WO2003099322A2 - Il-11 derivatives and therapeutic uses thereof - Google Patents

Abstract

The present invention is concerned with IL-11 signalling. In particular, the invention is concerned with IL-11 based assays; additionally novel variants of IL-11 and their uses in fertility treatment are provided. The invention provides a specific modulator of IL-11 signalling which is an IL-11 mutant at position 147. This mutant may be used as a contraceptive agent. The invention also provides for a method of contraception comprising administering an effective amount of W147A IL-11 mutant to a female mammalian subject.

Description

IL -11 Derivatives and Therapeutic Uses Thereof

The present invention is concerned with IL-11 signalling. In particular, the invention is concerned with IL-11 based assays; additionally novel variants of IL-11 and their uses in fertility treatment are provided.

The complex cellular and molecular changes that take place during the menstrual cycle in response to steroid hormones are mediated by a number of cytokines. Recent evidence from targeted gene knockout experiments in mice revealed that interleukin 11 receptorα (IL-llRα) is a key regulator of the decidualization of the murine endometrial stroma (11; 12) . Female mice lacking the IL-llRα gene were infertile due to defective differentiation of the stroma in response to an implanting blastocyst leading to resorption of the embryo. The expression of IL-11 in the mouse endometrium is maximal during decidualization, suggesting that IL-11-receptor interactions in the decidua are important in this process .

Further recent reports have demonstrated that IL- 11 RNA and protein are expressed in the human endometrium throughout the menstrual cycle, with highest levels reported to be in stromal cells in the late secretory phase when the cells undergo spontaneous decidual transformation (Dimitriadis et al, Mol. Hum. Reprod. , 6, 907-914, 2000; Cork et al, J. Reprod. Immunol. 50, 3-17, 2001).

It seems therefore that IL-11 receptor is a key signalling pathway in mammalian female fertility.

Based on the above observations, IL-11R is a potential target for compounds capable of modulating fertility. It is therefore desirable to identify IL- 11R modulators which would be potent and selective for IL-11R. However, the mechanism of action of activation of IL-11R has not yet been fully understood.

Interleukin-11 (IL-11) is a secreted polypeptide cytokine (1) , originally identified on the basis of its ability to stimulate the proliferation of the murine plasmacytoma cell line T1165 (1). The nucleotide and amino acid sequences of hIL-11 (1; 5; McKinley et al, Genomics 13(3), 814-819, 1992) and mIL-11 (Morris et al, J.Exp. He atol . 24(12), 1369- 1376, 1996) have been described.

Cytokine antagonists have proved to be powerful tools in the dissection of ligand-dependant signalling processes in vitro and in vivo . For example, IL-6 antagonists have been generated which bind to IL-6R but fail to form a hexameric complex with gpl30 (40) and CNTF antagonists with reduced binding to the LIFR (36) . These are both thought to act as antagonists by sequestering the ligand specific receptor component only.

Studies have revealed a number of in vi tro biological effects that IL-11 exerts on a diverse range of cell types. These include induction of acute phase response proteins in hepatocytes (2), induction of differentiation in immortalized hippocampal neurons (3), suppression of lipoprotein lipase activity in adipocytes (4) and inhibition of adipogenesis in cultured 3T3 Ll fibroblasts (5) . In association with other cytokines, IL-11 has a wide range of effects on both the proliferation and differentiation of a variety of hemopoietic cells (6) . For example, administration of IL-11 in vivo results in the stimulation of megakaryopoiesis and increased platelet counts (7), while recombinant human IL-11 (hIL-11) is now used for the treatment of chemotherapy-induced thrombocytopenia (8) . Further potential uses of IL-11 include chemotherapy induced oral mucositis (9), Crohn' s disease and rheumatoid arthritis (10).

Recent studies have also shown that IL-11 can induce multipotential hemopoietic stem cell amplification in vi tro in a concentration dependent fashion (50) .

IL-11 is a member of the gpl30 family of cytokines (13) , characterised by the use of a common transmembrane signal transducing receptor gpl30 (14- 18) . Other members of this family include interleukin-6 (IL-6), oncostatin M (OSM) , leukaemia inhibitory factor (LIF) , cardiotrophin-1 (CT-1) , ciliary neurotrophic factor (CNTF) , and a viral homologue of IL-6 encoded by the Kaposi's sarcoma associated herpesvirus (KSHV-IL-6) . The gpl30 family of cytokines exhibit overlapping and unique biological activities (13), therefore, the signal exerted by a cytokine and its biological response is dependent upon the exact composition of the signalling receptor complex. This signalling specificity can be conferred by the use of ligand specific receptors. These receptors are not directly involved in signalling, but aim to promote the formation of a high affinity complex between the respective ligand and gpl30 (13) . For example, IL-11 has been shown to initiate intracellular signal transduction in a manner similar to that previously shown for IL-6 (19) . The ligand specific IL-11 receptor (IL-11R) (20) promotes the formation of a high affinity complex between IL-11 and gpl30 (21) . Upon stimulation gpl30 is phosphorylated on tyrosine residues, leading to the stimulation of intracellular signal transduction pathways involving JAK1, JAK2 and TYK2 in the Janus kinase (JAK) family, mitogen-activated protein kinase and Src families (22- 25) .

Although biochemical analysis has suggested that IL-6 induces the formation of a hexameric complex, composed of two molecules each of IL-6, its ligand specific receptor (IL-6R) and gpl30 (26), published data regarding the composition of the ternary IL-11 receptor complex is not yet conclusive. Barton et al have shown that multiple copies of gpl30, IL-11 and IL-llR are present in the ternary IL-11 receptor complex (26) . Homodimerization of gpl30 was demonstrated to require both IL-11 and IL-llR (26) . Therefore, the high affinity IL-11 receptor complex must contain at least two copies each of IL-11, IL-llR and gpl30. This would indicate the formation of a hexameric complex similar to that observed for IL-6. By comparison, immunopreciptation experiments performed by Neddermann et al (27) describe the ternary IL-11 receptor complex as being pentameric consisting of two IL-11 and IL-llR molecules but only one gpl30, thus suggesting that gpl30 homodimerizatlon is not involved in IL-11 mediated signalling but another unidentified signalling receptor component is required (27) . Grotzinger et al have suggested that the IL-ll/IL-llR/gpl30 receptor complex may be a tetramer. In this instance, the ternary complex consists of one IL-11, one IL-llR and two gpl30 molecules (28) .

The gpl30 cytokines share a common four α-helix bundle fold in an up-up-down-down topology (29, 30), which is common to all members of the hematopoietin family of cytokines. Structural analysis and mutagenesis studies have revealed apparent receptor binding epitopes conserved among the gpl30 family of cytokines (31). IL-11 (32), as well as IL-6 (33, 34) and CNTF (35, 36) have been shown to have three receptor binding sites termed I, II and III. Recent studies have focussed upon the three distinct receptor-binding sites of murine IL-11 (mIL-11) (32). In this instance site I enables IL-11 to bind to IL- llR, while sites II and III mediate binding to gpl30. These are analogous in location to sites I, II and III of IL-6 (32) . Site I is formed by residues in the COOH-terminal end of helix D and the AB loop (37-39) . Site II is formed by exposed residues on helices A and C (40), while site III is composed of residues in the CD loop-NH₂-terminal end of the D helix (34) . It is therefore proposed that the hexameric ternary receptor signalling complex is formed by the dimerisation of two trimeric complexes containing one molecule of each component. Dimerisation of these trimers involves the simultaneous interaction of gpl30 with two molecules of IL-11. In this way, the cytoplasmic domains of gpl30 are brought into opposition leading to the activation of intracellular signalling processes. Recent studies by Barton et al have demonstrated residues critical for the binding of mIL-11 to the ϊL- 11R and gpl30 (32). A tryptophan residue (Trp 147) was identified as a crucial site III residue. A variant with an alanine substitution of 147 showed normal binding to IL-llR with reduced binding to gpl30 via site III and therefore is unable to form a complex capable of initiating signal transduction (32) .

However, although these residues seem critical in the IL-11 signalling pathway, there is no suggestion that compounds exhibiting these specific mutations can lead to selective and potent modulation. The prior art ((32) in particular) merely describes mutants which define these different regions of IL-11 involved in molecule recognition. It does not predict which would produce an effective, high affinity, IL-11 selective antagonist.

Moreover, identification of selective, potent modulators is further complicated by conflicting theories of IL-11 signals. The tetrameric model taught by the prior art indicates that the identified mutations would not be able to lead to high affinity modulators, since the mechanism of action of any modulator would have to be primarily to dilute out wild type mutant rather than directly disrupt signalling complexes containing wild type IL-11.

Consequently, knowledge of the complex IL-11 signalling pathway has taught away from providing selective potent modulators.

The inventors have now further characterised the IL-11 signalling pathway, thus allowing the identification of specific modulators.

In particular, they have demonstrated that the modulation of IL-11 signalling is mediated via a - ternary complex including IL-llR and gpl30. This is further described in the experimental part below.

The inventors have now shown that the IL-11 mutant 147A, which is unable to interact with gpl30 via site III, is a high affinity selective antagonist of IL-11 function. This activity arises from the ability of W147A to disrupt the formation of the high affinity hexameric signalling complex in the presence of T IL-11 and IL11-R. The selectivity of W147A arises from retention of the unique high affinity interaction between IL-11 and IL-llR. Also, they have now shown that the competitive addition of W147A in solution leads to the disruption of this hexameric receptor complex and induces the formation of a trimeric receptor complex containing just one molecule of the IL-11/IL-llR subunit and one molecule of gpl30 (figure 1) . This activity is paralleled by the ability of W147A to competitively inhibit IL-11 signalling in transfected cells as measured by cell multiplication and activation of STAT3 phosphorylation. These effects on IL-11 signalling are most easily rationalised by supposing that the antagonist action of W147A requires the concurrent presence of T IL-11 in a signalling complex. This would suggest that, in live cells, the functional signalling unit is a hexamer. It has been argued on the basis of molecular modelling by Grotzinger et al (28) that the hexamer represents an inert form of the receptor complex. They propose instead that the active complex is a tetramer with two gpl30 molecules and one molecule of IL-11/IL-11R (28) . In this model the inactive hexameric complex is generated by addition of another subnit of IL-ll/L- 11R. The inventors' findings suggest the opposite conclusion namely that two molecules of IL-11/IL-11R are required to elicit signal transduction via dimerisation of gpl30.

Also, the inventors have identified a function for IL-11 in decidual transformation of the human endometrium.

Based on these observations, the inventors have developed assays for screening compounds capable of modulating the activity of IL-11, its receptors and interactions therewith. The elucidation of the mechanisms involved in endometrial stroma decidualization has enabled the inventors to develop assays specifically designed to identify potential contraceptive or fertility agents, exhibiting specific activity in respect of the above target mechanisms. The screened compounds are potential fertility and/or contraceptive agents.

Accordingly, in a first aspect, the invention provides a method for identifying fertility and/or contraceptive agents, which method comprises: culturing cells expressing IL-11 receptor and gpl30 or biologically active portions thereof with IL- 11 or a biologically active portion thereof, in the absence or presence of the test compound; determining the effect of the test compound on IL-llR activation thereby identifying a compound which modulates IL-llR activation.

Preferably, the assay is carried out on physiologically-relevant cells expressing the desired IL-11 and gpl30 receptors. The cell, for example, can be a yeast cell or a cell of mammalian origin optionally transfected, such as Ba/F₃ cells transfected with BC neo/mgpl30 (43) and pcDNA3/mIL-HR (32) . More preferably, the cell is an isolated human female endometrial cell, such as a stromal cell.

According to a first embodiment the method of the invention can advantageously be based upon a phosphorylation assay or upon a proliferation assay or upon a decidualization assay.

Protocols for performing phosphorylation or proliferation assays are well known in the art, such as described in Sambrook et al. (Molecular Cloning: a Laboratory Manual, 1989) ; representative examples are given in the experimental part included herein. According to a first embodiment, the effect of the test compound can be assessed by determining its effect on IL-11 induced tyrosine phosphorylation. As shown by the inventors, IL-11 induces tyrosine phosphorylation of STAT3 in cells expressing IL-llR and gpl30.

Compounds which modulate IL-11 function can be identified by detecting the effect of said compounds on IL-11 induced phosphorylation.

Generally, the phosphorylation assay involves immunoprecipitation of cell lysates (previously exposed to IL-11 and the test compound) with an antibody followed by Western blotting of the immuno precipitate with specific antibodies.

More specifically, the tissue culture plates (6 well-plate) are coated with isolated mammalian female endo etrial cells or with transfected cells expressing the desired receptors, IL-llR and gpl30, or biologically active portions thereof. Samples containing IL-11 (or fragment of the protein which is still capable of binding to IL-llR and gpl30) with and without the test compound are then added to the wel^'-ls and the plates are incubated to allow time for binding of the test compound to the receptors. The wells are washed; the cells are lysed, antibodies specific for the receptors (such as anti-gpl30 antibody) are added and complexes are collected. Receptors which have become phosphorylated can be detected via the antibodies specific for the phosphorylated complex and the type of receptor can be identified via an antibody specific for said receptor; antibodies suitable for immuno detection include: anti-phospho STAT3 and anti- STAT3 (cell^' signalling technology) , anti- phosphotyrosine 4G10 (Upstate Biotechnology Inc.) and P20 (ICN Biomedicals Inc.) and anti-gpl30 (Upstate Biotechnology Inc.). This may easily be done by amino blotting or Western blotting. Western blotting is a well known technique for the analysis and identification of proteins. Generally, the complexes are separated by polyacrylamide gel electrophoresis and then transferred to a cellulose membrane or chemically treated paper to which the proteins bind; preferably, the complexes are transferred to a nitrocellulose membrane or a PVDF (Polyvinylidene

Difluoride) membrane. Phosphotyrosines bound to the membrane are detected by overlaying the appropriate antibody, and bound specific antibodies are detected by overlaying with HRP-conjugated secondary antibody followed by standard chem-immunescent detection procedures .

Antagonists will result in decreasing or inhibiting phosphorylation; agonists will result in increasing phosphorylation, as assessed by the intensity of the bands.

According to a second embodiment, the effect of the test compound can be assessed by determining the effect on IL-11 induced proliferation. As shown by- the inventors, IL-11 induces cell proliferation.

The method of the invention can be based upon a proliferation assay. The effects of agonists/ antagonists of IL-11 can be identified by measuring the effects on cellular growth (decrease or increase) , after treatment with the compound. Cellular proliferation assays either directly measure effects on cell growth by counting the cells under a microscope or using an electronic particle counter or indirectly, by measuring incorporation of radioactive cDNA precursors, by using chromogenenic dyes to quantitate total protein or by measuring the metabolic activity of cellular enzymes.

The uptake of ³[H] thymidine is a common method to indirectly determine cell number after treatment. This method requires a pulse of ³ [H] thymidine followed by washing and counting in a scintillation counter. In these experiments, the wells of microtiter plates are coated with cells expressing the desired receptors. A sample containing IL-11 (or fragment of the protein which is still capable of binding to IL-llR and gpl30)in the absence or presence of the test compound is then added to the wells and the plates are incubated to allow time for binding. ³[H] thymidine is added to each well and then the cells are washed and harvested. The amount of incorporation of ³[H] thymidine is determined by scintillation counting. Antagonists will result in decreased or inhibited thymidine uptake; agonists will result in increasing thymidine uptake.

According to a third embodiment, the effect of the test compound can be assessed by determining the effect on decidualization of isolated mammalian endometrial stromal cells. The method of the invention can be based upon a biological marker of decidualization; preferably, the marker is prolactin.

The determination of the effect of the test compound on decidualization may advantageously be based upon an immunosassay, well know in the art; protocols can be found for instance in Sambrook et al (Molecular Cloning, a Laboratory Manual, 1989) . An agonist will result in increasing decidualization, while antagonists will result in decreasing or inhibiting decidualization. Also, suitable ready-to-use kits are commercially available such as the solid-phase, two-site Immulite^® prolactin kit from DPC.

In short, the effect of the test compound can be measured according to a chemiluminescent immunometric assay wherein the marker levels are determined by detecting the chemiluminescent reaction products of the marker with the appropriate antibody.

The above-described methods can be used to screen for agonists or antagonists of IL-llR, and hence for compounds capable of enhancing or inhibiting fertility. The screened compounds can respectively be useful as fertility agents or as contraceptive agents.

It will be appreciated that a wide variety of candidate compounds may be tested in the screening methods of the invention. Suitable test compounds may include, for example, compounds having a known pharmacological or biochemical activity, compounds having no such identified activity and completely new molecules or libraries of molecules such as might be generated by combinatorial chemistry. Compounds which are nucleic acids, including naturally occurring nucleic acids and synthetic analogues, polypeptides or proteins are not excluded.

Typically, compound screening assays involve running a plurality of assay mixtures in parallel with different concentrations of the compound under test. Typically, one of these concentrations serves as a negative control, i.e. zero concentration of test compound.

Also provided by the present invention are kits for drug screening comprising cells expressing IL-llR and gpl30 and IL-11 or nucleic acid molecules encoding the proteins or vectors comprising the nucleic acid or cells comprising the vectors and other reagents.

Compounds which are identifiable as having potential pharmacological activity using the methods of the invention may be used as lead compounds in the further development of drugs with pharmaceutical potential or may themselves be formulated into pharmaceutical compositions.

According to a further aspect, the invention refers to the compounds identifiable by the above described methods.

According to a still further aspect, the invention provides a pharmaceutical composition comprising one or more of the above compounds as an active ingredient.

According to a still further aspect, the invention provides the use of the above compounds in the preparation of medicaments for modulating fertility in mammals.

In a preferred embodiment, said medicaments are suitable for contraceptive purposes.

The inventors have identified IL-11 mutants that have affinity for IL-llR. Said mutants may have reduced affinity for gpl30.

The inventors have shown that a specific IL-11 mutant, the W147A mutant, is capable of disrupting IL- 11 signalling through the IL-ll/IL-llR/gpl30 receptor complex. The W147A mutant can not be incorporated into the hexameric signalling complex, rather a trimeric complex appears to be formed when mutant as opposed to wild type IL-11 is used. The mutation at position 147 is thought to inhibit binding of gpl30 to site III of the IL-11 protein.

Furthermore, the inventors have shown that, unlike wild type IL-11, W147A can not induce cell proliferation of certain cells. This implies that a hexameric IL-ll/IL-llR/gpl30 signalling complex is required in order to mediate the signalling events leading to IL-11 induced cell proliferation. In this regard, IL-11 has been shown by the inventors to induce tyrosine phosphorylation of STAT3, a protein which is part of the Jak/STAT signalling pathway. STAT3 regulates gene expression in the nucleus following phosphorylation. The W147A mutant antagonises wild type IL-11 signalling through STAT3, as shown by the decreased levels of STAT3 phosphorylation as W147A levels are increased.

The inventors have also discovered that the W147A mutant can inhibit IL-11 and cAMP induced decidualization of human stromal cells.

Due to these newly discovered antagonistic properties of the W147A mutant the invention provides for use of the W147A mutant as a contraceptive agent. By inhibiting specific IL-11 mediated signalling events the W147A mutant could modulate fertility in a subject. The invention therefore provides for a method contraception comprising administering an effective amount of W147A IL-11 mutant to a female mammalian subject. The subject will preferably be mammalian and most preferably human. The W147A mutant may be the active ingredient in a suitable pharmaceutical composition together with a pharmacutically acceptable carrier or diluent. Such carriers and diluents are well known in the art.

According to a still further aspect, the invention provides IL-11 mutants comprising the following substitutions:

- site II IL-11 mutant at position 111 and 115 such as R111A/L1115A;

- site III IL-11 mutant at position 147 such as W147A; or proteins which differ therefrom only in conservative amino acid changes.

Mutations of residue 147 are preferred.

The above positions are given in respect of the urine form of IL-11. Obviously, the present invention concerns mutants of both murine and human IL-11. It is to be understood that substitutions at equivalent position (s) in the human form of IL-11 are also contemplated. According to a preferred aspect, human IL-11 mutants are contemplated. In particular, the human equivalent of IL-11 W147A is preferred.

The wild type upon which the mutants are preferably based has been deposited (accession number: NM 008350) .

For purposes herein, conservative amino acid substitutions may be made, provided that the resulting protein is IL-llR antagonist. Amino acid substitutions are preferably made in accordance with those set forth in Table 1 as follows:

TABLE 1

Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions.

According to a preferred aspect, mutations which are not non-polar in nature are contemplated by the present invention.

In particular, the following substitutions of Trpl47 are contemplated: Alanine, Glycine, Valine, Serine, Asparagine, Aspartic Acid, Glutamic Acid.

Also provided by the invention are nucleic acid sequences which encode the proteins of the invention.

The nucleic acid molecules according to the invention may, advantageously, be included in a suitable expression vector to express the proteins encoded therefrom in a suitable host. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of the transformed cells is well known to those skilled in the art as provided in Sambrook et al . (1989), molecular cloning, a laboratory manual, Cold Spring Harbour Laboratory Press.

An expression vector according to the invention includes a vector having a nucleic acid according to the invention operably linked to regulatory sequences, such as promoter regions, that are capable of effecting expression of said DNA fragments. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. Such vectors may be transformed into a suitable host cell to provide for expression of a protein according to the invention. Thus, in a further aspect, the invention provides a process for preparing proteins according to the invention which comprises cultivating a host cell, transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and recovering the expressed protein.

The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, and optionally a promoter for the expression of said nucleotide sequence and optionally a regulator of the promoter. The vectors may contain one or more selectable markers, such as, for example, antibiotic resistance. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and to direct an appropriate level of transcription initiation and also translation initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and for translation initiation the Shine- Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or be assembled from the sequences described by methods well known in the art.

Nucleic acid molecules according to the invention may be inserted into the vectors described in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense nucleic acids, including antisense peptide nucleic acid (PNA) , may be produced by synthetic means .

In accordance with the present invention, a defined nucleic acid includes not only the identical nucleic acid but also any minor base variations including in particular, substitutions in cases which result in a synonymous codon (a different codon specifying the same amino acid residue) due to the degenerate code in conservative amino acid substitutions. The term "nucleic acid sequence" also includes the complementary sequence to any single stranded sequence given regarding base variations.

The nucleic acid sequences according to the invention may be produced using recombinant or synthetic techniques, such as for example using PCR which generally involves making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which brings about amplification of the desired region, isolating the amplified region or fragment and recovering the amplified DNA. Generally, such techniques are well known in the art, such as described in Sambrook et al . (Molecular Cloning: a Laboratory Manual, 1989) .

The nucleic acids according to the invention may carry a revealing label. Suitable labels include radioisotopes such as ³²P or ³⁵S, enzyme labels or other protein labels such as biotin or fluorescent markers. Such labels may be added to the nucleic acids or oligonucleotides of the invention and may be detected using known techniques per se .

The protein according to the invention includes all possible amino acid variants encoded by the nucleic acid molecule according to the invention including a protein encoded by said molecule and having conservative amino acid changes. Proteins or polypeptides according to the invention further include variants of such sequences, including naturally occurring allelic variants which are substantially homologous to said proteins or polypeptides. In this context, substantial homology is regarded as a sequence which has at least 70%, preferably 80 or 90% and preferably 95% amino acid homology with the proteins or polypeptides encoded by the nucleic acid molecules according to the invention. The protein according to the invention may be recombinant, synthetic or naturally occurring, but is preferably recombinant.

A further aspect of the invention provides a host cell or organism, transformed or transfected with an expression vector according to the invention. The host cell or organism may advantageously be used in a method of producing protein, which comprises recovering any expressed protein from the host or organism transformed or transfected with the expression vector.

According to a further aspect of the invention there is also provided a transgenic cell, tissue or organism comprising a transgene capable of expressing a protein according to the invention. The term "transgene capable of expressing" as used herein encompasses any suitable nucleic acid sequence which leads to expression of proteins having the same function and/or activity. The transgene, may include, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromoso al state. Preferably, the transgene comprises the nucleic acid sequence encoding the proteins according to the invention as described herein, or a functional fragment of said nucleic acid. A functional fragment of said nucleic acid should be taken to mean a fragment of the gene comprising said nucleic acid coding for the proteins according to the invention or a functional equivalent, derivative or a nonfunctional derivative such as a dominant negative variant, or bioprecusor of said proteins.

The protein expressed by said transgenic cell, tissue or organism or a functional equivalent or bioprecusor of said protein also forms part of the present invention. Recombinant proteins may be recovered and purified from host cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose, chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.

The protein of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the expressed protein may lack the initiating methionine residue as a result of post-translational cleavage. Proteins which have been modified in this way are also included within the scope of the invention.

In a still further aspect the invention provides an antibody capable of specifically binding to a protein according to the invention. Preferably the antibody is capable of specifically binding to mutant W147A. An antibody according to the invention may be raised according to standard techniques well known to those skilled in the art by using the protein of the invention or a fragment or single epitope thereof as the challenging antigen.

A further aspect of the invention comprises a nucleic acid capable of hybridising to the nucleic acids encoding the proteins of the invention, and preferably capable of hybridising to the sequence of nucleotides coding for W147A, under high stringency conditions. Conditions of stringency are well known to those skilled in the art.

Stringency of hybridisation as used herein refers to conditions under which hybrids of polynucleic acids are stable. The stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. Tm can be approximated by the formula:

81.5°C+16.6(log₁₀[Na⁺]+0.41 (%G&C) -600/1 wherein 1 is the length of the hybrids in nucleotides. Tm decreases approximately by 1-1.5°C with every 1% decrease in sequence homology.

The nucleic acid capable of hybridising to nucleic acid molecules according to the invention will generally be at least 70%, preferably at least 80 or

90% and more preferably at least 95% homologous to the nucleotide sequences according to the invention.

The present invention also advantageously provides oligonucleotides consisting essentially of at least 10 consecutive nucleotides of a nucleic acid coding for the proteins of the invention and preferably from 10 to 50 consecutive nucleotides of a nucleic acid according to the invention, in particular oligonucleotides fragments from the sequence of nucleotides coding for W147A. These oligonucleotides may, advantageously be used as probes or primers to initiate replication, or the like. Oligonucleotides having a defined sequence may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with the sample under hybridising conditions and detecting the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample .

The invention will be further understood with reference to the following experimental examples, together with the accompanying figures in which:

Figure 1 shows non-denaturing PAGE of receptor complexes formed by IL-11 and W147A. Equimolar concentrations (300nM) of mIL-11, mIL-llR and mgpl30 were mixed together in various combinations, in the presence of increasing amounts of W147A. After incubation, complexes were subjected to nondenaturing PAGE and detection was carried out using silver staining;

Figure 2 shows the response of BA/F3-mgpl30/mIL- 11R cells to mIL-11 and W147A. An increasing concentration of mIL-11 (■) or W147A (A.) was applied to BA/F3 cells. Results are expressed as the A5O value of cells assayed for proliferation by MTT . Values are the mean of triplicate samples, and error bars represent the S.E;

Figure 3 shows the response of BA/F3-mgpl30/mIL- 11R cells to mIL-11 in the presence of W147A. A' constant concentration of mIL-11 (lOng/ml) was applied in the presence (A) or absence (■) of increasing concentrations of W147A. Results are expressed as the A570 value of cells assayed for proliferation by MTT. Values represent the mean of triplicate samples, and error bars represent the S.E;

Figure 4 shows mIL-11 mediated activation of STAT3 in Ba/F3-mgpl30/mIL-llR cells. Ba/F3- mgpl30/mIL-llR cells were incubated for 15 minutes in the presence of increasing concentrations of mIL-11. Cells were lysed with 1% Triton X-100 lysis buffer. Immunoblots were probed with the following antibodies: (top row) anti-phosphotyrosine STAT3 antibody; (bottom row) anti-STAT3 antibody;

Figure 5 show the receptor phosphorylation is inhibited by W147A. Ba/F3-gpl30/mIL-llR cells were incubated for 15 minutes in the presence of a constant concentration of mIL-11 (0.5ng/ml) and increasing concentrations of mIL-ll-Wl47A. Cells were processed as previously described. Top row, anti-phosphotyrosine STAT3 antibody; bottom row anti-STAT3 antibody.

Figure 6 shows that Wi47A inhibits phosphorylation of P-Stat3 in endometrial stromal cells in the presence of 40nm progesterone (P) and 0.35nm estrogen (E₂) . In Figure 6, A represents P-Stat3 and B shows Stat3 (loading control) . Results are expressed in respect of lOng/ml IL-11 (1), lOng/ml IL-11 plus O.lμg/ml W147A (2), lOng/ml IL-11 plus

P/E_z(3), lOng/ml IL-11 plus P/E₂ plus O.lμg/ml W147A (4), medium only (5) and positive control (HeLa cells treated with IFN) (6) .

Figure 7 shows the dose-dependent inhibition of rh IL-11 induced STAT3 phosphorylation (Tyr 705) by W147A in cultured human endometrial stromal cells determined by immunoblotting. (A) Lanes 1-6 - cells treated with lOng/ml IL-11 and the following concentrations of W147A: lanes 1-0; 2-100ng/ml;

3-250ng/ml; 4-500ng/ml; 5-lug/ml; 6-2ug/ml; lane 7- medium only; lane 8-positive control (Hela cells treated with IFNγ) . (B) Densitometric analysis of the gel in A. The relative density of the bands was expressed as the ration of P-STAT:STAT.

Figure 8 shows the inhibition of IL-11 and cAMP- induced decidualization of human endometrial stromal cells by W147A.

Examples

Plasmid constructs

The design and construct of pIG/mlL-llR-Fc, pIG/mgpl30-Fc and pGEX/mIL-11 plasmids (IL-11 wild type and W147A mutant) have been previously described (26,32) .

Expression and purification of proteins

Both mIL-llR-Fc and mgpl30-Fc were expressed in human embryonic kidney 293T cell (41) by transient expression as described previously (21) . Five days post transfection, the conditioned media, containing the Fc fusion receptors, was harvested and subsequently subjected to Protein A affinity chromatography. Soluble forms of mIL-llR and mgpl30 were cleaved from the Fc portion using human rhinovirus 3c protease as described by Karrow et al (21) . The purified soluble proteins were examined using SDS-polyacrylamide gel electrophoresis.

mIL-11 and the site III mutant, W147A, were expressed as glutathione S-transferase fusion proteins in the Escherichia coli strain JM109. Expression, purification and cleavage of the proteins were carried out as previously described (42) . Protein concentrations were determined using the Coomassie Blue Protein assay (Pierce) .

Cell culture and ligand bioassays Bioassays were carried out using Ba/F₃ cells transfected with BC neo/mgpl30 (43) and pcDNA3/mIL-HR as previously described (32) . mIL-11 and the mutant W147A were tested for biological activity on the Ba/F₃ - mgpl30/mIL-llR cells using the 3- (4, 5-dimethyl thiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) proliferation assay as previously described (21) .

Na tive PAGE assay.

Equimolar concentrations of soluble IL-llR and gpl30 components (50nM) were mixed together in the presence of various concentrations of wild type mIL-11 and the mutant W147A, in a total volume of lβμl of PBS, 0.05 % Tween 20. Complexes were allowed to form overnight at 18 - 20°C. 4μl of native gel loading buffer (120mM Tris, pH 6.8, 745nM glycine, 50 % glycerol, 0.5 % bromphenol blue) was added and each sample loaded onto a 4-20 % precast Tris-glycine gel (Novex) . The gel was run in native gel running buffer (24mM Tris, 149mM glycine) at 15mA for approximately 2 hours. The presences of proteins were detected using the silver-staining assay as previously described (44) .

Immunoprecipi ta tion and Western blot analysis

Following serum starvation for 4 hrs, Ba/F3- mgpl30/mIL-llR cells were incubated in the presence of varying concentrations of mIL-11 or mIL-11 plus site III mutant, W147A, for 15 mins at 37°C. Cells were solubilized in ice-cold lysis buffer (50mM Tris HCL, 150mM NaCl, ImM EDTA, ImM EGTA, 50mM NaF, ImM sodium orthovanadate (Na₃V0₄) , 1% Triton X-100 plus 1 complete protease inhibitor tablet (Roche Diagnostics GmbH) per 10 ml lysis buffer) . For immunoprecipitations cell lysate was precleared with protein-A/G-sepharose for 2 hrs at 4°C before overnight incubation with anti-gpl30 antibody. Immunocomplexed protein was isolated by 4 hr incubation with protein-A or G-sepharose beads and washed in lysis buffer.

Even amounts of immunoprecipitates or whole cell lysates were loaded onto 4-20% Tris-glycine gel and electrophoresed at 100V for 1 hour. For Western blotting, proteins were transferred onto PVDF

(Millipore) using a standard protocol. Membranes were blocked overnight in TBS buffer (20mM Tris pH7.5, 150mM

NaCl, 0.1% Tween-20) plus 5% BSA and subjected to immunodetection using specific antibodies: anti-phospho STAT3 and anti-STAT3 (Cell Signaling Technology) , anti- phosphotyrosine 4G10 (Upstate Biotechnology Inc.) and PY20 (ICN Bio edicals Inc.) and anti-gpl30 (Upstate Biotechnology Inc.). Membranes were then probed with secondary anti-mouse, sheep or rabbit horseradish peroxidase-conjugated antibodies (Amersham Life Science) and blots developed using Super Signal West-Pico Enhanced Chemiluminescence (ECL) (Pierce) .

Results

mIL-11 si te III mutant , W147A, inhibi ts mlL- H/sIL-llR/gpl30 hexamer forma tion Previous investigations suggest that IL-11 functions in a manner similar to interleukin-6 (26) . It is known that IL-6 signals through the formation of a hexameric complex consisting of two IL-6 ligands and two each of IL-6R and gpl30 (45,46). Recent studies have identified residues crucial for the binding of mIL-11 to both IL-llR and gpl30 (26) . This investigation has revealed three distinct binding sites analogous to the three IL-6 binding epitopes (32) . Mutagenesis studies confirmed that all three binding sites are required for the formation of a stable ternary receptor complex (26) thus strengthening the hypothesis that a mIL-11- induced hexameric receptor complex is required for the initiation of signalling events leading to a cellular response. Substitution of a critical site III amino acid, tryptophan-147, to an alanine residue leads to the inability of mIL-11 to dimerize gpl30 despite showing normal affinity for the IL-11 receptor (26,32) .

To test the effect of the mIL-11 site III antagonist, W147A, on the ability of mIL-11 to form a hexameric ligand-receptor complex, a non-denaturing PAGE was performed on various combinations of mIL-11 and W147A in the presence of soluble receptors, mlL- 11R and mgpl30. The results, as observed in figure 1, show that only the wild-type mIL-11, and not the mutant W147A, can induce the formation of a hexameric complex when mixed with soluble mIL-llR and mgpl30 (Fig. 1, lane 1) . However, the site III mutant does appear to form a trimeric-complex as displayed by silver-stained bands of a lower molecular weight (Fig. 1, lane 2) .

Decreasing the amount of mutant mIL-11 while simultaneously increasing the level of wild type IL-11 (Fig. 1, lanes 3 to 7) resulted in a differential increase in the level of the hexameric complex formed. In this case, the trimeric complex and free receptor components disappear as the amount of hexameric complex increases (increasing intensity of protein band) .

Proliferative response of Ba/F3-mgpl30/mIL-llR cells to mIL-11 and N147A The functional properties of mIL-11 and mutant were evaluated using a transfected Ba/F3 model system. Ba/F3 cells co-transfected with mgpl30 and mIL-llR are able to proliferate in the presence of mIL-11 alone. We therefore measured the activity of either wild-type mIL-11 or W147A on Ba/F3-mgpl30/mIL-llR cell proliferation (Fig. 2) . As expected, mIL-11 stimulated the growth of Ba/F3-mgpl30/mIL-llR cells in a concentration dependent manner. In contrast, W147A displayed no proliferative activity on these cells (Fig2) . This result confirms that mutation of tryptophan-147 abrogates mIL-11 agonistic activity thus is critically involved in mIL-11-induced proliferation of Ba/F3-mgpl30/mIL-llR cells. As W147A only forms a trimeric complex (Fig. 1, lane 2) then hexameric ligand/receptor complex formation may be a prerequisite for the initiation of intracellular events that eventually lead to cell proliferation.

W147A antagonises mIL-11 mediated Ba/F3-mgpl30/mIL-l lR cell proliferation

To assess the effect of W147A on mIL-11 induced Ba/F3-mgpl30/mIL-llR cell mitogenesis, cells were co- cultured in the presence of lOng/ml mIL-11 with increasing concentrations of W147A (Fig. 3) . At concentrations above 50ng/ml, W147A antagonised the proliferative activity of wild-type mIL-11. This block appeared to be concentration-dependent with 250ng/ml of W147A (25-fold) enough to cause a 50% reduction in the proliferative response of Ba/F3- mgpl30/mIL-llR cells mIL-11. A higher level of W147A (lg/ml) almost completely abolished mIL-11 induced mitogenic activity. This data suggests that inhibition of IL-ll/IL-llR/gpl30 hexameric complex formation is linked to a reduction in mIL-11-induced Ba/F3-mgpl30/mIL-llR cell proliferation and therefore reduced cellular responses. This prompted a further look at events downstream of receptor heterodimerisation.

mIL-11 induces STAT3 tyrosine phosphorylation in Ba/F3- mgpl30/mIL-llR cells in a dose-dependent manner

STAT monomers are tyrosine-phosphorylated allowing their homo- or hetero-dimerization and translocation to the nucleus where they regulate expression of various target genes (25,46,47). STAT3 is strongly activated by IL-6 cytokines (48) thus to test the effect of W147A, on the Jak/STAT pathway we first had to verify that wild-type mIL-11 was capable of inducing tyrosine-phosphorylation of STAT3 in the Ba/F3-mgpl30/mIL-llR cell line (Fig. 4). Phosphorylation of tyrosine-705, essential for dimerization and DNA-binding, was detected after stimulation with 0. Ing/ml mIL-11 (Fig. 4, top). The level of STAT3 phosphorylation increases with increasing mIL-11 concentration, with a dose of 5ng/ml and above appearing to cause maximal phosphorylation. Levels of STAT3 remained constant confirming even protein loading of samples (Fig. 4, bottom) . Stimulation of the Ba/F3-mgpl30/mIL-llR cell line with lOng/ml mIL-11 resulted in activation of STAT3, within 5 minutes, which remains tyrosine-phosphorylated for over 4 hours (data not shown) .

W147A antagonises mIL-11 induced STAT3 tyrosine phosphorylation in Ba/F3-mgpl30/mIL-llR cells .

To determine the effect of W147A on mIL-11- induced STAT3 phosphorylation, Ba/F3-mgpl30/mIL-llR cells were stimulated with 0.5ng/ml mIL-11 in the presence of increasing concentrations of the mutant W147A (Fig. 5) . As antagonist levels increased, the level of STAT3 phosphorylation decreased (Fig. 4, top) despite a constant level of STAT3 (Fig. 5, bottom) . This data confirms that the mIL-11 site III mutant, W147A, antagonises mIL-11 induced STAT3 pathway activation.

Screening assay for test compounds capable of modula ting STAT 3 phosphoryla tion

Western Immunoblotting Protocol for P-STAT3 assay (NEB Ltd)

Solutions and Reagents:

Transfer Buffer:

25 mM Tris base, 0.2M glycine, 20% ethanol (pH8.5)

SDS Sample Buffer:

62.5mM Tris-HCl(pH 6.8 at 25°C) 2% w/v SDS, 10%glycerol 50 mMDTT, 0.1%w/v bromphenol blue

Blocking Buffer:

IX TBS, 0.1% Tween-20 with 5%w/v nonfat dry milk; for

150 ml, add 15 ml

10X TBS to 135ml water, mix. Add 7.5g nonfat dry milk and mix well

While stirring, add 0.15ml Tween-20 (100%)

10X TBS (Tris-buffered saline) :

To prepare 1 liter of 10X TBS:24.2g Tris base, 80g

NaCl; adjust pH to 7.6 with HCl (use at IX) Primary Antibody Dilution Buffer:

IX TBS, 0.1% Tween-20 with 5% BSA; for 20 ml, add 2ml

10X TBS to 18 ml water; mix. Add l.Og BSA and mix well. While stirring, add 20 ml Tween-20 (100%). If background is high, a better signal to noise ratio can be obtained by diluting the primary antibody in 50% milk (in place of 5% BSA) in IX TBST.

Phototope®-HRP Western Blot Detection: Biotinylated protein marker, secondary anti-rabbit antibody conjugated to horseradish peroxidase (HRP) , anti-biotin antibody conjugated to HRP, Reneissanse® chemiluminescent reagent, peroxide. Wash Buffer TBS/T:

IX TBS, 0.1% Tween-20

Blotting Membrane

This protocol has been optimized for nitrocellulose membranes but PVDF membranes may also be used

Protein Blotting

1. Plate and culture 0.5 x 10⁶ cells in DMEM containing 0.5%FCS in 6-well plate for 2 days. This is recommended in order to reduce basal levels of Stat3 phosphorylation.

2. Aspirate media. Add fresh DMEM without FCS. Culture for 2-4 hours.

3. Aspirate media. Treat cells by adding fresh DMEM

(without FCS) containing either 100 ng or 10 ng/ml rhIL-11 with or without the test compound (10 times excess) for 15 min. Controls are treated with medium with or without the test compound (lμg/ml) .

4. Aspirate media from cultures; wash cells with IX PBS; aspirate.

5. Lyse cells by adding 100 ml of SDS Sample Buffer and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice. 6. Sonicate for 10-15 seconds.

7. Heat to 95-100°C for 5 minutes; cool on ice.

8. Microcentrifuge for 5 minutes.

9. Load 1 μl sample onto SDS-PAGE gel (7.5%). 1 μl of IFN-g treated and untreated Hela cells were loaded as controls.

10. Electrotransfer to nitrocellulose or PVDF membrane .

Membrane Blocking Gel & Antibody Incubations

1. (optional) After transfer, wash nitrocellulose membrane with TBS for 5 minutes at room temperature.

2. Incubate membrane in Blocking Buffer for 1 hour at room temperature.

3. Incubate membrane and primary antibody (at 1:1000 dilution) in Primary Antibody Dilution Buffer with gentle agitation overnight at 4°C.

4. Wash 5 times for 5 minutes each with TBST.

5. Incubate membrane with HRP-conjugated secondary antibody (1:2000) and HRP-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein markers in Blocking Buffer with gentle agitation for 1 hour at room temperature.

6. Wash membrane as in step 4. Detection of Proteins

1. Incubate membrane with Reneissanse for 1 minute at room temperature.

2. Drain membrane of excess developing solution, do not let dry, wrap in Saran Wrap and expose to x- ray film. An initial ten second exposure should indicate the proper exposure time. Note:

Due to the kinetics of the detection reaction, signal is most intense immediately following

Reneissanse incubation and declines over the following

2 hours .

Screening assay for test compounds capable of modulating endometrial proliferation

Test protocol for modulators of endometrial proliferation

1. Plate human endometrial stromal cells (10⁴ cells/well) in 96 well plate in DMEM supplemented with 10% FCS, lOOIU/ml penicillin and lOOng/ml of streptomycin and incubate for 24 h.

2. Change the medium to serum-free and incubate for 18 hours.

3. Stimulate cells with either - 11IL plus the test compounds as above for 24 hours.

4. Add I Ci H³-thymidine (Amersham Pharmacia Biotech, UK) to each well for the last 4 hours of incubation and wash cells 3 times in PBS, harvest and determine the amount of incorporated H³- thymidine using a β-plate counter (Wallac Ltd., Finland) .

Screening assay for test compounds capable of modulating decidualization of human endometrial cells

W147A and other test compounds were tested according to the following assay: 1. 10⁵ human endometrial stromal cells are plated in each well of a 4-well tissue culture plate in DMEM/F12 + 10% foetal calf serum (fcs) and incubated at 37°C in 5% C0₂ until confluent.

2. The culture medium is removed and cells are treated with 10-lOOng IL-11 with or without lOx excess of the test compound in DMEM/F12 with or without fcs.

3. Culture medium is changed and supernatant are collected at days 0-21 at 3 day intervals and stored at -20°C.

4. Prolactin levels are measured in the culture supernatant with the Immulite^® prolactin kit from DPC. The sample is added to the assay tube containing monoclonal prolactin-antibody-coated beads and incubated for 30 minutes at 37°C with intermitted agitation. Unbound sample is removed by centrifugation followed by an incubation for further 10 minutes with the chemiluminescent substrate PPD. The photon output is measured via a luminometer.

W147A inhibits IL-11- and cAMP-induced decidualization of human endometrial stromal cells

Cells were plated at IxlO⁵ / well into 4-well plates in duplicate in serum-free DMEM/F12 supplemented with 100 IU/ml penicillin and 100 mg/ml streptomycin and incubated until confluent. In order to induce in vitro decidualization, the cells were transferred to DMEM/F12 supplemented with 100 IU/ml penicillin and 100 mg/ml streptomycin and one of the following inducers for 16 days: 1 mM progesterone plus 36 nM estradiol (P+E₂) ; 0.5 mM 8-Bromo-cAMP (cAMP) ; 10 ng/ml rhIL-11 (R&D Systems) plus lμM progesterone plus 36 nM estradiol (P+E₂+IL-11) . W147A, a signalling inhibitor of IL-11, was added to the media containing either P+E₂+IL-11 at 100 ng/ml (P+E₂+IL- 11+W) , or cAMP at 250 ng/ml (cAMP+W) . The culture medium was changed every 3 days with continuous supplementation of P+E, IL-11, W147A and cAMP, and the supernatants stored and frozen at -20 °C. Concentrations of prolactin were measured using an Immulite Prolactin Kit (DPC) according to the manufacturer's instructions. Concentrations of IL-11 were measured in supernatants from the cells treated with P+E₂, cAMP and cAMP+W, by sandwich ELISA using antibodies from R&D Systems. Concentrations of prolactin and IL-11 were normalised against total protein measured by Coomassie Plus protein assay reagent (Pierce) according to the manufacturer's instructions. Levels of prolactin and IL-11 were expressed as ng/μg total protein and pg/μg total protein.

NB. All culture medium reagents were from Sigma unless indicated otherwise.

Accession Numbers

GenBank: mIL-11 : NM_008350 hIL-11 : NM 000641

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Claims

1. A pharmaceutical composition comprising as an active ingredient a modulator of IL-11 signalling for use as a contraceptive or fertility agent.

2. Use of a modulator of IL-11 signalling in the preparation of a medicament for modulating fertility in a mammalian female.

3. A method for modulating fertility comprising the administration of a modulator of IL-11 signalling to a mammalian female.

4. A composition, use or method according to any one of claims 1 to 3 wherein said modulator competitively inhibits IL-11 signalling by disrupting the IL-ll/IL- HR/gpl30 signalling complex.

5. A composition, use or method according to any one of claims 1 to 4 wherein said modulator is a murine or human IL-11 mutant or a biologically active portion thereof.

6. A composition, use or method according to claim 5 wherein said mutant has a substitution at position 147 from Tryptophan to any one of Alanine, Glycine, Valine, Serine, Asparagine, Aspartic acid or Glutamic acid, or the human equivalent (s) thereof.

7. A composition, use or method according to claim 5 or 6 wherein said mutant is W147A murine IL-11 or any of its derivatives which differ from said mutant in conservative amino acid changes only, or the human equivalent (s) thereof.

8. A composition, use or method according to claim 5 wherein said mutant is R111A/L115A murine IL-11 or any of its derivatives which differ from said mutant in conservative amino acid changes only, or the human equivalent (s) thereof.

9. A composition, use or method according to claim 7 or 8 wherein said derivatives include mutations which are not non-polar in nature.

10. A composition, use or method according to any one of claims 7 to 9 wherein said conservative substitutions are made in accordance with the following: Alanine replaced with Glycine or Serine Arginine replaced with Lysine Asparagine replaced with Glutamine or Histidine Cysteine replaced with Serine Glutamine replaced with Asparagine Gylcine replaced with Alanine or Proline Histidine replaced with Asparagine or Glutamine Isoleucine replaced with Leucine or Valine

11. A method of identifying compounds which are capable of modulating an IL-11 receptor or a biologically active portion thereof, which method comprises:

- culturing cells which express IL-11 receptor and gpl30 or biologically active portions thereof with IL-11 or a biologically active portion thereof, in the absence or presence of the test compound;

- determining the effect of the test compound on the IL-11 receptor activation.

12. A method according to claim 11 wherein the compounds which are capable of modulating an IL-11 receptor or a biologically active portion thereof comprise fertility and/or contraceptive agents.

13. A method according to claim 12, wherein said cells comprise isolated mammalian female endometrial stromal cells or yeast cells or cells of mammalian origin transfected so as to express the desired receptor on their surface.

14. A method according to any one of claims 11 to 13 wherein the IL-11 receptor is of the murine or human form.

15. A method according to any one of claims 11 to 14, wherein the IL-11 is of the murine or human form.

16. A method according to any one of claims 11 to 15, wherein the effect of the test compound comprises the effect on tyrosine phosphorylation induced by IL-11.

17. A method according to claims 16, wherein said tyrosine phosphorylation is that of STAT3.

18. A method according to claims 16 or 17, wherein the effect of the test compound comprises the inhibition of IL-11 induced STAT3 tyrosine phosphorylation.

19. A method according to any one of claims 16 to 18, wherein said effect of the test compound is assessed by carrying out a Western blotting assay.

20. A method according to any one of claims 11 to 14, wherein the effect of the test compound comprises the effect on IL-11 induced proliferation.

21. The method according to claim 20, wherein said effect of the test compound is assessed by carrying out proliferation assays.

22. A method according to any of claims 11 to 14 wherein the effect of the test compound comprises the effect on decidualisation of isolated mammalian female endometrial cells.

23. A method according to claim 22, wherein the effect is determined on the basis of a biological marker for decidualisation .

24. A method according to claim 23, wherein the said marker is prolactin.

25. A method according to any of claims 22 to 24, wherein the effect is assessed by carrying out an immunoassay.

26. A compound identifiable by the method according to any one of claims 11 to 25.

27. A medicament comprising as an active ingredient a compound according to claim 26.

28. Use of a compound according to claim 26 for preparation of a medicament for modulating fertility in a mammalian female.

29. A method for modulating fertility comprising the administration of a compound according to claim 26.

30. The use or the method of claim 2 or claim 3, wherein said modulation of fertility comprises contraception .

31. The use or the method of claim 2 or claim 3, wherein the mammalian female comprises female of the human or livestock species.

32. The use of the method according to claim 13, wherein the mammalian female is a human female.

33. A method of producing a pharmaceutical composition suitable for use as a contraceptive agent in a mammalian female, which method comprises: a) carrying the screening method according to any one of claims 11 to 25; and b) formulating any compound identified as capable for modulation of IL-llR activation in a pharmaceutical composition with a pharmaceutical acceptable carrier or diluent

34. A kit comprising cells expressing IL-llR and gpl30, IL-11 or nucleic acid molecules encoding the proteins or vectors comprising said nucleic acid molecules, or cells comprising the vectors, and optionally reagents for drug screening.