GB2375604A

GB2375604A - Methods for screening using interleukin soluble trimolecular complex

Info

Publication number: GB2375604A
Application number: GB0112251A
Authority: GB
Inventors: Francois Bertelli; Jason Peter Brown; Nicolas Steven Gee
Original assignee: Warner Lambert Co LLC
Current assignee: Warner Lambert Co LLC
Priority date: 2001-05-18
Filing date: 2001-05-18
Publication date: 2002-11-20
Also published as: EP1395832A1; WO2002095414A1; JP2004533612A; GB0112251D0

Abstract

A soluble trimolecular complex comprising interleukin (IL), soluble interleukin receptor (sIL-R) and a soluble interleukin receptor accessory protein (sIL-RAcP), and assays for the determination of the ability of a test compound to modulate the formation of said complex.

Description

METHODS FOR SCREENING USING INTERLEUKIN SOLUBLE TRIMOLECULAR COMPLEX FIELD OF INVENTION The present invention relates to screening methods, assays and reagents based on the unexpected discovery that interactions between interleukin (IL), the membrane-bound interleukin receptor (IL-R) and membrane-bound interleukin receptor accessory protein (IL-RAcP), which occur in vivo at the cell surface, can be modelled in solution. This provides for assays suitable for high throughput screening.

BACKGROUND OF THE INVENTION Interleukin-1 (IL-1) plays a central role in the mediation of immune and inflammatory responses (Dinarello, 1996).

The IL-1 may be: IL-la or IL-1ss receptor agonists which have comparable biological activities, or IL-1 receptor antagonist IL-lra. These cytokines are involved in a large number of biological effects such as fever, sleep, anorexia or hypotension by binding to specific receptors on the surface of responsive cells.

Two IL-1 receptors with different pharmacological characteristics, termed type I (Sims et al. 1988) and type

II (McMahan et al. 1991) have been cloned. IL-1a, IL-1ss and IL-lra all bind with comparable affinity to the 80kDa type I receptor (IL-1RI) (Dinarello, 1996). On the other hand, IL-1ss binds with much higher affinity and selectivity to the 68kDa type II receptor (IL-1RII) than the IL-1 receptor type I (Colotta et al. 1993). Furthermore IL-1RI is necessary for IL-1 signal transduction (Sims et al. 1993).

IL-1RII appears to act as a decoy receptor (Colotta et al.

1993).

Another component of the receptor complex, Interleukin 1 receptor accessory protein (IL-1RAcP), has also been cloned (Greenfeder et al. 1995). The IL-lRAcP forms, a ternary complex with IL-1RI and either IL-la or IL-1ss, but not with IL-lra. Formation of this trimolecular complex increases the binding affinity of IL-ljS for IL-1RI. Although IL-1RI and IL-1RI I may bind IL-1, IL-lRAcP does not bind IL-1 (Greenfeder et al. 1995). However transfection with IL- 1RAcP restores IL-1 responsiveness in mammal cells not expressing IL-lRAcP (Korherr et al. 1997). Therefore although IL-1RAcP is not required for the binding of IL-1 to IL-1RI, it is essential for IL-1 signal transduction.

Endogenous cell-surface receptor and accessory proteins are membrane-bound and contain hydrophobic trans-membrane domains. Such proteins are not soluble and are not suitable for use in in vitro high throughput screening.

SUMMARY OF THE INVENTION The present invention provides screening methods, assays and reagents based on interactions between interleukin (IL), the membrane-bound interleukin receptor (IL-R) and membrane-bound interleukin receptor accessory protein (ILRAcP) modelled in solution.

In one general aspect, the present invention provides an assay method for determining the ability of a test compound to modulate the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising:

(a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, a soluble IL-RAcP polypeptide and a test compound; and (b) determining the amount of said trimolecular complex formed.

In a most preferred embodiment the present invention also provides an assay method for determining the ability of a test compound to disrupt or interfere with the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a test compound; (b) bringing the test compound into contact with a defined amount of a soluble IL-R polypeptide and a soluble IL-RAcP polypeptide; (c) adding an IL polypeptide to the mixture obtained in test (b); and (d) determining the amount of said trimolecular complex formed.

Another general aspect of the present invention provides an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, a soluble IL antagonist, a soluble IL-RAcP polypeptide and a test compound; (b) determining the amount of said trimolecular complex formed; and (c) comparing the amount of said trimolecular complex formed at step (a) with the amount of said trimolecular complex formed in the absence of

test compound.

A preferred embodiment of the present invention is an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a test compound; (b) bringing the test compound into contact with a soluble IL-R polypeptide, a soluble IL antagonist and a soluble IL-RAcP polypeptide; (c) adding an IL polypeptide to the mixture obtained at step (b); (d) determining the amount of said trimolecular complex formed; and (e) comparing the amount of said trimolecular complex formed at step (c) with the amount of said trimolecular complex formed in the absence of test compound.

The present invention also concerns an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, an IL-R antagonist, a soluble IL-RAcP polypeptide and a test compound; (b) determining the amount of said trimolecular complex formed; and (c) comparing the amount of said trimolecular complex formed at step (a) with the amount of said trimolecular complex formed in the absence of test compound.

A preferred embodiment of the present invention is an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a test compound; (b) bringing the test compound into contact with an IL polypeptide, an IL-R antagonist and a soluble IL-RAcP polypeptide ; (c) adding a soluble IL-R polypeptide to the mixture obtained in step (b); (d) determining the amount of said trimolecular complex formed; and (e) comparing the amount of said trimolecular complex formed at step (c) with the amount of said trimolecular complex formed in the absence of test compound.

A further aspect of the present invention relates to an assay method for determining the ability of a test compound to disrupt or interfere with the stability of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a defined amount of a trimolecular complex comprising an IL polypeptide, a soluble IL-R polypeptide and a soluble IL-RAcP polypeptide; (b) contacting a test compound with said trimolecular complex ; and (c) comparing the amount of said trimolecular complex present after step (b) to the amount of said trimolecular complex initially present at step (a).

In a preferred assay method of the invention the IL, sIL-R and sIL-RAcP are of mammalian origins.

In a most preferred assay method of the invention the IL, sIL-R and sIL-RAcP are from human, mouse or rat.

A general aspect of the invention provides an assay method wherein the IL polypeptide is in its mature form and has a barrel shaped 3-dimensional structure composed of 12 to 13 beta-strands and a cellular activity mediated by a ternary (i. e. trimolecular) complex at the cell surface.

In a preferred embodiment of the invention, the assay method is an assay method wherein the IL polypeptide has an amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or a fragment thereof.

Another general aspect of the invention concerns an assay method wherein the soluble IL-R polypeptide comprises 3 Iglike domains which are included in three structural domains identified as domain 1, domain 2 and domain 3 starting from the N-terminal end of the sequence, said domain 1 containing one Ig-like domain and two disulfide bonds; said domain 2 containing one Ig-like domain and two overlapping disulfide bonds; said domain 3 containing one Ig-like domain and one disulfide bond.

In a preferred embodiment of the invention, the assay method is an assay method wherein the soluble IL-R polypeptide has an amino acid sequence of SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ

ID NO: 17, or SEQ ID NO: 18, or a fragment thereof.

A further general aspect of the invention relates to an assay method wherein the soluble IL-RAcP polypeptide comprises 3 Ig-like domains which are included in three structural domains identified as domain 1, domain 2 and domain 3 starting from the N-terminal end of the sequence, said domain 1 containing one Ig-like domain and two disulfide bonds; said domain 2 containing one Ig-like domain and two overlapping disulfide bonds; said domain 3 containing one Ig-like domain and one disulfide bond.

In a preferred embodiment of the invention, the assay method is an assay method wherein the soluble IL-RAcP polypeptide has an amino acid sequence of SEQ ID NO: 21, or SEQ ID NO: 22, or SEQ ID NO: 23, or SEQ ID NO: 24, or SEQ ID NO: 25, or SEQ ID NO: 26, or a fragment thereof.

The present invention also concerns a soluble tri-molecular complex comprising or consisting of the following elements: (a) an IL polypeptide (b) a soluble IL-R polypeptide which binds said IL polypeptide, and (c) a soluble IL-RAcP polypeptide which binds to the IL-R polypeptide/IL polypeptide binary complex.

A preferred soluble complex of the invention is a soluble complex wherein the IL, sIL-R and sIL-RAcP are of mammalian origins.

A most preferred soluble complex of the invention is a soluble complex wherein the IL, sIL-R and sIL-RAcP are from human, mouse or rat.

The most preferred soluble complex is a soluble complex wherein at least one of the IL, sIL-R or sIL-RAcP is from human.

In a particularly preferred soluble complex of the invention the IL polypeptide has an amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or a fragment thereof.

In another particularly preferred soluble complex of the invention the soluble IL-R polypeptide has an amino acid sequence of SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or a fragment thereof.

In a further particularly preferred soluble complex of the invention the soluble IL-RAcP polypeptide has an amino acid sequence of SEQ ID NO: 21, or SEQ ID NO: 22, or SEQ ID NO: 23, or SEQ ID NO: 24, or SEQ ID NO: 25, or SEQ ID NO: 26, or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A, 1B and 1C show the interaction respectively of IL-lp (A), sIL-lRI (B) and sIL-lRAcP (C) purified proteins with separate flow cells of the BIAcore chip which have been respectively coupled with sIL-lRI, IL-lp or sIL-lRAcP.

Figures 2A and 2B show the interaction of previously formed IL-1ss/sIL-1RI complex with an excess of IL-lp (A) or sIL-lRI (B) being added on separate flow cells of the BIAcore chip

which have been respectively coupled with sIL-RI, IL-lp or IL-lRAcP.

Figure 3A shows the sequential injection of IL-lp and sILlRAcP on separate flow cells of the BIAcore chip which have been respectively coupled with sIL-lRI, sIL-lp or sILlRAcP-6His. Figure 3B shows the capture of sIL-lRAcP-6His on a NiNTA chip and injection of premixed IL-lp/sIL-lRI binary complex or IL-1 alone.

Figure 4 shows a comparison of the IL-lfl and IL-lra interactions with separate flow cells of the BIAcore chip which have been respectively coupled with IL-lp, sIL-lRAcP or sIL-lRI when sIL-RAcP is added during the dissociation process.

Figures 5A and 5B show the binding of the FEWTPGYWQPYALPL peptide (SEQ ID NO 35) on separate flow cells of the BIAcore chip which have been respectively coupled with sIL- lp, sIL-lRAcP or sIL-lRI.

In figure 5A the FEWTPGYWQPYALPL peptide is added alone. In figure 5B a premixed FEWTPGYWQPYALPL peptide/sIL-lRI complex is added with an excess of sIL-lRI.

Figure 6A shows the sequential addition of IL-lp and sILlRAcP on separate flow cells of the BIAcore chip which have been respectively coupled with sIL-lR Type I, sIL-lR Type II or sIL-lRAcP. Figure 6B shows the sequential addition of sIL-lRII and sIL-lRAcP on separate BIAcore flow cells coupled respectively with IL-1p, sIL-lRI or sIL-lRAcP.

For figures 1 to 6 the Flow cell 1 is coupled with BSA

(Bovine Serum Albumin) and this control flow cell is substracted from the other in the displayed sensorgrams.

Figure 7 shows respectively the binding kinetics of the IL- l/sIL-lRI binary complex (figure 7A) and IL-Iss/sIL-lRI/sIL- lRAcP ternary complex (figure 7B).

Figure 8 is a graphical representation of a direct ternary complex HTRF assay format of the invention.

Figure 9 is a graphical representation of a direct binary complex HTRF assay format of the invention.

Figure 10 shows the kinetics of ternary complex formation in a direct ternary complex HTRF assay format of the invention.

Figure 11 shows the influence of various IL-ljS and sILlRAcP-Cy5 concentrations on the signal obtained using a direct ternary complex HTRF assay format of the invention.

Figure 12 shows the influence of various concentrations of unlabelled sIL-lRI and sIL-lRAcP on the signal obtained using a direct ternary complex HTRF assay format of the invention.

Figure 13 shows the influence of various concentrations of IL-lra and the FEWTPGYWQPYALPL peptide (of SEQ ID NO 35) on the inhibition of ternary complex formation in a direct ternary HTRF assay format of the invention.

Figure 14 shows a sequence alignment of the amino acid sequences of IL-la from human (Genbank accession number: x02531), mouse (Genbank accession number: NM010554) and

rat (Genbank accession number: D00403).

Figure 15 shows a sequence alignment of the amino acid

sequences of IL-ljS from human (Genbank accession number : x02532), mouse (Genbank accession number : NM~008361) and rat (Genbank accession number: M98820).

Figure 16 shows a sequence alignment of the amino acid sequences of IL-18 from human (Genbank accession number : AF077611) and mouse (Genbank accession number: Nom-008360).

Figure 17 shows a sequence alignment of the amino acid sequences of IL-1R Type I from human (Genbank accession number x16896), mouse (Genbank accession number NM008362) and rat (full length protein (Genbank accession number m95578), naturally occuring soluble protein (Genbank accession number NM013123)), IL-1R Type II from human (Genbank accession number Nom004633) and mouse (Genbank accession number NM010555), and IL-18R from human (Genbank

accession number NM003855) and mouse (Genbank accession number NM008365). The underlined sequence indicates the signal sequence of the human IL-1RI protein, which is cleaved after protein maturation.

Figure 18 shows a sequence alignment of the amino acid sequences of IL-lRAcP from Human (full length protein (Genbank accession number AF029213), naturally occuring soluble protein (Genbank accession number AF167343)), mouse (Genbank accession number NM~008364), and rat (Genbank

accession number NM012968) and IL-18RAcP from human (Genbank accession number NM003853) and mouse (Genbank accession number nom~010553). The underlined sequence indicates the signal sequence of the human IL-lRAcP protein, which is cleaved after maturation of the protein.

Figure 19 shows the sequence alignment of human IL-1 (Genbank accession number x16896) and IL-18 receptors (Genbank accession number NM003855). Figure 20 shows the sequence alignment of human IL-1 receptor accessory protein (Genbank accession number AF029213) and IL-18 (Genbank accession number NM003853) receptor accessory protein.

In figures 19 and 20 the bold and underlined sequence indicates the signal sequences of the proteins; the transmembrane domains are outlined by shaded boxes.

Figures 21 and 22 show hydropathy plots of the full length sequence of human IL-1RI (figure 21A) and IL-18R (figure 21B), IL-lRAcP (figure 22A) and IL-18RAcP (figure 22B).

Figure 23 is a graphical representation of the structural organization of human extracellular Interleukin-1 receptor type I and Interleukin-1 receptor accessory protein.

Figure 24 shows the sequence alignment of the human Interleukin-1 type I receptor (IL-lRI ; Genbank accession number x16896) and the human Interleukin receptor accessory protein (IL-lRAcP ; Genbank accession number af029213). The first and the last cysteine of each structural domain are

indicated by shading and boding. Underlined portions of sequence (and (~) indicate the localization of domains 1, 2 and 3 respectively.

Figure 25 is a graphical representation of flashplate assays formats of the method of the invention. Figure 25A shows a Nickel-flashplate format, figure 25B shows a

Streptavidin-flashplate format.

DETAILED DESCRIPTION OF THE INVENTION As used herein the term soluble when applied to a polypeptide means any polypeptide which isn't membrane- bound.

The ability of IL-1ss, IL-1RI and IL-lRAcP to interact in solution is shown for the first time in the present application. It was surprising that the binding activity of the mature membrane-bound proteins IL-1R and IL-lRAcP could be replicated by truncated proteins which lacked the cytosolic and transmembrane domains and comprise only the extracellular domains. The interaction of soluble fragments of these proteins allows the development of assays suitable for high throughput screening, which enable direct measurement of the protein binding characteristics.

The pro-inflammatory cytokines IL-la and IL-lp interact with the IL-1 receptor and IL-1 receptor accessory protein at the cell-surface leading to further pro-inflammatory responses via gene activation through the IRAK, NIK, NFKB signalling cascade (Dinarello, 1996).

Current protein-based therapies include i. v. dosing of the soluble form of the IL-1 receptor and/or the IL-1 receptor antagonist which act as functional antagonists of IL activity (Rosenwasser, 1998).

Limitations of such therapies include the expense of producing the large quantities of these recombinant proteins required for sufficient biological activity and the requirement for i. v. dosing.

The identification of small molecule inhibitors of the IL

trimolecular complex and hence of IL-1 biological activity using the assay method described herein may provide a significant advantage over the above routes since it may be possible to dose orally and to reduce the cost of production of such compounds compared to the production cost of recombinant proteins.

Interferon-y inducing factor or IL-18 is a member of the T

helper type I cell (Thi)-inducing family of cytokines and has many structural and functional similarities with IL-lp.

It also forms a ternary complex at the cell surface together with an IL-18-receptor (or ILlRrp ; IL-1 receptor related protein also known as IL-18Ra) and an IL-18 accessory protein like receptor (AcPL) or IL-18Rp (also known as IL-18RAcP). As with IL-1, IL-18 signalling occurs via the IRAK, NIK, NFKB pathway leading to a proinflammatory stimulus (Dinarello, 1999a).

Crohn's disease is marked by chronic inflammation of the gastrointestinal tract. The upregulation of IFN-y in Crohn's disease due to overproduction of IL-18 may be involved in the pathogenesis of the disease (Monteleone et al. , 1999).

Hence, inhibition of IL-18 activity through an appropriate antagonist represents a potential treatment for this disease. Other potential uses of an antagonist of IL-18 activity include the treatment of asthma, autoimmune demyelinating diseases, rheumatoid arthritis (Gracie et al. , 1999) and psoriasis (Dinarello, 1999b).

There are currently no therapies able to reduce IL-18 activity. Hence, there is a need for small molecule inhibitors, antagonists of IL-18 activity through its interaction with IL-18Ra and IL-18Rss.

Conversely, potentiation of IL-18 activity may also have

clinical benefits. For example, IL-18 appears to have antiangiogenic and antitumour activity on primary T241 fibrosarcomas implanted subcutaneously into C57B16/J mice IL-18 agonists could potentially-but not exclusively-be orally administered. In addition, physiological'agonists' of IL-18 activity may also be useful therapies for protection against fungal and bacterial infections (Akira, 2000).

Inhibitor molecules of IL activity may be obtained with the screening assay of the invention, designed and used for therapeutic purposes as described above, for example, for treatment of inflammation-related diseases such as Crohn's disease, osteoarthritis, inflammatory pain, and rheumatoid arthritis.

Activators or"agonists"of IL activity may also be identified and appropriate agents may be obtained, designed and used for therapeutic purposes as described herein above, for example in the treatment of immunodeficiency.

IL trimolecular complex One general aspect of the present invention is a soluble trimolecular complex consisting of the following elements: (a) an IL polypeptide (b) a soluble IL-R polypeptide which binds said IL polypeptide, and (c) a soluble IL-RAcP polypeptide which binds to the IL-R polypeptide/IL polypeptide binary complex.

In the present application the term IL means IL polypeptide, the term sIL-R means soluble IL-R polypeptide, the term sIL-RAcP means soluble IL-RAcP polypeptide.

The three members of the IL ternary complex are highly conserved between mammalian species. As a result, in an assay of the present invention, the components of the ternary complex, IL, sIL-R and sIL-RAcP, may be derived from the same species, eg. human, mouse, rat or may be from different species. For example, human sIL-lRAcP may bind to a bimolecular complex of rat IL-1/murine sIL-lR to form a trimolecular complex.

In a preferred assay of the present invention, the components of the ternary complex, IL, sIL-R and sIL-RAcP, are derived from the same mammalian species.

In a most preferred assay of the present invention, the components of the ternary complex, IL, sIL-R and sIL-RAcP, are derived from human, mouse or rat sequences.

In the most preferred assay of the present invention, the components of the ternary complex, IL, sIL-R and sIL-RAcP, are derived from human sequences.

IL Polypeptide In the present invention, IL polypeptide is understood as a polypeptide whose mature form has a barrel shaped 3dimensional structure composed of 12-14 beta-strands and whose cellular activity is mediated by a ternary (i. e. trimolecular) complex at the cell surface. IL polypeptide includes members of the IL-1 and IL-18 families, such as human (Genbank accession number: x02531), mouse (Genbank accession number: NM010554) and rat (Genbank accession number: D00403) immature IL-la, human (Genbank accession number: x02532), mouse (Genbank accession number: NM~008361) and rat (Genbank accession number: M98820) immature IL-1ss and human (Genbank accession number :

AF077611) and mouse (Genbank accession number: NM~008360) immature IL-18, human (Genbank accession number AF200492) and mouse immature IL-lHl (Genbank accession number AF200493), human immature IL-lH2 (Genbank accession number AF200494), mouse immature IL-lH3 (Genbank accession number AF200495), human immature IL-lH4 (Genbank accession number AF200496), human (Genbank accession number AF230377) and mouse immature IL-18 (Genbank accession number AF230378), human (Genbank accession number AJ242738) and mouse immature IL-lLl (Genbank accession number AJ250429), human immature FIL1 delta (Genbank accession number AF201830), human immature FIL1 eta (Genbank accession number XM002375), human immature FIL1 zeta (Genbank accession number XM010759), human immature FIL1 epsilon (Genbank accession number XM010757), human (Genbank accession number AF206696) and mouse (Genbank accession number AF206697) immature IL-1 epsilon.

The amino acid sequences of IL-la and IL-lp from human, rat and mouse and IL-18 from human and mouse are shown respectively on the sequence alignments of figures 14,15 and 16.

An IL polypeptide may also be an IL amino acid sequence or a fragment, derivative, analog or active portion thereof which retains the biological activity of IL. What is intended by"biological activity"is to designate the ability of the polypeptide to form a trimolecular complex with the corresponding receptor and receptor accessory protein.

Preferably IL is IL-1 or IL-18.

Preferred IL polypeptides for use in the assays of the

invention are mature IL polypeptides which form a soluble ternary complex with a soluble IL receptor and a soluble IL Receptor Accessory Protein, such as human (SEQ ID No: 1), mouse (SEQ ID No: 5) and rat (SEQ ID No: 7) mature IL-la, human (SEQ ID No: 4), mouse (SEQ ID No: 6) and rat (SEQ ID No: 8) mature IL-L6, and human (SEQ ID No: 9) and mouse (SEQ ID No: 10) mature IL-18.

Most preferred IL polypeptides for use in the assays of the invention are the human mature IL-lp sequence of SEQ ID N04 and the human mature IL-18 sequence of SEQ ID N09. hIL indicates a human form of IL.

IL-R Polypeptides 'Full length IL-R The mouse IL-1 receptor was cloned by expression cloning techniques (Sims et al. , 1988). This gene encodes a receptor containing an NH2-extracellular domain, a transmembrane domain, and an intracellular domain. Sequence analysis further revealed the extracellular portion of the IL-1 receptor to be organised into three domains (Ig-like domains, see Figure 23), similar to those of the immunoglobulin gene superfamily. Co-crystallisation of the extracellular domain of the human IL-1 receptor with the cytokine IL-lp confirmed the above structural prediction for this portion of the protein (Vigers et al. , 1997). X- ray crystal structure of a small antagonist peptide (ETPFTWEESNAYYWQPYALPL, SEQ ID NO 37) bound to the Type-1 Interleukin-1 Receptor was recently disclosed (Vigers et al. , 2000). This peptide, close to the sequence of SEQ ID

N035, mimicked the interaction of IL-lp and IL-lra with domains 1 and 2 of the soluble IL-1R.

A second group who crystallised the IL-1RI with IL-lra describes three Ig-like domains (Schreuder et al. , 1997) with very similar ranges to those described in Vigers et al. (1997).

In the present application, IL-R polypeptide means a polypeptide comprising an intracellular domain, one single transmembrane domain and an extracellular domain. The extracellular domain comprises three Ig-like domains and allows the binding of IL-R to an IL and an IL-RAcP. The signalling form of the IL-R polypeptide is membrane bound.

IL-R polypeptides may include members of the IL-1R and IL- 18R families, such as for example, human IL-1RI (Genbank accession number x16896), mouse IL-1RI (Genbank accession number NM008362), rat IL-1RI (Genbank accession number m95578), the naturally occuring soluble rat IL-1R type 1 (Genbank accession number NM~013123), human IL-18R (Genbank accession number NM003855), mouse IL-18R (Genbank accession number NM008365), human IL-lRrp2 (Genbank accession number AF284434), mouse IL-lRrp2 (Genbank accession number AF284433), human TIGIRR-1 (Genbank accession number AF284436), mouse TIGIRR-1 (Genbank accession number AF284437) (Born et al, (2000), JBC, 275, 29946-29954) Except the naturally occuring soluble rat IL-1R type 1 (Genbank accession number NM~013123) the above defined full length IL-R are not usually suitable for an assay of the invention.

'Soluble IL-R Polypeptides Soluble IL-R polypeptide (sIL-R) means any soluble polypeptide fragment of a full-length IL-R which retains the extracellular binding activity of the full length protein (i. e. the ability to form a trimolecular complex with its corresponding IL and with its corresponding receptor accessory protein which may be soluble or membrane bound).

Preferred soluble IL-R for use according to the present invention comprise or consist of the extracellular domain of the corresponding endogenous protein or a portion or fragment thereof which retains its binding capability. A suitable portion or fragment binds to the same ligands as the endogenous protein. The extracellular domain of a membrane-bound receptor or accessory protein has been identified by a hydropathy analysis of the mature sequence as described in Example 1. The extracellular domain of IL-R comprises three Ig-like domains which are included in three structural domains identified herein as domain 1, domain 2 and domain 3 starting from the N-terminal end of the sequence. Domain 1 contains one Ig-like domain and two disulfide bonds. Domain 2 contains one Ig-like domain and two overlapping disulfide bonds. Domain 3 contains one Iglike domain and one disulfide bond. See Figure 23 for domains organization of IL-1RI.

Domain 3 of IL-1RI is critical for high-affinity binding of IL-lp to the IL-1RI (Schreuder et al., 1997). Thus a soluble form of the IL-1RI receptor consisting of domains 1 and 2 only has a significantly reduced affinity for IL-1ss (lOM).

Preferred soluble IL-R polypeptides For example purposes the sIL-R polypeptides described herein are human sIL-lRI and human sIL-18R but with the help of figure 17 one skilled in the art can extrapolate it to other sIL-lR polypeptides.

In one embodiment of the invention, the soluble IL-R polypeptide is a soluble IL-1 receptor polypeptide, preferably a soluble IL-1 type I receptor polypeptide.

In a preferred embodiment of the invention, a soluble receptor polypeptide is a polypeptide which retains the binding properties of the full length receptor and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 13.

A most preferred soluble receptor polypeptide is a polypeptide which comprises or consist of an amino acid sequence of SEQ ID NO: 13 or an analog, derivative, active portion or fragment thereof which retains the binding properties of the full length protein.

In further embodiments of the invention when the IL polypeptide is IL-1, a soluble IL-R polypeptide which retains the binding properties of the full length receptor and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

In further preferred embodiments of the invention, when the IL polypeptide is IL-1, a soluble IL-R polypeptide is a

polypeptide which comprises or consist of an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16, or an analog, derivative, active portion or fragment thereof which retains the binding properties of the full length protein.

In another embodiment of the invention, the soluble IL-R polypeptide is a soluble IL-18R polypeptide.

In a preferred embodiment of the invention, a soluble receptor polypeptide is a polypeptide which retains the binding properties of the full length protein and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 17.

A most preferred soluble receptor polypeptide is a polypeptide which comprises or consist of an amino acid sequence of SEQ ID NO: 17 or an analog, derivative, active portion or fragment thereof which retains the binding properties of the full length protein.

In further embodiments of the invention, when the IL polypeptide is IL-18, a soluble IL-R polypeptide which retains the binding properties of the full length receptor and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 18.

In further preferred embodiments of the invention, when the IL polypeptide is IL-18, a soluble IL-R polypeptide is a polypeptide which comprises or consist of an amino acid sequence of SEQ ID NO: 18, or an analog, derivative, active

portion or fragment thereof which retains the binding properties of the full length protein.

Deletion ranges The deletion of the C-terminus segment of the extracellular domain of IL-R after the final cysteine residue of domain 3 would yield a protein which is likely to retain the binding capabilities of the full length protein.

Furthermore, deletions from the N-terminal portion of the extracellular domain of IL-R up to the first cysteine of domain 1 would also yield a protein which is likely to retain the binding capabilities of the full length protein.

In a preferred embodiment of the invention, deletions of between 1 and 30 amino acids, preferably between 1 and 20 amino acids, more preferably between 1 and 15 amino acids, most preferably between 1 and 10 amino acids from the Cterminus segment of the extracellular domain of IL-R yield a polypeptide which is likely to retain the binding properties of the full length protein.

A most preferred sIL-R of the invention comprises or consists of the extracellular domain (of the corresponding endogenous full length IL-R) to which no further deletion has been made.

IL-RAcP polypeptides Full length Interleukin receptor accessory proteins In the present application, IL-RAcP polypeptide means a polypeptide comprising an intracellular domain, one single transmembrane domain and an extracellular domain. The

extracellular domain comprises three Ig-like domains and allows the binding of IL-RAcP to an IL-R/IL binary complex.

The signalling form of the IL-RAcP polypeptide is membrane bound.

The prediction for the location of these three Ig-like domains in the IL-1RAcP has been made by Greenfeder et al.

(Greenfeder et al. , 1995). He defined the boundaries of the Ig-like domains of the IL-lRAcP by the location of unique Cystyl ('C') residues, which can be found close to the N- and-C termini of each domain as outlined in figure 24.

Figure 24 shows the sequence alignment of the human Interleukin-1 type I receptor (IL-lRI ; Genbank accession

number x16896) and the human Interleukin receptor accessory protein (IL-lRAcP ; Genbank accession number af029213). The first and last cysteine of each domain are indicated by shading and boding. Underlined portions of sequence identified by (), () and (~) indicate the localization of domains 1, 2 and 3 respectively.

Yoon and Dinarello (1998) showed that monoclonal antibodies directed to a segment of the third domain of IL-lRAcP were able to prevent IL-ljS signalling.

The Ig-like domains for the IL-1RI were determined according to those presented in Greenfeder et al. , 1995 for the IL-lRAcP.

IL-RAcP polypeptides may include members of the IL-1RAcP- like family polypeptides and IL-18RAcP family like polypeptides, for example, human (Genbank accession number AF029213), mouse (Genbank accession number NM008364), or rat (Genbank accession number NM012968) immature IL-lRAcP, human (Genbank accession number NM003853) or mouse

(Genbank accession number NM010553) immature IL-18RAcP, human IL-1R9 (Genbank accession number AF212016) (Carrie et al, Nature genetics, (1999), 23,25-31 and Sana et al., (2000) Genomics, 69, 252-262), human TIGIRR-2 (Genbank accession number AF284435), human IL1RAPLl (Genbank accession number NM014271), naturally occuring soluble

human IL-lRAcP (Genbank accession number AF167343). Except the naturally occuring soluble human IL-lRAcP (Genbank accession number AF167343) the above defined full length IL-RAcP are not usually suitable for an assay of the invention.

'Soluble IL-RAcP Polypeptides Soluble IL-RAcP polypeptide (sIL-RAcP) means any soluble polypeptide fragment of a full-length IL-RAcP which retains the extracellular binding activity of the full length protein (i. e. the ability to form a trimolecular complex with its corresponding IL-R/IL complex which may be soluble or membrane bound).

Preferred soluble IL-RAcP for use according to the present invention comprises or consists of the extracellular domain of the endogenous protein or a portion or fragment thereof which retains its binding capability. A suitable portion or fragment binds to the same ligands as the endogenous protein.

The extracellular domains of IL-RAcP comprise three Ig-like domains which are included in three structural domains identified herein as domain 1, domain 2 and domain 3 starting from the N-terminal end of the sequence. Domain 1 contains one Ig-like domain and two disulfide bonds. Domain 2 contains one Ig-like domain and two overlapping disulfide

bonds. Domain 3 contains one Ig-like domain and one disulfide bond. See Figure 23 for the domain organization of IL-lRAcP.

* Preferred soluble IL-RAcP polypeptides For illustration purposes, the preferred soluble IL-RAcP polypeptides described herein are human sIL-lRAcP and human sIL-18RAcP but with the help of figure 18 one skilled in the art can extrapolate it to other sIL-lRAcP polypeptides.

In one embodiment of the invention the sIL-RAcP is a soluble IL-LEAP polypeptide.

In a preferred embodiment of the invention, the soluble ILRAcP polypeptide is a polypeptide which retains the binding properties of the full length receptor accessory protein and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 21.

A most preferred soluble IL-RAcP polypeptide is a polypeptide which comprises or consist of an amino acid sequence of SEQ ID NO: 21 or an analog, derivative, active portion or fragment thereof which retains the binding properties of the full length protein.

In further embodiments of the invention when the IL polypeptide is IL-1, a soluble IL-RAcP polypeptide is a polypeptide which retains the binding properties of the full length receptor accessory protein and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably

95% identity with the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 26.

In further preferred embodiments of the invention when the IL polypeptide is IL-1, a soluble IL-RAcP polypeptide is a polypeptide which comprises or consist of an amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 26 or an analog, derivative, active portion or fragment thereof which retains the binding properties of the full length protein.

In another embodiment of the invention the sILRAcP is a soluble IL-18RAcP polypeptide In another preferred embodiment of the invention the soluble IL-RAcP polypeptide is a polypeptide which retains the binding properties of the full length receptor accessory protein and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 24.

A most preferred soluble IL-RAcP polypeptide is a polypeptide which comprises or consist of an amino acid sequence of SEQ ID NO: 24 or an analog, derivative, active portion or fragment thereof which retains the binding properties of the full length protein.

In further embodiments of the invention when the IL polypeptide is IL-18, a soluble IL-RAcP polypeptide is a polypeptide which retains the binding properties of the full length receptor accessory protein and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably

95% identity with the amino acid sequence of SEQ ID NO: 25.

In further preferred embodiments of the invention when the IL polypeptide is IL-18, a soluble IL-RAcP polypeptide is a polypeptide which comprises or consist of an amino acid sequence of SEQ ID NO: 25 or an analog, derivative, active portion or fragment thereof which retains the binding properties of the full length protein.

Deletion ranges The deletion of the C-terminus segment of the extracellular domain of IL-RAcP after the final cysteine residue of domain 3 would yield a protein which is likely to retain the binding capabilities of the full length protein.

Furthermore, deletions from the N-terminal portion of the extracellular domain of IL-RAcP up to the first cysteine of domain 1 would also yield a protein which is likely to retain the binding capabilities of the full length protein.

In a preferred embodiment of the invention, deletions of between 1 and 30 amino acids, preferably between 1 and 20 amino acids, more preferably between 1 and 15 amino acids, most preferably between 1 and 10 amino acids from the Cterminus segment of the extracellular domain of IL-RAcP yield a polypeptide which is likely to retain the binding properties of the full length protein.

A most preferred sIL-RAcP of the invention comprises or consists of the extracellular domain (of the corresponding endogenous full length IL-RAcP) to which no further deletion has been made.

Family members The three members of the IL ternary complex are highly conserved among mammalian species. This high level of sequence identity among the various species has at least two practical consequences: a) The three structural portions of the extracellular domains of IL-R and IL-RAcP can be selected from various species. It is therefore possible to construct IL-R and IL-RAcP chimeras which can include one member of the IL complex from a species and at least another member of the IL complex from another species. The chimeras can also involve a combination of nucleic acid sequences such that one generates a cross-species chimera in a single protein by reference to the cross-species sequence alignment of figures 14 to 18. See Born et al., 2000 for a selection of IL-1R family chimera. b) The prediction of the locations of the structural domains and hence the Ig-like domains in any ILR or IL-RAcP and the determination of the portion of structural domains 1 and 3 which can be deleted from any IL-R and IL-RAcP to yield a polypeptide with appropriate binding capabilities can be determined by one skilled in the art, for example from the sequence alignment comparisons of figures 17 and 18 coupled with the data of figure 24.

Figure 24 shows the sequence alignment of the human Interleukin-1 type I receptor (IL-lRI ; x16896) and the human Interleukin receptor accessory protein (IL-lRAcP ; af029213). The first and last cysteine of each domain are indicated by shading and boding. Underlined portions of sequence identified by (), () and () indicate the

positions of domains 1,2 and 3 respectively.

As a consequence, the deletion ranges provided above can also be applied to other mammalian IL-R and IL-RAcP sequences such as rat or mouse sequences.

Signal sequence for peptide production A secretory signal sequence is defined as a sequence at the N-terminus of a protein sequence which permits the protein to translocate outside the cell membrane via the endoplasmic reticulum and golgi apparatus. Through which the secretory signal sequence is specifically cleaved by enzymes, ultimately yielding a secreted and processed protein. Signal sequence and cleavage sites vary with proteins. General features of signal sequences are the hydropathy of their composition (F, L, I, V, M, A, C and P residues are generally present). The average length of a signal sequence is twenty amino-acid residues. This appears to be the length necessary for the protein to go through the membrane and be secreted. In order to produce an extracellular protein, a skilled person can fuse a mature protein and effective signal sequences to obtain a fully secreted protein. In the context of the present invention any signal sequence can be used in order to produce a mature IL polypeptide or/and an s1L-R polypeptide or/and an sIL-RAcP polypeptide for use in the invention. Preferred signal sequences are known signal sequences such as Melittin sp (MKFLVNVALVFMVVYISYIYA, SEQ ID N038), Human pancreatic Lipase sp (MWLLLTMASLISVLGTTHG, SEQ ID NO39), human IL-RI sp (MKVLLRLICFIALLISSLEA, SEQ ID N040) or human IL-lRAcP sp (MTLLWCVVSLYFYGILQSDA, SEQ ID NO41). By inserting the mature gene of a target protein downstream

from the DNA sequence coding region of the signal in a eukaryotic expression system (e. g. the insectcell/baculovirus system), a secreted protein may be produced.

Screening assays The main advantage of using soluble forms of the proteins is the ease with which these reagents enable one to format and run High Throughput Screening (HTS) assays. Homogenous and soluble assays are also very amenable to miniaturisation which can aid HTS formatting. Such assays often give more reliable data due to their homogenous nature. This is very important in HTS modes as one wishes to be able to detect with confidence the small number of compounds which may be active in the assay (often around 0. 3% of the total number of compounds screened in a typical HTS).

IL-R polypeptides used in the assays of the present invention are soluble. Endogenous active functional IL/IL-R/IL-RAcP complex involves membrane bound IL-R which contains a hydrophobic transmembrane domain and a cytoplasmic domain. Such membrane-bound IL-R polypeptides are not soluble and are not usually suitable for use in the present invention.

IL-RAcP polypeptides used in assays of the present invention are soluble. Endogenous active functional IL/IL-R/IL-RAcP complex involves membrane bound IL-RAcP which contains a hydrophobic transmembrane domain. Such membrane-bound IL-RAcP polypeptides are not soluble and are not usually suitable for use in the present invention.

In one particular embodiment of the invention the assay can be carried out with soluble IL-R and IL-RAcP polypeptides still comprising a signal sequence.

A preferred signal sequence is the naturally occuring signal sequence of the protein.

However most preferred sIL-R and sIL-RAcP polypeptides used in the assay of the present invention do not contain a signal sequence.

In another embodiment of the present invention, soluble polypeptides may be bound to a support for use in screening. This support could be through anchorage to a cell membrane or anchorage to a BIAcore sensor chip.

In a further embodiment of the invention the IL polypeptide may contain an ATG Methionine start codon in replacement of the naturally occurring signal sequence.

The present invention concerns the use of a trimolecular IL/sIL-R/sIL-RAcP complex of the invention in screening methods and assays for agents which modulate the interaction between IL and IL-R, and/or the interaction between IL-RAcP and the IL-R/IL bimolecular complex, thereby modulating the formation and/or stability of the trimolecular complex including IL, IL-R and IL-RAcP.

Methods of obtaining agents able to modulate the interaction between IL and sIL-R, or the interaction between sIL-RAcP and the sIL-R/IL bimolecular complex include methods wherein a suitable end-point is used to assess the interaction of the soluble components in the presence and absence of a test substance. Such assay systems may be used in an assay of the present invention to determine IL binding to sIL-R, sIL-RAcP binding to the sIL-

R/IL bimolecular complex or disruption of an already formed trimolecular complex. Generally of most interest is modulation of the formation of the ternary complex including IL, sIL-R and sIL-RAcP.

Various types of screening assay technologies which can be used in the present invention are briefly described below.

* Scintillation Proximity Assay (SPA) In a scintillation proximity assay, a biotinylated protein fragment may be bound to streptavidin coated scintillantimpregnated beads (e. g. as produced by Amersham). Binding of a radiolabelled polypeptide is then measured by determination of radioactivity induced scintillation as the radioactive peptide binds to the immobilized fragment.

Agents which cause a modulation (i. e. reduction or enhancement) in the measured scintillation are thus modulators (inhibitors, activators) of the interaction.

* Flashplate technology (NEN Life Sciences)

Flashplate technology (NEN Life Sciences) may also be used as an end point determination method. The surfaces of wells in a Flashplate microtitre plate contain scintillant and may also be coated with a material such as Ni-NTA or streptavidin which enables a molecule to be anchored to the surface of the well, for example via a 6-His tag or a biotin group.

If a [H] labelled molecule interacts with a molecule anchored to the surface, the proximity of the [3H] to the scintillant leads to the activation of the scintillant and the emission of light. This emission, which is directly related to the amount of interaction of the molecules, can

be measured in a microplate scintillation counter.

Inhibitors or enhancers of the interaction reduce or enhance respectively the amount of [3H] which is in proximity with the scintillant and hence reduce or enhance respectively the signal.

Test compounds may be assayed by measuring the effect on the cpm (counts per minute) output of the test assay when compared to that under identical conditions without the test compound.

In one preferred embodiment of the present invention, biotin-sIL-lR is anchored to a streptavidin flashplate, free sIL-lRAcP, and free 1251 IL-lp (or more preferably 3H IL-lp) are then added.

In another preferred configuration, sIL-lRAcP-6His is anchored to a Ni-flashplate via the 6his tag and a binary complex comprising [3H] labelled sIL-lR associated with ILlss is then added.

* Homogeneous Time Resolved Fluorescence (HTRF) Homogeneous time resolved fluorescence (HTRF) is a dual label fluorescent technique which is particularly suitable for end point determination in assays of the present invention. The technique involves labelling each of the interacting polypeptides or complexes with a fluorescent label, such that a donor label on one molecule is brought into proximity with an acceptor label when the molecules interact.

A problem with many dual-label fluorescent technologies is the background fluorescence of the assay components. Signal from an energy acceptor molecule proximal to an energy

donor is partially obscured by signal from other components of the assay, such as acceptor molecules distant from a donor molecule.

HTRF is based on the discovery that lanthanide cryptate molecules, such as europium cryptate fluoresce over a long time period when excited by light of a particular wavelength (Kolb et al 1996, Prat et al 1995). When a lanthanide cryptate such as europium cryptate is used as an energy donor label in a bioassay, energy is transferred to acceptor molecules over a long time period. Short-lived signal from distant acceptors and other assay components rapidly dies away after excitation, so signal measured a short time after excitation will be almost entirely produced by acceptors which are in close proximity to a donor molecule and are still being excited by the long lived fluorescence of the donor. Thus the assay can be used to determine the proximity of two species of biomolecules labelled with the donor and acceptor molecules respectively. Suitable energy donor labels include europium cryptate, and suitable energy acceptor labels include Cyanine 5 (Cy-5) and XL665TM (obtainable from CIS-Bio international, France).

Thus, sIL-lRAcP is for example labelled with an energy acceptor such as Cy-5 and sIL-lRI may be labelled with an energy donor such as europium cryptate. The proximity of the receptor and accessory protein can thereby be determined by measuring the delayed fluorescent emission at 665nm (for Cy-5) after excitation at 337 nm. The close proximity of the receptor and accessory protein is indicative of the presence of a trimolecular complex of IL-

1, sIL-lRI and sIL-lRAcP.

a Origentm Technology (Igen) origen technology (Igen) may also be used as an end point method in assays of the present invention. One molecule of an interacting pair is attached to a magnetic bead, for example by Ni-NTA/6-His or biotin/streptavidin linkage. A second molecule labelled with ruthenium (II) tris- (bipyridine) is added and the interaction between the molecules brings the ruthenium (II) tris- (bipyridine) label into proximity with a stimulating electrode. The electrochemical stimulus causes the label to chemioluminesce. The light output is related to the extent of interaction between the molecules.

For example, in one suitable configuration, sIL-lRAcP-6His is anchored to a Ni-NTA magnetic bead via the 6His tag and a binary complex comprising ruthenium (II) tris- (bipyridine) labelled sIL-lR associated with IL-lss is then added. Light output from the label is then measured following electrochemical stimulation.

The amount of test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used.

Typically, concentrations from about 0. 001 nM to 1 mM of putative inhibitor compound may be used. Prefered

concentrations are for example from 0. 01 nM to 100AM, most preferably 0. 1 to 50 11M, and particularly about 10 11M.

Greater concentrations may be used when a peptide is the test substance.

It has to be noted that the use of BIAcore (surface plasmon resonance) or Flashplate is more physiological than the use of HTRF. Use of the C-terminal 6His tag to anchor sIL-1RAcP

to either a Ni-NTA BIAcore sensor chip or flashplate surface orientates the protein in a manner similar to that it would be predicted to take when anchored to a membrane in its membrane-anchored full-length form. This makes such assays slightly more representative of the physiological situation compared to the assay of the proteins in free solution.

Primary screening assays Screening assay for IL modulators 'Modulation'as used herein means enhancement, disruption or interference with the formation and/or stability of a trimolecular complex consisting of IL, sIL-R and sIL-RAcP.

A modulator may activate, enhance, disrupt, reduce, interfere with or wholly or partially abolish interaction between IL and sIL-R, thereby affecting the formation of the sIL-R/IL bimolecular complex and/or activate, enhance, disrupt, reduce, interfere with or wholly or partially abolish the interaction between the sIL-R/IL bimolecular complex and sIL-RAcP, thereby affecting the formation of the trimolecular complex including IL, sIL-R and sIL-RAcP.

A modulator may also disrupt an already formed trimolecular complex including IL, sIL-R and sIL-RAcP.

In one general aspect, the present invention provides an assay method for determining the ability of a test compound to modulate the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) bringing into contact an IL polypeptide, a

soluble IL-R polypeptide, a soluble IL-RAcP polypeptide and a test compound ; and (b) determining the amount of said trimolecular complex formed.

In a most preferred embodiment, the present invention is an assay method for determining the ability of a test compound to modulate the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a test compound ; (b) bringing the test compound into contact with a defined amount of a soluble IL-R polypeptide and a soluble IL-RAcP polypeptide ; (c) adding an IL polypeptide to the mixture obtained in step (b) ; and (d) determining the amount of said trimolecular complex formed.

In another aspect, the present invention is an assay method for determining the ability of a test compound to disrupt or interfere with the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, a soluble IL-RAcP polypeptide and a test compound ; and (b) determining the amount of said trimolecular complex formed.

It is important to note that the order in which IL, the sIL-R, the sIL-RAcP and the test compounds are contacted in step (a) can vary. In other words, any order of addition of the various components is possible as long as there is no

trimolecular complex formation before the test compound is added. Furthermore, any preformation of bimolecular complexes is also acceptable as long as no trimolecular complex is preformed before the incorporation of the test compound.

In most preferred embodiments of the invention an IL used in the assay is a human mature IL-1 or a human mature IL- 18, sIL-R used in the assay is a human sIL-lRI or a human sIL-18R, sIL-RAcP used in the assay is a human sIL-lRAcP or a human sIL-18RAcP.

In the most preferred embodiment of the invention IL is the human mature IL-lss, sIL-R is a human sIL-lRI, sIL-RAcP is a human sIL-lRAcP.

* Screening assay for IL activators using an IL antagonist In a reaction medium comprising IL, an IL antagonist, sIL-R and sIL-RAcP, the presence of an entity which binds the IL

antagonist freeing IL from its interaction with the IL antagonist leads to an IL activator like effect. In fact IL can then bind sIL-R, leading to an IL/sIL-R/sIL-RAcP ternary complex formation.

The present invention therefore also concerns an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, a soluble IL antagonist, a soluble IL-RAcP polypeptide and a test compound ; (b) determining the amount of said trimolecular complex formed ; and (c) comparing the amount of said trimolecular complex formed at step (a) with the amount of said trimolecular complex formed in the absence of test compound.

A preferred embodiment of the present invention is an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a test compound; (b) bringing the test compound into contact with a soluble IL-R polypeptide, a soluble IL antagonist and a soluble IL-RAcP polypeptide; (c) adding an IL polypeptide to the mixture obtained at step (b) ; (d) determining the amount of said trimolecular complex formed; and

(e) comparing the amount of said trimolecular complex formed at step (c) with the amount of said trimolecular complex formed in the absence of test compound.

A most preferred embodiment of the present invention is an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) bringing into contact a test compound, an IL polypeptide and a soluble IL antagonist; (b) adding a soluble IL-R polypeptide and a soluble IL-RAcP polypeptide; (c) determining the amount of said trimolecular complex formed; and (d) comparing the amount of said trimolecular complex formed at step (b) with the amount of said trimolecular complex formed in the absence of test compound.

In preferred embodiments of this screening assay the soluble IL antagonist is a soluble IL antagonist polypeptide.

A most preferred soluble IL antagonist polypeptide is soluble IL-1RII or IL-18BP.

'IL-1RII Suitable soluble IL-1RII polypeptides include polypeptides which retain the binding properties of the full length ILRII and which have at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90%

identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 42.

A most preferred soluble receptor type II polypeptide for use in those assays is the polypeptide of SEQ ID NO: 42 or an analog, derivative, active portion or fragment of said sequences which retains the binding capabilities of the full length protein.

In other embodiments of the invention another signal sequence as mentionned above may be added to the IL-RII polypeptide.

'IL-18 binding protein (IL-18BP) A suitable IL-18BP polypeptide is a polypeptide which retains the binding properties of the full length IL-18BP and which has at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 43.

A most preferred soluble IL-18BP polypeptides for use in this assay is the polypeptide of SEQ ID NO: 43 or an analog, derivative, active portion or fragment of said sequences which retains the binding capabilities of the full length protein.

Screening assay for IL activators using an IL-R antagonist In a reaction medium comprising IL, an IL-R antagonist, IL- R and IL-RAcP, the presence of an entity which binds the IL-R antagonist freeing IL-R from its interaction with the

IL-R antagonist leads to an IL activator like effect. In fact sIL-R can then bind IL, leading to an IL/sIL-R/sIL- RAcP ternary complex formation.

The present invention therefore also concerns an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, an IL-R antagonist, a soluble IL-RAcP polypeptide and a test compound; (b) determining the amount of said trimolecular complex formed; and (c) comparing the amount of said trimolecular complex formed at step (a) with the amount of said trimolecular complex formed in the absence of test compound.

A preferred embodiment of the present invention is an assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising : (a) providing a test compound; (b) bringing the test compound into contact with an IL polypeptide, an IL-R antagonist and a soluble IL-RAcP polypeptide; (c) adding a soluble IL-R polypeptide to the mixture obtained in step (b); (d) determining the amount of said trimolecular complex formed; and (e) comparing the amount of said trimolecular complex formed at step (c) with the amount of said

trimolecular complex formed in the absence of test compound.

In preferred embodiments of this screening assay the IL-R antagonist is an IL-R antagonist polypeptide.

A most preferred IL-R antagonist polypeptide is IL-ra.

'IL-Ira

Suitable IL-1ra polypeptides are polypeptides which retain the binding properties of the full length IL-1ra and which have at least 60% identity, or at least 80% identity, preferably 85% identity, more preferably 90% identity, most preferably 95% identity with the amino acid sequence of SEQ ID NO: 44.

One of the most preferred soluble IL-1 ra polypeptides for use in this assay is the polypeptide of SEQ ID NO: 44 or an analog, derivative, active portion or fragment of said sequence which retains the binding capabilities of the full length protein.

Other suitable IL-lra polypeptides include human IL1HYl (Genbank accession number AF186094), mouse IL1HYl (Genbank accession number NM 019451) and mouse IL-1rn (Genbank accession number M74294).

The assay methods set forth above are considered by the inventors as preferably being primary assays for the screening of test compounds capable of disrupting, interfering with or enhancing the formation of a ternary complex including IL, IL-R and IL-RAcP.

An agent identified using one or more of the primary screens as having ability to modulate the formation of the

trimolecular complex may be further assessed using one or more of the secondary screens described below.

* Secondary Screening assays A secondary screen assay method according to the invention involves testing for the effect of the test compound that was found to be positive in one of the primary screening assays on the bimolecular complex between IL and the soluble IL-R polypeptide. This allows the interaction which is affected by the test compound to be identified as either the IL interaction with sIL-R or the sIL-RAcP interaction with the sIL-R/IL bimolecular complex.

In particular embodiments of the invention, a secondary screening assay method comprises: (a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, and a test compound that was found to be positive in one of the primary screens described above; and, (b) determining the formation of a bimolecular complex consisting of the IL polypeptide and the soluble IL-R polypeptide.

For example, in the case of a test compound being an inhibitor, the interaction disrupted by the test compound can then be determined by comparing the formation of bimolecular complex with that of trimolecular complex.

Where only trimolecular complex formation is inhibited, the compound disrupts the sIL-RAcP interaction with the sILR/IL bimolecular complex. Where the formation of both complexes is inhibited, either both interactions are inhibited or the interaction between IL and sIL-R is

inhibited.

A particular embodiment of the invention concerns a secondary screening assay method comprising: (a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, and a test compound; and, (b) assaying the presence of a bimolecular complex consisting of the IL polypeptide and the soluble IL-R polypeptide in the reaction mixture of step (a).

A further aspect of the present invention relates to a secondary screening assay method for determining the ability of a test compound to disrupt or interfere with the stability of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a defined amount of a trimolecular complex comprising an IL polypeptide, a soluble IL-R polypeptide and a soluble IL-RAcP polypeptide ; (b) contacting a test compound with said trimolecular complex; and (c) comparing the amount of said trimolecular complex present after step (b) to the amount of trimolecular complex initially present at step (a).

In most preferred embodiments of the invention an IL used in the assay is a human mature IL-1 or a human mature IL- 18, sIL-R used in the assay is a human sIL-lRI or a human sIL-18R, sIL-RAcP used in the assay is a human sIL-lRAcP or

a human sIL-18RAcP.

In the most preferred embodiment of the invention IL is the human mature IL-1, sIL-R is a human sIL-lRI, sIL-RAcP is a human sIL-lRAcP.

Secondary assays may also include testing for induction of downstream IL effectors. For example, the activity of a NFkB activation gene may be assayed after IL-1 stimulation using a commercially available NFKB driven-p-lac reporter (Aurora technology).

Agents able to modulate the interaction between IL and sILR, or the interaction between sIL-RAcP and the sIL-R/IL bimolecular complex as described above may also be used to modulate the interaction between IL and the membrane bound IL-RAcP and the membrane bound IL-R/IL bimolecular complex.

The present invention is further illustrated, but in no case limited, by the figures and the examples below.

EXAMPLES MATERIALS Oligonucleotide primers were synthesized using an ABI 392 DNA Synthesizer or obtained directly from PE Applied Biosystems. Human brain Quick Clone cDNAs were obtained from Clontech. A synthetic gene, in plasmid pDR540, encoding the mature form of human IL-1 with optimal E. coli codon usage was employed. Restriction enzymes and other DNA modifying enzymes were obtained from either Boehringer Mannheim or Stratagene. Recombinant human IL-lra, IL-1 , and soluble IL-1 receptor type II (sIL-lRII) were purchased from R & D Systems. Peptides were synthesised by Abachem Ltd.

Tissue culture media and reagents were obtained from Life Technologies Ltd. A BIAcore# 2000 was used to measure binding kinetics and affinity constants for the interactions between proteins. CM5 and NTA-chips, N-

hydroxysuccinimide, N-ethyl-N'- (3-diethylaminopropyl) carbodiimide, and ethanolamine coupling reagents (BIAcore) were used to immobilize proteins to the sensor surface using a standard amine-coupling procedure (Jonsson et al.

1991).

EXAMPLE 1: Identification of IL-1R and IL-lRAcP extracellular domains The extracellular domain of a membrane-bound receptor or accessory protein has been identified by a hydropathy analysis of the mature sequence. Trans-membrane regions are indicated in a hydropathy plot as regions with an average of twenty continuous amino acids residues with a high

hydropathy index.

The hydropathy index provides indication of the transmembrane region. Following this analysis, the precise boundaries of the region are then mapped using standard techniques known to a skilled person.

Figures 21 and 22 show a hydropathy plot of the full-length sequence of human IL-1RI and IL-18R (Figure 21) and human IL-lRAcP and IL-18RAcP (Figure 22).

The TMpred program makes a prediction of membrane-spanning regions and their orientation. The algorithm is based on the statistical analysis of TMbase, a database of naturally occuring transmembrane proteins. The prediction is made using a combination of several weight-matrices for scoring (K. Hofmann & W. Stoffel, 1993).

The vertical dashed lines delimit the region that is predicted to be the single transmembrane domain of the protein (as can be seen from the strongly positive hydropathy index in this region of the protein sequence).

The residue immediately prior to the start of the transmembrane domain of the human IL-1R and human IL-lRAcP was thus selected to be the final residue encoding the soluble form of these proteins (and indeed these Cterminally deleted proteins were secreted upon expression in the insect-cell/baculovirus system). These C-terminally deleted proteins could also be expressed in bacteria or yeast.

Figures 17 and 18 show a sequence alignment of the amino acid sequence of respectively IL-1R type I and IL-1R type II, IL-lRAcP and IL-18RAcP from human, rat and mouse.

In Figures 17 and 18, the predicted transmembrane domains of the Human Interleukin I receptor type I and II, Human

Interleukin i receptor accessory protein, Human Interleukin 18 receptor and Human Interleukin 18 receptor accessory protein are outlined in a transparent box which contains the appropriate bolded residues in accordance with the transmembrane domain determination obtained by plotting the hydropathy.

Figures 17 and 18 show a conserved location in the primary sequence of the transmembrane domain (boxed) and thus it is very likely that similar C-terminally deleted forms of other full length IL-R and full length IL-RAcP, including

other members of the IL-1 family like receptor polypeptide and IL-1 family like receptor accessory protein polypeptide are also secreted upon expression in the insectcell/baculovirus system.

EXAMPLE 2: Cloning, Expression, and Purification of Recombinant Proteins 1) Methods 1.1) IL-l The synthetic gene (SEQ ID NO 2) encoding IL-lfl (SEQ ID NO 3) was modified by PCR using Pfu DNA polymerase to introduce a Nco I restriction site at the 5'start codon (Primers: 5'-GTCCCATGGCACCGGTTAGATCTCTG-3', with the Nco I site shown in bold (SEQ ID No: 27) and 51CAGCTTATCGGCGTAGAGGAT-3', which corresponds to a region of pDR540 distal to the IL-l gene (SEQ ID No: 28). The product was digested with Nco I and Bam HI (a site present immediately after 2 tandem stop codons in the gene) and subcloned into pQE-60 (Qiagen). The resulting construct

(pQE-hrIL-1 ), which was sequenced for confirmation, is designed for expression of the mature form of the IL-1 protein (sequence of SEQ ID NO 4) with the addition of an N-terminal methionine residue. pQE-hrIL-1 was transformed into M15 E. coli containing the plasmid pREP4, which encodes the lac repressor for tight regulation of IL-1 expression (Qiagen QIAexpress system).

Transformed M15 E. coli were cultured using standard methods and then induced with Isopropyl-1-thio-beta-D- galactopyranoside (Sigma) at a final concentration of 2mM.

After 4h of growth, cells were harvested by centrifugation, 4000g (Beckman 21, JA14 rotor, 5500rpm) for 30 minutes at 4 C. The supernatant was discarded and pellets were stored at-70 C until required. One litre of culture produced approximately 4g of cells (wet weight). The cell pellet was resuspended in 50ml of 50mM Tris-HCl, pH 8.0, 5mM EDTA, O. lmM PMSF and 250) Jg lysozyme/ml (buffer A). The suspension was mixed gently and incubated at 250C for 20 minutes. Five ml of Buffer B (1. 5M NaCl, 0. 1M CaCl2, 0.1M MgCl2, and 1mM PMSF) containing 25 l of DNAse I (Stratagene) were added.

The mixture was incubated until the cell lysate showed low viscosity.

The mixture was centrifuged at 17,000 g for 1 hour at 40C and the supernatant recovered and diluted with 400ml of 10mM sodium acetate, pH 5.1 (buffer C). After centrifugation at 17,000 g for 30 minutes at 40C (to remove the precipitate) the supernatant was loaded onto an SP- Sepharose Fast-Flow (Pharmacia Biotech Inc. ) column (5cm i. d. 2.5cm) equilibrated with buffer C at a flow rate of 250ml/h. Elution was performed with 220mM sodium acetate, pH 5.1. The eluate was concentrated to 3ml and loaded at

30ml/h onto a G75 Sephadex column (2. 5x90cm) equilibrated with buffer (PBS, pH 7.4 or 100mM NaHC03, pH 8.3).

Fractions containing IL-1 were pooled and concentrated to 5ml. The last peak fraction from the G75-Sephadex yielded a single band on a 4-20 % SDS-PAGE gel under reducing conditions at the expected Mr of hIL-ljS, which is-17, 400Da.

An IL-lss affinity column was generated by cross-linking human recombinant IL-lss (20mg) purified as above to 19 of CNBr-activated Sepharose-4B matrix (Pharmacia) as described by the manufacturer.

1.2) sIL-lRI A full-length cDNA (SEQ ID Noel) encoding the human IL-1RI (SEQ ID N 12) (Hammond et al. 1999) was modified by PCR using Pfu DNA polymerase to introduce an EcoRI site prior to the start codon, and a stop codon in place of the codon for His336 (the first residue in the predicted transmembrane region) followed by an Xho I site.

Primer sequences: 5'-TGCGAATTCATGAAAGTGTTACTCAGACTTATTTG-3' (Eco RI site in bold) (SEQ ID NO: 29) and 5'-

TGACTCGAGTTACTTCTGGAAATTAGTGACTGG-3' (Xho I site in bold) (SEQ ID NO : 30). The PCR product was cloned into pBluescript II SK (+) and sequenced on both strands. The Eco RI to Xho I fragment was then subcloned into pFastBacl and used to generate recombinant baculovirus bacmid DNA by site specific transposition in DHlOBac (Bac-to-Bac System, Life Technologies). The resulting bacmid DNA from several clones was purified and used to transfect sf9 cells using Cellfectin.

For optimal expression of the Human Interleukin I receptor protein in insect cells, PCR was utilized as described herein to insert the following Kozak translation initiation consensus sequence immediately prior to the ATG start codon :"GCCACC".

Variations on this consensus sequence may also effect efficient expression, indeed other sequences may be even more effective at directing high-level translation.

Cells were routinely maintained between 1 and 5xl06 cells/ml in 1 litre polycarbonate shake flasks (Corning) containing 400ml of SF-900 II SFM serum-free insect cell media.

Culturing conditions were 270C with rotation at 110rpm in a shaking incubator. For protein production, cells were grown to a density of 5x106 cells/ml then diluted to 2x106 cells/ml immediately prior to infection with a 1: 100 dilution of the appropriate baculovirus P3 stock. Cells infected with the sIL-lRI expressing baculovirus were cultured for a further four days and then filtered serially through 100,70 and 50mm nylon sieves prior to dilution in three volumes of 0.2M glucose. A cocktail of protease inhibitors containing 1mM PMSF, 1mg/ml Leupeptin, 1mg/ml Aprotinin, 10mg/ml phosphoramidon, 1mg/ml E64, and 1mM EDTA was added into the mixture.

The crude broth was diluted 1/3 with 10mM MES pH 6.0 and applied at 25ml/min to an SP-Sepharose XL Streamline-50 (Amersham Pharmacia Biotech. ) expanded-bed chromatography column (i. d. 2. 5cm x h. 100cm, 20cm packed bed) preequilibrated in 10mM MES pH 6.0. The column was washed before elution with 350mM NaCl/lOmM HEPES pH 7. 5. The eluted material containing the soluble IL-1RI was applied to an IL-1ss-Sepharose column (2. 5mg IL-1ss/ml of gel),

equilibrated in PBS at a flow rate of 5ml/h. The column was then washed extensively with PBS and eluted with lOOmM Glycine-HCl, 150mM NaCl, pH 3.0. Fractions (2ml) were neutralised immediately with 2501 of 1M Tris-HCl, pH 8.0.

Aliquots of each step were analysed by SDS-PAGE and Western blotting using an IL-RI (N-20) polyclonal antibody (Santa Cruz Biotechnology Inc.). Glycine-HCl pH 3.0 elution fractions yielded a single band on a 4-20 % SDS-PAGE gel under reducing conditions at-44, 000 Da due to the glycosylation of the receptor. The expected Mr of nonglycosylated human sIL-lRI is-36, 381 Da.

Protein concentrations were measured by U. V. spectrophotometry using a theoretical molar extinction coefficient of 50,810 M-1. cm-1 confirmed by the Bradford assay using bovine serum albumin as standard.

The sIL-lRI produced has the sequence of SEQ ID N013.

1.3) sIL-lRAcP Many tags may be introduced either to the C-terminus or Nterminus of the protein using standard molecular biological techniques. Examples of purification tags that may be used include, c-myc, FLAG, Hemagluttinin A, V5, E-tag, BirA-tag and 6His.

For the human soluble IL-lRAcP (generated from GenBank AF029213, nucleic sequence of SEQ ID N020, amino acid sequence of SEQ ID NO 19), a 6His tag was utilized. Thus the following sequence was added after the final GAA (glutamic acid) triplet codon sequence :

CAT CAC CAT CAC CAT CAC (SEQ ID NO : 36) The final CAC was followed by a'TGA'STOP codon.

*The following oligonucleotide primers were used in PCR reactions to amplify human IL-lRAcP sequences. The primers were based on the published IL-lRAcP cDNA sequence (Genbank accession number AF029213, (Huang et al. 1997)), 51GGATGACACTTCTGTGGTGTG-31 (SEQ ID NO: 31) and 5'- TCCTTTTCATTATTCCTTTCATACA-3' (SEQ ID NO: 32). PCR products were amplified from human cortex cDNA (Clontech) using Taq polymerase. The resulting products were cloned into pCRScript (Stratagene) and a number of clones characterized by DNA sequencing. Three mismatches were found in the fulllength clone, two of which lead to changes in the encoded amino acids. These were corrected by site directed mutagenesis.

Using the corrected clone as the template, the extracellular portion of the IL-lRAcP (residues 1-359) was generated by PCR using Pfu DNA polymerase and the primers 5'-TCGCCACCATGGACACTTCTGTGGTGTG-3' (5'primer) (SEQ ID NO:

33) and 5'TCGGAATTCCTCAGTGATGGTGATGGTGATGTTCCACTGTGTATCTTGGAGC-31 (3' primer) (SEQ ID NO : 34). The 5'primer includes a Kozak consensus sequence flanking the start codon while the 3' primer encodes six Histidine residues, a stop codon and an EcoRI site following the final extracellular residue (Glu359) of the IL-1RAcP. The PCR product was 5' phosphorylated with polynucleotide kinase, purified then blunt-end cloned into the StuI site of the baculovirus transfer vector pFastBac 1 (Life Technologies). A clone was isolated with the gene ligated in the positive orientation with respect to the polyhedrin promoter and confirmed by

sequencing. The resulting bacmid DNA from several clones were purified and used to transfect sf9 cells using Cellfectin as described by the manufacturer (Life Technologies).

For optimal expression of the Human Interleukin I receptor accessory protein in insect cells, PCR was utilized as described herein to insert the following Kozak translation initiation consensus sequence immediately prior to the ATG start codon :"GCCACC".

The soluble IL-lRAcP (sIL-lRAcP) was produced in insect cells essentially as described above for sIL-lR except that the cells were cultured for five days after infection.

Prior to chromatography on the streamline column the crude broth was diluted two-fold with glucose and elution was performed with 0.5M KCl/lOmM Imidazole/20mM Tris-HCl pH 8.0 at 10ml/min. This material was loaded at 0.5ml/min onto a Ni2+ charged NTA (Qiagen) column (lcm i. d. x 4cm h. ). The column was then washed sequentially at 40C with 0. 5M KCl/20mM Tris-HCl pH 8.0, 20mM Imidazole, then 1M KCl/20mM Tris-HCl pH 8.0, and finally 0.5M KCl/20mM Tris-cl pH 8.0, 20mM Imidazole until a stable Assonm baseline was reached. The 6-His tagged sIL-lRAcP was eluted with 0. 5M Imidazole/100mM KCI/20mM Tris-HCl pH 8.0 and dialysed extensively against PBS.

The protein concentration was determined using a theoretical molar extinction coefficient of 59,630 M-1. cm' and confirmed by Bradford protein assay.

2) Results: SDS-PAGE gels (4-20%) of the samples coupled with colloidal blue staining and Western blotting and hybridization with the appropriate commercially available antibodies were performed to check the purity and identity of the proteins respectively from the purification schemes. Molecular weights were verified by Western blotting. The biological activity of the purified IL-1ss was checked by microphysiometry as described (Hammond et al., 1999) and was similar to material from commercial sources. The yield of recombinant IL-1ss was -5mg/litre of culture with a purity of > 95% as determined by SDS-PAGE. Mass spectrometry of ILlss revealed partial processing of the initiating methionine.

Aliquots from each step of the sIL-lRI purification were analysed by SDS-PAGE and Western blotting using an IL-RI (N-20) polyclonal antibody. The-44, OOODa band confirms the presence of the soluble glycosylated IL-1RI. The predicted molecular weight of the receptor lacking the signal sequence is 36,381 Da.

Using Penta-His monoclonal antibodies for detection (Qiagen) sIL-lRAcP gave a molecular weight of-45, OOODa indicating a significant glycosylation of the protein. The predicted molecular weight of sIL-lRAcP with the 6-His tag is 39,935Da. A yield of 1.5mg/L culture supernatant was generally obtained for both proteins.

The sIL-lRAcP produced has the sequence of SEQ ID N021.

EXAMPLE 3: Surface Plasmon Resonance 1) Methods: 1.1) Immobilization of proteins The BIAcore running buffer was 10mM HEPES, pH 7.4, 150mM NaCl, 1mM EDTA, and 0. 005% P20 surfactant (Polyoxyethylenesorbitan) (HBS, BIAcore). Equal volumes of 0. 1M N-hydroxysuccinimide and 0. 1M N-ethyl-N'- (3diethylaminopropyl) carbodiimide were mixed, and zu were injected over the surface of the sensor chip to activate the carboxymethylated dextran at 5I/min. hIL-1ss 50 g/ml in lOmM acetate, pH 5, sIL-lRI 10 g/ml in 10mM acetate, pH 5.5, and sIL-1RAcP 10 g/ml in 10mM acetate, pH 4.5, were coupled respectively in flow cells 2,3, and 4. Each coupling was followed by 351 of ethanolamine to block remaining active carboxyl groups. The immobilization

procedure was carried out at 25 OC and at a constant flow rate of 51/min. Flow cell 1, immobilization control, has been coupled with BSA 10 g/ml in 10mM acetate, pH 5.0.

1.2) Kinetic Assays on the BIAcore All experiments were carried out at 250C with a constant flow rate of 20 g/min in HBS buffer. 40Al of the analyte were injected for 2 min (association phase), followed by HBS for 5-min (dissociation phase). Equal volumes of each protein dilution were also injected over a mock blocked surface to serve as a blank sensorgram for subtraction of bulk refractive index background and non-specific binding of the analyte.

All kinetic assays were followed by an injection of 20 Al of 100mM HCl to dissociate any remaining bound ligand.

Experiments were carried out with a low level of immobilization, high flow rate (2081/min), and appropriate concentrations of analyte to limit mass transport effects. Furthermore, mass transport limitations were checked using the BIAsimulation program. Curves derived from these assays were used to generate kinetic constants. Five concentrations of ligand (in duplicate) were injected over the four flow cells in a random sequence.

All protein surfaces displayed a high capacity for ligands relative to surface RU (resonance units) density as well as long stability. Typically, an average of more than 85% binding capacity was preserved after 100 cycles of ligand binding and regeneration with HCl.

The amount of IL-lss on flow cell 2 was 763 RU. The amount of SIL-LORI on flow cell 3 was 2413 RU, and the amount of sIL-lRAcP on flow cell 4 was 1092 RU.

1.3) Data Analysis Sensorgrams were analyzed by non-linear least-squares curve fitting using the BIAevaluation program 3.0 (BIAcore).

Kinetic constants were generated from the association and dissociation curves from the BIAcore experiments by fitting to a single-site binding model (A + B = AB). This model gave a single exponential fit with a X2 < 0. 5. Comparison fitting with more complicated models did not give a better

interpretation of the data. The equation (1)

Rt = Roe') (Eq. 1)

was used for the dissociation phase, where Rt was the amount

of ligand remaining bound in RU at time t and to was the beginning of dissociation phase. The final dissociation rate constant, koff, was calculated from the mean of the

values obtained from a series of injections. To analyze the association phase, the equation (2)

Rt = . 0-e-- (Eq. 2)

was employed where Req was the amount of bound ligand (in RU) at equilibrium, to was the starting time of injection, and kg = kon. C +koff, where C was the concentration of analyte injected over the sensor chip surface. The association rate constant, kon, was determined from the slope of a plot of ks versus C. The apparent equilibrium dissociation constant KD was determined from the ratio of these two kinetic constants (keff/ken).

Data were first subtracted from controls and zeroed using BIAevaluation 3.0 software (BIAcore) before global fitting to a bimolecular reaction model. Rate equations were generated and then numerically integrated for the entire data set simultaneously. Association (kon) and dissociation (koff) rate constants were obtained for the entire data set along with residual S. D. (standard deviation) values, replication S. D. values, and correlation coefficients. Fits were not improved by using a mass transport model.

2) Results: 2.1) Interactions between the members of the IL trimolecular complex Interaction of IL-1ss and sIL-lRI on the sensor chip Figures 1A, 1B and 1C show the interaction respectively of IL-lp (A), sIL-lRI (B) and sIL-lRAcP (C) purified proteins with separate flow cells of the BIAcore chip which have been respectively coupled with sIL-lRI, IL-lp or sIL-lRAcP.

Channels 2,3, and 4 coupled with IL-1&num;, sIL-lRI, and sILlRAcP are represented respectively with (........), (--),

and (- -) lines. The concentrations of proteins used for free ligands are 50g/ml, lOg/ml, and lOg/ml respectively for IL-1ss, sIL-1RI, and sIL-lRAcP. Figure 1A, 1B, and 1C represent interactions of IL-ljS, sIL-lRI, and sIL-lRAcP respectively.

Figure lA-C indicates the ability of each ligand to bind specifically to its cognate receptor on the chip. Thus, hIL-lss recognises sIL-lRI immobilized on flow cell 3, while free sIL-lRI recognises IL-1ss on flow cell 2. Neither protein interacts with immobilized sIL-lRAcP. Moreover, free sIL-lRAcP does not recognize immobilized IL-1ss or sIL- 1RI (Flow cells 2 and 3, respectively), though it can interact to some extent with itself (flow cell 4).

Formation of an IL-1ss/sIL-1RI/sIL-RAcP ternary complex Figures 2A and 2B show the interaction of previously formed IL-lj8/sIL-lRI complex with an excess of IL-lp (A) or sIL-lRI (B) being added on separate flow cells of the BIAcore chip

which have been respectively coupled with sIL-RI, IL-lp or IL-lRAcP.

The two proteins were incubated at room temperature for 15 minutes in HBS buffer (10mM HEPES, 150mM NaCl, pH 7.4) at different ratios. Figures 2A and 2B represent the complex interaction with the sensor chip when an excess (approximately 2: 1) of IL or IL-R was applied respectively ), (), and () lines represent channels 2,3, and 4 coupled with IL-10, sIL-lRI, and sIL-lRAcP respectively.

As shown in figure 2, the pre-formed IL-l/sIL-lRI binary complex interacts with immobilized sIL-lRAcP to generate a ternary complex.

In order to demonstrate the sequential mechanism of complex formation, IL-10, sIL-lRI, and sIL-1RAcP were serially injected. With both methods, ternary complexes were obtained on the IL-10 and sIL-lRI chip surfaces, thus mimicking the predicted interaction of these molecules at

the cell surface : i. e. first IL-1 ? binds to sIL-lRI and sIL-RAcP binds to the IL-lp/IL-RI complex.

Figure 3 shows the formation of the ternary complex using two different methods. Figure 3A shows the sequential injection of IL-lp and sIL-1RAcP on separate flow cells of the BIAcore chip which have been respectively coupled with sIL-lRI, sIL-lp or sIL-lRAcP- 6His. IL-Iss and sIL-lRI (not shown) were bound to their respective specific molecule on the chip to form a binary complex and then sIL-lRAcP was injected before the dissociation phase to form the ternary complex (coinject command on the BIAcore). Concentrations of IL-10, sIL-lRI, and sIL-lRAcP used were 10 g/ml. The analysis of the binding of sIL-lRAcP on the binary complex gave an affinity constant (KD) of 1.37nM and 4.28nM

respectively for IL-1ss on sIL-1Rlchip (figure 3A) and sIL- 1RI on IL-lp chip (figure not shown). Figure 3B shows the capture of sIL-lRAcP6His on a NiNTA chip followed by the addition of binary complex IL-1ss/sIL-1RI (-) or IL-1ss alone (----).

Figure 3B examined whether immobilization via the Cterminal 6-His tag prevents the formation of the ternary complex. When sIL-lRAcP was captured on a Ni-NTA (nui2± Nitriloacetic acid) sensor chip and pre-formed IL-1ss/sIL-1RI binary complex was injected, the formation of a ternary complex was observed, while no signal was observed if IL-1ss or sIL-lRI (not shown) were injected alone.

Interactions with IL-ira The IL-ira inhibits the biological effects of IL-1α and IL- 1ss by competing with these agents for cell surface receptors. IL-lfl, IL-1α, and IL-ira all bind with comparable affinity to the IL-1RI (Dinarello, 1996). The IL-lRAcP forms a ternary complex with IL-1RI and either IL-la or IL- 1ss, but not with IL-ira (Greenfeder et al. 1995). This was confirmed in figure 4 where the inability of the IL- 1ra/sIL-1RI to form a ternary complex with the sIL-lRAcP is shown.

Figure 4 shows a comparison of the IL-1ss and IL-ira interactions with separate flow cells of the BIAcore chip which have been respectively coupled with sil sIL-lRAcP or sIL-lRI when sIL-RAcP is added during the dissociation process.

Concentrations of cytokine were 0. 5 g/ml and lg/ml for IL-

1ss and IL-1ra respectively. Concentration of sIL-lRAcP was 10u.g/ml.(), (---), and (----) lines represent channels 2,3, and 4 coupled respectively with IL-1ss, sIL- 1RI, and sIL-lRAcP.

In the same way, the addition of sIL-lRAcP during the dissociation phase of the IL-lra/sIL-lRI complex has no effect on the rate of dissociation. However, the dissociation rate (koff) is much slower than that of the ILlss/sIL-lRI complex (see Table 2).

Interaction of the peptide of SEQ ID NO : 35 with sIL-lRI The peptide FEWTPGYWQPYALPL (AF11733) SEQ ID No: 35, which was identified by phage display, is able to form a tight complex with the sIL-lRI (Yanofsky et al. 1996).

Figures 5A and 5B show the binding of FEWTPGYWQPYALPL peptide on a BIAcore sensor chip. Figure 5A represents the interaction of the peptide with the different channels.

Figure 5B shows the interaction of a pre-mixed complex between the peptide and sIL-lRI in excess. (), (-), and (----) lines represent channels 2,3, and 4 coupled with IL-lss, sIL-lRI, and sIL-lRAcP respectively.

Figure 5 A confirms the interaction between the sIL-lRI and the peptide of SEQ ID NO 35. A pre-formed Peptide/sIL-lRI complex was unable to interact with immobilized IL-ljS (Figure 5B) nor was it able to form a ternary complex with sIL-lRAcP.

Interaction with the soluble IL-1R Type II Figure 6 shows the involvement of the soluble form of the decoy IL-1R type II in the IL-1R complex. Figure 6A shows a comparison of binding of IL-lp on a sIL-1RII flow cell (- --), on a sIL-lRI flow cell (---) and on a sIL-lRAcP flow cell (), with a coinjection of sIL-lRAcP following cytokine injection. Figure 6B uses the same chip as the previous experiment and shows the interaction of sIL-lRII with coupled IL-113, sIL-lRI and sIL-lRAcP. This shows the ability of sIL-lRII to form a binary complex with IL-1ss, but this binary complex with soluble receptor was unable to form a ternary complex with sIL-lRAcP. It has to be noted that although the kon of sIL-lRII for IL-lp is slower than for sIL-lRI, the koff of sIL-lRII for IL-lp is much slower than that for sIL-lRI. In physiological terms, this decoy receptor type II is slower than IL-1RI to capture circulating IL-lp but once type II binary complex is made, this complex is very slow to dissociate and there is no need for an accessory protein to stabilize it.

2.2) Kinetic studies

Figure 7 shows binding kinetics of the IL-Iss/sIL-1RI binary complex (figure 7A) and IL-1ss/sIL-1RI/sIL-1RAcP (figure 7B). In figure 7A, free sIL-lRI was run on the IL-1ss chip using five different concentrations (119,59. 5,29. 8,14. 9, and 7.4nM, upper to lower curve respectively). In figure 7B, binding kinetics of sIL-lRAcP on the IL-1ss/sIL-1RI binary complex is shown. Figure 7B represents a range of sIL-lRAcP concentrations on the receptor chip following an IL-lss capture step with free IL-1ss (50g/ml). The range of sIL-

lRAcP concentrations was 119nM, 29.8nM, 14.9nM, and 7. 4nM from upper to lower curves respectively. The sensorgrams shown (corresponding to figures 7A and 7B) were subtracted from controls. See table 2 for results.

IL-IjS/sIL-lRI binary complex Figure 7A presents kinetic data for the interaction of the sIL-lRI (concentration range 7.4-119 nM) with the IL-1ss

chip. The KD observed was 1. 53 nM. A similar value was observed in the reverse experiment with free IL-1ss on the receptor chip (concentration range 0.12-50 nM).

IL-Iss/sIL-1RI/sIL-1RAcP ternary complex Two protocols were used to determine the kinetic constants

of the sIL-1RAcP on the IL-1ss/sIL-IRI binary complex. First, the pre-formed binary complex (2 : 1 molar excess of IL-1ss) was tested over a range of concentrations on the sIL-lRAcP chip. Second, IL-lss was captured on the receptor chip prior to the application of the sIL-lRAcP (Figure 7B). The receptor chip was preferred over the IL-1ss chip in this kinetic analysis since the formation of the ternary complex was more pronounced when the receptor was immobilized.

Kinetics of molecules involved in IL-1 signalling Data obtained from the SPR experiments using the BIAcore sensor chip are shown in table 2. The two first rows (experiments 1 and 2) indicate rate constants (Kon and Koff) and equilibrium binding constants (KD) for the simple kinetics between IL or sIL-R as shown in figure 7A. Rows 3 & 4 indicate the same kinetics in the presence of a constant concentration of sIL-lRAcP (10 Ilg/ml -250nM) in

each sample of free IL-ljS or sIL-R. Rows 5, and 6 represent binding constants of kinetics of a pre-formed binary complex (protein in excess in bold) on the sIL-lRAcP chip.

Ranges of concentrations of the binary complex were 7. 4nM- 119nM and 2.9-45. 8nM for experiments 5 and 6 respectively.

Experiment 7 (figure 7B) represents a two-step kinetic with a first capture of a constant concentration of IL-l (5g/ml) on the sIL-R chip followed by the injection of a range of concentrations of sIL-lRAcP (7. 4nM-119nM). Binding analyses of molecules interacting with the IL-lfl/sIL- lRI/sIL-lRAcP complex: Row 8 represents binding data for the Interleukin-1 receptor antagonist on the sIL-R chip.

Concentration range applied was 1.82-29. 2nM. Row 9 represents binding data for the soluble IL-1 receptor type

II on the IL-1ss chip. Concentration range applied was 4. 2- 67. 8nM. Row 10 represents binding data for AF11733 peptide (FEWTPGYWQPYALPL-OH, Seq NO: 35) on the sIL-R chip.

Concentration range applied was 240-7760nM. All constants were calculated as described herein. The standard deviation was less than 0.1 and the X2 parameter is lower than 0.1 except for the experiment 10 (row 10 of table 2).

TABLE2

Exp IL-1ss/sIL-1RI Sensor Kon Koff KD X2 interaction surface (l/Ms) (l/s) (nM) 1 IL-lp sIL-lRI 6. 9x105 2. 5x10-3 3. 61 0.03 +0. 06 2 sIL-lRI IL-lp 3. 9x106 6. OX10-3 1.53 0.08 +0. 03 3 IL-lp + sIL-sIL-lRI 6. 5x105 1. 9x10-3 2.32 0.09 1RAcP (10 g/ml) : tu. 05 4 sIL-1RI + sIL-IL-lp 2. lxl06 2. 3xlO-3 1.10 0.08 1RAcP (10 g/ml) ~0. 03 Exp Interactions between IL-lp/sIL-lRI and sIL-lRAcP 5 IL-lp/sIL-lRI sIL-5. 1x105 2. 1x10-3 4.10 0. 05 (2/1 excess) 1RAcP +0. 08 6 IL-lp/sIL-lRI sIL- 9.1x105 7. 7x10-4 0. 85 0. 03 (1/2 excess) 1RAcP +0. 01 7 IL-lp (50flg/ml) sIL-lRI 9. 7x105 7. 7x10-4 0.77 0.05 + sIL-lRAcP : tu. 02 Exp Additional interactions 8 IL-1ra sIL-lRI 2. 6x105 1. 9x10-4 0. 75 0.03 ~0.01 9 sIL-lRII IL-lp 2. 8x104 3. 8x10-4 13. 5 0. 10 ~1. 2 10 Peptide of sequence of sIL-1RI 3. 2x105 1. 1x10-3 3.37 1-12 SEQ ID NO : 35 ~0. 26 Table 2 shows the results of binding analysis of the IL- ImsIL-IRI/sIL-lRAcP interaction using surface plasmon resonance and with molecules involved in this interaction.

'IL-Ira IL-lra interacts with the sIL-lRI but does not form a ternary complex with the sIL-lRAcP (Fig. 4). IL-lra over the concentration range 1.82-29. 2nM was applied to the sIL-R

chip. Kinetic differences were apparent between IL-lra and IL-lss. The major difference is due to the dissociation process which is significantly slower in the case of IL-lra compared to IL-1ss (Koff of antagonist 1. 9x10-4 s-1 compared to agonist 2. 5xlO-3 S-l, see table 2, compare rows 1 and 8). However, the affinity of IL-1) 3 for the sIL-R in the presence of sIL-lRAcP is similar to that of the IL-lra/sIL-lRI

binary complex (table 2).

* sIL-lRII IL-1RII interacts with IL-1ss but no signalling results from the formation of this complex in cells (Liu et al. 1996), due essentially to the lack of a cytoplasmic domain on IL- 1RII. A range of soluble IL-1RII concentrations (4.2- 67. 8nM) was applied to the IL-1ss chip. This enables the comparison of the interaction of IL-1ss with these two receptor types and its implications for the signal transduction mechanism. The interaction of IL-lwith sILlRII is about 10-fold weaker (KD = 13.5 nM) than with the sIL-lRI. This is consistent with the fact that the type II receptor acts as a decoy receptor competing weakly with type I for agonist binding. e Peptide of sequence SEQ ID NO : 35 The peptide FEWTPGYWQPYALPL (Genbank accession number: AF11733), which was obtained by phage display against immobilized sIL-lRI (Yanofsky et al. 1996), was applied to

the receptor chip (240-7760nM). As observed in Figure 5, this peptide inhibits binary complex formation with IL-ljS

(see Table 2). Binding constants for the peptide/sIL-lRI and IL-Iss/sIL-1RI complexes were similar. The lower KD for the IL-lra/sIL-lRI complex is due primarily to a slower off rate (table 2).

In the present application, the ability of recombinant extracellular domains involved in the IL-Iss/IL-lRI/IL-lRAcP complex to interact and form stable binary and ternary complexes in solution, or on a sensor chip (Figures 1-3) has been demonstrated. As reported previously, for full length proteins (Greenfeder et al. (1995), Yoon and Dinarello, (1998) ), there was no interaction between IL-ljS and sIL-lRAcP, and no direct interaction between sIL-lRI and sIL-lRAcP. A weak interaction was shown between free sIL-lRAcP and immobilized sIL-lRAcP (Figure 1C).

The ternary complex could be generated after sequential injection of IL-lfl and sIL-lRAcP over the sIL-lRI chip or after injection of sIL-lRI and sIL-lRAcP over the IL-Iss chip. In the former case, the formation of the ternary complex was more pronounced (figure 3B), although the chip surfaces had the same level of immobilization in molar terms. This probably reflects coupling of IL-1ss in an unfavourable orientation when IL-lp is anchored on the chip. A ternary complex was also obtained after addition of a pre-made IL-IjS/sIL-lRI binary complex to the sIL-lRAcP chip (Figure 2).

The KD for the IL-Iss/IL-1RI binary complex in cells lacking accessory protein was 2 nM (Greenfeder et al. 1995 ; Laye et al. 1998). A significant stabilization of the complex was observed in cells co-expressing the IL-1RI and sIL-1RAcP,

with a five-fold increase in affinity (KD - 0. 4 nM) (Greenfeder et al. 1995). Kinetic data from our SPR experiments with sIL-lRI and sIL-lRAcP yielded a KD of 1.53nM and 3. 61nM for the free sIL-lRI on anchored-IL-ljO and free IL-lss on anchored-sIL-lRI respectively. When the analytes were loaded in the presence of sII.. -lRAcP the apparent Ko values were 1.1 nM and 2.32 nM, thus confirming the stabilizing effect of the accessory protein on the binary complex.

The interaction of pre-formed IL-1ss/sIL-IRI complex with the sIL-lRAcP sensor chip gave a significantly higher affinity (KD = 0. 85 nM). To further study the formation of the ternary complex, we used a two-step protocol. IL-lss was first captured on the receptor chip and varying concentrations of sIL-lRAcP were immediately applied (see Table 2, row 7). The KD obtained, 0. 77nM, correlates well with values obtained in different cell lines expressing the IL-1RI and IL-lRAcP by Greenfeder et al. 1995.

The IL-lra interacts strongly with IL-1RI but is unable to induce signal transduction and acts as a competitive antagonist of IL-1. Crystallographic studies have demonstrated that IL-lra interacts with the first two domains of the receptor in the same manner as IL-1ss and ILla (Schreuder et al., 1997). However, unlike the agonists, it is unable to interact with the third domain, a crucial step for the formation of the ternary complex with accessory protein. As expected (Figure 4), IL-lra interacts strongly (KD = 0. 75nM) with the soluble receptor type I, however a ternary complex with IL-lRAcP cannot be formed.

The IL-1RII is known as a decoy receptor, it does not

contain a significant intracellular domain, crucial for signal transduction. Our data provides evidence that there is no interaction between the IL-l/sIL-lRII binary complex and sIL-lRAcP (figure 6). Using conditions identical to those adopted previously for sIL-lRI, which gave a ternary complex with the sIL-lRAcP chip, no signal was observed when a pre-formed sIL-lRII/IL-lss complex was injected. To confirm this result, sIL-lRII was passed over the IL-10 chip and IL-lp on sIL-lRII chip, both gave a binary complex, and injection of sIL-lRAcP gave no further signal. Lang et al.

(1998) showed that chimeric IL-lRIIextra-membranar/ILlRIcytoplasmic receptor was able to form a ternary complex with IL-lRAcP and induces signal after IL-lp stimulation.

Malinowsky et al. (1998) suggested that the presence of ILlRAcP was required for binding of an agonist to the ILlRII. In contrast, the present application provides evidence that soluble IL-1RII binds IL-10, but does not form a ternary complex with sIL-1RAcP. A recent publication (Neumann et al. , (2000) ) demonstrates how a cell-surface IL-lRII/IL-lp binary complex is not able to form a ternary complex with the IL-lRAcP thus resulting in no intracellular signalling. This finding supports the hypothesis that the role of the IL-1RII is as an IL-1 'scavenger'-competitor of the IL-1R-leading ultimately to the prevention of IL-lp to form a ternary complex (and hence signal) with the IL-1RI and the IL-lRAcP.

Our results confirm that sIL-lRII has the potential to modulate IL-1 signalling by recruiting agonists to form a non-signalling complex with a 9-fold higher KD (13. 5nM)

compared to that for the sIL-lR/IL-ljS binary complex (KD 1. 53nM). However, some type II receptors have shown a higher affinity with a preference for IL-lfl over IL-la and

IL-1ra (Liu et al. 1996). But the koff of the sIL-lRII/IL-lp binary complex is significantly slower than that of a type I binary complex, indicating a physiological role for the type II receptor.

EXAMPLE 4: Optimization of the labelling of the proteins for the HTRF Assays 1) Method The interactions characterized above were adapted and developed for use in an assay based on the HTRFO technology.

Proteins Proteins at the following concentrations were used in the development of assays.

- IL-1ss (Mw 17,400) Concentration=1. 42mg/ml - Extracellular domain of IL-1R type I (sIL-lRI) (MW 44,000) Concentration=1. 49mg/ml - Extracellular domain of IL-lRAcP (sIL-1RAcP) with a C- terminal 6-His tag (MW 45,000) Concentration=1.58mg/ml - Interleukin-1 receptor antagonist (IL-Ira) (MW 17,100) Concentration=20g/ml - Peptide AF11377 (Seq No: 35) (MW 1,858) Concentration=lmM

Labelling of Reagents The reagents were labelled with activated-Europium Cryptate, Activated-XL665 or Activated-CY5 through amine groups using heterobifunctional reagents and purified using gel filtration chromatography to remove unreacted reagents, according to standard protocols for amine coupling via EDC/NHS activation.

Cryptate labelling - sIL-1 Receptor type I Labelled sIL-1 Receptor at a concentration of 0.130 mg/ml and a Rmf=2. 05 K/Receptor (Ratio of Dye Cy5/Protein). The product was stored in Phosphate buffer 100mM pH 7.0 with 0.1% BSA and 0.1% Tween 20 and stored at-80 G under aliquots of 1val.

CY5 labelling - sIL-1 Receptor Accessory protein: Labelled sIL-lRAcP at a concentration of 0. 065mg/ml and Rmf=2.6 CYS/sIL-lRAcP was stored in Phosphate buffer 100mM pH 7.0 with 0.1% BSA and stored at-80 C in 101 aliquots.

1.1) Semi direct assay The semi-direct system consisted of tagging the sIL-1RAcP with a 6His affinity-tag. However, this protein was unable to bind to its anti6His-K conjugate.

1.2) Direct assays Then several direct assay formats were tested.

In the first direct assay to be tested the SIL-IRAI is directly labelled with cryptate and the sIL-lRAcP is

labelled with XL665.

However, when the trimolecular complex was formed, maximum signal obtained with XL665 was very low (around 30%). The low signal being attributed to high steric hindrance.

In the second assay system the sIL-lRAcP was directly labelled with cryptate and the sIL-lR was labelled with XL665. In these conditions no signal was detected.

The third assay system to be tested corresponds to the first direct assay system in which XL665 is replaced by CY5. In these conditions a maximum specific signal of around 1200% was obtained.

This third assay system has been choosen.

Example 5: HTRF assay Direct Assay System A direct assay format as shown in Figure 8 was designed involving sIL-lRI directly labelled with Europium cryptate and sIL-lRAcP directly labelled with CY5.

The interaction reaction may be carried out in a one step or a two step procedure: binary complex formation followed by ternary complex formation. After testing as described below, the two procedures were shown to give equivalent results so only the one step procedure was used subsequently.

All the reagents were diluted in HEPES buffer 10mM, pH 7.4, O. 1% BSA, 0. 005% Tween 20 and 0.2M KF.

1) Protocol The assay was set up as follows: 501 IL-1ss (or buffer in the negative test) 501 Cryptate conjugate 501 CY5 conjugate 50Al buffer The reaction was incubated at room temperature for at least 15 minutes, the equilibrium being reached after about 1 hour (see figure 10). Readings were taken on a Discovery instrument (Packard) under standards conditions for HTRF measurement (delay 50s, gate 400ILs) 2) Results The results were expressed as DeltaF: R=ratio= (F665nm/F62onm) xl0" DeltaF (%) = [ (Rpositive-Rnegative)/Rnegative]x100 Negative=all reagents except IL-1ss ligand Positive=all reagents with IL-1ss ligand Evaluation of the Assay Performances The first experiment was performed in the following conditions:

IL-1ss was used at a final concentration of 50 nM. sIL-lRI-Eur was used at final concentration of 1. 25 or 5 nM. sIL-lRAcP-CY5 was used at final concentration of 10 or 50 nM.

The results obtained are given in table 3 below.

Table 3:

[sIL-1RI-Eur] nM 1. 25 5 [sIL-lRAcP-CYS] nM 10 50 10 50 Delta F% 1404.9 1034.9 1420. 9 1213.1 As expected we observe that in the absence of IL-lfl ligand, sIL-lRI-Eur and sIL-1RAcP-CY5 do not interact with each other thus no energy transfer signal was generated in the negative.

In presence of the ligand, the formed ternary complex gives rise to a high-energy transfer signal. A signal of around 1200% was observed after a 10min incubation.

Specificity of the signal In the absence of the ligand no energy transfer signal was obtained.

We also investigated the absence of signal by replacing either sIL-lRI-Eur by an antibody-Eur conjugate or sIL- 1RAcP-CY5 by an antibody-CY5 conjugate both in negative (without ligand) and positive (with ligand) wells. These reagents were used at about the same concentration in cryptate or CY5 than the reference reagents we are using in the assay.

No non-specific signal was detected using antibody-Eur instead of sIL-lRI-Eur. No non-specific signal was detected using antibody-CY5 instead of sIL-lRAcP.

Kinetics and stability of the signal sIL-1ss was used at a final concentration of 10 nM sIL-lRI-Eur was used at a final concentration of 1.25 nM sIL-lRAcP-CY5 was used at a final concentration of 10 nM Reading after 10 mins, 30 mins, 3 hours and overnight.

The results obtained are shown in Figure 10.

The equilibrium is reached after 10 mins at room temperature. After an overnight incubation at room temperature less than 10% of the signal is lost.

Influence of the KF concentration IL-1ss was used at a final concentration of 10 nM sIL-lRI-Eur was used at a final concentration of 1.25 nM sIL-lRAcP-CY5 was used at a final concentration of 10 nM All reagents were diluted in HEPES 10mM, pH 7.0, 0. 1% BSA, 0.005% Tween 20 and KF O. 1M, 0.2M or 0.4M.

The results obtained are shown in the table 4 below.

Table 4:

KF concentration (M) 0. 1 0. 2 0. 4 Delta F% 1214.4 1211 1233.9 KF concentration between O. 1M and 0. 4M has no effect on the signal.

Binding Analysis Influence of IL-1ss concentration ILIp was used at increasing final concentrations from 0. 156 to 10 nM sIL-lRI-Eur was used at a final concentration of 1.25 nM (data not shown). sIL-lRAcP-CY5 was used at a final concentration of 10 nM The results obtained are shown in Figure 11.

There is a direct relationship between the Delta F obtained and IL-1ss concentration. The signal plateau is reached at 5 nM final concentration of IL-1ss. From the saturation binding curve the Kd for IL-1ss was estimated at around 1.0

nM.

Influence of sIL-lRAcP-CY5 concentration IL-l&num; was used at a final concentration of 10 nM sIL-lRI-Eur was used at a final concentration of 1.25 nM sIL-lRAcP-CY5 was used at increasing final concentrations from 0.078 to 10 nM The results obtained are shown in Figure 11.

There is a direct relationship between the Delta F obtained and sIL-lRAcP-CY5 concentration. A plateau is obtained from a 10 nM final concentration of sIL-lRAcP-CY5. From the saturation binding curve the Kd for sIL-lRAcP was estimated at around 1.5 nM.

Influence of the sIL-lRI-Eur concentration IL-ljS was used at a final concentration of 10 nM. sIL-lRI-Eur was used at increasing final concentrations from 0.312 to 20 nM. sIL-lRAcP-CY5 was used at a final concentration of 10 nM.

There is a direct relationship between the specific signal (at 665 nm) obtained and sIL-lRI-Eur concentration. A plateau is obtained from 10nM final concentration of sILlRI-Eur. From the saturation binding curve the Kd for sIL- 1RI was estimated at around 3.5 nM.

The maximum delta F% was obtained using sIL-1RI-Eur from 1.25 to 2.5 nM as shown in the table 5 below.

Table 5 :

[sIL-lRI-Cps Cps Specific signal Delta F% Eur] 665 nm 620 nm at 665 nm nM Neg 0, 312 le+07 1, 5e+09 825 Pos 8573 Neg0, 625 2e+08 2, 9e+09 1043 Pos 8744 Neg 1, 25 3e+08 5, 3e+09 1167 Pos 29572 Neg 2, 5 5e+08 1, le+10 1310 Pos 61240 Neg 5 1e+10 2,2e+11 1173 Pos 102670 Neg 10 2e+10 4,0e+11 878 Pos 151264 Neg 20 4e+10 6,3e+11 878 Pos 163302 Influence of unlabelled sIL-lRI concentration ILl-beta was used at a final concentration of 10 nM.

sIL-1RI-Eur was used at a final concentration of 1. 25 nM. sIL-lRAcP-CY5 was used at a final concentration of 10 nM. Unlabelled sIL-lRI was used at increasing final concentrations from 0.098 to 50 nM.

The reaction was set up as follows : 50gel sIL-lRI-Eur

501 unlabelled sIL-1RI 501 sIL-1RAcP-CY5 501 IL-1ss (or buffer in the negative test) The results are shown in Figure 12.

50% inhibition is measured with an unlabelled sIL-1RI concentration of 25 nM.

Influence of unlabelled sIL-lRAcP concentration IL-lwas used at a final concentration of 10 nM sIL-1RI-Eur was used at a final concentration of 1. 25 nM sIL-1RAcP-CY5 was used at a final concentration of 2 nM Unlabelled sIL-lRAcP was used at increasing final concentrations from 0.78 to 100 nM The reaction was set up as follows: 501 IL-ljS (or buffer in the negative test) 501 unlabelled sIL-lRAcP 501 sIL-lRAcP-CY5 501 sIL-lRI-Eur Results are shown in figure 12.

50% inhibition is measured with an unlabelled sIL-lRAcP concentration of 10 nM.

Effect of ILl-ra IL-1ss was used at a final concentration of 2 nM sIL-lRI-Eur was used at a final concentration of 1.25 nM sIL-lRAcP-CY5 was used at a final concentration of 10 nM IL-l-ra antagonist was used at increasing final concentrations from 0.078 to 20 nM

The reaction was set up as follows: 50 l IL-1ss (or buffer in the negative test) 501 IL-lra 501 sIL-lRAcP-CY5 501 sIL-IRI-Eur Readings were taken after 15 mins, 1,3 and 5 hours.

Equilibrium is reached after about 3 hours. At the equilibrium, 50% inhibition is measured with a IL-1ra concentration of around InM.

These results are shown on figure 13.

Effect of AF11377 inhibitor peptide

IL-ljS was used at a final concentration of 2nM sIL-lRI-Eur was used at a final concentration of 1. 25nM sIL-lRAcP-CY5 was used at a final concentration of lOnM AF11377 inhibitor peptide (Seq No: 35) was used at increasing final concentrations from 0.78 to 20 nM The reaction was set up as follows: 50Al IL-10 (or buffer in the negative test) 501 AF11377

50/il sIL-lRAcP-CY5 50its sIL-lRI-Eur Readings were taken after 15 mins, 1, 3 and 5 hours. Equilibrium is reached after about 2 hours. At the equilibrium, 50% inhibition is measured with an AF11377 peptide concentration of around 20 nM.

These results are shown on figure 13.

Signal obtained at about KD concentration for each reagent The assay was performed using the three reagents at a concentration near the KD.

IL-ljS was used at a final concentration of 2 nM sIL-lRI-Eur was used at a final concentration of 1. 25 nM sIL-lRAcP-CY5 was used at a final concentration of 2nM The following table 6 shows the signal obtained. (cps means counts per second) Table 6:

cps cps Ratio R Delta R Delta F% 665 nm 620 nm Negative25135581545011 test (without ligand) Positive 15131 52749 2868 2418 537 test (with ligand)

HTRF Assay Summary Table 7 below shows the preferred reagent concentrations for use in the HTRF ternary complex assay, on the basis of the developmental work described herein.

Table 7:

Reagents Stock solution Initial Final concentration concentration concentration before addition in the reaction IL-lss 5 M 8 nM 2 nM sIL-lRI-Eur 3.09 AM 5 nM 1.25 nM sIL-lRAcP-CY5 1.5 M 8 nM 2 nM Buffer or--Range of cone inhibitor Further optimizations were performed in-house in order to reduce the final assay volume from 20a1 to 161 and to transfer the assay from 96-well to 384-well format. Table 7 concentrations were conserved for the assay but volumes of each component were reduced from 50 l to 41 in order to obtain 16 l as a final volume. The signal and stability of the complex were identical to original format (data not shown).

Example 6: Best in-house optimization of the HTRF direct assay system l) Materials and Methods Assay Buffer lOmM Hepes 0. 2M KF

0. 1% BSA pH to 7.4 with NaOH at 21 C Required for all reagent and compound dilutions Assay components 1. Soluble ILl-receptor-l-labelled with Europium Cryptate (sIL-lRl-Eur) Batch 002 (labelled by Cis Bio). Store at-80 C.

Stock concentration 200pg/ml. (4. 762M) Dilute 1: 1190 to give 4nM (diluted 4 fold in assay to give a final concentration of 1nM) 2. Soluble IL-1 receptor accessory protein labelled with Cy5 (sIL-1RAcP-Cy5) Batch 7 (Labelled by PGRD Cambridge).

Stock concentration 3. 6uM Dilute 1: 90 to give 40nM (diluted 4 fold in assay to give a final concentration of lOnM) 3. IL1ss Batch 1 (Produced at PGRD Cambridge). Store at-80 C.

Stock Concentration 5.0 M Dilute 1: 625 to give 8nM (diluted 4 fold in assay to give a final concentration of 2nM) 4. Test compounds/buffer The assay was set forth as follow in order to use the biggest possible volumes: - 1. 3 pl of compound in 10 % DMSO (250M daughter plates) - 6. 7 pl of IL1ss, initial concentration = 4. 8nM, final concentration 2nM

- 4 Al of sIL-lRI-Eur, initial concentration = 2nM, final concentration InM - 4 pl of sIL-lRAcP-Cy5, initial concentration = 20nM, final concentration lOnM Microtitre Plates All assays were performed in black plates.

Black Optiplate (96 well), Packard Catalogue No 6005207 60 plates per box Assay volume 200gel Black Optiplate (384 well) Packard Catalogue No 6005256 30 plates per box Assay volume 641 Black Proxiplate (384 well), Packard Catalogue No 6006260 50 plates per box Assay volume 16 l Blanks and system controls Blanks/System Controls were incorporated into each plate, in duplicate as follows: (All concentrations indicate final concentration in assay) 1. Buffer 2. sIL-lRAcP-Cy5 (lOnM) 3. sIL-1R1-Eur (l. OnM) 4. sIL-lRAcP-Cy5 (lOnM) + sIL-1R1-Eur (l. OnM) 5. sIL-lRAcP-CyS (lOnM) + sIL-1RI-Eur (l. 0nM) + IL-1ss (2nM) Control 4 was used as the system blank for all

calculations.

Control 5 produces the maximum possible signal.

2) Protocol Assays were set up as indicated in the Table below.

Reagents and test compounds were all diluted to the appropriate concentration with assay buffer. The % DMSO in each well was < 1% in all assays performed.

Reagents were added into the assay in order of appearance in the table.

Formation of the ternary complex was initiated by addition of IL-lp (2nM for competition experiments).

Plates were sealed with TopSeal A (Packard) to prevent evaporation and incubated for a minimum of 3 hours, but generally overnight, prior to reading. The signal was stable for at least 24 hours.

Assay Composition See table 8 below.

Table 8

384 well 384 well (64pl (16pl assay) assay) Test compound 16Jll (x4 41il (x4 stock) stock) sIL-lRl-Eur 16 l 4 l sIL-lRAcP-Cy5 16 l 4 l IL1ss 16 l 4 l Plate Reading All validation studies experiments (using current

reagents) were read using a Wallac Victor2 Plate Reader.

Early assay validation was performed using default settings on the Discovery Plate Reader. Note that a minimum assay volume of 601 is required for optimal reading in standard 384 well plates.

Excitation wavelenth 337nm.

Emission wavelengths 620 and 665nm.

Example 7 HTRF assay Semi-direct System A semi-direct assay format was designed utilizing biotinylated sIL-lRI and sIL-lRAcP directly labelled with CY5. Europium Cryptate coupled to Streptavidin was also used.

The interaction reaction was carried out in a one step procedure just by adding all components. All the reagents were diluted in HEPES buffer lOmM, pH 7.4, 0. 1% BSA, 0.005% Tween 20 and 0. 2M KF.

1) Protocol The assay was set up as follows:

50gel IL-lss (or buffer in the negative test) 501 Biotinylated-sIL-lRI and Streptavidin-Cryptate conjugate 501 sIL-lRAcP-CY5 conjugate 501 buffer The reaction was incubated at room temperature for at least 15 minutes, the equilibrium being reached after about 1 hour (see figure 10). Readings were taken on a Discovery instrument (Packard) under standards conditions for HTRF

measurement (delay 50s, gate 400vs).

2) Results The results were expressed as DeltaF: R=ratio= (F665nm/F62onm) x104 DeltaF (%) = [(Rpositive-Rnegative)/Rnegative]x100 Negative=all reagents except IL-1ss ligand Positive=all reagents with IL-1ss ligand Evaluation of the Assay Performances The first experiment was performed in the following conditions: IL-10 was used at a final concentration of 5nM.

Biotin-sIL-lRI was used at a final concentration of 1. 25 or 5nM. Streptavidin-Cryptate was used at 20ng/well. sIL-lRAcP-CY5 was used at a final concentration of 10 or 50nM.

EXAMPLE 8: Flashplate assay The C-terminaly 6His tagged sIL-lRAcP is used to anchor the protein to a Ni-NTA coated flashplate (manufactured by NEN). We know already from the BIAcore data that anchored in this orientation the sIL-lRAcP can interact with the ILlp/sIL-lRI binary complex to form a ternary complex. Thus this interaction is detected through the use of either tritium ([3H]) labelled IL-lp or sIL-lRI. Light, which can be detected on an instrument such as a Packard TOPcount ss- scintillation counter, is emitted from the flashplate upon

binding of the radiolabelled binary complex to the sILlRAcP. Excitation of the scintillant embedded in the flashplate well occurs only when the radionuclide is in sufficient proximity to the well-surface i. e. when the radiolabelled binary complex has formed a ternary complex with the anchored sIL-lRAcP.

It is also possible to use the C-terminaly 6His tagged sIL- 1RI in order to anchor the protein to a Ni-NTA coated flashplate (manufactured by NEN). The ability of the IL-1RI to form firstly a binary complex with IL-lp and subsequently a ternary with sIL-lRAcP has been demonstrated on the BIAcore with anchored sIL-lRI (not necessarily anchored via the C-terminus of the protein). Thus in this format one may assay both for the formation of the binary and the ternary complex. When the binary complex formation is assayed [3H] labelled IL-lp is used which results in scintillation upon interaction with sIL-lRI anchored to a flashplate. With the sIL-lRAcP labelled with jH] the formation of the ternary complex is assayed.

Thus using an appropriate combination of the above assay formats one assays for molecules which may inhibit either the formation of the binary or ternary complex.

Alternatively the assay described above is run using either biotinylated sIL-lRI or biotinylated sIL-1RAcP. Thus one exploits the presence of these biotin moieties on the protein to anchor it to a streptavidin flashplate.

Biotinylation is also used as a method for [3H] labelling these molecules using commercially available [H]-biotin.

Labelling using [3H]-amino acids in the expression media is also a method for labelling these proteins with a high-

specific activity.

Similarly SPA bead technology (Amersham) could be used as an alternative to the Flashplate method described above.

Thus the flashplate would be replaced by a standard plastic plate (96 or 384-well) containing an appropriate quantity of SPA beads Example 9 Origen Technology Assay (Igen) sIL-lRAcP-6His is anchored to a Ni-NTA magnetic bead via the 6his tag and binary complex comprising ruthenium (II) tris- (bipyridine) labelled sIL-lR associated with IL-10 is then added. Light output from the label is then measured following electrochemical stimulation.

Another possibility is to have sIL-IR-6His anchored to a Ni-NTA magnetic bead via the 6his tag and binary complex comprising ruthenium (II) tris- (bipyridine) labelled sIL-lRAcP associated with IL-10 or labelled IL-1 associated with IL-lRAcP is then added. Light output from the label is then measured following electrochemical stimulation. A futher method for anchoring the appropriate protein to a magnetic bead would be to use streptavidincoated beads and to biotinylate either sIL-lR or sIL-lRAcP as appropriate.

Table 9 shows the GenBank Accession numbers of nucleotide sequences which encode proteins involved in IL-1 bimolecular and/or trimolecular complex.

TABLE 9, Table of Interleukin-l relevant accession numbers

IL-1 Receptor type I Receptor Accessory protein Receptor type II Soluble AcP Soluble receptor type I Human x02532 x16896 AF029213 NM~004633 AF167343 Human X02531 Mouse NM~008361 NM~008362 NM~008364 NM~010555 Mouse NM010554 Rat D00403 m95578 NM~012968 Rat M98820 NM~013123

Table 10 shows the GenBank Accession numbers of nucleotide sequences which encode proteins involved in IL-18 bimolecular and/or trimolecular complex.

TABLE 10

Table of Interleukin-18 relevant accession numbers IL-18 Receptor type I Receptor Accessory protein IL-18 binding protein Human AF077611 NM003855 NM003853 AF215907 Mouse NM~008360 NM~008365 NM~010553

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SEQUENCE LISTING < 110 > Warner-Lambert < 120 > Methods for screening using interleukin soluble trimolecular complex < 130 > A212 < 140 > < 141 > < 160 > 44 < 170 > PatentIN Ver. 2.1 < 210 > 1 < 211 > 159 < 212 > PRT < 213 > Homo sapiens < 400 > 1 Ser Ala Pro Phe Ser Phe Leu Ser Asn Val Lys Tyr Asn Phe Met Arg 1 5 10 15 lIe lIe Lys Tyr Glu Phe Ile Leu Asn Asp Ala Leu Asn Gln Ser lIe 20 25 30 lIe Arg Ala Asn Asp Gln Tyr Leu Thr Ala Ala Ala Leu His Asn Leu 35 40 45

Asp Glu Ala Val Lys Phe Asp Met Gly Ala Tyr Lys Ser Ser Lys Asp 50 55 60 Asp Ala Lys Ile Thr Val Ile Leu Arg Ile Ser Lys Thr Gln Leu Tyr 65 70 75 80 Val Thr Ala Gln Asp Glu Asp Gln Pro Val Leu Leu Lys Glu Met Pro 85 90 95 Glu lIe Pro Lys Thr Ile Thr Gly Ser Glu Thr Asn Leu Leu Phe Phe 100 105 110 Trp Glu Thr His Gly Thr Lys Asn Tyr Phe Thr Ser Val Ala His Pro 115 120 125 Asn Leu Phe lIe Ala Thr Lys Gln Asp Tyr Trp Val Cys Leu Ala Gly 130 135 140 Gly Pro Pro Ser lIe Thr Asp Phe Gln lIe Leu Glu Asn Gln Ala 145 150 155 < 210 > 2 < 211 > 1124 < 212 > DNA < 213 > Homo sapiens < 400 > 2 ttcgaggcac aaggcacaac aggctgctct gggattctct tcagccaatc ttcattgctc 60

aagtgtctga agcagccatg gcagaagtac ctgagctcgc cagtgaaatg atggcttatt 120 acagtggcaa tgaggatgac ttgttctttg aagctgatgg ccctaaacag atgaagtgct 180 ccttccagga cctggacctc tgccctctgg atggcggcat ccagctacga atctccgacc 240 accactacag caagggcttc aggcaggccg cgtcagttgt tgtggccatg gacaagctga 300 ggaagatgct ggttccctgc ccacagacct tccaggagaa tgacctgagc accttctttc 360 ccttcatctt tgaagaagaa cctatcttct ttgacacatg ggataacgag gcttatgtgc 420 acgatgcacc tgtacgatca ctgaactgca cgctccggga ctcacagcaa aaaagcttgg 480 tgatgtctgg tccatatgaa ctgaaagctc tccacctcca gggacaggat atggagcaac 540 aagtggtgtt ctccatgtcc tttgtacaag gagaagaaag taatgacaaa atacctgtgg 600 ccttgggcct caaggaaaag aatctgtacc tgtcctgcgt gttgaaagat gataagccca 660 ctctacagct ggagagtgta gatcccaaaa attacccaaa gaagaagatg gaaaagcgat 720 ttgtcttcaa caagatagaa atcaataaca agctggaatt tgagtctgcc cagttcccca 780 actggtacat cagcacctct caagcagaaa acatgcccgt cttcctggga gggaccaaag 840 gcggccagga tataactgac ttcaccatgc aatttgtgtc ttcctaaaga gagctgtacc 900 cagagagtcc tgtgctgaat gtggactcaa tccctagggc tggcagaaag ggaacagaaa 960 ggtttttgag tacggctata gcctggactt tcctgttgtc tacaccaatg cccaactgcc 1020 tgccttaggg tagtgctaag aggatctcct gtccatcagc caggacagtc agctctctcc 1080 tttcagggcc aatccccagc ccttttgttg agccaggcct ctct 1124 < 210 > 3 < 211 > 269 < 212 > PRT < 213 > Homo sapiens < 400 > 3 Met Ala Glu Val Pro Glu Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser 1 5 10 15 Gly Asn Glu Asp Asp Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln Met 20 25 30 Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile 35 40 45 Gln Leu Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala 50 55 60 Ala Ser Val Val Val Ala Met Asp Lys Leu Arg Lys Met Leu Val Pro 65 70 75 80 Cys Pro Gln Thr Phe Gln Glu Asn Asp Leu Ser Thr Phe Phe Pro Phe 85 90 95

Ile Phe Glu Glu Glu Pro He Phe Phe Asp Thr Trp Asp Asn Glu Ala 100 105 110 Tyr Val His Asp Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp 115 120 125 Ser Gln Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala 130 135 140 Leu His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met 145 150 155 160 Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys He Pro Val Ala Leu 165 170 175 Gly Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp 180 185 190 Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys 195 200 205 Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu He Asn Asn 210 215 220 Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr 225 230 235 240 Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly 245 250 255 Gln Asp He Thr Asp Phe Thr Met Gln Phe Val Ser Ser 260 265 < 210 > 4 < 211 > 153 < 212 > PRT < 213 > Homo sapiens < 400 > 4 Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gln Lys 1 5 10 15 Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln 20 25 30 Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln 35 40 45 Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Lys Glu 50 55 60 Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu 65 70 75 80 Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu 85 90 95 Lys Arg Phe Val Phe Asn Lys lIe Glu lIe Asn Asn Lys Leu Glu Phe 100 105 110 Glu Ser Ala Gln Phe Pro Asn Trp Tyr He Ser Thr Ser Gln Ala Glu

115 120 125 Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp Ile Thr 130 135 140 Asp Phe Thr Met Gln Phe Val Ser Ser 145 150 < 210 > 5 < 211 > 161 < 212 > PRT < 213 > Mus musculus < 400 > 5 Met Asn Glu Phe Gly Ser Ala Pro Tyr Thr Tyr Gln Ser Asp Leu Arg 1 5 10 15 Tyr Lys Leu Met Lys Leu Val Arg Gln Lys Phe Val Met Asn Asp Ser 20 25 30 Leu Asn Gln Thr Ile Tyr Gln Asp Val Asp Lys His Tyr Leu Ser Thr 35 40 45 Thr Trp Leu Asn Asp Leu Gln Gln Glu Val Lys Phe Asp Met Tyr Ala 50 55 60 Tyr Ser Ser Gly Gly Asp Asp Ser Lys Tyr Pro Val Thr Leu Lys Ile 65 70 75 80 Ser Asp Ser Gln Leu Phe Val Ser Ala Gln Gly Glu Asp Gln Pro Val 85 90 95 Leu Leu Lys Glu Leu Pro Glu Thr Pro Lys Leu Ile Thr Gly Ser Glu 100 105 110 Thr Asp Leu lIe Phe Phe Trp Lys Ser He Asn Ser Lys Asn Tyr Phe 115 120 125 Thr Ser Ala Ala Tyr Pro Glu Leu Phe He Ala Thr Lys Glu Gln Ser 130 135 140 Arg Val His Leu Ala Arg Gly Leu Pro Ser Met Thr Asp Phe Gln He 145 150 155 160 Ser < 210 > 6 < 211 > 152 < 212 > PRT < 213 > Mus musculus < 400 > 6 Val Pro He Arg Gln Leu His Tyr Arg Leu Arg Asp Glu Gln Gln Lys 1 5 10 15 Ser Leu Val Leu Ser Asp Pro Tyr Glu Leu Lys Ala Leu His Leu Asn 20 25 30

Gly Gln Asn lIe Asn Gln Gln Val He Phe Ser Met Ser Phe Val Gln

35 40 45 Gly Glu Pro Ser Asn Asp Lys He Pro Val Ala Leu Gly Leu Lys Gly 50 55 60 Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Gly Thr Pro Thr Leu 65 70 75 80 Gln Leu Glu Ser Val Asp Pro Lys Gln Tyr Pro Lys Lys Lys Met Glu 85 90 95 Lys Arg Phe Val Phe Asn Lys He Glu Val Lys Ser Lys Val Glu Phe 100 105 110 Glu Ser Ala Glu Phe Pro Asn Trp Tyr He Ser Thr Ser Gln Ala Glu 115 120 125 His Lys Pro Val Phe Leu Gly Asn Asn Ser Gly Gln Asp He Ile Asp 130 135 140 Phe Thr Met Glu Ser Val Ser Ser 145 150 < 210 > 7 < 211 > 156 < 212 > PRT < 213 > Rattus sp.

< 400 > 7 Ser Ala Pro His Ser Phe Gln Asn Asn Leu Arg Tyr Lys Leu lIe Arg 1 5 10 15 He Val Lys Gln Glu Phe He Met Asn Asp Ser Leu Asn Gln Asn He 20 25 30 Tyr Val Asp Met Asp Arg Ile His Leu Lys Ala Ala Ser Leu Asn Asp 35 40 45 Leu Gln Leu Glu Val Lys Phe Asp Met Tyr Ala Tyr Ser Ser Gly Gly 50 55 60 Asp Asp Ser Lys Tyr Pro Val Thr Leu Lys Val Ser Asn Thr Gln Leu 65 70 75 80 Phe Val Ser Ala Gln Gly Glu Asp Lys Pro Val Leu Leu Lys Glu He 85 90 95 Pro Glu Thr Pro Lys Leu He Thr Gly Ser Glu Thr Asp Leu He Phe 100 105 110 Phe Trp Glu Lys He Asn Ser Lys Asn Tyr Phe Thr Ser Ala Ala Phe 115 120 125 Pro Glu Leu Leu He Ala Thr Lys Glu Gln Ser Gln Val His Leu Ala 130 135 140 Arg Gly Leu Pro Ser Met He Asp Phe Gln He Ser 145 150 155 < 210 > 8

< 211 > 152 < 212 > PRT < 213 > Rattus sp.

< 400 > 8 Val Pro lIe Arg Gln Leu His Cys Arg Leu Arg Asp Glu Gln Gln Lys 1 5 10 15 Cys Leu Val Leu Ser Asp Pro Cys Glu Leu Lys Ala Leu His Leu Asn 20 25 30 Gly Gln Asn lIe Ser Gln Gln Val Val Phe Ser Met Ser Phe Val Gln 35 40 45 Gly Glu Thr Ser Asn Asp Lys He Pro Val Ala Leu Gly Leu Lys Gly 50 55 60 Leu Asn Leu Tyr Leu Ser Cys Val Met Lys Asp Gly Thr Pro Thr Leu 65 70 75 80 Gln Leu Glu Ser Val Asp Pro Lys Gln Tyr Pro Lys Lys Lys Met Glu 85 90 95 Lys Arg Phe Val Phe Asn Lys He Glu Val Lys Thr Lys Val Glu Phe 100 105 110 Glu Ser Ala Gln Phe Pro Asn Trp Tyr lIe Ser Thr Ser Gln Ala Glu 115 120 125 His Arg Pro Val Phe Leu Gly Asn Ser Asn Gly Arg Asp Ile Val Asp 130 135 140 Phe Thr Met Glu Pro Val Ser Ser 145 150 < 210 > 9 < 211 > 157 < 212 > PRT < 213 > Homo sapiens < 400 > 9 Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val lIe Arg Asn Leu Asn 1 5 10 15 Asp Gln Val Leu Phe lIe Asp Gln Gly Asn Arg Pro Leu Leu Glu Asp 20 25 30 Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr lIe Phe Ile 35 40 45 lIe Arg Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile 50 55 60 Ser Val Lys Cys Glu Lys lIe Ser Thr Leu Ser Cys Glu Asn Lys Ile 65 70 75 80 He Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys 85 90 95 Ser Asp He Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys 100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu 115 120 125 Lys Glu Arg Asp Leu Phe Lys Leu He Leu Lys Lys Glu Asp Glu Leu 130 135 140 Gly Asp Arg Ser lIe Met Phe Thr Val Gln Ser Glu Asp 145 150 155 < 210 > 10 < 211 > 157 < 212 > PRT < 213 > Mus musculus < 400 > 10 Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val He Arg Asn lIe Asn 1 5 10 15 Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met 20 25 30 Thr Asp He Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu He He 35 40 45 Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser 50 55 60 Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys He He 65 70 75 80 Ser Phe Glu Glu Met Asp Pro Pro Glu Asn lIe Asp Asp He Gln Ser 85 90 95 Asp Leu He Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu 100 105 110 Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu 115 120 125 Asp Asp AlaPhe Lys Leu He Leu Lys Lys Lys Asp Glu Asn Gly Asp 130 135 140 Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser 145 150 155 < 210 > 11 < 211 > 569 < 212 > PRT < 213 > Homo sapiens < 400 > 11 Met Lys Val Leu Leu Arg Leu He Cys Phe He Ala Leu Leu lIe Ser 1 5 10 15 Ser Leu Glu Ala Asp Lys Cys Lys Glu Arg Glu Glu Lys He He Leu 20 25 30 Val Ser Ser Ala Asn Glu He Asp Val Arg Pro Cys Pro Leu Asn Pro 35 40 45

Asn Glu His Lys Gly Thr He Thr Trp Tyr Lys Asp Asp Ser Lys Thr 50 55 60 Pro Val Ser Thr Glu Gln Ala Ser Arg He His Gln His Lys Glu Lys 65 70 75 80 Leu Trp Phe Val Pro Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys 85 90 95 Val Val Arg Asn Ser Ser Tyr Cys Leu Arg He Lys He Ser Ala Lys 100 105 110 Phe Val Glu Asn Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala He Phe 115 120 125 Lys Gln Lys Leu Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr 130 135 140 Met Glu Phe Phe Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp 145 150 155 160 Tyr Lys Asp Cys Lys Pro Leu Leu Leu Asp Asn He His Phe Ser Gly 165 170 175 Val Lys Asp Arg Leu He Val Met Asn Val Ala Glu Lys His Arg Gly 180 185 190 Asn Tyr Thr Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro 195 200 205 He Thr Arg Val He Glu Phe He Thr Leu Glu Glu Asn Lys Pro Thr 210 215 220 Arg Pro Val He Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu 225 230 235 240 Gly Ser Gln He Gln Leu He Cys Asn Val Thr Gly Gln Leu Ser Asp 245 250 255 He Ala Tyr Trp Lys Trp Asn Gly Ser Val He Asp Glu Asp Asp Pro 260 265 270 Val Leu Gly Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg 275 280 285 Arg Ser Thr Leu He Thr Val Leu Asn He Ser Glu He Glu Ser Arg 290 295 300 Phe Tyr Lys His Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly He 305 310 315 320 Asp Ala Ala Tyr He Gln Leu He Tyr Pro Val Thr Asn Phe Gln Lys 325 330 335 His Met He Gly He Cys Val Thr Leu Thr Val He He Val Cys Ser 340 345 350 Val Phe He Tyr Lys He Phe Lys He Asp He Val Leu Trp Tyr Arg 355 360 365 Asp Ser Cys Tyr Asp Phe Leu Pro He Lys Ala Ser Asp Gly Lys Thr

370 375 380 Tyr Asp Ala Tyr lIe Leu Tyr Pro Lys Thr Val Gly Glu Gly Ser Thr 385 390 395 400 Ser Asp Cys Asp lIe Phe Val Phe Lys Val Leu Pro Glu Val Leu Glu 405 410 415 Lys Gln Cys Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val 420 425 430 Gly Glu Asp Ile Val Glu Val Ile Asn Glu Asn Val Lys Lys Ser Arg 435 440 445 Arg Leu lIe Ile Ile Leu Val Arg Glu Thr Ser Gly Phe Ser Trp Leu 450 455 460 Gly Gly Ser Ser Glu Glu Gln He Ala Met Tyr Asn Ala Leu Val Gln 465 470 475 480 Asp Gly Ile Lys Val Val Leu Leu Glu Leu Glu Lys Ile Gln Asp Tyr 485 490 495 Glu Lys Met Pro Glu Ser Ile Lys Phe He Lys Gln Lys His Gly Ala 500 505 510 He Arg Trp Ser Gly Asp Phe Thr Gln Gly Pro Gln Ser Ala Lys Thr 515 520 525 Arg Phe Trp Lys Asn Val Arg Tyr His Met Pro Val Gln Arg Arg Ser 530 535 540 Pro Ser Ser Lys His Gln Leu Leu Ser Pro Ala Thr Lys Glu Lys Leu 545 550 555 560 Gln Arg Glu Ala His Val Pro Leu Gly 565 < 210 > 12 < 211 > 1710 < 212 > DNA < 213 > Homo sapiens < 400 > 12 atgaaagtgt tactcagact tatttgtttc atagctctac tgatttcttc tctggaggct 60 gataaatgca aggaacgtga agaaaaaata attttagtgt catctgcaaa tgaaattgat 120 gttcgtccct gtcctcttaa cccaaatgaa cacaaaggca ctataacttg gtataaagat 180 gacagcaaga cacctgtatc tacagaacaa gcctccagga ttcatcaaca caaagagaaa 240 ctttggtttg ttcctgctaa ggtggaggat tcaggacatt actattgcgt ggtaagaaat 300 tcatcttact gcctcagaat taaaataagt gcaaaatttg tggagaatga gcctaactta 360 tgttataatg cacaagccat atttaagcag aaactacccg ttgcaggaga cggaggactt 420 gtgtgccctt atatggagtt ttttaaaaat gaaaataatg agttacctaa attacagtgg 480

tataaggatt gcaaacctct acttcttgac aatatacact ttagtggagt caaagatagg 540 ctcatcgtga tgaatgtggc tgaaaagcat agagggaact atacttgtca tgcatcctac 600 acatacttgg gcaagcaata tcctattacc cgggtaatag aatttattac tctagaggaa 660 aacaaaccca caaggcctgt gattgtgagc ccagctaatg agacaatgga agtagacttg 720 ggatcccaga tacaattgat ctgtaatgtc accggccagt tgagtgacat tgcttactgg 780 aagtggaatg ggtcagtaat tgatgaagat gacccagtgc taggggaaga ctattacagt 840 gtggaaaatc ctgcaaacaa aagaaggagt accctcatca cagtgcttaa tatatcggaa 900 attgaaagta gattttataa acatccattt acctgttttg ccaagaatac acatggtata 960 gatgcagcat atatccagtt aatatatcca gtcactaatt tccagaagca catgattggt 1020 atatgtgtca cgttgacagt cataattgtg tgttctgttt tcatctataa aatcttcaag 1080 attgacattg tgctttggta cagggattcc tgctatgatt ttctcccaat aaaagcttca 1140 gatggaaaga cctatgacgc atatatactg tatccaaaga ctgttgggga agggtctacc 1200 tctgactgtg atatttttgt gtttaaagtc ttgcctgagg tcttggaaaa acagtgtgga 1260 tataagctgt tcatttatgg aagggatgac tacgttgggg aagacattgt tgaggtcatt 1320 aatgaaaacg taaagaaaag cagaagactg attatcattt tagtcagaga aacatcaggc 1380 ttcagctggc tgggtggttc atctgaagag caaatagcca tgtataatgc tcttgttcag 1440 gatggaatta aagttgtcct gcttgagctg gagaaaatcc aagactatga gaaaatgcca 1500 gaatcgatta aattcattaa gcagaaacat ggggctatcc gctggtcagg ggactttaca 1560 cagggaccac agtctgcaaa gacaaggttc tggaagaatg tcaggtacca catgccagtc 1620 cagcgacggt caccttcatc taaacaccag ttactgtcac cagccactaa ggagaaactg 1680 caaagagagg ctcacgtgcc tctcgggtag 1710 < 210 > 13 < 211 > 316 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : soluble IL-R < 400 > 13 Asp Lys Cys Lys Glu Arg Glu Glu Lys Ile He Leu Val Ser Ser Ala 1 5 10 15 Asn Glu He Asp Val Arg Pro Cys Pro Leu Asn Pro Asn Glu His Lys 20 25 30 Gly Thr He Thr Trp Tyr Lys Asp Asp Ser Lys Thr Pro Val Ser Thr 35 40 45

Glu Gln Ala Ser Arg He His Gln His Lys Glu Lys Leu Trp Phe Val 50 55 60 Pro Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys Val Val Arg Asn 65 70 75 80 Ser Ser Tyr Cys Leu Arg lIe Lys He Ser Ala Lys Phe Val Glu Asn 85 90 95 Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala He Phe Lys Gln Lys Leu 100 105 110 Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr Met Glu Phe Phe 115 120 125 Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp Tyr Lys Asp Cys 130 135 140 Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly Val Lys Asp Arg 145 150 155 160 Leu He Val Met Asn Val Ala Glu Lys His Arg Gly Asn Tyr Thr Cys 165 170 175 His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro Ile Thr Arg Val 180 185 190 Ile Glu Phe He Thr Leu Glu Glu Asn Lys Pro Thr Arg Pro Val He 195 200 205 Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu Gly Ser Gln He 210 215 220 Gln Leu He Cys Asn Val Thr Gly Gln Leu Ser Asp Ile Ala Tyr Trp 225 230 235 240 Lys Trp Asn Gly Ser Val He Asp Glu Asp Asp Pro Val Leu Gly Glu 245 250 255 Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg Arg Ser Thr Leu 260 265 270 Ile Thr Val Leu Asn He Ser Glu He Glu Ser Arg Phe Tyr Lys His 275 280 285 Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly He Asp Ala Ala Tyr 290 295 300 He Gln Leu He Tyr Pro Val Thr Asn Phe Gln Lys 305 310 315 < 210 > 14 < 211 > 317 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : soluble IL-R < 400 > 14 Asp Val Cys Thr Glu Tyr Pro Asn Gln He Val Leu Phe Leu Ser Val

1 5 10 15 Asn Glu lIe Asp He Arg Lys Cys Pro Leu Thr Pro Asn Lys Met His 20 25 30 Gly Asp Thr He He Trp Tyr Lys Asn Asp Ser Lys Thr Pro He Ser 35 40 45 Ala Asp Arg Asp Ser Arg He His Gln Gln Asn Glu His Leu Trp Phe 50 55 60 Val Pro Ala Lys Val Glu Asp Ser Gly Tyr Tyr Tyr Cys He Val Arg 65 70 75 80 Asn Ser Thr Tyr Cys Leu Lys Thr Lys Val Thr Val Thr Val Leu Glu 85 90 95 Asn Asp Pro Gly Leu Cys Tyr Ser Thr Gln Ala Thr Phe Pro Gln Arg 100 105 110 Leu His He Ala Gly Asp Gly Ser Leu Val Cys Pro Tyr Val Ser Tyr 115 120 125 Phe Lys Asp Glu Asn Asn Glu Leu Pro Glu Val Gln Trp Tyr Lys Asn 130 135 140 Cys Lys Pro Leu Leu Leu Asp Asn Val Ser Phe Phe Gly Val Lys Asp 145 150 155 160 Lys Leu Leu Val Arg Asn Val Ala Glu Glu His Arg Gly Asp Tyr He 165 170 175 Cys Arg Met Ser Tyr Thr Phe Arg Gly Lys Gln Tyr Pro Val Thr Arg 180 185 190 Val He Gln Phe He Thr He Asp Glu Asn Lys Arg Asp Arg Pro Val 195 200 205 He Leu Ser Pro Arg Asn Glu Thr He Glu Ala Asp Pro Gly Ser Met 210 215 220 He Gln Leu He Cys Asn Val Thr Gly Gln Phe Ser Asp Leu Val Tyr 225 230 235 240 Trp Lys Trp Asn Gly Ser Glu He Glu Trp Asn Asp Pro Phe Leu Ala 245 250 255 Glu Asp Tyr Gln Phe Val Glu His Pro Ser Thr Lys Arg Lys Tyr Thr 260 265 270 Leu He Thr Thr Leu Asn He Ser Glu Val Lys Ser Gln Phe Tyr Arg 275 280 285 Tyr Pro Phe He Cys Val Val Lys Asn Thr Asn He Phe Glu Ser Ala 290 295 300 His Val Gln Leu He Tyr Pro Val Pro Asp Phe Lys Asn 305 310 315 < 210 > 15 < 211 > 317

< 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: soluble IL-R < 400 > 15 Asp Lys Cys Thr Glu Tyr Pro Asn Glu Val lIe Ser Phe Ser Ser Val 1 5 10 15 Asn Glu He Asp He Arg Ser Cys Pro Leu Thr Pro Asn Glu Met His 20 25 30 Gly Gly Thr lIe He Trp Tyr Lys Asn Asp Ser Lys Thr Pro He Ser 35 40 45 Ala Asp Lys Asp Ser Arg He His Gln Gln Asn Glu His Leu Trp Phe 50 55 60 Val Pro Ala Lys Met Glu Asp Ser Gly Tyr Tyr Tyr Cys He Met Arg 65 70 75 80 Asn Ser Thr Tyr Cys Leu Lys Thr Lys He Thr Met Ser Val Leu Glu 85 90 95 Asn Asp Pro Gly Leu Cys Tyr Asn Thr Gln Ala Ser Phe He Gln Arg 100 105 110 Leu His Val Ala Gly Asp Gly Ser Leu Val Cys Pro Tyr Leu Asp Phe 115 120 125 Phe Lys Asp Glu Asn Asn Glu Leu Pro Lys Val GIn Trp Tyr Lys Asn 130 135 140 Cys Lys Pro Leu Pro Leu Asp Asp Gly Asn Phe Phe Gly Phe Lys Asn 145 150 155 160 Lys Leu Met Val Met Asn Val Ala Glu Glu His Arg Gly Asn Tyr Thr 165 170 175 Cys Arg Thr Ser Tyr Thr Tyr Gln Gly Lys Gln Tyr Pro Val Thr Arg 180 185 190 Val He Thr Phe He Thr He Asp Asp Ser Lys Arg Asp Arg Pro Val 195 200 205 He Met Ser Pro Arg Asn Glu Thr Met Glu Ala Asp Pro Gly Ser Thr 210 215 220 He Gln Leu lIe Cys Asn Val Thr Gly Gln Phe Thr Asp Leu Val Tyr 225 230 235 240 Trp Lys Trp Asn Gly Ser Glu He Glu Trp Asp Asp Pro He Leu Ala 245 250 255 Glu Asp Tyr Gln Phe Leu Glu His Pro Ser Ala Lys Arg Lys Tyr Thr 260 265 270 Leu He Thr Thr Leu Asn Val Ser Glu Val Lys Ser Gln Phe Tyr Arg 275 280 285 Tyr Pro Phe Ile Cys Phe Val Lys Asn Thr His He Leu Glu Thr Ala

290 295 300 His Val Arg Leu Val Tyr Pro Val Pro Asp Phe Lys Asn 305 310 315 < 210 > 16 < 211 > 261 < 212 > PRT < 213 > Rattus sp.

< 400 > 16 Asp Lys Cys Thr Glu Tyr Pro Asn Glu Val Ile Ser Phe Ser Ser Val 1 5 10 15 Asn Glu Ile Asp Ile Arg Ser Cys Pro Leu Thr Pro Asn Glu Met His 20 25 30 Gly Gly Thr Ile Ile Trp Tyr Lys Asn Asp Ser Lys Thr Pro Ile Ser 35 40 45 Ala Asp Lys Asp Ser Arg Ile His Gln Gln Asn Glu His Leu Trp Phe 50 55 60 Val Pro Ala Lys Met Glu Asp Ser Gly Tyr Tyr Tyr Cys Ile Met Arg 65 70 75 80 Asn Ser Thr Tyr Cys Leu Lys Thr Lys lie Thr Met Ser Val Leu Glu 85 90 95 Asn Asp Pro Gly Leu Cys Tyr Asn Thr Gln Ala Ser Phe lie Gln Arg 100 105 110 Leu His Val Ala Gly Asp Gly Ser Leu Val Cys Pro Tyr Leu Asp Phe 115 120 125 Phe Lys Asp Glu Asn Asn Glu Leu Pro Lys Val Gln Trp Tyr Lys Asn 130 135 140 Cys Lys Pro Leu Pro Leu Asp Asp Gly Asn Phe Phe Gly Phe Lys Asn 145 150 155 160 Lys Leu Met Val Met Asn Val Ala Glu Glu His Arg Gly Asn Tyr Thr 165 170 175 Cys Arg Thr Ser Tyr Thr Tyr Gln Gly Lys Gln Tyr Pro Val Thr Arg 180 185 190 Val lie Thr Phe Ile Thr Ile Asp Asp Ser Lys Arg Asp Arg Pro Val 195 200 205 Ile Met Ser Pro Arg Asn Glu Thr Met Glu Ala Asp Pro Gly Ser Thr 210 215 220 Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Phe Thr Asp Leu Val Tyr 225 230 235 240 Trp Lys Trp Asn Gly Ser Glu Ile Glu Trp Asp Asp Pro Ile Leu Ala 245 250 255 Glu Asp Tyr Gln Leu 260

< 210 > 17 < 211 > 312 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: soluble IL-R < 400 > 17 Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val Val Glu Gly Glu Pro 1 5 10 15 Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His Glu Ile Glu Thr 20 25 30 Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser Gln Glu His Val Glu 35 40 45 Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His Asp Cys Val Leu 50 55 60 Glu Phe Trp Pro Val Glu Leu Asn Asp Thr Gly Ser Tyr Phe Phe Gln 65 70 75 80 Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu Asn Val Ile Arg Arg Asn 85 90 95 Lys His Ser Cys Phe Thr Glu Arg Gln Val Thr Ser Lys Ile Val Glu 100 105 110 Val Lys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser Tyr Tyr Gln Thr 115 120 125 Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys Leu Leu Leu 130 135 140 Glu Asn Asn Lys Asn Pro Thr lIe Lys Lys Asn Ala Glu Phe Glu Asp 145 150 155 160 Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His His Asn Gly Lys Leu 165 170 175 Phe Asn Ile Thr Lys Thr Phe Asn lIe Thr lIe Val Glu Asp Arg Ser 180 185 190 Asn He Val Pro Val Leu Leu Gly Pro Lys Leu Asn His Val Ala Val 195 200 205 Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser Ala Leu Leu Asn Glu 210 215 220 Glu Asp Val He Tyr Trp Met Phe Gly Glu Glu Asn Gly Ser Asp Pro 225 230 235 240 Asn He His Glu Glu Lys Glu Met Arg He Met Thr Pro Glu Gly Lys 245 250 255 Trp His Ala Ser Lys Val Leu Arg lIe Glu Asn He Gly Glu Ser Asn 260 265 270

Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser Thr Gly Gly Thr Asp 275 280 285 Thr Lys Ser Phe Ile Leu Val Arg Lys Ala Asp Met Ala Asp Ile Pro 290 295 300 Gly His Val Phe Thr Arg Gly Met 305 310 < 210 > 18 < 211 > 309 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: soluble IL-R < 400 > 18 Lys Ser Cys Ile His Arg Ser Gln He His Val Val Glu Gly Glu Pro 1 5 10 15 Phe Tyr Leu Lys Pro Cys Gly He Ser Ala Pro Val His Arg Asn Glu 20 25 30 Thr Ala Thr Met Arg Trp Phe Lys Gly Ser Ala Ser His Glu Tyr Arg 35 40 45 Glu Leu Asn Asn Arg Ser Ser Pro Arg Val Thr Phe His Asp His Thr 50 55 60 Leu Glu Phe Trp Pro Val Glu Met Glu Asp Glu Gly Thr Tyr lIe Ser 65 70 75 80 Gln Val Gly Asn Asp Arg Arg Asn Trp Thr Leu Asn Val Thr Lys Arg 85 90 95 Asn Lys His Ser Cys Phe Ser Asp Lys Leu Val Thr Ser Arg Asp Val 100 105 110 Glu Val Asn Lys Ser Leu His He Thr Cys Lys Asn Pro Asn Tyr Glu 115 120 125 Glu Leu lIe Gln Asp Thr Trp Leu Tyr Lys Asn Cys Lys Glu He Ser 130 135 140 Lys Thr Pro Arg He Leu Lys Asp Ala Glu Phe Gly Asp Glu Gly Tyr 145 150 155 160 Tyr Ser Cys Val Phe Ser Val His His Asn Gly Thr Arg Tyr Asn Ile 165 170 175 Thr Lys Thr Val Asn Ile Thr Val He Glu Gly Arg Ser Lys Val Thr 180 185 190 Pro Ala He Leu Gly Pro Lys Cys Glu Lys Val Gly Val Glu Leu Gly 195 200 205 Lys Asp Val Glu Leu Asn Cys Ser Ala Ser Leu Asn Lys Asp Asp Leu 210 215 220 Phe Tyr Trp Ser He Arg Lys Glu Asp Ser Ser Asp Pro Asn Val Gln

225 230 235 240 Glu Asp Arg Lys Glu Thr Thr Thr Trp Ile Ser Glu Gly Lys Leu His 245 250 255 Ala Ser Lys lIe Leu Arg Phe Gln Lys lIe Thr Glu Asn Tyr Leu Asn 260 265 270 Val Leu Tyr Asn Cys Thr Val Ala Asn Glu Glu Ala lIe Asp Thr Lys 275 280 285 Ser Phe Val Leu Val Arg Lys Glu Ile Pro Asp Ile Pro Gly His Val 290 295 300 Phe Thr Gly Gly Val 305 < 210 > 19 < 211 > 570 < 212 > PRT < 213 > Homo sapiens < 400 > 19 Met Thr Leu Leu Trp Cys Val Val Ser Leu Tyr Phe Tyr Gly He Leu 1 5 10 15 Gln Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met 20 25 30 Arg Gln He Gln Val Phe Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro 35 40 45 Leu Phe Glu His Phe Leu Lys Phe Asn Tyr Ser Thr Ala His Ser Ala 50 55 60 Gly Leu Thr Leu He Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu 65 70 75 80 Glu Pro lIe Asn Phe Arg Leu Pro Glu Asn Arg lIe Ser Lys Glu Lys 85 90 95 Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr 100 105 110 Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro 115 120 125 Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu 130 135 140 Pro Val His Lys Leu Tyr lIe Glu Tyr Gly He Gln Arg He Thr Cys 145 150 155 160 Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr He Thr 165 170 175 Trp Tyr Met Gly Cys Tyr Lys Ile Gln Asn Phe Asn Asn Val lIe Pro 180 185 190 Glu Gly Met Asn Leu Ser Phe Leu Ile Ala Leu He Ser Asn Asn Gly 195 200 205

Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His 210 215 220 Leu Thr Arg Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala 225 230 235 240 Val Pro Pro Val Ile His Ser Pro Asn Asp His Val Val Tyr Glu Lys 245 250 255 Glu Pro Gly Glu Glu Leu Leu He Pro Cys Thr Val Tyr Phe Ser Phe 260 265 270 Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr lIe Asp Gly Lys Lys 275 280 285 Pro Asp Asp lIe Thr He Asp Val Thr He Asn Glu Ser He Ser His 290 295 300 Ser Arg Thr Glu Asp Glu Thr Arg Thr Gln He Leu Ser He Lys Lys 305 310 315 320 Val Thr Ser Glu Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser 325 330 335 Ala Lys Gly Glu Val Ala Lys Ala Ala Lys Val Lys Gln Lys Val Pro 340 345 350 Ala Pro Arg Tyr Thr Val Glu Leu Ala Cys Gly Phe Gly Ala Thr Val 355 360 365 Leu Leu Val Val He Leu He Val Val Tyr His Val Tyr Trp Leu Glu 370 375 380 Met Val Leu Phe Tyr Arg Ala His Phe Gly Thr Asp Glu Thr He Leu 385 390 395 400 Asp Gly Lys Glu Tyr Asp He Tyr Val Ser Tyr Ala Arg Asn Ala Glu 405 410 415 Glu Glu Glu Phe Val Leu Leu Thr Leu Arg Gly Val Leu Glu Asn Glu 420 425 430 Phe Gly Tyr Lys Leu Cys He Phe Asp Arg Asp Ser Leu Pro Gly Gly 435 440 445 He Val Thr Asp Glu Thr Leu Ser Phe He Gln Lys Ser Arg Arg Leu 450 455 460 Leu Val Val Leu Ser Pro Asn Tyr Val Leu Gln Gly Thr Gln Ala Leu 465 470 475 480 Leu Glu Leu Lys Ala Gly Leu Glu Asn Met Ala Ser Arg Gly Asn He 485 490 495 Asn Val He Leu Val Gln Tyr Lys Ala Val Lys Glu Thr Lys Val Lys 500 505 510 Glu Leu Lys Arg Ala Lys Thr Val Leu Thr Val He Lys Trp Lys Gly 515 520 525 Glu Lys Ser Lys Tyr Pro Gln Gly Arg Phe Trp Lys Gln Leu Gln Val

530 535 540 Ala Met Pro Val Lys Lys Ser Pro Arg Arg Ser Ser Ser Asp Glu Gln 545 550 555 560 Gly Leu Ser Tyr Ser Ser Leu Lys Asn Val 565 570 < 210 > 20 < 211 > 1713 < 212 > DNA < 213 > Homo sapiens < 400 > 20 atgacacttc tgtggtgtgt agtgagtctc tacttttatg gaatcctgca aagtgatgcc 60 tcagaacgct gcgatgactg gggactagac accatgaggc aaatccaagt gtttgaagat 120 gagccagctc gcatcaagtg cccactcttt gaacacttct tgaaattcaa ctacagcaca 180 gcccattcag ctggccttac tctgatctgg tattggacta ggcaggaccg ggaccttgag 240 gagccaatta acttccgcct ccccgagaac cgcattagta aggagaaaga tgtgctgtgg 300 ttccggccca ctctcctcaa tgacactggc aactatacct gcatgttaag gaacactaca 360 tattgcagca aagttgcatt tcccttggaa gttgttcaaa aagacagctg tttcaattcc 420 cccatgaaac tcccagtgca taaactgtat atagaatatg gcattcagag gatcacttgt 480 ccaaatgtag atggatattt tccttccagt gtcaaaccga ctatcacttg gtatatgggc 540 tgttataaaa tacagaattt taataatgta atacccgaag gtatgaactt gagtttcctc 600 attgccttaa tttcaaataa tggaaattac acatgtgttg ttacatatcc agaaaatgga 660 cgtacgtttc atctcaccag gactctgact gtaaaggtag taggctctcc aaaaaatgca 720 gtgccccctg tgatccattc acctaatgat catgtggtct atgagaaaga accaggagag 780 gagctactca ttccctgtac ggtctatttt agttttctga tggattctcg caatgaggtt 840 tggtggacca ttgatggaaa aaaacctgat gacatcacta ttgatgtcac cattaacgaa 900 agtataagtc atagtagaac agaagatgaa acaagaactc agattttgag catcaagaaa 960 gttacctctg aggatctcaa gcgcagctat gtctgtcatg ctagaagtgc caaaggcgaa 1020 gttgccaaag cagccaaggt gaagcagaaa gtgccagctc caagatacac agtggaactg 1080 gcttgtggtt ttggagccac agtcctgcta gtggtgattc tcattgttgt ttaccatgtt 1140 tactggctag agatggtcct attttaccgg gctcattttg gaacagatga aaccatttta 1200 gatggaaaag agtatgatat ttatgtatcc tatgcaagga atgcggaaga agaagaattt 1260 gtattactga ccctccgtgg agttttggag aatgaatttg gatacaagct gtgcatcttt 1320 gaccgagaca gtctgcctgg gggaattgtc acagatgaga ctttgagctt cattcagaaa 1380

agcagacgcc tcctggttgt tctaagcccc aactacgtgc tccagggaac ccaagccctc 1440 ctggagctca aggctggcct agaaaatatg gcctctcggg gcaacatcaa cgtcatttta 1500 gtacagtaca aagctgtgaa ggaaacgaag gtgaaagagc tgaagagggc taagacggtg 1560 ctcacggtca ttaaatggaa aggggaaaaa tccaagtatc cacagggcag gttctggaag 1620 cagctgcagg tggccatgcc agtgaagaaa agtcccaggc ggtctagcag tgatgagcag 1680 ggcctctcgt attcatcttt gaaaaatgta tga 1713 < 210 > 21 < 211 > 339 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: soluble IL-RAcP < 400 > 21 Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met Arg Gln He Gln 1 5 10 15 Val Phe Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro Leu Phe Glu His 20 25 30 Phe Leu Lys Phe Asn Tyr Ser Thr Ala His Ser Ala Gly Leu Thr Leu 35 40 45 He Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu Glu Pro He Asn 50 55 60 Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys Asp Val Leu Trp 65 70 75 80 Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr Thr Cys Met Leu 85 90 95 Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro Leu Glu Val Val 100 105 110 Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu Pro Val His Lys 115 120 125 Leu Tyr He Glu Tyr Gly He Gln Arg He Thr Cys Pro Asn Val Asp 130 135 140 Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr He Thr Trp Tyr Met Gly 145 150 155 160 Cys Tyr Lys He Gln Asn Phe Asn Asn Val He Pro Glu Gly Met Asn 165 170 175 Leu Ser Phe Leu He Ala Leu He Ser Asn Asn Gly Asn Tyr Thr Cys 180 185 190 Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His Leu Thr Arg Thr 195 200 205

Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala Val Pro Pro Val 210 215 220 Ile His Ser Pro Asn Asp His Val Val Tyr Glu Lys Glu Pro Gly Glu 225 230 235 240 Glu Leu Leu lIe Pro Cys Thr Val Tyr Phe Ser Phe Leu Met Asp Ser 245 250 255 Arg Asn Glu Val Trp Trp Thr He Asp Gly Lys Lys Pro Asp Asp Ile 260 265 270 Thr He Asp Val Thr Ile Asn Glu Ser He Ser His Ser Arg Thr Glu 275 280 285 Asp Glu Thr Arg Thr Gln He Leu Ser He Lys Lys Val Thr Ser Glu 290 295 300 Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser Ala Lys Gly Glu 305 310 315 320 Val Ala Lys Ala Ala Lys Val Lys Gln Lys Val Pro Ala Pro Arg Tyr 325 330 335 Thr Val Glu < 210 > 22 < 211 > 339 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: soluble IL-RAcP < 400 > 22 Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met Arg Gln He Gln 1 5 10 15 Val Phe Glu Asp Glu Pro Ala Arg He Lys Cys Pro Leu Phe Glu His 20 25 30 Phe Leu Lys Tyr Asn Tyr Ser Thr Ala His Ser Ser Gly Leu Thr Leu 35 40 45 He Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu Glu Pro He Asn 50 55 60 Phe Arg Leu Pro Glu Asn Arg He Ser Lys Glu Lys Asp Val Leu Trp 65 70 75 80 Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr Thr Cys Met Leu 85 90 95 Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro Leu Glu Val Val 100 105 110 Gln Lys Asp Ser Cys Phe Asn Ser Ala Met Arg Phe Pro Val His Lys 115 120 125 Met Tyr He Glu His Gly He His Lys He Thr Cys Pro Asn Val Asp

130 135 140 Gly Tyr Phe Pro Ser Ser Val Lys Pro Ser Val Thr Trp Tyr Lys Gly 145 150 155 160 Cys Thr Glu Ile Val Asp Phe His Asn Val Leu Pro Glu Gly Met Asn 165 170 175 Leu Ser Phe Phe Ile Pro Leu Val Ser Asn Asn Gly Asn Tyr Thr Cys 180 185 190 Val Val Thr Tyr Pro Glu Asn Gly Arg Leu Phe His Leu Thr Arg Thr 195 200 205 Val Thr Val Lys Val Val Gly Ser Pro Lys Asp Ala Leu Pro Pro Gln 210 215 220 lIe Tyr Ser Pro Asn Asp Arg Val Val Tyr Glu Lys Glu Pro Gly Glu 225 230 235 240 Glu Leu Val lIe Pro Cys Lys Val Tyr Phe Ser Phe Ile Met Asp Ser 245 250 255 His Asn Glu Val Trp Trp Thr Ile Asp Gly Lys Lys Pro Asp Asp Val 260 265 270 Thr Val Asp lIe Thr Ile Asn Glu Ser Val Ser Tyr Ser Ser Thr Glu 275 280 285 Asp Glu Thr Arg Thr Gln Ile Leu Ser lIe Lys Lys Val Thr Pro Glu 290 295 300 Asp Leu Arg Arg Asn Tyr Val Cys His Ala Arg Asn Thr Lys Gly Glu 305 310 315 320 Ala Glu Gln Ala Ala Lys Val Lys Gln Lys Val Ile Pro Pro Arg Tyr 325 330 335 Thr Val Glu < 210 > 23 < 211 > 339 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: soluble IL-RAcP < 400 > 23 Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met Arg Gln Ile Gln 1 5 10 15 Val Phe Glu Asp Glu Pro Ala Arg lIe Lys Cys Pro Leu Phe Glu His 20 25 30 Phe Leu Lys Tyr Asn Tyr Ser Thr Ala His Ser Ser Gly Leu Thr Leu 35 40 45 lIe Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu Glu Pro Ile Asn 50 55 60

Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys Asp Val Leu Trp 65 70 75 80 Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr Thr Cys Met Leu 85 90 95 Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro Leu Glu Val Val 100 105 110 Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Arg Leu Pro Val His Arg 115 120 125 Leu Tyr Ile Glu Gln Gly Ile His Asn lIe Thr Cys Pro Asn Val Asp 130 135 140 Gly Tyr Phe Pro Ser Ser Val Lys Pro Ser Val Thr Trp Tyr Lys Gly 145 150 155 160 Cys Thr Glu lIe Val Asn Phe His Asn Val Gln Pro Lys Gly Met Asn 165 170 175 Leu Ser Phe Phe lIe Pro Leu Val Ser Asn Asn Gly Asn Tyr Thr Cys 180 185 190 Val Val Thr Tyr Leu Glu Asn Gly Arg Leu Phe His Leu Thr Arg Thr 195 200 205 Met Thr Val Lys Val Val Gly Ser Pro Lys Asp Ala Val Pro Pro His 210 215 220 lIe Tyr Ser Pro Asn Asp Arg Val Val Tyr Glu Lys Glu Pro Gly Glu 225 230 235 240 Glu Leu Val Ile Pro Cys Lys Val Tyr Phe Ser Phe Ile Met Asp Ser 245 250 255 His Asn Glu Ile Trp Trp Thr Ile Asp Gly Lys Lys Pro Asp Asp Val 260 265 270 Pro Val Asp He Thr lIe He Glu Ser Val Ser Tyr Ser Ser Thr Glu 275 280 285 Asp Glu Thr Arg Thr Gln He Leu Ser He Lys Lys Val Thr Pro Glu 290 295 300 Asp Leu Lys Arg Asn Tyr Val Cys His Ala Arg Asn Ala Glu Gly Glu 305 310 315 320 Ala Glu Gln Ala Ala Lys Val Lys Gln Lys Val He Pro Pro Arg Tyr 325 330 335 Thr Val Glu < 210 > 24 < 211 > 338 < 212 > PRT < 213 > Artificial Sequence < 220 >

< 223 > Description of Artificial Sequence: soluble IL-RAcP < 400 > 24 Phe Asn lIe Ser Gly Cys Ser Thr Lys Lys Leu Leu Trp Thr Tyr Ser 1 5 10 15 Thr Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp Leu Pro Glu Pro 20 25 30 Gln Lys Ser His Phe Cys His Arg Asn Arg Leu Ser Pro Lys Gln Val 35 40 45 Pro Glu His Leu Pro Phe Met Gly Ser Asn Asp Leu Ser Asp Val Gln 50 55 60 Trp Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp lIe Arg Lys 65 70 75 80 Ser Tyr Pro His lIe Ile Gln Asp Lys Cys Thr Leu His Phe Leu Thr 85 90 95 Pro Gly Val Asn Asn Ser Gly Ser Tyr lIe Cys Arg Pro Lys Met lIe 100 105 110 Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met He Leu Glu Val 115 120 125 Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser His Lys Gln 130 135 140 Asp Leu Leu Leu Gly Ser Thr Gly Ser He Ser Cys Pro Ser Leu Ser 145 150 155 160 Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp Tyr Lys Asn Gly 165 170 175 Lys Leu Leu Ser Val Glu Arg Ser Asn Arg lIe Val Val Asp Glu Val 180 185 190 Tyr Asp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr Gln Ser Asp 195 200 205 Thr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val Arg Thr He 210 215 220 Val Gly Asp Thr Lys Leu Lys Pro Asp He Leu Asp Pro Val Glu Asp 225 230 235 240 Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr He Ser Cys Lys Ala 245 250 255 Arg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys Trp Tyr He 260 265 270 Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu Ala Lys Ser 275 280 285 Ile Lys Ser Thr Leu Lys Asp Glu He He Glu Arg Asn He Ile Leu 290 295 300 Glu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val Cys Phe Val 305 310 315 320

Gln Asn Ser lIe Gly Asn Thr Thr Gln Ser Val Gln Leu Lys Glu Lys 325 330 335 Arg Gly < 210 > 25 < 211 > 337 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : soluble IL-RAcP < 400 > 25 Phe Asn His Ser Ala Cys Ala Thr Lys Lys Leu Leu Trp Thr Tyr Ser 1 5 10 15 Ala Arg Gly Ala Glu Asn Phe Val Leu Phe Cys Asp Leu Gln Glu Leu 20 25 30 Gln Glu Gln Lys Phe Ser His Ala Ser Gln Leu Ser Pro Thr Gln Ser 35 40 45 Pro Ala His Lys Pro Cys Ser Gly Ser Gln Lys Asp Leu Ser Asp Val 50 55 60 Gln Trp Tyr Met Gln Pro Arg Ser Gly Ser Pro Leu Glu Glu lIe Ser 65 70 75 80 Arg Asn Ser Pro His Met Gln Ser Glu Gly Met Leu His lIe Leu Ala 85 90 95 Pro Gln Thr Asn Ser lIe Trp Ser Tyr lIe Cys Arg Pro Arg Ile Arg 100 105 110 Ser Pro Gln Asp Met Ala Cys Cys Ile Lys Thr Val Leu Glu Val Lys 115 120 125 Pro Gln Arg Asn Val Ser Cys Gly Asn Thr Ala Gln Asp Glu Gln Val 130 135 140 Leu Leu Leu Gly Ser Thr Gly Ser He His Cys Pro Ser Leu Ser Cys 145 150 155 160 Gln Ser Asp Val Gln Ser Pro Glu Met Thr Trp Tyr Lys Asp Gly Arg 165 170 175 Leu Leu Pro Glu His Lys Lys Asn Pro He Glu Met Ala Asp He Tyr 180 185 190 Val Phe Asn Gln Gly Leu Tyr Val Cys Asp Tyr Thr Gln Ser Asp Asn 195 200 205 Val Ser Ser Trp Thr Val Arg Ala Val Val Lys Val Arg Thr Ile Gly 210 215 220 Lys Asp He Asn Val Lys Pro Glu He Leu Asp Pro He Thr Asp Thr 225 230 235 240

Leu Asp Val Glu Leu Gly Lys Pro Leu Thr Leu Pro Cys Arg Val Gln 245 250 255 Phe Gly Phe Gln Arg Leu Ser Lys Pro Val He Lys Trp Tyr Val Lys 260 265 270 Glu Ser Thr Gln Glu Trp Glu Met Ser Val Phe Glu Glu Lys Arg He 275 280 285 Gln Ser Thr Phe Lys Asn Glu Val He Glu Arg Thr He Phe Leu Arg 290 295 300 Glu Val Thr Gln Arg Asp Leu Ser Arg Lys Phe Val Cys Phe Ala Gln 305 310 315 320 Asn Ser He Gly Asn Thr Thr Arg Thr He Arg Leu Arg Lys Lys Glu 325 330 335 Glu < 210 > 26 < 211 > 336 < 212 > PRT < 213 > Homo sapiens < 400 > 26 Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met Arg Gln He Gln 1 5 10 15 Val Phe Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro Leu Phe Glu His 20 25 30 Phe Leu Lys Phe Asn Tyr Ser Thr Ala His Ser Ala Gly Leu Thr Leu 35 40 45 He Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu Glu Pro He Asn 50 55 60 Phe Arg Leu Pro Glu Asn Arg He Ser Lys Glu Lys Asp Val Leu Trp 65 70 75 80 Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr Thr Cys Met Leu 85 90 95 Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro Leu Glu Val Val 100 105 110 Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu Pro Val His Lys 115 120 125 Leu Tyr He Glu Tyr Gly He Gln Arg He Thr Cys Pro Asn Val Asp 130 135 140 Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr He Thr Trp Tyr Met Gly 145 150 155 160 Cys Tyr Lys He Gln Asn Phe Asn Asn Val He Pro Glu Gly Met Asn 165 170 175 Leu Ser Phe Leu He Ala Leu He Ser Asn Asn Gly Asn Tyr Thr Cys

180 185 190 Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His Leu Thr Arg Thr 195 200 205 Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala Val Pro Pro Val 210 215 220 lIe His Ser Pro Asn Asp His Val Val Tyr Glu Lys Glu Pro Gly Glu 225 230 235 240 Glu Leu Leu lIe Pro Cys Thr Val Tyr Phe Ser Phe Leu Met Asp Ser 245 250 255 Arg Asn Glu Val Trp Trp Thr lIe Asp Gly Lys Lys Pro Asp Asp He 260 265 270 Thr He Asp Val Thr He Asn Glu Ser He Ser His Ser Arg Thr Glu 275 280 285 Asp Glu Thr Arg Thr Gln He Leu Ser He Lys Lys Val Thr Ser Glu 290 295 300 Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser Ala Lys Gly Glu 305 310 315 320 Val Ala Lys Ala Ala Lys Val Lys Gln Lys Gly Asn Arg Cys Gly Gln 325 330 335 < 210 > 27 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: primer < 400 > 27 gtcccatggc accggttaga tctctg 26 < 210 > 28 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: primer < 400 > 28 cagcttatcg gcgtagagga t 21 < 210 > 29 < 211 > 35 < 212 > DNA

< 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : primer < 400 > 29 tgcgaattca tgaaagtgtt actcagactt atttg 35 < 210 > 30 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : primer < 400 > 30 tgactcgagt tacttctgga aattagtgac tgg 33 < 210 > 31 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : primer < 400 > 31 ggatgacact tctgtggtgt g 21 < 210 > 32 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : primer < 400 > 32 tccttttcat tattcctttc ataca 25 < 210 > 33 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : primer < 400 > 33 tcgccaccat ggacacttct gtggtgtg 28

< 210 > 34 < 211 > 52 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: primer < 400 > 34 tcggaattcc tcagtgatgg tgatggtgat gttccactgt gtatcttgga gc 52 < 210 > 35 < 211 > 15 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: peptide < 400 > 35 Phe Glu Trp Thr Pro Gly Tyr Trp Gln Pro Tyr Ala Leu Pro Leu 1 5 10 15 < 210 > 36 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: 6His tag < 400 > 36 catcaccatc accatcac 18 < 210 > 37 < 211 > 21 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: peptide < 400 > 37 Glu Thr Pro Phe Thr Trp Glu Glu Ser Asn Ala Tyr Tyr Trp Gln Pro 1 5 10 15 Tyr Ala Leu Pro Leu 20 < 210 > 38 < 211 > 21 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: signal sequence

< 400 > 38 Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr lIe 1 5 10 15 Ser Tyr lIe Tyr Ala 20 < 210 > 39 < 211 > 19 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence : signal sequence < 400 > 39 Met Trp Leu Leu Leu Thr Met Ala Ser Leu lIe Ser Val Leu Gly Thr 1 5 10 15 Thr His Gly < 210 > 40 < 211 > 20 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: signal sequence < 400 > 40 Met Lys Val Leu Leu Arg Leu lIe Cys Phe lIe Ala Leu Leu lIe Ser 1 5 10 15 Ser Leu Glu Ala 20 < 210 > 41 < 211 > 20 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: signal sequence < 400 > 41 Met Thr Leu Leu Trp Cys Val Val Ser Leu Tyr Phe Tyr Gly lIe Leu 1 5 10 15 Gln Ser Asp Ala 20 < 210 > 42 < 211 > 321 < 212 > PRT < 213 > Artificial Sequence

< u < 223 > Description of Artificial Sequence : soluble IL-1R type II < 400 > 42 Arg Ser Cys Arg Phe Arg Gly Arg His Tyr Lys Arg Glu Phe Arg Leu 1 5 10 15 Glu Gly Glu Pro Val Ala Leu Arg Cys Pro GIn Val Pro Tyr Trp Leu 20 25 30 Trp Ala Ser Val Ser Pro Arg He Asn Leu Thr Trp His Lys Asn Asp 35 40 45 Ser Ala Arg Thr Val Pro Gly Glu Glu Glu Thr Arg Met Trp Ala Gln 50 55 60 Asp Gly Ala Leu Trp Leu Leu Pro Ala Leu Gln Glu Asp Ser Gly Thr 65 70 75 80 Tyr Val Cys Thr Thr Arg Asn Ala Ser Tyr Cys Asp Lys Met Ser He 85 90 95 Glu Leu Arg Val Phe Glu Asn Thr Asp Ala Phe Leu Pro Phe He Ser 100 105 110 Tyr Pro Gln He Leu Thr Leu Ser Thr Ser Gly Val Leu Val Cys Pro 115 120 125 Asp Leu Ser Glu Phe Thr Arg Asp Lys Thr Asp Val Lys He Gln Trp 130 135 140 Tyr Lys Asp Ser Leu Leu Leu Asp Lys Asp Asn Glu Lys Phe Leu Ser 145 150 155 160 Val Arg Gly Thr Thr His Leu Leu Val His Asp Val Ala Leu Glu Asp 165 170 175 Ala Gly Tyr Tyr Arg Cys Val Leu Thr Phe Ala His Glu Gly Gln Gln 180 185 190 Tyr Asn He Thr Arg Ser He Glu Leu Arg Ile Lys Lys Lys Lys Glu 195 200 205 Glu Thr He Pro Val He He Ser Pro Leu Lys Thr lIe Ser Ala Ser 210 215 220 Leu Gly Ser Arg Leu Thr He Pro Cys Lys Val Phe Leu Gly Thr Gly 225 230 235 240 Thr Pro Leu Thr Thr Met Leu Trp Trp Thr Ala Asn Asp Thr His He 245 250 255 Glu Ser Ala Tyr Pro Gly Gly Arg Val Thr Glu Gly Pro Arg Gln Glu 260 265 270 Tyr Ser Glu Asn Asn Glu Asn Tyr He Glu Val Pro Leu He Phe Asp 275 280 285

Pro Val Thr Arg Glu Asp Leu His Met Asp Phe Lys Cys Val Val His ? aqs Rnn

Asn Thr Leu Ser Phe Gln Thr Leu Arg Thr Thr Val Lys Glu Ala Ser 305 310 315 320 Ser < 210 > 43 < 211 > 135 < 212 > PRT < 213 > Homo sapiens < 400 > 43 Arg Ala Thr Pro Val Ser Gln Thr Thr Thr Ala Ala Thr Ala Ser Val 1 5 10 15 Arg Ser Thr Lys Asp Pro Cys Pro Ser Gln Pro Pro Val Phe Pro Ala 20 25 30 Ala Lys Gln Cys Pro Ala Leu Glu Val Thr Trp Pro Glu Val Glu Val 35 40 45 Pro Leu Asn Gly Thr Leu Ser Leu Ser Cys Val Ala Cys Ser Arg Phe 50 55 60 Pro Asn Phe Ser Ile Leu Tyr Trp Leu Gly Asn Gly Ser Phe Ile Glu 65 70 75 80 His Leu Pro Gly Arg Leu Trp Glu Gly Ser Thr Ser Arg Glu Arg Gly 85 90 95 Ser Thr Gly Trp Ala Glu Gly Asn Leu Ala Pro His Pro Arg Ser Pro 100 105 110 Ala Leu Gln Pro Gln Gln Ser Thr Ala Ala Gly Leu Arg Leu Ser Thr 115 120 125 Gly Pro Ala Ala Ala Gln Pro 130 135 < 210 > 44 < 211 > 156 < 212 > PRT < 213 > Homo sapiens < 400 > 44 Glu Thr lIe Cys Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala 1 5 10 15 Phe Arg lIe Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn 20 25 30 Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu 35 40 45 Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile 50 55 60 His Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr 65 70 75 80

Arg Leu Gln Leu Glu Ala Val Asn He Thr Asp Leu Ser Glu Asn Arg 85 90 95 Lys Gln Asp Lys Arg Phe Ala Phe He Arg Ser Asp Ser Gly Pro Thr 100 105 110 Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala 115 120 125 Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly 130 135 140 Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu 145 150 155

Claims

CLAIMS: 1) Assay method for determining the ability of a test compound to modulate the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) bringing into contact an IL polypeptide, a soluble IL-R polypeptide, a soluble IL-RAcP polypeptide and a test compound ; and (b) determining the amount of said trimolecular complex formed.
2) Assay method for determining the ability of a test compound to disrupt or interfere with the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a test compound ; (b) bringing the test compound into contact with a defined amount of a soluble IL-R polypeptide and a soluble IL-RAcP polypeptide ; (c) adding an IL polypeptide to the mixture obtained in step (b) ; and (d) determining the amount of said trimolecular complex formed.
3) Assay method according to claim 1 for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method further comprising: - the addition of a soluble IL antagonist at step (a); and - an additional step (c) in which the amount of said trimolecular complex formed at step (a) is compared with the amount of said trimolecular

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complex formed in the absence of test compound.
4) Assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: : (a) providing a test compound; (b) bringing the test compound into contact with a soluble IL-R polypeptide, a soluble IL antagonist and a soluble IL-RAcP polypeptide ; (c) adding an IL polypeptide to the mixture obtained at step (b) ; (d) determining the amount of said trimolecular complex formed ; and (e) comparing the amount of said trimolecular complex formed at step (c) with the amount of said trimolecular complex formed in the absence of test compound.
5) Assay method according to claim 1 for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method further comprising: - the addition of an IL-R antagonist at step (a) ; and - an additional step (c) in which the amount of said trimolecular complex formed at step (a) is compared with the amount of said trimolecular complex formed in the absence of test compound.
6) Assay method for determining the ability of a test compound to enhance the formation of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: : (a) providing a test compound ;

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(b) bringing the test compound into contact with an IL polypeptide, an IL-R antagonist and a soluble IL-RAcP polypeptide; (c) adding a soluble IL-R polypeptide to the mixture obtained in step (b); (d) determining the amount of said trimolecular complex formed; and (e) comparing the amount of said trimolecular complex formed at step (c) with the amount of said trimolecular complex formed in the absence of test compound.
7) Assay method for determining the ability of a test compound to disrupt or interfere with the stability of a trimolecular complex including IL, a soluble IL-R and a soluble IL-RAcP, the method comprising: (a) providing a defined amount of a trimolecular complex comprising an IL polypeptide, a soluble IL-R polypeptide and a soluble IL-RAcP polypeptide; (b) contacting a test compound with said trimolecular complex; and (c) comparing the amount of said trimolecular complex present after step (b) to the amount of said trimolecular complex initially present at step (a).
8) Assay method according to any one of claims 1 to 7 wherein the IL, sIL-R and sIL-RAcP are of mammalian origins.
9) Assay method according to any one of claims 1 to 8 wherein the IL, sIL-R and sIL-RAcP are from human, mouse or rat.

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10) Assay method according to any one of claims 1 to 9 wherein the IL polypeptide is an IL amino acid sequence or a fragment, derivative, analog or active portion thereof which retains the biological activity of IL.
11) Assay method according to any one of claims 1 to 10 wherein the IL polypeptide is in its mature form and has a barrel shaped 3-dimensional structure composed of 12 to 13 beta-strands and a cellular activity mediated by a ternary (i. e. trimolecular) complex at the cell surface.
12) Assay method according to claim 11 wherein the IL polypeptide has an amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or a fragment thereof.
13) Assay method according to any one of claims 1 to 12 wherein the soluble IL-R polypeptide is any soluble polypeptide fragment of a full-length IL-R which retains the extracellular binding activity of the full length protein.
14) Assay method according to any one of claims 1 to 13 wherein the soluble IL-R polypeptide comprises 3 Ig-like domains which are included in three structural domains identified as domain 1, domain 2 and domain 3 starting from the N-terminal end of the sequence, said domain 1 containing one Ig-like domain and two disulfide bonds; said domain 2 containing one Ig-like domain and two overlapping disulfide bonds; said domain 3 containing one Ig-like domain and one disulfide bond.

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15) Assay method according to claim 14 wherein the soluble IL-R polypeptide has an amino acid sequence of SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or a fragment thereof.
16) Assay method according to any one of claims 1 to 15 wherein the soluble IL-RAcP polypeptide is any soluble polypeptide fragment of a full-length IL-RAcP which retains the extracellular binding activity of the full length protein.
17) Assay method according to any one of claims 1 to 16 wherein the soluble IL-RAcP polypeptide comprises 3 Ig-like domains which are included in three structural domains identified as domain 1, domain 2 and domain 3 starting from the N-terminal end of the sequence, said domain 1 containing one Ig-like domain and two disulfide bonds; said domain 2 containing one Ig-like domain and two overlapping disulfide bonds; said domain 3 containing one Ig-like domain and one disulfide bond.
18) An assay method according to claim 17 wherein the soluble IL-RAcP polypeptide has an amino acid sequence of SEQ ID NO: 21, or SEQ ID NO: 22, or SEQ ID NO: 23, or SEQ ID NO: 24, or SEQ ID NO : 25, or SEQ ID NO: 26, or a fragment thereof.
19) A soluble tri-molecular complex comprising or consisting of the following elements: (a) an IL polypeptide (b) a soluble IL-R polypeptide which binds said IL polypeptide, and (c) a soluble IL-RAcP polypeptide which binds to the IL-R polypeptide/IL polypeptide binary complex.

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20) A soluble complex according to claim 19 wherein the IL, sIL-R and sIL-RAcP are of mammalian origins.
21) A soluble complex according to claim 19 wherein the IL, sIL-R and sIL-RAcP are from human, mouse or rat.
22) A soluble complex according to claim 19 wherein at least one of the IL, sIL-R or sIL-RAcP is from human.
23) A soluble complex according to any one of claims 19 to 22 wherein the IL polypeptide has an amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO : 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or a fragment thereof.
24) A soluble complex according to any one of claims 19 to 23 wherein the soluble IL-R polypeptide has an amino acid sequence of SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or a fragment thereof.
25) A soluble complex according to any one of claims 19 to 24 wherein the soluble IL-RAcP polypeptide has an amino acid sequence of SEQ ID NO: 21, or SEQ ID NO: 22, or SEQ ID NO: 23, or SEQ ID NO: 24, or SEQ ID NO: 25, or SEQ ID NO: 26, or a fragment thereof.