WO2003031468A1

WO2003031468A1 - Therapeutic and diagnostic molecules that are capable of interacting with socs proteins

Info

Publication number: WO2003031468A1
Application number: PCT/AU2002/001353
Authority: WO
Inventors: Douglas James Hilton; Nicos A. Nicola; Warren Scott Alexander; Robyn Starr; Sandra Elaine Nicholson; Tracy Willson; Elizabeth Viney; Steven Rakar; Danielle Krebs; Manuel Baca; Rachel Uren
Original assignee: Walter and Eliza Hall Institute of Medical Research
Current assignee: Walter and Eliza Hall Institute of Medical Research
Priority date: 2001-10-05
Filing date: 2002-10-04
Publication date: 2003-04-17
Anticipated expiration: 2004-04-05

Abstract

The present invention relates generally to molecules which are capable of interacting with members of the family of suppressors of cytokine signalling (SOCS) proteins. The molecules range from intracellular proteinaceous targets for which the SOCS is a ligand to chemicals including proteinaceous entities identified inter alia by screening of natural products, chemical libraries and/or through rational drug design. The identification of intracellular targets of a SOCS protein and/or the identification of other interactors of the SOCS molecule permits the development of a range of therapeutic and diagnostic applications. The present invention particularly relates to SOCS-6 and its involvement in various physiological processes such as those mediated by growth factor or hormone signalling.

Description

THERAPEUTIC AND DIAGNOSTIC MOLECULES THAT ARE CAPABLE OF INTERATING WITH SOCS PROTEINS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

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

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

Cells continually monitor their environment in order to modulate physiological and biochemcial processes which in turn affects future behaviour. Frequently, a cell's initial interaction with its surroundings occurs via receptors expressed on the plasma membrane. Activiation of these receptors, whether through binding endogenous ligands (such as cytokines) or exogenous ligands (such as antigens), triggers a biochemical cascade from the membrane through the cytoplasm to the nucleus. Of the endogenous ligands, cytokines represent an important and versatile group. However, of particular importance are molecules which regulate cytokine function. An example of this class of molecules are members of the family of suppressors of cytokine signalling (SOCS).

SOCS proteins contain aC-terminal homology domain which is termed the "SOCS box" [Starr, R., et al., Nature (London) 387: 917-921 1997]. The first member of this family was called CIS (cytokine-inducible SH2-containing protein) [Yoshimura, A. et al., EMBO J. 14: 2816-2826 1995] and was shown to inhibit erythropoietin and interleukin-3 receptor signalling. SOCS-1 was cloned from a retro viral expression library as a cDNA whose constitutive expression inhibited interleukin-6-induced differentiation of Ml [Starr, R., et al, Nature (London) 387: 917-921 1997] cells and it was simultaneously cloned as a protein that interacted with activated JAK kinases (JAK-binding protein, JAB) [Endo, T.A., et al. Nature (London) 387: 921-924, 1997] and as a protein with antigenic similarity to STATs (STAT-inducible STAT inhibitor, SSI) [Naka, T., et al., Nature (London) 387: 924-929 1997]. The sequence similarity of SOCS-1 and CIS led to the identification of six additional members of this family (SOCS-2-7) each with an SH2 domain and a C-terminal SOCS box [Starr, R., et al., Nature (London) 387: 917-921 1997; Hilton, D.J., et al. Proc. Natl. Acad. Sci. USA 95: 114-119, 1998; Masuhara, M., et al., Biochem. Biophys. Res. Commun. 239: 429-446, 1997; Minamoto, S., et al. Biochem. Biophys. Res. Commun. 237: 79-83, 1996]. An additional twelve proteins have been described that contain a C- terminal SOCS box but instead of an SH2 domain they contain different protein-protein interaction domains including WD40, ankyrin repeats, SPRY or small GTPase domains [Hilton, D.J., et al., Proc. Natl. Acad. Sci. USA 95: 114-119 1998].

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

SOCS-6 is one member of the suppressor of cytokine signalling (SOCS) family of proteins and comprises the characteristic central SH2 domain and C-terminal SOCS box. The physiological function of many SOCS proteins including SOCS-6 is not clearly understood. A biochemical analysis of SOCS-6 has revealed that it is a ubiquitously expressed cytosolic protein. Like other proteins containing a SOCS box, SOCS-6 binds to elongins B and C. This suggests that SOCS-6 may act as an E3 ubiquitin ligase, targeting proteins which interact through its SH2 domain for ubiquitination and proteasomal degradation.

In work leading up to the present invention, the inventors sought to identify molecules which interact with the SOCS-6 SH2 domain. Such molecules and in particular endogenous proteinaceous molecules enable elucidation of signalling pathways involving

SOCS-6 and related SOCS molecules. The inventors have surprisingly identified that

SOCS-6 interacts with wter alia insulin receptor substrate-4, insulin receptor substrate-2 and the p85 regulatory subunit of P13K (p85α and p85β) (IRS-4, IRS-2 and p85) and, hence, is involved in insulin-like growth factor (IGF) and insulin receptor (IR) signalling.

In accordance with the present invention, it is proposed that the SOCS-6 and related SOCS molecules are involved in suppression of IR signalling in the presence of insulin and/or

IGF-mediated stimulation or activation. This indicates that SOCS-6 and related SOCS molecules have a role in the pathogenesis of diabetes and other related conditions. 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, <400>2, etc. A sequence listing is provided after the Bibliography.

The present invention is predicated in part on the surprising observation that SOCS-6 associates with tyrosine-phosphorylated proteins in response to IGF-1 stimulation. These proteins were identified to include inter alia IRS-4, IRS-2 and p85. It is proposed, in accordance with the present invention, that SOCS-6 interacts with IRS-4, IRS-2 and p85 before, during and/or after stimulation mediated by IGF and/or its isoforms or homologues and in particular IGF-1. The identification of SOCS-6's involvement in IGF-mediated signalling indicates that this molecule is involved in inhibition of ER signalling thereby contributing to cytokine or growth factor-mediated insulin resistance amongst other conditions. The identification of the involvement of the SH2 domain of SOCS-6 provides a target for the identification of chemical and proteinaceous molecules capable of modulating SOCS-6 activity and, more particularly, their use as therapeutic agents in the treatment of insulin resistance, amongst other conditions such as those associated with IGF-mediated signalling.

Accordingly, one aspect of the present invention contemplates a method for modulating IGF-mediated and/or insulin-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and insulin receptor or an insulin receptor substrate or a component in an IGF-mediated signalling pathway. Another aspect of the present invention contemplates a method for modulating IGF-1- mediated and/or insulin-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and insulin receptor or an insulin receptor substrate or a component in an IGF-1 -mediated signalling pathway.

A further aspect of the present invention provides a method for modulating IGF-1 and/or insulin-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and insulin receptor or an insulin receptor substrate or a component in an IGF-1 -mediated signalling pathway.

Yet another aspect of the present invention contemplates' a method of ameliorating the effects of insulin resistance in an animal cell, said method comprising introducing to said animal cell or animal comprising said cell an antagonist of SOCS-6 for a time and under conditions sufficient to inhibit, reduce or otherwise suppress SOCS-6 inhibition of IGF-1- and/or insulin-mediated signalling pathways or a component in an IGF-1 mediated signalling pathway.

Still another aspect of the present invention contemplates a method of modulating activity of SOCS-6 in an animal such as a human, said method comprising administering to said human or mammal an effective amount of a molecule for a time and under conditions sufficient to inhibit SOCS-6 binding to an intracellular ligand such as IRS-2 or IRS-4.

Still yet another aspect of the present invention contemplates a method of modulating levels of SOCS-6 in a cell, said method comprising contacting a cell containing a SOCS gene with an effective amount of an inhibitor of expression of the SOCS-6 gene for a time and under conditions sufficient to modulate levels of said SOCS protein. Even yet another aspect of the present invention contemplates a method of modulating signal transduction in a cell containing a SOCS-6 gene comprising contacting said cell with an effective amount of an inhibitor of SOCS-6 gene expression for a time sufficient to modulate levels of SOCS protein with the cell.

Another aspect of the present invention contemplates a method of modulating the activity of a cytokine or cytokine-like molecule, said method comprising administering to a subject a modulating effective amount of a molecule for a time and under conditions sufficient to decrease the biological activity of SOCS-6 or the suppressing effects of SOCS-6. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of a polypeptide of the complex or its ligand.

A further aspect of the present invention contemplates a composition and in particular a pharmaceutical composition comprising an effector molecule as defined above and one or more pharmaceutically acceptable carriers and/or diluents.

Yet another aspect of the present invention further provides genetically modified animals in which one or both alleles of SOCS-6 are mutated alone or in combination with another mutation in one or both alleles for another gene such as encoding another SOCS molecule.

Still another aspect of the present invention provides a method of producing a genetically modified non-human animal, said method comprising introducing into embryonic stem cells of an animal a genetic construct comprising a SOCS-6 nucleotide sequence carrying a single or multiple nucleotide substitution, addition and/or deletion or inversion or insertion wherein there is sufficient SOCS-6 nucleotide sequences to promote homologous recombination with a SOCS-6 gene within the genome of said embryonic stem cells selecting for said homologous recombination and selecting embryonic stem cells which carry a mutated SOCS-6 gene and then generating a genetically modified animal from said embryonic stem cell. Still another aspect of the present invention contemplates a method of screening for an effector molecule which is capable of modulating the functional interaction between a SOCS molecule and an intracellular ligand in an animal cell, said method comprising contacting in the presence of said intracellular ligand or analogue or derivative thereof a polypeptide comprising an SH2 domain of said SOCS molecule, or derivative of said SH2 domain, with a test agent; and detecting a reduced level of interaction between said ligand or analogue or derivative thereof and said SH2 domain or derivative relative to a reference level of said interaction in the absence of said test agent, wherein said reduced level is indicative of said agent being an effector molecule.

BRIEF DESCRIPTION OF THE FIGURES

Figure la is a diagrammatic representation of the SOCS-6 molecule which identifies the region used for immunization to generate anti-SOCS-6 monoclonal antibodies.

Figure lb is a summary of the characteristics of the anti-SOCS-6 monoclonal antibodies.

Figure 2 is a diagrammatic representation showing the targeted disruption of the SOCS-6 gene by homologous recombination, (a) Genomic map of SOCS-6 gene; (b) Targeting vector; (c) Targeted allele.

Figure 3 is a photographic representation showing expression of SOCS-6 mRNA in tissues from wild-type (A and B) and SOCS-6^7" (B) mice.

Figure 4 is a photographic representation showing expression of SOCS-6 in tissues from wild-type and SOCS-6^{" "} mice, (a) Lysates were prepared from various organs taken from either wild-type (+/+) or SOCS-6^^'1'^ (-/-) mice and immunoprecipitations were carried out using the ICI antiSOCS-6 mAb. Western blotting was carried out using the 3A7 anti- SOCS-6 mAb. (b) This figure shows that an equal amount of protein was used for each immunoprecipitation. 40 μg of lysate from each sample was subjected to Western blotting using an anti-heat shock-70 pAb.

Figure 5 is a graphical representation showing that SOCS-6^"Λ mice are 10% smaller than wild-type littermates.

Figure 6 is a photographic and tabulated representation showing identification of proteins which associate with the SOCS-6 SH2 domain in response to IGF-1 stimulation. 293T cells were stimulated with 200 ng/mL IGF-1, lysed and precipitated using the SOCS-6 SH2 domain (S6-SH2) or control resin. Precipitates were separated on a SDS-PAGE gel and stained with Coomassie blue. Bands of interest were excised and subjected to mass spectrometric analysis. Figure 7 is a photographic representation showing that SOCS-6 associates with tyrosine- phosphorylated proteins in response to IGF-1 stimulation.

Figure 8 is a photographic representation showing that SOCS-6 associates with IRS-4 in response to stimulation with IGF-1. 293T cells were left untransfected or were transiently transfected with either a FLAG epitope tagged version of SOCS-6 (S6) or a FLAG-tagged version of WSB-1. Cells were left unstimulated (-) or were stimulated with 200 ng/mL IGF-1 (+). FLAG-SOCS-6 and FLAG-WSB-1 were immunoprecipitated with anti-FLAG Abs. Alternatively, non-transfected lysates were precipitated with the SOCS-6-SH2 domain, the SOCS-7-SH2 domain or control resin. Western blotting was then carried out using an anti-IRS-4 Ab (Figure 8a). The blot was stripped and reprobed using an anti- FLAG Ab (Figure 8b).

Figure 9 is a photographic representation showing that SOCS-6 associates with p85 of PI 3 kinase in response to IGF-1 stimulation.

Figure 10 is a graphical representation showing a comparison of insulin production following glucose injection in wild-type and SOCS-6^{" "} mice.

Figure 11 is a graphical representation of the glucose tolerance test in wild-type and SOCS-6^" mice following glucose injection.

Figure 12 shows sequences of the degenerate libraries used to determine the sequences of preferred SOCS-6 and SOCS-7 phosphopeptide ligands. Each X position is an equal mixture of 19 L-amino acids, with cysteine omitted to simplify the N-terminal sequencing analysis, (a) Library used to map preferred residues at the pY+1 to pY+3 positions (i.e. 1^st through 3^rd amino acid positions downstream of the phosphotyrosine). (b) Library used to map preferred residues at the pY+3 to pY+5 positions.

Figure 13 are graphical representations showing selection of phosphopeptides that bind the (a) SOCS-6 and (b) SOCS-7 SH2 domains. The degenerate phosphopeptide mixtures (Figure 12) were incubated with Sepharose resin containing either immobilized SOCS-6 or SOCS-7 SH2 domain. The column was washed and bound peptides were eluted with 1% aqueous TFA. The eluted peptide mixture was analyzed by N-terminal sequencing and the results compared to those from the eluate of Sepharose resin alone. Data is shown for the relative amino acid composition at each of the randomized positions within the libraries. The data has been normalized so that the sum of the values of all amino acids equals the number of amino acids (i.e. 19).

The following are abbreviations used in the specification:

ABBREVIATIONS

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification that SOCS-6 is involved in the IGF-1 -mediated signalling pathway and in the regulation of insulin production. It is proposed, in accordance with the present invention, that in the presence of IGF-1 and/or insulin, SOCS-6, induced by cytokines, associates with IRS-2, IRS-4, p85 and/or the ER thereby suppressing its function. This may contribute to, in conditions involving high cytokine involvement such as inflammation, acromegally and/or obesity, insulin resistance and development of diabetes such as type 2 diabetes. The present invention provides a target, therefore, to identify therapeutic molecules capable of modulating expression of SOCS-6 and related SOCS molecules, or capable of modulating the interaction between SOCS-6 and related SOCS molecules and ERS-2, ER.S-4 and p85 for use in manipulating IGF-1 and insulin-mediated pathways.

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

The term "modulating" means that the effector molecule may be an agonist or antagonist of SOCS interaction with its ligand. An agonist would promote suppression of signalling mediated by the SOCS molecule. An antagonist would inhibit suppression of signalling. Consequently, in conditions where IGF- and/or insulin-mediated signalling is down regulated such as in insulin resistance, then an antagonist is preferred. In conditions resulting in excess IGF- and/or insulin-mediated signalling, then an agonist is preferred.

Reference herein to "IGF" or "insulin growth factor" includes IGF-1 or its functionally equivalent homologues and isoforms. IGF-1, however, is the most preferred form of IGF. Accordingly, another aspect of the present invention contemplates a method for modulating IGF-1 -mediated and/or insulin-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and insulin receptor or an insulin receptor substrate or a component in an IGF-1 -mediated signalling pathway.

The present invention is particularly directed to SOCS-6 but extends to any SOCS molecule capable of interacting with and suppresses the function of the IGF- and/or insulin-mediated signalling pathways. Examples of other SOCS molecules contemplated herein include SOCS-1, SOCS-2 and SOCS-3 amongst others. However, SOCS-6 is most preferred.

Accordingly, another aspect of the present invention provides a method for modulating IGF-1 and/or insulin-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS-6 molecule and insulin receptor or an insulin receptor substrate or a component in an IGF-1 -mediated signalling pathway.

A "component" in the IGF-1- or insulin-mediated signalling pathways includes ERS-2,

IRS-4 and p85.

Accordingly, another aspect of the present invention provides an effector molecule capable of modulating interaction between a SOCS molecule and an intracellular ligand in an animal cell wherein said effector molecule modulates IGF- and/or insulin-mediated signalling.

Preferably, the effector molecule is an antagonist.

Usefully, the antagonist interacts with the SH2 domain of the SOCS molecule. Preferably, the SOCS molecule is SOCS-6.

Preferably, the effector molecule is capable of inhibiting SOCS-6 function and is useful in treating insulin resistance.

Accordingly, another aspect of the present invention is directed to a SOCS-6 antagonist wherein said antagonist inhibits SOCS-6-mediated suppression of IGF- and/or insulin- mediated signalling pathways.

The effector molecules of the present invention may be identified, for example, by screening a library of synthetic or natural agents. Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Dalton. Candidate agents typically comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.

Small (non-peptide) molecule modulators of a SOCS molecule are particularly preferred. In this regard, small molecules are particularly preferred because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, and/or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect, for example, the interaction between the SOCS molecule and its intracellular ligand. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Thus, the present invention contemplates "natural product screening" including screening environmental and biological locations such as coral, river beds, plants, microorganisms, rock formations, Antarctic or Arctic regions or sea water or sea beds for chemical molecules which are capable of interacting with a target SOCS molecule thereby modulating its function. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterifϊcation, amidification, etc. to produce structural analogues.

Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.

In a particular embodiment, the effector molecule is capable of interacting with the SH2 domain of a SOCS molecule.

Suitably, the effector molecule is an antagonist of said SOCS molecule.

In one embodiment, the effector molecule comprises a phosphopeptide sequence. For example, in the case of SOCS-6, the phosphopeptide sequence is preferably selected from:

VE(pY)XιX₂X₃NHSGR [SEQ ED ΝO:3]; or RN(pY)VFX₃X₄X₅LK [SEQ ED NO:4]

wherein

pY is phosphotyrosine; Xi is Y, M, V, W, F or I, preferably V; X₂ is Y, M, V, W, F, I or L, preferably Y;

X₃ is M, V, W, F, I or L, preferably M, V, F or I; X is any amino acid, suitably N or W; and X₅ is any amino acid, suitably W.

Alternatively, in the case of SOCS-7, the phosphopeptide sequence is preferably selected from:-

NE(pY)X]X₂X₃VHSGR [SEQ ED ΝO:5]; or RN(pY)VFX₃X₄X₅LK [SEQ ED NO:6]

wherein

pY is phosphotyrosine; Xi is M, V, W, I or L, preferably V;

X₂ is any amino acid, suitably Q, T, E, Y, M, V, W, F, I or L; X₃ is Y, M, W, F, I or L, preferably W or F; X is any amino acid, suitably D, T, Y, V, W, or F; and

X₅ is any amino acid, suitably D, H, Y, P, W, or F.

The invention encompasses any effector molecules which are capable of modulating the interaction between the SH2 domain of a SOCS molecule and its intracellular ligand.

Accordingly, another aspect of the present invention contemplates a method of screening for an effector molecule which is capable of modulating the functional interaction between a SOCS molecule and an intracellular ligand in an animal cell, said method comprising contacting in the presence of said intracellular ligand or analogue or derivative thereof a polypeptide comprising an SH2 domain of said SOCS molecule, or derivative of said SH2 domain, with a test agent; and detecting a reduced level of interaction between said ligand or analogue or derivative thereof and said SH2 domain or derivative relative to a reference level of said interaction in the absence of said test agent, wherein said reduced level is indicative of said agent being an effector molecule. Preferably, the SOCS molecule is selected from SOCS-6 or SOCS-7. In a preferred embodiment, the polypeptide, which is contacted with the test agent, preferably comprises the sequence set forth in SEQ ED NO: 7 or SEQ ED NO:8. In an especially preferred embodiment, the polypeptide comprises the sequence set forth in SEQ ED NO: 9 or SEQ ED NO: 10.

Preferably, the SH2 domain is contacted with the test agent in the presence of a phosphopeptide sequence as broadly described above.

In one specific example, the effector molecules is useful in treating or otherwise ameliorating the symptoms of insulin resistance.

Accordingly, another aspect of the present invention contemplates a method of ameliorating the effects of insulin resistance in an animal cell, said method comprising introducing to said animal cell or animal comprising said cell an antagonist of SOCS-6 for a time and under conditions sufficient to inhibit, reduce or otherwise suppress SOCS-6 inhibition of IGF-1- and/or insulin-mediated signalling pathways or a component in an IGF-1 mediated signalling pathway.

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

As stated above, the effector molecule may be found following natural product screening or screening of chemical libraries or may be generated by preparing derivatives of analogues of SOCS (e.g. SOCS-6) itself or a SOCS ligand (e.g. ERS-2, ERS-4 or p85).

The terms "derivatives" or its singular form "derivative" in relation to a SOCS molecule and in particular SOCS-6 includes parts, mutants, fragments and analogues as well as hybrid or fusion molecules and glycosylation variants of the SOCS. Particularly useful derivatives comprise single or multiple amino acid substitutions, deletions and/or additions to the SOCS SH2 domain and/or the SOCS box amino acid sequence.

Preferably, the derivatives act as antagonists but agonists are also contemplated by the present invention. The present invention further extends to homologues of SOCS-6 which include the functionally or structurally related molecules from different animal species. The present invention also encompasses analogues and mimetics. Mimetics include a class of molecule generally but not necessarily having a non-amino acid structure and which functionally are capable of acting in an analogous or antagonistic manner to the protein for which it is a mimic, in this case, SOCS-6. Mimetics may comprise a carbohydrate, aromatic ring, lipid or other complex chemical structure or may also be proteinaceous in composition.

The present invention further extends to a range of deletion mutants of SOCS and in particular SOCS-6 carrying deletions in the SH2 or the SOCS box. Molecules are also contemplated by the present invention which encompasses only the carboxy terminal region or amino terminal region or fused to another peptide, polypeptide or protein.

As stated above, the present invention contemplates agonists and antagonists of SOCS-6. Antibodies are one example of an antagonist although are more useful in diagnostic applications or in the purification of SOCS peptides.

In a preferred embodiment, the present invention provides antagonists of SOCS-6. Such antagonists may be used, for example, in the treatment or prophylaxis of cytokine mediated dysfunction such as autoimmunity, immune suppression or hyperactive immunity or other condition including but not limited to dysfunctions in the haemopoietic, endocrine, hepatic and neural systems. Dysfunctions mediated by other signal transducing elements such as hormones or endogenous or exogenous molecules, antigens, microbes and microbial products, viruses or components thereof, ions, hormones and parasites are also contemplated by the present invention. They may also be useful in promoting degradation or inhibiting degradation.

In particular, antagonists of SOCS-6 are useful in ameliorating the symptoms or effects of insulin resistance.

Analogues of SOCS-6, especially those acting as antagonists contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.

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 NaBH₄; 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 NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

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

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

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

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

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown below:-

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

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

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

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

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

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

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

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

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

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

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

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-θ!-methylarginine Dmarg o;-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-Q!-methylhistidine Dmhis N-(3-aminopropyl) glycine Norn

D-α-mefhylisoleucine Dmile N-arnino-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-cϋ-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmfhr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmt 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)glycine 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 D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-mefhyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

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

N-(l-methylpropyl)glycine Nile D-N-mefhylserine 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-napfhylalanine 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-Q!-methylalanine Mala L-α-methylarginine Marg L-αi-methylasparagine Masn

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

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-mefhylglutamine Mgln L-cϋ-methylglutamate Mglu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-θ!-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-θ!-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-G!-methyltyrosine Mtyr

L-Q!-mefhylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 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=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and N _α-methylamino acids, introduction of double bonds between C_α and Cρ atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

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

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

Another aspect of the present invention contemplates a method of modulating activity of SOCS-6 in an animal such as a human, said method comprising administering to said human or mammal an effective amount of a molecule for a time and under conditions sufficient to inhibit SOCS-6-mediated binding to an intracellular ligand such as ERS-2 or ERS-4.

Still a further aspect of the present invention contemplates a method of modulating levels of SOCS-6 in a cell, said method comprising contacting a cell containing a SOCS gene with an effective amount of an inhibitor of expression of the SOCS-6 gene for a time and under conditions sufficient to modulate levels of said SOCS protein.

Yet a further aspect of the present invention contemplates a method of modulating signal transduction in a cell containing a SOCS-6 gene comprising contacting said cell with an effective amount of an inhibitor of SOCS-6 gene expression for a time sufficient to modulate levels of SOCS protein with the cell.

Still a further aspect of the present invention contemplates a method of modulating the activity of a cytokine or cytokine-like molecule, said method comprising administering to a subject a modulating effective amount of a molecule for a time and under conditions sufficient to decrease the biological activity of SOCS-6 or the suppressing effects of SOCS-6. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of a polypeptide of the complex or its ligand.

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

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of superfactants. The preventions of the action of microoganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmersol and the like. In many cases, it will be preferable to include isotinic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, baccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound. Altemative dosage amounts include from about 1 μg to about 1000 mg and from about 10 μg to about 500 mg.

The present invention also extends to forms suitable for topical application such as creams, lotions and gels as well as a range of "paints" which are applied to skin and through which the active ingredients are absorbed. In addition, the complex or components thereof may be associated with penetration or the TAT protein of HIN. Pharmaceutically acceptable barriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and except insofar as any conventional media or agent is incompatible with the active ingredient, their use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

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

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable barrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts range from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. Dosages may also be expressed per body weight of the recipient. For example, from about 10 ng to about 1000 mg/kg body weight, from about 100 ng to about 500 mg/kg body weight and for about 1 μg to above 250 mg/kg body weight may be administered.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating levels of polypeptides involved in the complex. The vector, may, for example, be a viral vector.

The present invention further provides an animal model useful for assessing potential agonists and antagonists of SOCS-6 function.

Accordingly, the present invention further provides genetically modified animals in which one or both alleles of SOCS-6 are mutated alone or in combination with another mutation in one or both alleles for another gene such as encoding another SOCS molecule. Preferably, the genetically modified animals are laboratory test animals such as murine species (e.g. mice, rats), rabbits, guinea pigs or hamsters, livestock animals such as sheep, pigs, horses or cows or non-human mammals such as primates. Conveniently, and preferably, the genetically modified animal is a murine species such as a mouse or rat.

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

Genetically modified animals and in particular knock-out murine animals may be prepared by any number of means. In one method, a targeting DNA construct is prepared comprising a nucleotide sequence which is sufficiently homologous to a target sequence such a SOCS-6 to permit homologous recombination. The SOCS-6 targeting sequence may be isogenic or non-isogenic to the target SOCS-6 sequence. The targeting DNA construct generally comprises a selectable marker within the targeting sequence such that by homologous recombination, the target SOCS-6 gene is disrupted by an insertional mutation. The targeting DNA construct is generally introduced into an embryonic stem cell or embryonic stem cell line.

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

Accordingly, another aspect of the present invention provides a method of producing a genetically modified non-human animal, said method comprising introducing into embryonic stem cells of an animal a genetic construct comprising a SOCS-6 nucleotide sequence carrying a single or multiple nucleotide substitution, addition and/or deletion or inversion or insertion wherein there is sufficient SOCS-6 nucleotide sequences to promote homologous recombination with a SOCS-6 gene within the genome of said embryonic stem cells selecting for said homologous recombination and selecting embryonic stem cells which carry a mutated SOCS-6 gene and then generating a genetically modified animal from said embryonic stem cell.

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

The SOCS-6 nucleotide sequence may be isogenic or non-isogenic to the SOCS-6 gene in the embryonic stem cell.

The term "isogenic" means that the SOCS-6 nucleotide sequence in the construct is derived from the same animal strain from which the embryonic stem cell has been derived or a strain exhibiting the same genotype.

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

A range of selectable markers may be employed and reference may be made to U.S. Patent No. 5,789,215 for general methodologies. Breeding protocols may also be adopted to introduce mutations or other genetic modifications into SOCS-6. In one approach, an EMS or other mutagenized mouse is cross with a non-mutagenized mouse to produce a Gl generation. The Gl generation may then be crossed with an index strain to produce GEFI kindreds which are then screened phenotypically for mutation in SOCS-6. Mutations in SOCS-6 may be dominant or recessive and mutations may be detected directly on SOCS-6 or by its effect on another gene or on its effect in alleviating the effects of a first mutation on another gene.

All genetically modified animals including knock-out mice carrying mutations in one or both SOCS-6 alleles alone or in combination with mutations in other genes.

The present invention further contemplates antibodies to SOCS-6.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, refs. 9,10,11).

Another aspect of the present invention contemplates a method for detecting SOCS-6 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for SOCS-6 or its derivatives or homologues for a time and under conditions sufficient for an antibody-SOCS-6 complex to form, and then detecting said complex.

The presence of SOCS-6 may be accomplished in a number of ways such as by Western blotting and ELIS A procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non- competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control ample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is one which might contain SOCS-6 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.

In a typical forward sandwich assay, a first antibody having specificity for the SOCS-6 or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about 37°C including 25°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

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

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

By "reporter molecule", as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.

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

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

As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody- hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The ability to identify SOCS-6 immunologically permits diagnostic assays for SOCS-6 alone or in a complex with other molecules such as molecules involved in growth factor or hormone signalling.

Diagnostic assays may also involve immobilizing SOCS-6 and screening for molecules which interact with SOCS-6. Alternatively, particular ligands may be immobilized to screen for SOCS-6. The ability to quantitate SOCS-6 or its ligands or complexes thereof provides significant diagnostic and therapeutic value.

SOCS-6 quantitation may also be conducted at the genetic level where mRNA transcript levels may be determined for the SOCS-6 gene. Such genetic assays may also be conducted at the DNA level especially to identify polymorphisms of SOCS-6.

In one embodiment, differential hybridization or differential priming is useful in distinguishing between polymorphisms of the SOCS-6 gene. Single nucleotide polymorphism (SNP) technology may also be employed.

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

EXAMPLE 1 SOCS-6

SOCS molecules are described in International Patent Application No. PCT/AU97/00729 [WO 98/20023]. The nucleotide and amino acid sequences of SOCS-6 are shown in SEQ ED NOS:l and 2, respectively.

EXAMPLE 2 Generating anti-SOCS-6 monoclonal antibodies

Mice were immunized with a GST-SOCS-6 N-terminal peptide, as defined in Figure 1A. This peptide is referred to as "SOCS-6 N(B)". Splenocytes from immunized mice were then fused with myeloma cells and hydridomas were selected. Hybridoma supernatant fluids were then screened by ELISA to detect monoclonal antibodies (mAbs) which recognized GST SOCS6 N(B) but not GST alone.

The mAbs identified were then characterized by isotype, Ippt, Western blot and epitope to which it binds (defined by amino acid residue sequence). The results are shown in Figure lb. Two antibodies, ICl and IC3, were directed to amino acids numbers 250-290 of SOCS6 and 3A7 was directed to amino acid residues 290-330.

EXAMPLE 3 Generation of knock-out mice

The SOCS-6 gene was disrupted in mouse embryonic stem (ES) cells by homologous recombination of a targeting vector carrying a selectable marker. The targeting vector is shown in Figure 2b, the genomic target is shown in Figure 2a and the resulting targeted allele is shown in Figure 2c. Four ES cell clones were identified carrying the targeted allele. SOCS-6 knock-out mice generated using these ES cells were shown to be genotypically SOCS-6^" (see Example 4 and Figure 3). EXAMPLE 4 Expression of SOCS-6 mRNA

The expression of SOCS-6 mRNA was determined in tissues from wild-type mice and from mice genotypically SOCS-6^7". The results are shown in Figure 3. In Figure 3a, eye, testes, bone shaft, large intestine, muscle, bladder, spleen, liver, lung, bone marrow, heart, kidney, brain, thymus and salivary gland tissue was tested using a coding region corresponding to SOCS-6. SOCS-6 mRNA was identified as a 2.33 kb band. SOCS-6^7" mice lack this band in heart, kidney, brain, thymus and salivary gland tissue.

EXAMPLE 5 Expression of SOCS-6 protein in tissue

Figure 4 shows the results of Western blot analysis using anti-SOCS-6 antibodies generated in Example 2 in wild-type and SOCS-6^7" mice.

In Figure 4a, lysates were prepared from various organs taken from either wild-type (+/+) or SOCS-&^"' (-/-) mice and immunoprecipitations were carried out using the ICl antiSOCS-6 mAb. Western blotting was carried out using the 3A7 anti-SOCS-6 mAb. Figure 4b shows that an equal amount of protein was used for each immunoprecipitation. 40 μg of lysate from each sample was subjected to Western blotting using an anti-heat shock-70 pAb.

These data support the genotype of the SOCS-6^7" and tissue distribution shown in Example 4.

EXAMPLE 6

Physiological effects ofSOCS-6^~A deletion

SOCS-6^{" "} mice are born in normal mendelian ratio and survived normally for at least six months. Male and female mice appeared fertile. The weight versus age of SOCS-6^{' "} mice was determined and compared to wild-type mice. The results presented in Figure 5 show that SOCS-6^{" "} mice are approximately 10% smaller than wild-type littermates.

EXAMPLE 7 Identification of proteins which associate with SOCS-6

293T cells were stimulated with 200 ng/mL IGF-1, lysed and precipitated using the SOCS- 6 SH2 domain (S6-SH2) or control resin. Precipitates were separated on an SDS-PAGE gel and stained with Coomassie blue. Bands of interest were excised and subjected to mass spectrometric analysis.

The results are shown in Figure 6 and Table 1.

TABLE 1

Number

Database | Molecular of

Sample Name Protein(s) identified Accession weight peptides

Number (kDa) identified

#1 Insulin receptor substrate 4 TrEMBL | 133.6 26 - homosapiens 014654

Histone deacetylase 6 SwissProt | 131.3 (HD6) - homosapiens Q9UBN7

Hypothetical protein SwissProt | 95.1 Kl AA0310 - homosapiens O15027

Three proteins were identified, i.e. insulin receptor substrate 4 (ERS-4) [TrEMBLO 14654], histone deacetylase 6 (HD6) [SwissProt Q9UBN7] and hypothetical protein KIAA0310 [SwissProt )15027]. EXAMPLE 8 Association of tyrosine-phosphorylated proteins with SOCS-6

293T cells were transiently transfected with a FLAG epitope tagged version of SOCS-6 on a FLAG-tagged version of WSB-1. Cells transfected with FLAG-SOCS-6 were stimulated with 200 ng/mL IGF-1 and all samples were taken after 0, 5, 10, 20, 30, 60, 120, 180 and 300 mins of stimulation, respectively. Cells transfected with FLAG- WSB-1 were stimulated with 200 ng/mL IGF-1 for 20 mins. FLAG-SOCS-6 and FLAG-WSB-1 were immunoprecipitated with anti-FLAG antibodies and Western blotting was then carried out using an anti-phosphotyrosine Ab or an anti-phosophoMAP kinase Ab. The anti- phosphotyrosine Ab blot was stripped and reprobed using an anti-FLAG Ab. The anti- phosphoMAP kinase blot was stripped and reprobed using an anti-MAP kinase Ab. The results shown in Figure 7 demonstrate tyrosine-phosphorylated proteins with SOCS-6 in response to IGF-1 stimulation.

EXAMPLE 9 SOCS-6-IRS-4 interaction

293T cells were left untransfected or were transiently transfected with either a FLAG epitope tagged version of SOCS-6 (S6) or a FLAG-tagged version of WSB-1. Cells were left unstimulated (-) or were stimulated with 200 ng/mL IGF-1 (+). FLAG-SOCS-6 and FLAG-WSB-1 were immunoprecipitated with anti-FLAG Abs. Alternatively, non- transfected lysates were precipitated with the SOCS-6-SH2 domain, the SOCS-7-SH2 domain or control resin. Western blotting was then carried out using an anti-ERS-4 Ab (Figure 8a). The blot was stripped and reprobed using an anti-FLAG Ab (Figure 8b).

The results in Figure 8 clearly shows that SOCS-6 associates with IRS-4 in response to IGF-1 stimulation. EXAMPLE 10 SOCS-6 association with P13 kinase (p85)

Anti-p85 antibodies were used to test SOCS-6 association with this molecule in response to IGF-1 stimulation. The results are shown in Figure 9. The results show that in response to IGF-1 stimulation, SOCS-6 is capable of interacting with PI 3 kinase (p85) which is part of the IGF-1 signalling pathway.

EXAMPLE 11 Physiological characteristics of SOCS-6 knock-out mice

Given that the previous Examples clearly show involvement of SOCS-6 in the IGF-1 pathway, insulin was determined between wild-type mice and SOCS-6^{" "} mice post glucose injection. The results are shown in Figure 10 and indicate that SOCS-6^7" mice have impaired insulin generating capacity.

A glucose tolerance test was then performed and the results are shown in Figure 11. Again, the results support a reduction in the tolerance of SOCS-6^{" "} mice to glucose.

The results presented herein indicate that SOCS-6 is involved in modulation of the IGF-1 pathway and in insulin-dependent signal transduction.

EXAMPLE 13 Recombinant expression ofSH2 domains and immobilization onto Sepharose resin

Fragments of the murine SOCS-6 and SOCS-7 proteins encompassing the SH2 domains were recombinantly expressed in E. coli. These were expressed as hexahistidine-tagged proteins using a modified form of the pET15b vector (Novagen). The amino acid sequences of the recombinant protein constructs were:- SOCS-6:

MCSHHHHHHGARQ [SEQ ED NO:9]

SAPGVARVYDSVQSSGPMVVTSLTEELKKLAKQGWYWGPITRWEAEGKLANV PDGSFLVRDSSDDRYLLSLSFRSHGKTLHTRIEHSNGRFSFYEQPDVEGHTSIVDL IHSERDSENGAFCYSRSRLPGSATYPVRLTNPVSRFMQ [SEQ ID NO:7]

SOCS-7:

MCSHHHHHHGARQ [SEQ ED NO: 10]

LYRPDSSSFAASLRELEKCGWYWGPMN EDAEMKLKGKPDGSFLVRDSSD PRYILSLSFRSQGITHHTRMEHYRGTFSLWCHPKFEDRCQSVVEFEKRAIMHSK NGKFLYFLRSRVPGLPPTPVQLLYPVSRFSN [SEQ ID NO: 8]

The recombinant proteins were purified from bacterial cells lysed in 7 M guanidinium hydrochloride using immobilised nickel ion affinity chromatography. Purified proteins were refolded by dialysis into phosphate-buffered saline containing 0.02% w/v Tween-20, 0.5 mM tris(2-carboxyethyl)-phosphine. The refolded proteins were covalently immobilized onto NHS-activated Sepharose at a density of 3 mg of protein per mL of activated resin. "Control" resin was prepared by derivatizing NHS-activated Sepharose resin with ethanolamine.

EXAMPLE 14

Design and synthesis of phosphopeptide libraries

Partially degenerate phosphopeptide libraries were designed to probe the sequence specificity of the phosphopeptide-binding sites from the SH2 domains from SOCS-6 and SOCS-7. Two such libraries were synthesized, VE(pY)XιX₂X₃VHSGR and RN(pY)VF

X₃X₄X₅LK, where pY represents phosphotyrosine and each X position was an equal mixture all possible L-amino acids with the exception of cysteine (Figure 12). The total degeneracy of each library was 19³ = 6859 peptides. This library was synthesized on a Rink amide MBHA resin using Fmoc amino acids and HATU activation. All non- degenerate positions were double-coupled and following amino acid coupling, the resin was treated with acetic anhydride to acetylate any remaining free amine groups. Phosphotyrosine was coupled as the Fmoc-O-benzyl-L-phophotyrosine derivative. For each degenerate position, the terminal Fmoc group was removed, the resin dried, and split into 19 equal lots. To each resin aliquot an appropriate activated Fmoc amino acid was coupled for 2 hours, after which time the resin samples were recombined, washed with dimethylformamide and treated with acetic anhydride. This procedure was repeated for each degenerate position. Cleavage and deprotection of the peptide library was effected by treatment with 95% v/v trifluoroacetic acid (TFA) containing 5% v/v triisopropylsilane. TFA was removed by evaporation with a stream of nitrogen, the peptide library mixture was precipitated with di ethyl ether, solubilized in 50% v/v aqueous acetonitrile containing 0.1% v/v TFA, filtered and lyophilized. The sequences of the synthetic libraries were confirmed by N-terminal sequencing and each degenerate position was found to contain an approximately equal proportion of each of the 19 different amino acids.

EXAMPLE 15 Phosphopeptide library screening

VEpYXXXVHSGR library screening: 400 μL of immobilized protein resin was incubated with 4 mg of phosphopeptide library solubilized in 2 mL 50 mM sodium phosphate (pH 7.5) containing 150 mM NaCl, 2 mM DTT and 0.2% w/v Tween-20. The mixture was gently shaken at room temperature for 1 hour, then transferred to a disposable 10 mL chromatography column. After allowing the peptide solution to drain, the resin was washed with 5 x 8 mL phosphate-buffered saline containing 0.2% w/v Tween-20 and 1 x 8 mL 2 mM sodium phosphate (pH 7.5), using nitrogen pressure to reduce the total washing time to ~7 minutes. Bound phosphopeptides were eluted from the resin with 3 x 400 μL of 1% v/v aqueous TFA, and the combined eluate was lyophilized. A control experiments was performed in the same way using "control" resin in place of the SH2 protein resin. RNpYVFXXXLK library screening: 180 μL of immobilized protein resin was incubated with 3.6 mg of phosphopeptide library solubilized in 1.8 mL 50 mM sodium phosphate (pH 7.5) containing 150 mM NaCl, 2 mM DTT and 0.2% w/v Tween-20. The mixture was gently shaken at room temperature for 1 hour, then transferred to a disposable 10 mL chromatography column. After allowing the peptide solution to drain, the resin was washed under gravity with 18 x 8 mL phosphate-buffered saline containing 0.2% w/v Tween-20 and 1 x 8 mL 2 mM sodium phosphate (pH 7.5). The total washing time was -60 minutes. Bound phosphopeptides were eluted from the resin with 2 x 180 μL of 1% v/v aqueous TFA, and the combined eluate was lyophilized. A control experiment was performed in the same way using "control" resin in place of the SH2 protein resin.

Dried samples were resuspended in 60 μL of 30% v/v aqueous acetonitrile containing 0.1% v/v TFA and sequenced on an Applied Biosystems 494 Procise Protein Sequencer. To correct for the differential recoveries during sequencing, variable abundance of amino acids in the degenerate mixture, and potential differences in nonspecific adsorption to the control resin, the ratio of each amino acid at a given cycle of the phosphopeptide mixture retained on the SH2 resin to the same amino acid in the same cycle of the peptide mixture from the control experiment is calculated and shown in Figure 13.

Synthetic phosphopeptide libraries were used to explore optimal sequences for binding to the SH2 domains from SOCS-6 and SOCS-7. These data (Figure 13) reveals that for SOCS-6, the most important determinant is a valine residue in the pY+1 position (the first amino acid downstream from the essential phosphotyrosine residue). Of lesser significance are preferences for residues with a hydrophobic character at pY+2 and pY+3. There is only weak selection of specific amino acid types at the pY+4 and pY+5 positions and the predominance of tryptophan at these positions suggests that it may have been selected nonspecifically. Thus the pY+4 and +5 positions are probably not very important for interaction with SOCS-6. For SOCS-7, the most significant position is also pY+1 where valine is again the preferred amino acid, with lesser specificity for other hydrophobic residues. The next most important position was at pY+3 where tryptophan and phenylalanine were the preferred amino acids types, with lesser specificity for other hydrophobic residues. The ρY+2, +4 and +5 positions showed weak selection of certain amino acids suggesting that these positions are probably not that critical to phosphopeptide binding. The specificity of the SH2 domains from SOCS-6 and SOCS-7 is, therefore, similar.

This information can be used to predict possible SOCS-6 or SOCS-7 docking sites in naturally occurring tyrosine phosphorylated proteins. For example, possible -pTyr-Val- docking sites within the human insulin receptor substrate proteins occur at Y896 in ERS-1, Y823 in ERS-2 and Y700, Y828, Y921, Y959 and Y1046 in ERS-4.

The specificity information can also be used to design artificial phosphopeptide ligands which bind to the SH2 domains of SOCS-6 and SOCS-7. Such a peptide can then be used to set up an assay for screening the abililty of other peptide or non-peptide molecules to compete for binding to SOCS-6 or SOCS-7. Molecules identified in this way may act as antagonists of the biological activity of SOCS-6 and SOCS-7.

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.

Claims

CLAIMS:

1. A method for modulating IGF-mediated and or insulin-mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and insulin receptor or an insulin receptor substrate or a component in an IGF-mediated signalling pathway.

2. The method of claim 1 wherein the IGF is IGF-1.

3. The method of claim 1 or 2 wherein the SOCS molecule is SOCS-6.

4. The method of claim 1 or 2 wherein the SOCS molecule is SOCS-7.

5. A method for modulating IGF-1- and SOCS-6-mediated and/or insulin- mediated signalling in an animal cell or in an animal comprising said cell, said method comprising administering to said cell or said animal comprising said cell an amount of an effector molecule capable of modulating the functional interaction between a SOCS molecule and insulin receptor or an insulin receptor substrate or a component in an IGF-1- mediated signalling pathway.

6. The method of claim 5 wherein the component of the IGF-1- or insulin- mediated signalling pathway is ERS-2 or P85.

7. The method of claim 1 or 2 or 3 or 4 or 5 or 6 wherein the animal is a mammal.

8. The method of claim 7 wherein the mammal is a human.

9. An effector molecule capable of modulating interaction between a SOCS molecule and an intracellular ligand in an animal cell wherein the effector molecule modulates IGF- and/or insulin-mediated signalling.

10. The effector molecule of claim 9 wherein the effector molecule is an antagonist.

11. The effector molecule of claim 10 wherein the antagonist interacts with the SH2 domain of SOCS-6.

12. The effector molecule of claim 11 wherein the SOCS molecule is SOCS-6.

13. The effector molecule of claim 11 wherein the SOCS molecule is SOCS-7.

14. A SOCS-6 antagonist wherein said antagonist inhibits SOCS-6-mediated suppresion of IGF- and/or insulin-mediated signalling pathways.

15. A SOCS-7 antagonist wherein said antagonist inhibits SOCS-7-mediated suppresion of IGF- and/or insulin-mediated signalling pathways.

16. The effector molecule of claim 12 or the antagonist of claim 14 comprising a phosphopeptide sequence is preferably selected from:

VE(pY)X_!X₂X₃VHSGR [SEQ ED NO:3]; or RN(pY)VFX₃X₄X₅LK [SEQ ED NO:4]

wherein

pY is phosphotyrosine; Xi is Y, M, V, W, F, or I, preferably V; X₂ is Y, M, V, W, F, I or L, preferably Y; X₃ is M, V, W, F, I or L, preferably M, V, F or I;

X is any amino acid, suitably V or W; and X₅ is any amino acid, suitably W.

17. The effector molecule of claim 13 or the antagonist of claim 15 comprising phosphopeptide sequence selected from:

NE(pY)XιX₂X₃VHSGR [SEQ ID ΝO:5]; or RN(pY)NFX₃X₄X₅LK [SEQ ED ΝO:6]

wherein

pY is phosphotyrosine;

Xi is M, V, W, I or L, preferably V;

X₂ is any amino acid, suitably Q, T, E, Y, M, V, W, F, I or L;

X₃ is Y, M, W, F, I or L, preferably W or F;

X₄ is any amino acid, suitably D, T, Y, V, W, or F; and X is any amino acid, suitably D, H, Y, P, W, or F.

18. A method of screening for an effector molecule which is capable of modulating the functional interaction between a SOCS molecule and an intracellular ligand in an animal cell, said method comprising contacting in the presence of said intracellular ligand or analogue or derivative thereof a polypeptide comprising an SH2 domain of said SOCS molecule, or derivative of said SH2 domain, with a test agent; and detecting a reduced level of interaction between said ligand or analogue or derivative thereof and said SH2 domain or derivative relative to a reference level of said interaction in the absence of said test agent, wherein said reduced level is indicative of said agent being an effector molecule.

19. The method of claim 18 wherein the SOCS molecule is SOCS-6.

20. The method of claim 18 wherein the SOCS molecule is SOCS-7.

21. The method of claim 18 or 19 or 20 wherein the SOCS molecule is selected from SEQ ED No. 7, SEQ ED No. 8, SEQ ED No. 9 and SEQ ED No. 10.

22. A method of ameliorating the effects of insulin resistance in an animal cell, said method comprising introducing to said animal cell or animal comprising said cell an antagonist of SOCS-6 for a time and under conditions sufficient to inhibit, reduce or otherwise suppress SOCS-6 inhibition of IGF-1- and/or insulin-mediated signalling pathways or a component in an IGF-1 mediated signalling pathway.

23. The method of claim 22 wherein the animal is a mammal.

24. The method of claim 23 wherein the mammal is a human.

25. A genetically modified animal in which one or both alleles of SOCS-6 are mutated alone or in combination with another mutation in one or both alleles for another gene such as encoding another SOCS molecule.

26. A method of producing a genetically modified non-human animal, said method comprising introducing into embryonic stem cells of an animal a genetic construct comprising a SOCS-6 nucleotide sequence carrying a single or multiple nucleotide substitution, addition and/or deletion or inversion or insertion wherein there is sufficient SOCS-6 nucleotide sequences to promote homologous recombination with a SOCS-6 gene within the genome of said embryonic stem cells selecting for said homologous recombination and selecting embryonic stem cells which carry a mutated SOCS-6 gene and then generating a genetically modified animal from said embryonic stem cell.

27. Use of an antagonist of a SOCS molecule in the manufacture of a medicament for the treatment of a dysfunction.

28. Use of claim 27 wherein the SOCS molecule is SOCS-6.

29. Use of claim 27 wherein the SOCS molecule is SOCS-7.

30. Use of claim 27 wherein the dysfunction is autoimmunity, immune suppression or hyperactive immunity.

31. Use of claim 27 wherein the dysfunction is a dysfunction of the haemopoietic, endocrine, hepatic or neural systems.

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