274(50): 35553-35561, 1999;

Roberts, et al, Proc Natl Acad Sci USA 98(16): 9324-9329, 2001;

Sambrook et al, Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring

Harbor Laboratories, Cold Spring Harbor, NY, 1990 Schultheis, et al, Blood 99(5): 1766-75, 2002; -53

Stofega etal, Mol. Endo.14(9): 1338-1350,2000; Teglundetα/., Cell 93(5): 841-50, 1998; Udy etal, Proc Natl Acad Sci USA 94(14): 7239-7244, 1997 Yasukawa et al, Annu Rev Immunol 18: 143-164, 2000; Yasukawa et al, EMBO J 18: 1309-1320, 1999;

Claims

- 54 -CLAIMS:

1. A method for identifying an agent which modulates a cytokine-mediated signalling pathway or event, said method comprising contacting candidate agents with a receptor for said cytokine and/or with a molecule which binds to said cytokine receptor, and selecting agents which bind or otherwise interact with said receptor or molecule.

2. The method of claim 1 wherein the cytokine is growth hormone or a functional equivalent or homolog thereof.

3. The method of claim 1 or 2 wherein said agent modulates the interaction between said molecule and said cytokine receptor.

4. The method of claim 3 wherein the molecule is a SOCS protein.

5. The method of claim 4 wherein the agent modulates the interaction between the SOCS-2 protein and a growth hormone receptor.

6. The method of claim 5 wherein the agent modulates the interaction between the SH2 domain of the SOCS-2 protein and a growth hormone receptor.

7. The method of claim 4 or 5 wherein the agent modulates the interaction between a phosphorylated tyrosine in the cytoplasmic domain of the growth hormone receptor and said SOCS-2 protein, or domain thereof.

8. The method of claim 6 wherein the phosphorylated tyrosine is selected from the residues: Tyr 595, Tyr 534 and Tyr 487 in the human growth hormone receptor, or equivalent residue thereof in another animal species.

9. A method for identifying an agent which modulates a growth hormone-mediated signal, pathway or event , said method comprising: - 55 -

(i) immobilizing one of a SOCS-2 molecule or the growth hormone receptor or their parts, fragments, derivatives, homologs or analogs to a solid support via binding partners wherein one of said binding partners is attached or otherwise anchored to said solid support and the other of said binding partners is linked to said SOCS-2 or receptor molecule or their parts, fragments, derivatives, homologs or analogs;

(ii) contacting said immobilized SOCS-2 or growth hormone receptor molecule or their parts, fragments, derivatives, homologs or analogs with the other of receptor or SOCS-2 molecule or their parts, fragments, derivatives, homologs or analogs in the presence of a candidate agent; and

(iii) measuring qualitatively or quantitatively a change in signal emission indicative of enhanced or diminished binding between said SOCS-2 and the growth hormone receptor or their parts, fragments, derivatives, homologs or analogs;

wherein the agent is identified as a potential agonist or antagonist by an enhanced or diminished interaction between the SOCS-2 protein and the growth hormone receptor.

10. The method of claim 9 wherein the immobilized SOCS-2 molecule or growth hormone receptor or their parts, fragments, derivatives, homologs or analogs is attached 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-2 or receptor molecule or their parts, fragments, derivatives, homologs or analogs.

11. The method of claim 9 or 10 wherein the SOCS-2 fragment or part is the SH2 domain. - 56

12. The method of any one of claims 9 to 11 wherein enhanced or diminished binding between said SOCS-2 and growth hormone receptor or their parts, fragments, derivatives, homologs or analogs is measured by Surface Plasmon Resonance (SPL).

13. The method of any one of claims 9 to 11 wherein the enhanced or diminished binding between said SOCS-2 and growth hormone receptor or their parts, fragments, derivatives, homologs or analogs is measured using an alpha screen.

14. An agent that modulates the interaction between a SOCS protein and the growth hormone receptor identified by the method of any one of claims 1 to 13.

15. The agent of claim 14 wherein the agent modulates the interaction of the SOCS-2 protein with the growth hormone receptor.

16. The agent of claim 15 wherein the agent interacts with one or more phosphorylated tyrosine residues on the growth hormone receptor.

17. The agent of claim 16 wherein the agent interacts with at least one of the following residues on the growth hormone receptor: Tyr 595, Tyr 534 and Tyr 487 on the human growth hormone receptor, or equivalent residues thereof in the growth hormone receptor from another animal species.

18. The agent of claim 14 or 15 wherein said agent interacts with two or more of said phosphorylated tyrosine residues.

19. The agent of any one of claims 14 to 18 identified by the method of any one of claims 1 to 13.

20. A pharmaceutical composition comprising the agent of any one of claims 14 to 19 together with a pharmaceutically acceptable carrier or diluent. 57

21. A method for regulating signaling of a cytokine or related molecule in an animal subject, said method comprising administering to said subject, or to a site within said subject, an effective amount of an agent that modulates the interaction between said cytokine receptor and a molecule that interacts with said receptor.

22. The method of claim 21 wherein the cytokine is growth hormone or a functional equivalent or homolog thereof.

23. The method of claim 21 or 22 wherein the molecule that interacts with said cytokine receptor is a SOCS protein.

24. The method of any one of claims 21 to 23 wherein the agent modulates the interaction between the SOCS-2 protein and a growth hormone receptor.

25. The method of claim 24 wherein the agent modulates the interaction between the SH2 region of the SOCS-2 protein and a growth hormone receptor.

26. The method of any one of claims 21 to 25 wherein the agent modulates the interaction between a phosphorylated tyrosine in the cytoplasmic domain of the growth hormone receptor and said SOCS protein.

27. The method of claim 26 wherein the phosphorylated tyrosine is selected from the residues: Tyr 595, Tyr 534 and Tyr 487 in the human growth hormone receptor, or equivalent residues thereof in the growth hormone receptor of another animal species.

28. The method of any one of claims 21 to 27 wherein the animal subject is selected from the list of: humans, mice, rats, guinea pigs, pigs, sheep or goats.

29. The method of any one of claims 21 to 28 wherein said animal subject is a human. - 58

30. An isolated mutant growth hormone receptor wherein said receptor exhibits decreased or eliminated SCOS-2 mediated modulation of signalling and/or binding affinity with SOCS-2.

31. The mutant receptor of claim 30 wherein said mutant receptor comprises an amino acid substitution at one or more phosphorylated tyrosine residues.

32. The mutant receptor of claim 31 wherein said mutant receptor comprises an amino acid substitution at Y487, Y534 and/or Y595 of the human growth hormone receptor, or equivalent residues thereof in the growth hormone receptor of another animal species.

33. The mutant receptor of any one of claims 30 to 32 wherein the mutant receptor comprises amino acid substitutions at two or more phosphorylated tyrosine residues.

34. The mutant receptor of any one of claims 30 to 33 wherein the mutant receptor comprises amino acid substitutions at Y487 and Y595 of the human growth hormone receptor, or equivalent residues thereof in the growth hormone receptor of another animal species.

35. A vector comprising a genetic sequence encoding the mutant growth hormone of any one of claims 30 to 34.

36. The vector of claim 35 wherein the vector is an expression vector.

37. The vector of claim 35 wherein the vector is a "knock-in" vector.

38. A method for mutating or modifying the amino acid sequence of the growth hormone receptor in a eukaryotic cell, said method comprising transforming the vector of claim 37 into said cell and selecting for the marker, wherein the mutant growth hormone - 59 -

receptor gene in the transformants is incorporated into the genome by homologous recombination.

39. A eukaryotic cell generated using the method of claim 38.

40. The cell of claim 39 wherein the cell is from a species selected from the list of: humans, mice, rats, fish, guinea pigs, pigs, sheep or goats.

41. An embryo or animal comprising one or more cells of claims 39 or 40.

42. A transgenic animal that has expresses the mutant growth hormone receptor of any one of claims 30 to 34.

43. The transgenic animal of claim 42 wherein the animal is from a species selected from the list of: humans, mice, rats, fish, guinea pigs, pigs, sheep or goats.

44. The transgenic animal of claim 42 or 43 wherein said animal is a mouse.