IL-1 receptor based cytokine traps and method of using Number:7,417,134 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides a fusion polypeptide capable of binding interleukin-1 (O:-1) to form a nonfunctional complex. It also provides a nucleic acid sequence encoding the fusion polypeptide and methods of using the fusion polypeptide.<H cytokine, receptor, IL, 1, receptor, b, 7417134.html, Patent, pto, 7417134

Title: IL-1 receptor based cytokine traps and method of using

Abstract: The present invention provides a fusion polypeptide capable of binding interleukin-1 (O:-1) to form a nonfunctional complex. It also provides a nucleic acid sequence encoding the fusion polypeptide and methods of using the fusion polypeptide.

Patent Number: 7,417,134 Issued on 08/26/2008 to Stahl, et al.

Inventors: Stahl; Neil (Carmel, NY), Yancopoulos; George D. (Yorktown Heights, NY)
Assignee: Regeneron Pharmaceuticals, Inc. (Tarrytown, NY)

Appl. No.: 11/134,114

Filed: May 20, 2005

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
10282162	Oct., 2002	6927044
09787835
PCT/US99/22045	Sep., 1999
09313942	May., 1999	6472179
60101858	Sep., 1998

Current U.S. Class: 536/23.4 ; 424/85.2; 435/320.1; 435/69.1; 435/69.52; 435/69.7; 536/23.1; 536/23.5

Current International Class: C07H 21/04 (20060101); A61K 38/20 (20060101); C12N 15/00 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


5470952	November 1995	Stahl et al.
6610750	August 2003	Charbit et al.

Foreign Patent Documents


0835939	Jun., 1991	EP
0533006	Sep., 1992	EP
WO93/19163	Sep., 1993	WO
WO93/19777	Oct., 1993	WO
WO94/22914	Oct., 1994	WO
WO95/06737	Mar., 1995	WO
WO96/11213	Apr., 1996	WO
WO96/23881	Aug., 1996	WO
WO96/35783	Nov., 1996	WO
WO97/15669	May., 1997	WO
WO97/31946	Sep., 1997	WO
WO99/37772	Jul., 1999	WO

Other References

Greenfeeder, S.A., et al., (1995) J. Biol. Chem. 270(23):13757-13765. cited by other .
Seipelt, I., et al., (1997) Biochem. Biophys. Res. Comm. 293:534-542. cited by other .
Stahl, N., et al., (1999) FASEB J. Abstract, 1457. cited by other .
U.S. Appl. No. 60/101,858, filed Sep. 25, 1998, Stahl,N. & Yancopoulos,G.D. cited by other .
U.S. Appl. No. 09/313,942, filed May 19, 1999, Stahl,N. & Yancopoulos,G.D. cited by other .
J. of Biol. Chem., 1995, Greenfeeder, S.A., et. al., "Molecular Cloning and Characterization of a Second Subunit of the Interleukin 1 Receptor Complex", 270(23):13757-13765. cited by other .
Biochem and Biophys Res Comm, 1997, Seipelt, I., et al., "Overexpression, Purification, and Use of a Soluble Human Interleukin-4 Receptor .alpha.-chain/Ig.gamma.1 Fusion Protein for Ligand Binding Studies," 239:534-542. cited by other .
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Primary Examiner: Saoud; Christine J
Assistant Examiner: Seharaseyon; Jegatheesan
Attorney, Agent or Firm: Gregg, Esq.; Valeta

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/282,162 filed 28 Oct. 2002, now U.S. Pat. No. 6,927,044, which is a continuation-in-part of U.S. Ser. No. 09/787,835, filed 22 Mar. 2001, now abandoned, which is a U.S. National Stage Application of International Application No. PCT/US99/22045, filed 22 Sep. 1999, which is a continuation of U.S. Ser. No. 09/313,942, filed 19 May 1999, now U.S. Pat. No. 6,472,179, which claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional Application No. 60/101,858 filed Sep. 25, 1998. The disclosures of these publications are hereby incorporated by reference into this application in their entireties.

Claims

We claim:

1. A recombinant nucleic acid molecule encoding a fusion polypeptide which forms a multimer capable of binding interleukin-1 (IL-1) to form a nonfunctional complex, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:51.

2. The nucleic acid of claim 1 encoding a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:52.

3. A vector comprising the nucleic acid molecule of claim 1.

4. A host-vector system for the production of a fusion polypeptide, comprising the vector of claim 3 in a suitable host cell.

5. A method of producing a fusion polypeptide, comprising growing cells of the host-vector system of claim 4, under conditions permitting production of the fusion polypeptide and recovering the fusion polypeptide so produced.

6. A recombinant nucleic acid molecule encoding a fusion polypeptide which forms a multimer capable of binding interleukin-1 (IL-1) to form a nonfunctional complex, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:53.

7. The nucleic acid of claim 6 encoding a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:54.

8. A recombinant nucleic acid molecule encoding a fusion polypeptide which forms a multimer capable of binding interleukin-1 (IL-1) to form a nonfunctional complex, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:55.

9. The nucleic acid of claim 8 encoding a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:56.

10. A fusion polypeptide comprising the amino acid sequence of SEQ ID NO:52.

11. A composition comprising a multimer of the fusion polypeptide of claim 10.

12. The composition of claim 11 wherein the multimer is a dimer.

13. A method of treating arthritis, comprising administering a therapeutically effective amount of the composition of claim 12.

Description

FIELD OF THE INVENTION

The invention relates to receptor-based fusion proteins capable of binding and inhibiting the biological activity of a cytokine. More specifically, the invention relates to interleukin-1 (IL-1) fusion proteins capable of trapping and inhibiting the action of IL-1, and therapeutic uses thereof.

DESCRIPTION OF RELATED ART

Although discovered for varying biological activities, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM) and interleukin-6 (IL-6) comprise a defined family of cytokines (referred to herein as the "CNTF family" of cytokines). These cytokines are grouped together because of their distant structural similarities (Bazan et al. 1991 J. Neuron 7:197-208), and, perhaps more importantly, because they share ".beta." signal-transducing receptor components (Baumann et al. 1993 J. Biol. Chem. 265:19853-19862); Davis et al. 1993 Science 260:1805-1808; Gearing et al. 1992 Science 255:1434-1437; Ip et al. 1992 Cell 69: 1121-1132; Stahl et al. 1993 J. Biol. Chem. 268: 7628-7631; Stahl et al. 1993 Cell 74:587-590). Receptor activation by this family of cytokines results from either homo- or hetero-dimerization of these .beta. components.

In addition to the .beta. components, some of these cytokines also require specificity-determining ".alpha." components that are more limited in their tissue distribution than the .beta. components, and thus determine the cellular targets of the particular cytokines. Thus, LIF and OSM are broadly acting factors that may only require the presence of gp130 and LIFR.beta. on responding cells, while CNTF requires CNTFR.alpha.. Both CNTFR.alpha. and IL-6R.alpha. (Hibi et al. Cell 63:1149-1157) can function as soluble proteins, consistent with the notion that they do not interact with intracellular signaling molecules but that they serve to help their ligands interact with the appropriate signal transducing .beta. subunits.

Additional evidence from other cytokine systems also supports the notion that dimerization provides a common mechanism by which all cytokine receptors initiate signal transduction. Studies with the erythropoietin (EPO) receptor are also consistent with the importance of dimerization in receptor activation, as EPO receptors can be constitutively activated by a single amino acid change that introduces a cysteine residue and results in disulfide-linked homodimers (Watowich et al. 1992 Proc. Natl. Acad. Sci. USA 89:2140-2144).

In addition to homo- or hetero-dimerization of .beta. subunits as the critical step for receptor activation, a second important feature is that formation of the final receptor complex by the CNTF family of cytokines occurs through a mechanism whereby the ligand successively binds to receptor components in an ordered manner (Davis et al. 1993 supra). Thus CNTF first binds to CNTFR.alpha., forming a complex which then binds gp130 to form an intermediate (called here the .alpha..beta.1 intermediate) that is not signaling competent because it has only a single .beta. component, before finally recruiting LIFR.beta. to form a heterodimer of .beta. components which then initiates signal transduction. Altogether, these findings led to a proposal for the structure of a generic cytokine receptor complex in which each cytokine can have up to 3 receptor binding sites: a site that binds to an optional a specificity-determining component (a site), a site that binds to the first .beta. signal-transducing component (.beta.1 site), and a site that binds to the second .beta. signal-transducing component (.beta.2 site). These 3 sites are used in sequential fashion, with the last step in complex formation--resulting in .beta. component dimerization--critical for initiating signal transduction (Davis et al. 1993 supra). Knowledge of the details of receptor activation and the existence of the non-functional .beta.1 intermediate for CNTF has led to the finding that CNTF is a high affinity antagonist for IL-6 under certain circumstances, and provides the strategic basis for designing ligand or receptor-based antagonists for the CNTF family of cytokines as detailed below.

BRIEF SUMMARY OF THE INVENTION

An object of the invention is the construction of several specific interleukin-1 (IL-1) cytokine antagonists, termed "IL-1 Traps", each having different sequences but all being capable of blocking the binding of IL-1 to its receptor, thus functioning as IL-1 antagonists.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Ordered binding of receptor components in a model of a generic cytokine receptor. The model indicates that cytokines contain up to 3 receptor binding sites and interact with their receptor components by binding first the optional a component, followed by binding to b1, and then b2. The b components for many cytokine receptors interact through membrane proximal regions (shaded boxes) with the Jak/Tyk family of cytoplasmic protein tyrosine kinases. Only upon dimerization of b components is signal transduction initiated, as schematized by the tyrosine phosphorylations (P) of the b components and the Jak/Tyk kinases.

FIG. 2: CNTF inhibits IL-6 responses in a PC12 cell line (called PC12D) that expresses IL6Ra, gp130, CNTFRa, but not LIFRb. Serum-deprived PC12D cells were incubated +IL-6 (50 ng/mL) in the presence or absence of CNTF as indicated. Some plates also received soluble IL6Ra (1 mg/mL) or soluble CNTFRa (1 mg/mL) as indicated. Cell lysates were subjected to immunoprecipitation with anti-gp130 and immunoblotted with anti-phosphotyrosine. Tyrosine phosphorylation of gp130 is indicative of IL-6 induced activation of the IL-6 receptor system, which is blocked upon coaddition of CNTF.

FIG. 3: Scatchard analysis of iodinated CNTF binding on PC12D cells. PC12D cells were incubated with various concentrations of iodinated CNTF in the presence or absence of excess non-radioactive competitor to determine the specific binding. The figure shows a Scatchard plot of the amount of iodinated CNTF specifically bound, and gives data consistent with two binding sites with dissociation constants of 9 pM and 3.4 nM.

FIGS. 4A-4B. The amino acid sequence of human gp130-Fc-His.sub.6 (SEQ ID NO: 7). Amino acids 1 to 619 are from human gp130 (Hibi et al., Cell 63:1149-1157 (1990). Note that amino acid number 2 has been changed from a Leu to a Val in order to accommodate a Kozak sequence in the coding DNA sequence. The signal peptide of gp130-Fc-His.sub.6 has been italicized (amino acids 1 to 22). The Ser-Gly bridge is shown in bold type (amino acids 620, 621). Amino acids 662 to 853 are from the Fc domain of human IgG1 (Lewis, et al., J. Immunol. 151:2829-2838 (1993). (.dagger.) mark the two cysteines (amino acids number 632 and 635) of the IgG hinge preceding the Fc that form the inter-chain disulfide bridges that link two Fc domains. The hexahistine tag is shown in bold/italic type (amino acids 854 to 859). (.cndot.) shows the position of the STOP codon.

FIG. 5: The amino acid sequence of human IL-6Ra-Fc (SEQ ID NO: 8). Key: Amino acids 1 to 358 are from human IL-6Ra (Yamasaki et al. 1088 Science 241:825-828). Note that amino acid number 2 has been changed from a Leu to a Val in order to accommodate a Kozak sequence in the coding DNA sequence. The signal peptide of IL-6Ra-Fc has been italicized (amino acids 1 to 19). The Ala-Gly bridge is shown in bold type (amino acids 359, 360). Amino acids 361 to 592 are from the Fc domain of human IgG1 (Lewis et al., J. Immunol. 151:2829-2838 (1993). (.dagger.) mark the two cysteines (amino acids number 371 and 374) of the IgG hinge preceding the Fc that form the inter-chain disulfide bridges that link two Fc domains. (.cndot.) shows the position of the STOP codon.

FIG. 6: The CNTF/IL-6/IL-11 receptor system. The ordered formation of the hexameric signal transducing receptor complex is depicted schematically. The cytokine associates with the Ra component to form an obligatory cytokine.cndot.Ra complex (Kd is about 5 nM). This low affinity complex next associates with the first signal transducing component, marked b1, to form a high affinity cytokine.cndot.Ra.cndot.b1 complex (Kd is about 10 pM). In the case of IL-6Ra, this component is gp130. This trimeric high affinity complex subsequently associates with another such complex. Formation of this complex results in signal transduction as it involves dimerization of two signal transducing components, marked b1 and b2 respectively (adapted from (Ward et al., J. Bio. Chem. 269:23286-23289 (1994); Stahl and Yancopoulos, J. Neurobiology 25:1454-1466 (1994); Stahl and Yancopoulos, Cell 74:587-590 (1993).

FIG. 7: Design of heterodimeric receptor-based ligand Traps for IL-6. The heterodimeric ligand Trap is comprised of two interdisulfide linked proteins, gp130-Fc and IL-6Ra-Fc. The gp130-Fc.cndot.IL-6Ra-Fc complex (upper panel) is shown to mimic the high affinity cytokine.cndot.Ra.cndot.b1 complex (lower panel). The ligand Trap functions as an antagonist by sequestering IL-6 and thus rendering unavailable to interact with the native receptors on IL-6-responsive cells.

FIG. 8. Heteromeric immunoglobulin Heavy/Light Chain Receptor Fusions. An example of a heavy/light chain receptor fusion molecule is schematically depicted. The extracellular domain of gp130 is fused to Cg, whereas the extracellular domain of IL-6Ra is fused to the constant region of the kappa chain (k). The inter-chain disulfide bridges are also depicted (S-S).

FIGS. 9A-9B. Amino acid sequence of gp130-Cg1 (SEQ ID NO: 9). Key: Amino acids 1 to 619 are from human gp130 (Hibi, et al., Cell 63:1149-1157 (1990). Ser-Gly bridge is shown in bold type. Amino acids 662 to 651 are from the constant region of human IgG1 (Lewis et al., J. Immunol. 151:2829-2838 (1993). (*) shows the position of the STOP codon.

FIG. 10: Amino acid sequence of gp130D3fibro (SEQ ID NO: 10). Key: Amino acids 1 to 330 are from human gp130 (Hibi et al. Cell 63:1149-1157 (1990). Other symbols as described in FIG. 9.

FIG. 11: Amino acid sequence of J-CH1 (SEQ ID NO: 11). Key: The Ser-Gly bridge is shown in bold, the J-peptide is shown in italics, the C.sub.H1 domain is underlined.

FIG. 12: Amino acid sequence of Cg4 (SEQ ID NO: 12). Key: The Ser-Gly bridge is shown in bold type. Amino acids 2 to 239 comprise the Cg4 sequence.

FIG. 13: Amino acid sequence of k-domain (SEQ ID NO: 13). Key: The Ser-Gly bridge is shown in bold type. Amino acids 2 to 108 comprise the k domain. The C-terminal cysteine (amino acid 108) is that involved in the disulfide bond of the k domain with the C.sub.H1 domain of Cg.

FIG. 14: Amino acid sequence of 1-domain (SEQ ID NO: 14). Key: The Ser-Gly bridge is shown in bold type. Amino acids 2 to 106 comprise the 1 domain (Cheung, et al., J. Virol. 66: 6714-6720 (1992). The C-terminal cysteine (amino acid 106) is that involved in the disulfide bond of the 1 domain with the C.sub.H1 domain of Cg.

FIG. 15: Amino acid sequence of the soluble IL-6Ra domain (SEQ ID NO: 15). Key: Amino acids 1 to 358 comprise the soluble IL-6Ra domain (Yamasaki, et al., Science 241:825-828 (1988). The Ala-Gly bridge is shown in bold type.

FIG. 16: Amino acid sequence of the soluble IL-6R.alpha.313 domain (SEQ ID NO: 16): Key: Amino acids 1 to 313 comprise the truncated IL-6Ra domain (IL-6Ra313). The Thr-Gly bridge is shown in bold type.

FIG. 17: Purification of gp130-Cg1.cndot.IL-6Ra-k. 4% to 12% SDS-PAGE gradient gel run under non-reducing conditions. Proteins were visualized by staining with silver. Lane 1: approximately 100 ng of material purified over Protein A Sepharose (Pharmacia). Lane 2: Molecular size standards (Amersham). Lane 3: The Protein A-purified material shown here after further purification over an IL-6 affinity chromatography step. The positions of the gp130-Cg1 dimer [(gp130-Cg1).sub.2], the gp130-Cg1 dimer associated with one IL-6Ra-k [(gp130-Cg1).sub.2.cndot.(IL-6Ra-k).sub.1], and the gp130-Cg1 dimer associated with two IL-6Ra-k [(gp130-Cg1).sub.2.cndot.(IL-6Ra-k).sub.2] are shown, as well as the sizes for the molecular size standards in kilodaltons (200, 100, and 46).

FIG. 18: IL-6 dissociates slowly from the ligand Trap. The dissociation rate of IL-6 from a heavy/light chain receptor-based ligand Trap (gp130-Cg1.cndot.IL-6Ra-k) was compared to that obtained with the neutralizing monoclonal antibody B-E8 (BE8 MAb).

FIGS. 19A-19B: IL-6 can induce multimerization of the ligand Trap. (FIG. 19A) Two different ligand Traps are depicted schematically and listed according to their ability to bind protein A. gp130-Fc.cndot.IL-6Ra-Fc (GF6F) binds protein A via its Fc-domains, whereas gp130--C.sub.H1.cndot.IL-6Ra-k (G16K) does not bind to protein A. (FIG. 19B) Anti-kappa western blotting of proteins precipitated with Protein A-Sepharose from mixtures of GF6F.+-.IL-6, G16K.+-.IL-6, or GF6F plus G16K.+-.IL-6, as marked.

FIG. 20: Inhibition of IL-6-dependent XG-1 cell proliferation. XG-1 cells [Zhang, et al., Blood 83:3654-3663 (1994)] were prepared for a proliferation assay by starving the cells from IL-6 for 5 hours. Assays were set up in 96-well tissue culture dishes in RPMI+10% fetal calf serum+penicillin/streptomycin+0.050 nM 2-mercaptoethanol+glutamine. 0.1 ml of that media was used per well. Cells were suspended at a density of 250,000 per ml at the start of the assay. 72 hours post addition of IL-6.+-.ligands Traps or antibodies, an MTT assay was performed as described (Panayotatos et al. Biochemistry 33:5813-5818 (1994). The different ligand Traps utilized are listed.

FIGS. 21A-21D: Nucleotide sequence (SEQ ID NO: 17) encoding and deduced amino acid sequence (SEQ ID NO: 18) of fusion polypeptide designated 424 which is capable of binding the cytokine IL-4 to form a nonfunctional complex.

FIGS. 22A-22D: Nucleotide sequence (SEQ ID NO: 19) encoding and deduced amino acid sequence (SEQ ID NO: 20) of fusion polypeptide designated 603 which is capable of binding the cytokine IL-4 to form a nonfunctional complex.

FIGS. 23A-23D: Nucleotide sequence (SEQ ID NO: 21) encoding and deduced amino acid sequence (SEQ ID NO: 22) of fusion polypeptide designated 622 which is capable of binding the cytokine IL-4 to form a nonfunctional complex.

FIGS. 24A-24F: Nucleotide sequence (SEQ ID NO: 23) encoding and deduced amino acid sequence (SEQ ID NO: 24) of fusion polypeptide designated 412 which is capable of binding the cytokine IL-6 to form a nonfunctional complex.

FIGS. 25A-25F: Nucleotide sequence (SEQ ID NO: 25) encoding and deduced amino acid sequence (SEQ ID NO: 26) of fusion polypeptide designated 616 which is capable of binding the cytokine IL-6 to form a nonfunctional complex.

FIGS. 26A-26E: Nucleotide sequence (SEQ ID NO: 27) encoding and deduced amino acid sequence (SEQ ID NO: 28) of fusion polypeptide designated 569 which is capable of binding the cytokine IL-1 to form a nonfunctional complex.

FIG. 27: Shows that an IL-4 Trap designated 4SC375, which is a fusion polypeptide of IL-2Rg-scb-IL4Ra-Fc.DELTA.C1, is several orders of magnitude better as an IL-4 antagonist than IL4RaFc.DELTA.C1 alone in the TF1 cell bioassay.

FIG. 28: Shows that an IL-4 Trap designated 4SC375 displays antagonistic activity in the TF1 cell bioassay equivalent to an IL-4 Trap designated 4SC424 (described in FIGS. 21A-21D) which is a fusion polypeptide of IL-2Rg-IL4Ra-Fc.DELTA.C1 having the IL-2Rg component flush with the IL-4Ra component.

FIG. 29: Shows that the IL6 Trap (6SC412 IL6R-scb-gpx-Fc.DELTA.C1) described in FIGS. 24A-24F is a better antagonist of IL-6 in the XG1 bioassay than the neutralizing monoclonal antibody to human IL-6-BE8.

FIG. 30: Shows that the Trap 1SC569 (described in FIGS. 26A-26E) is able to antagonize the effects of IL-1 and block the IL-6 production from MRC 5 cells upon treatment with IL-1.

FIGS. 31A-31G: The nucleotide (SEQ ID NO: 29) and encoded amino acid (SEQ ID NO: 30) sequence of the IL-4Ra.IL-13Ra1.Fc single chain Trap construct is set forth.

FIGS. 32A-32G: The nucleotide (SEQ ID NO: 31) and encoded amino acid (SEQ ID NO: 32) sequence of the IL-13Ra1.IL-4Ra.Fc single chain Trap construct is set forth.

FIG. 33: Blocking of IL-13 by IL-4Ra.IL-13Ra1.Fc and IL-13Ra1.IL-4Ra.Fc. Addition of either IL-4Ra.IL-13Ra1.Fc or IL-13Ra1.IL-4Ra.Fc Trap at a concentration of 10 nM blocks IL-13-induced growth up to .about.2 nM. At an IL-13 concentration of .about.4-5 nM the growth of TF1 cells is inhibited by 50%.

FIG. 34: Blocking of IL-4 by IL-4Ra.IL-13Ra1.Fc and IL-13Ra1.IL-4Ra.Fc. Addition of either IL-4Ra.IL-13Ra1.Fc or IL-13Ra1.IL-4Ra.Fc at a concentration of 10 nM blocks IL-4-induced growth up to .about.1 nM. At an IL-4 concentration of .about.3-4 nM the growth of TF1 cells is inhibited by 50%.

FIG. 35: Human IL-1 Trap blocks the in vivo effects of exogenously administered huIL-1. BALB/c mice were given subcutaneous injection of huIL-1 (0.3 .mu.g/kg) at time 0. Twenty-four hours prior to huIL-1 injection, the animals were pre-treated with either vehicle or 150-fold molar excess of huIL-1 Trap. Two hours prior to sacrifice (26 hrs), the mice were re-challenged with a second injection of huIL-1 (0.3 .mu.g/kg, s.c.). Blood samples were collected at various time points and sera were assayed for IL-1 levels (expressed as mean+/-SEM; n=5 per group).

FIGS. 36A & 36B: Human IL-4 Trap antagonizes the effects of human IL-4 in monkeys. FIG. 36A: Cynomologus monkeys were treated in three parts as indicated. Human IL-4 (25 .mu.g/kg) was injected subcutaneously twice daily for 4 days and human IL-4 Trap (8 mg/ml) and vehicle were given intravenously daily for 5 days, beginning 1 day prior to human IL-4 administration. Plasma was collected daily and assayed for MCP-1 levels. Results were expressed as mean +/-SEM; n=4. (ANOVA p<0.0007; Tukey-Kramer: Part 2 vs. Part 1, p, 0.05; Part 2 vs. Part 3, p, 0.05; Part 1 vs. Part 3, not significant.) FIG. 36B: Cynomologus monkeys were treated in three parts as indicated. Human IL-4 (25 .mu.g/kg) was injected subcutaneously twice daily for 4 days and human IL-4 Trap (8 mg/ml) and vehicle were given intravenously daily for 5 days, beginning 1 day prior to human IL-4 administration. Whole blood was collected daily for flow cytometry analysis for CD16. Results were expressed as mean +/-SEM; n=4. (ANOVA p<0.042; Tukey-Kramer: Part 2 vs. Part 1, p<0.05; Part 2 vs. Part 3 and Part 1 vs. Part 3, not significant.)

FIG. 37: Murine IL-4 Trap partially prevented IL-4-mediated IgE increase in mice. BALB/C mice injected with anti-mouse IgD (100 .mu.l/mouse, s.c.) were randomly divided into 3 groups, each received (on days 3-5) either vehicle, murine IL-4 Trap (1 mg/kg, s.c.), or a monoclonal antibody to mouse IL-4 (1 mg/kg, s.c.). Sera were collected at various time points and assayed for IgE levels. Results were expressed as mean +/-SEM (n=5 per group). (ANOVA p=0.0002; Tukey-Kramer: vehicle vs. IL-4 Trap, p<0.01; vehicle vs. IL-4 antibody, p<0.001; IL-4 Trap vs. IL-4 antibody, not significant).

FIGS. 38A-38I: Nucleotide (SEQ ID NO: 33) and deduced amino acid (SEQ ID NO: 34) sequence of Human IL-1 Trap 570-FE.

FIG. 39A-39I: Nucleotide (SEQ ID NO: 35) and deduced amino acid (SEQ ID NO: 36) sequence of Human IL-1 Trap 570-FE.B.

FIGS. 40A-40I: Nucleotide (SEQ ID NO: 37) and deduced amino acid (SEQ ID NO: 38) sequence of Human IL-1 Trap 570-FE.C.

FIGS. 41A-41I: Nucleotide (SEQ ID NO: 39) and deduced amino acid (SEQ ID NO: 40) sequence of Human IL-1 Trap 823.

FIGS. 42A-42I: Nucleotide (SEQ ID NO: 41) and deduced amino acid (SEQ ID NO: 42) sequence of Human IL-1 Trap 823-1198.B.

FIGS. 43A-43I: Nucleotide (SEQ ID NO: 43) and deduced amino acid (SEQ ID NO: 44) sequence of Human IL-1 Trap 823-1267.C.

FIGS. 44A-44I: Nucleotide (SEQ ID NO: 45) and deduced amino acid (SEQ ID NO: 46) sequence of Human IL-1 Trap 1647-CtF.

FIGS. 45A-45I: Nucleotide (SEQ ID NO: 47) and deduced amino acid (SEQ ID NO: 48) sequence of Human IL-1 Trap 1647-CtF.B.

FIGS. 46A-46I: Nucleotide (SEQ ID NO: 49) and deduced amino acid (SEQ ID NO: 50) sequence of Human IL-1 Trap 1647-CtF.C.

FIGS. 47A-47I: Nucleotide (SEQ ID NO: 51) and deduced amino acid (SEQ ID NO: 52) sequence of Human IL-1 Trap 1649.

FIGS. 48A-48I: Nucleotide (SEQ ID NO: 53) and deduced amino acid (SEQ ID NO: 54) sequence of Human IL-1 Trap 1649-B.

FIGS. 49A-49I: Nucleotide (SEQ ID NO: 55) and deduced amino acid (SEQ ID NO: 56) sequence of Human IL-1 Trap 1646-C.

FIG. 50: Human IL-1 Trap blocks the in vivo effects of exogenously administered human IL-1. Male C57BL/6 mice were given a subcutaneous injection of recombinant human IL-1.beta. (rhIL-1.beta.; 0.3 mg/kg). Twenty four hours prior to rhIL-1.beta. administration, animals were treated with either vehicle, human IL-1 Trap 569 (50 or 150-fold molar excess; 0.18 or 0.54 mg/kg, respectively), or recombinant murine IL-1 receptor antagonist (rmIL-1ra; 150 or 750-fold molar excess; 45.8 or 229 .mu.g/kg, respectively). Blood samples were taken 2 h after administration of rhIL-1.beta. and the sera were assayed for IL-6 levels using a mouse IL-6 ELISA. Exogenous administration of rhIL-1.beta. significantly increased serum IL-6 levels. Pretreatment with either a 50 or 150-fold molar excess of hIL-1 Trap blocked the rhIL-1.beta.-induction of IL-6. In contrast, injection of rmIL-1ra at either a 150 or 750-fold molar excess did not block IL-6 induction.

FIG. 51: Human IL-1 Trap blocks the effects of IL-1 in Inflamed Joints. Anesthetized male C57BL/6 mice were given an intra-articular (i.a.) injection of Zymosan A (300 .mu.g in 10 .mu.l) into the right knee joint through the patellar ligament. Sterile PBS was injected i.a. (10 .mu.l) into the left knee joint through the patellar ligament. Twenty four hours prior to i.a. injections, animals were treated with either vehicle or hIL-1 Trap 569 (19 mg/kg, s.c.). The patellae were removed 24 h after injection of zymosan in order to measure proteoglycan synthesis, each patella and associated ligament were incubated for 3 h at 37.degree. C., 5% CO.sub.2 in media (RPMI with HEPES, HCO.sub.3, glutamine & penicillin/streptomycin) containing 10 uCi/ml .sup.35S-sulfate. Following incubation, tissue was washed and fixed overnight in 10% formalin. The tissue was then placed in Decalcifing Solution for 4 h prior to dissection of the patella from surrounding tissue. Each patella was then incubated overnight in Solvable at 50.degree. C. Ultima Gold liquid scintillation fluid was added and the samples were counted in a liquid scintillation counter. Values were reported as the ratio of cpm of zymosan patella/cpm of vehicle patella for each animal. Intra-articular injection of zymosan reduces proteoglycan synthesis by approximately 50% relative to vehicle injection. Administration of hIL-1 Trap prior to zymosan injection blocked the local action of IL-1.beta. and proteoglycan synthesis returned to approximately 90% of control.

FIGS. 52-53: Murine IL-1 Trap Reduces the Severity of Arthritis Symptoms in a Zymosan-Accelerated Collagen-Induced Arthritis (CIA) model. Male DBA-1 mice were immunized intradermally at the base of the tail with 100 .mu.g/50 .mu.l bovine Type II collagen (CII) emulsified with complete and incomplete Freund's adjuvant (2:1:1 ratio) and boosted intradermally with CII (100 .mu.g/50 .mu.l) emulsified with incomplete Freund's adjuvant on day 21. Since CIA in DBA-1 mice occurs gradually over a long time period with a low incidence, we synchronized the onset of arthritis symptoms by injecting the animals intraperitoneally on day 30 with 3 mg zymosan. Two hours prior to zymosan injection, the mice were randomly distributed into treatment groups and were injected with either vehicle or mIL-1 Trap (31 or 10 mg/kg, 3.times./week, 8 injections, s.c.). Arthritis symptoms (ASI scores) in the paws were evaluated 3.times./week by individuals who were blinded to the treatment group. Animals were sacrificed 24 h after the 8th injection at which time paw width along with ASI scores were measured. Within 5 days after i.p injection of zymosan, vehicle treated animals had an significant increase in ASI score relative to those receiving mIL-1 Trap with symptoms reaching a maximum 10 to 14 days after zymosan injection. Murine IL-1 Trap acted in a dose-dependent fashion such that animals receiving 10 mg/kg Trap had more arthritis symptoms (greater ASI score) than those receiving 31 mg/kg. However, both mIL-1 Trap treated groups had a significantly lower degree of arthritis symptoms than vehicle. This difference in ASI score is also reflected in the paw width at the time of sacrifice (FIG. 67). Animals receiving mIL-1 Trap had paw widths that were similar to those of naive, non-collagen immunized animals

FIG. 54: Various concentrations of IL-1 Trap 1649 were incubated in the presence of 5 pM human IL-1b overnight at room temperature. The mixtures were then added to duplicate wells of 293-NFkB cells (20,000 cells/well) for 5 hrs at 37.degree. C., 5% CO.sub.2. Steady-Glo Reagent (Promega) was added to the cells for 15 min at room temperature and luciferase gene expression was quantitated as relative light units (RLU) by luminometry. IL-1 Trap 1649 displays an IC.sub.50 of 32 pM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated nucleic acid molecule encoding a fusion polypeptide capable of binding a cytokine to form a nonfunctional complex comprising: (a) a nucleotide sequence encoding a first fusion polypeptide component comprising the amino acid sequence of the cytokine binding portion of the extracellular domain of the specificity determining component of a cytokine's receptor; (b) a nucleotide sequence encoding a second fusion polypeptide component comprising the amino acid sequence of the cytokine binding portion of the extracellular domain of the signal transducing component of a cytokine's receptor; and (c) a nucleotide sequence encoding a third fusion polypeptide component comprising the amino acid sequence of a multimerizing component.

By "cytokine binding portion" what is meant is the minimal portion of the extracellular domain necessary to bind the cytokine. It is accepted by those of skill in the art that a defining characteristic of a cytokine receptor is the presence of the two fibronectin-like domains that contain canonical cysteines and of the WSXWS box (Bazan 1990 supra). Sequences encoding the extracellular domains of the binding component of the cytokine's receptor and of the signal transducing component of the cytokine's receptor may also be used to create the fusion polypeptide of the invention. Similarly, longer sequences encoding larger portions of the components of the cytokine's receptor may be used. However, it is contemplated that fragments smaller than the extracellular domain will function to bind the cytokine and therefore, the invention contemplates fusion polypeptides comprising the minimal portion of the extracellular domain necessary to bind the cytokine as the cytokine binding portion.

The invention comprises a "specificity determining component" of a cytokine receptor and a "signal transducing component" of the cytokine receptor. Regardless of the nomenclature used to designate a particular component or subunit of a cytokine receptor, one skilled in the art would recognize which component or subunit of a receptor is responsible for determining the cellular target of the cytokine, and thus would know which component constitutes the "specificity determining component."

Similarly, regardless of the nomenclature used, one of skill in the art would know which component or subunit of a receptor would constitute the "signal transducing component." As used herein, the "signal transducing component" is a component of the native receptor which is not the specificity determining component and which does not bind or weakly binds the cytokine in the absence of the specificity determining component. In the native receptor, the "signal transducing component" may participate in signaling.

For example, while some cytokine receptors have components designated .alpha. and .beta., the IL-4 receptor has a signal transducing component referred to as IL-2R.gamma.. However, regardless of what name is associated with that component, one skilled in the art would know which component of the IL-4 receptor is the signal transducing component. Thus to practice the present invention and create a high affinity Trap for IL-4, one of skill in the art would create an isolated nucleic acid comprising a nucleotide sequence encoding a first fusion polypeptide component comprising the amino acid sequence of the cytokine binding portion of the extracellular domain of the specificity determining component of the IL-4 receptor (IL-4R.alpha.); a nucleotide sequence encoding a second fusion polypeptide component comprising the amino acid sequence of the cytokine binding portion of the extracellular domain of the signal transducing component of the IL-4 receptor (IL-2R.gamma.); and a nucleotide sequence encoding a third fusion polypeptide component comprising the amino acid sequence of a multimerizing component (for example, an Fc domain of IgG) to create a high affinity Trap for IL-4.

In preparing the nucleic acid sequence encoding the fusion polypeptide of the invention, the first, second, and third components of the fusion polypeptide are encoded in a single strand of nucleotides which, when expressed by a host vector system, produces a monomeric species of the fusion polypeptide. The monomers thus expressed then multimerize due to the interactions between the multimerizing components (the third fusion polypeptide components). Producing the fusion polypeptides in this manner avoids the need for purification of heterodimeric mixtures that would result if the first and second components were produced as separate molecules and then multimerized. For example, U.S. Pat. No. 5,470,952 describes the production of heterodimeric proteins that function as CNTF or IL-6 antagonists. The heterodimers are purified from cell lines co-transfected with the appropriate alpha (.alpha.) and beta (.beta.) components. Heterodimers are then separated from homodimers using methods such as passive elution from preparative, nondenaturing polyacrylamide gels or by using high pressure cation exchange chromatography. The need for this purification step is avoided by the methods of the present invention.

In addition, WO 96/11213 states that the applicant has prepared homodimers in which two IL-4 receptors are bound by a polymeric spacer and has prepared heterodimers in which an IL-4 receptor is linked by a polymeric spacer to an IL-2 receptor gamma chain. The polymeric spacer described is polyethylene glycol (PEG). The two receptor components, IL-4R and IL-2R.gamma. are separately expressed and purified. Pegylated homodimers and heterodimers are then produced by joining the components together using bi-functional PEG reagents. It is an advantage of the present invention that it avoids the need for such time consuming and costly purification and pegylation steps.

In one embodiment of the invention, the nucleotide sequence encoding the first component is upstream of the nucleotide sequence encoding the second component. In another embodiment of the invention, the nucleotide sequence encoding the first component is downstream of the nucleotide sequence encoding the second component. Further embodiments of the invention may be prepared in which the order of the first, second and third fusion polypeptide components are rearranged. For example, if the nucleotide sequence encoding the first component is designated 1, the nucleotide sequence encoding the second component is designated 2, and the nucleotide sequence of the third component is designated 3, then the order of the components in the isolated nucleic acid of the invention as read from 5' to 3' may be any of the following six combinations: 1,2,3; 1,3,2; 2,1,3; 2,3,1; 3,1,2; or 3,2,1.

In further embodiments of the invention, the cytokine bound by the fusion polypeptide may be a member of the hematopoietin family of cytokines selected from the group consisting of interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-11, interleukin-13, interleukin-15, granulocyte macrophage colony stimulating factor, oncostatin M, leukemia inhibitory factor, and cardiotrophin-1.

In additional embodiments of the invention, the cytokine bound by the fusion polypeptide may be a member of the interferon family of cytokines selected from the group consisting of IFN-.gamma., IFN-.alpha., and IFN-.beta..

In additional embodiments of the invention, the cytokine bound by the fusion polypeptide may be a member of the immunoglobulin superfamily of cytokines selected from the group consisting of B7.1 (CD80) and B7.2 (B70).

In still further embodiments of the invention, the cytokine bound by the fusion polypeptide may be a member of the TNF family of cytokines selected from the group consisting of TNF-.alpha., TNF-.beta.beta, LT-.beta., CD40 ligand, Fas ligand, CD 27 ligand, CD 30 ligand, and 4-1BBL.

In additional embodiments of the invention, the cytokine bound by the fusion polypeptide may be a cytokine selected from the group consisting of interleukin-1 (IL-1), IL-10, IL-12, IL-14, IL-18, and MIF.

Because specificity determination and signal transduction occurs by a similar mechanism in the TGF-.beta./BMP family of cytokines (see, for example, Kingsley 1994 Genes & Development 8:133-146) the present invention may be used to produce high affinity antagonists for cytokines that are members of the TGF-.beta./BMP family.

Therefore, in additional embodiments of the invention, the cytokine bound by the fusion polypeptide may be a member of the TGF-.beta./BMP family selected from the group consisting of TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, BMP-2, BMP-3a, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-15, BMP-16, endometrial bleeding associated factor (EBAF), growth differentiation factor-1 (GDF-1), GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-12, GDF-14, mullerian inhibiting substance (MIS), activin-1, activin-2, activin-3, activin-4, and activin-5.

In alternative embodiments of the invention, the specificity determining component, the signal transducing component, or both, may be substituted for by a single chain Fv. A single chain Fv (scFv) is a truncated Fab having only the V region of a heavy chain linked by a stretch of synthetic peptide to a V region of a light chain. (See, for example, U.S. Pat. Nos. 5,565,332; 5,733,743; 5,837,242; 5,858,657; and 5,871,907 incorporated by reference herein). Thus the present invention contemplates, for example, an isolated nucleic acid molecule encoding a fusion polypeptide capable of binding a cytokine to form a nonfunctional complex comprising a nucleotide sequence encoding a first fusion polypeptide component comprising the amino acid sequence of the cytokine binding portion of the extracellular domain of the specificity determining component of the cytokine receptor; a nucleotide sequence encoding a second fusion polypeptide component comprising the amino acid sequence of an scFv capable of binding the cytokine at a site different from the site at which the cytokine binding portion of the extracellular domain of the specificity determining component of the cytokine receptor binds; and a nucleotide sequence encoding a third fusion polypeptide component comprising the amino acid sequence of a multimerizing component. Alternatively, the specificity determining component may be substituted for by a scFv that binds to a site on the cytokine different from the site at which the signal transducing component binds. Thus the invention contemplates an isolated nucleic acid molecule encoding a fusion polypeptide capable of binding a cytokine to form a nonfunctional complex comprising a nucleotide sequence encoding a first fusion polypeptide component comprising the amino acid sequence of a scFv that binds to a site on the cytokine different from the site at which the cytokine binding portion of the extracellular domain of the signal transducing component of the cytokine receptor binds; a nucleotide sequence encoding a second fusion polypeptide component comprising the amino acid sequence of the cytokine binding portion of the extracellular domain of the signal transducing component of the cytokine's receptor; and a nucleotide sequence encoding a third fusion polypeptide component comprising the amino acid sequence of a multimerizing component.

In another embodiment, the invention contemplates an isolated nucleic acid molecule encoding a fusion polypeptide capable of binding a cytokine to form a nonfunctional complex comprising a nucleotide sequence encoding a first fusion polypeptide component comprising the amino acid sequence of a first scFv that binds to a site on the cytokine; a nucleotide sequence encoding a second fusion polypeptide component comprising the amino acid sequence a second scFv that binds to a site on the cytokine different from the site at which the first scFv binds; and a nucleotide sequence encoding a third fusion polypeptide component comprising the amino acid sequence of a multimerizing component.

In all of the above described embodiments comprising scFvs, the invention also contemplates embodiments in which the nucleotide sequence encoding the first component is upstream of the nucleotide sequence encoding the second component; embodiments in which the nucleotide sequence encoding the first component is downstream of the nucleotide sequence encoding the second component; and further embodiments of the invention in which the order of the first, second and third fusion polypeptide components is rearranged. For example, if the nucleotide sequence encoding the first component is designated 1, the nucleotide sequence encoding the second component is designated 2, and the nucleotide sequence of the third component is designated 3, then the order of the components in the isolated nucleic acid of the invention as read from 5' to 3' may be any of the following six combinations: 1,2,3; 1,3,2; 2,1,3; 2,3,1; 3,1,2; or 3,2,1.

In preferred embodiments of the invention, the multimerizing component comprises an immunoglobulin derived domain. More specifically, the immunoglobulin derived domain may be selected from the group consisting of the Fc domain or the heavy chain of IgG. Even more specifically, immunoglobulin domain may be selected from the group consisting of the Fc domain or the heavy chain of IgG.sub.1 or IgG.sub.4. In another embodiment, the multimerizing component may be an Fc domain from which the first five amino acids (including a cysteine) have been removed to produce a multimerizing component referred to as Fc(DCI). Alternatively, the multimerizing component may be an Fc domain in which a cysteine within the first five amino acids has been substituted for by another amino acid such as, for example, serine or alanine.

The present invention also provides for fusion polypeptides encoded by the isolated nucleic acid molecules of the invention. Preferably, the fusion polypeptides are in multimeric form, due to the function of the third component, the multimerizing component. In a preferred embodiment, the multimer is a dimer. Suitable multimerizing components are sequences encoding an immunoglobulin heavy chain hinge region (Takahashi et al. 1982 supra); immunoglobulin gene sequences, and portions thereof. In a preferred embodiment of the invention, immunoglobulin gene sequences, especially one encoding the Fc domain, are used to encode the multimerizing component.

The present invention also contemplates a vector which comprises the nucleic acid molecule of the invention as described herein.

A preferred embodiment of the invention is an isolated nucleic acid molecule having the sequence set forth in SEQ ID NO:33 encoding a fusion polypeptide having the sequence set forth in SEQ ID NO:34, wherein the fusion polypeptide forms a multimer that is capable of binding a cytokine to form a nonfunctional complex; an isolated nucleic acid molecule having the sequence set forth in SEQ ID NO:35 encoding a fusion polypeptide having the sequence set forth in SEQ ID NO:36, wherein the fusion polypeptide forms a multimer that is capable of binding a cytokine to form a nonfunctional complex; and an isolated nucleic acid molecule having the sequence set forth in SEQ ID NO:37 encoding a fusion polypeptide having the sequence set forth in SEQ ID NO:38, wherein the fusion polypeptide forms a multimer that is capable of binding a cytokine to form a nonfunctional complex; as well as fusion polypeptides encoded by the above-described nucleic acid molecules.

Other preferred embodiments of the invention are isolated nucleic acid molecules having the sequences set forth in SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, or 83 encoding fusion polypeptides having the sequences set forth in SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, or 84, respectively, wherein each fusion polypeptide forms a multimer that is capable of binding IL-1 to form a non-functional complex.

Also provided is an expression vector comprising a nucleic acid molecule of the invention as described herein, wherein the nucleic acid molecule is operatively linked to an expression control sequence. Also provided is a host-vector system for the production of a fusion polypeptide comprising the expression vector of the invention which has been introduced into a host cell suitable for expression of the fusion polypeptide. The suitable host cell may be a bacterial cell such as E. coli, a yeast cell, such as Pichia pastoris, an insect cell, such as Spodoptera frugiperda, or a mammalian cell, such as a COS, CHO, 293, BHK or NS0 cell.

The present invention also provides for methods of producing the fusion polypeptides of the invention by growing cells of the host-vector systems described herein, under conditions permitting production of the fusion polypeptide and recovering the fusion polypeptide so produced.

The present invention provides novel antagonists which are based on receptor components that are shared by cytokines such as the CNTF family of cytokines.

The invention described herein contemplates the production of antagonists to any cytokine that utilizes an a specificity determining component which, when combined with the cytokine, binds to a first .beta. signal transducing component to form a nonfunctional intermediate which then binds to a second .beta. signal transducing component causing .beta.-receptor dimerization and consequent signal transduction. According to the invention, the soluble .alpha. specificity determining component of the receptor (sR.alpha.) and the extracellular domain of the first .beta. signal transducing component of the cytokine receptor (.beta.1) are combined to form heterodimers (sR.alpha.:.beta.1) that act as antagonists to the cytokine by binding the cytokine to form a nonfunctional complex.

The invention described herein also contemplates the production of antagonists to any cytokine that utilizes an a specificity determining component which, when combined with the cytokine, binds to a .beta. signal transducing component to form a receptor complex which then initiates signal transduction. According to the invention, the soluble a specificity determining component of the receptor (sR.alpha.) and the extracellular domain of the .beta. signal transducing component of the cytokine receptor (b) are combined to form heterodimers (sR.alpha.:.beta.) that act as antagonists to the cytokine by binding the cytokine to form a nonfunctional complex.

As described in Example 1, CNTF and IL-6 share the .beta.1 receptor component gp130. The fact that CNTF forms an intermediate with CNTFR.alpha. and gp130 can be demonstrated (Example 1) in cells lacking LIFR.beta., where the complex of CNTF and CNTFR.alpha. binds gp130, and prevents homodimerization of gp130 by IL-6 and IL-6R.alpha., thereby blocking signal transduction. These studies provide the basis for the development of the IL-6 antagonists described herein, as they show that if, in the presence of a ligand, a nonfunctional intermediate complex, consisting of the ligand, its a receptor component and its b1 receptor component, can be formed, it will effectively block the action of the ligand. Other cytokines may use other .beta.1 receptor components, such as LIFR.beta., which may also be used to produce antagonists according to the present invention.

Thus for example, in one embodiment of the invention, effective antagonists of IL-6 or CNTF consist of heterodimers of the extracellular domains of the a specificity determining components of their receptors (sIL-6R.alpha. and sCNTFR.alpha., respectively) and the extracellular domain of gp130. The resultant heterodimers, which are referred to hereinafter as sIL-6R.alpha.:.beta.1 and sCNTFR.alpha.:.beta.1, respectively, function as high-affinity Traps for IL-6 or CNTF, respectively, thus rendering the cytokine inaccessible to form a signal transducing complex with the native membrane-bound forms of their receptors.

Although soluble ligand binding domains from the extracellular portion of receptors have proven to be somewhat effective as Traps for their ligands and thus act as antagonists (Bargetzi et al. 1993 Cancer Res 53:4010-4013; and 1992 Proc. Natl. Acad. Sci. USA 89:8616-8620; Mohler et al. 1993 J. Immunol. 151: 1548-1561; Narazaki et al. 1993 Blood 82:1120-1126), the IL-6 and CNTF receptors are unusual in that the .alpha. receptor components constitute ligand binding domains that, in concert with their ligands, function effectively in soluble form as receptor agonists (Davis et al. 1993 supra; Taga et al. 1989 Cell 58: 573-581). The sR.alpha.:.beta.1 heterodimers prepared according to the present invention provide effective Traps for their ligands, binding these ligands with affinities in the picomolar range (based on binding studies for CNTF to PC12D cells) without creating functional intermediates. The technology described herein may be applied to develop a cytokine Trap for any cytokine that utilizes an .alpha.-component that confers specificity, as well as a .beta. component which, when bound to the .alpha.-specificity component, has a higher affinity for the cytokine than either component alone. Accordingly, antagonists according to the invention include antagonists of IL-1 through IL-5 (IL-1: Greenfeder, et al. 1995 J Biol Chem 270:13757-13765; Guo et al. 1995 J Biol Chem 270:27562-27568), IL-2 (Taniguchi et al. EP 0386289-A and 0386304-A; Takeshita et al. 1992 Science 257:379-382); IL-3 (Kitamura et al. 1991 Cell 66:1165-1174), IL-4 (Idzerda et al. 1990 J Exp Med 171:861-873), IL-5 (Taverneir et al. 1991 Cell 66:1175-1184), IL-11 (Cherel et al. EMBL/GenBank/DDBJ databases Accession No. Z38102), IL-15 (Hemar et al. 1995 J Cell Biol 1295:55-64); Taniguchi et al. EP 0386289-A and 0386304-A); Takeshita et al. 1992 Science 257:379-382), granulocyte-macrophage colony stimulating factor (GM-CSF) Hayashida et al. 1990 Proc. Natl. Acad. Sci. U.S.A. 97:9655-9659), LIF, .gamma.-interferon (Aguet 1988 et al. Cell 55:273-280; Soh et al. 1994 Cell 76:793-802), and transforming growth factor beta (TGF.beta.) (Inagaki et al. 1993 Proc. Natl. Acad. Sci. USA 90:5359-5363).

The .alpha. and .beta. receptor extracellular domains may be prepared using methods known to those skilled in the art. The CNTFRa receptor has been cloned, sequenced and expressed (Davis et al. 1991 Science 253:59-63 which is incorporated by reference in its entirety herein). The cloning of LIFR.beta. and gp130 are described in Gearing et al. 1991 EMBO J. 10:2839-2848, Hibi et al. 1990 supra and WO 93/10151, all of which are incorporated by reference in their entirety herein.

The receptor molecules useful for practicing the present invention may be prepared by cloning and expression in a prokaryotic or eukaryotic expression system. The recombinant receptor gene may be expressed and purified utilizing any number of methods. The gene encoding the factor may be subcloned into a bacterial expression vector, such as for example, but not by way of limitation, pCP110.

The recombinant factors may be purified by any technique which allows for the subsequent formation of a stable, biologically active protein. For example, and not by way of limitation, the factors may be recovered from cells either as soluble proteins or as inclusion bodies, from which they may be extracted quantitatively by 8M guanidinium hydrochloride and dialysis. In order to further purify the factors, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration may be used.

The sR.alpha.:.beta. heterodimeric receptors may be engineered using known fusion regions, as described in WO 93/10151 which describes production of .beta. receptor heterodimers, or they may be prepared by crosslinking of extracellular domains by chemical means. The domains utilized may consist of the entire extracellular domain of the a and b components, or they may consist of mutants or fragments thereof that maintain the ability to form a complex with its ligand and other components in the sR.alpha.:.beta.1 complex. For example, as described below in Example 4, IL-6 antagonists have been prepared using gp130 that is lacking its three fibronectin-like domains.

In one embodiment of the invention, the extracellular domains are engineered using leucine zippers. The leucine zipper domains of the human transcription factors c-jun and c-fos have been shown to form stable heterodimers (Busch et al. 1990 Trends Genetics 6:36-40; Gentz et al. 1989 Science 243:1695-1699) with a 1:1 stoichiometry. Although jun-jun homodimers have also been shown to form, they are about 1000-fold less stable than jun-fos heterodimers. Fos-fos homodimers have not been detected.

The leucine zipper domain of either c-jun or c-fos are fused in frame at the C-terminus of the soluble or extracellular domains of the above mentioned receptor components by genetically engineering chimeric genes. The fusions may be direct or they may employ a flexible linker domain, such as the hinge region of human IgG, or polypeptide linkers consisting of small amino acids such as glycine, serine, threonine or alanine, at various lengths and combinations. Additionally, the chimeric proteins may be tagged by His-His-His-His-His-His (His6) (SEQ. ID NO. 1) to allow rapid purification by metal-chelate chromatography, and/or by epitopes to which antibodies are available, to allow for detection on western blots, immunoprecipitation, or activity depletion/blocking in bioassays.

In another embodiment, as described below in Example 3, the sR.alpha.:.beta.1 heterodimer is prepared using a similar method, but using the Fc-domain of human IgG1 (Aruffo et al. 1991 Cell 67:35-44). In contrast to the latter, formation of heterodimers must be biochemically achieved, as chimeric molecules carrying the Fc-domain will be expressed as disulfide-linked homodimers. Thus, homodimers may be reduced under conditions that favor the disruption of inter-chain disulfides but do not effect intra-chain disulfides. Then monomers with different extracellular portions are mixed in equimolar amounts and oxidized to form a mixture of homo- and heterodimers. The components of this mixture are separated by chromatographic techniques. Alternatively, the formation of this type of heterodimers may be biased by genetically engineering and expressing molecules that consist of the soluble or extracellular portion of the receptor components followed by the Fc-domain of hIgG, followed by either the c-jun or the c-fos leucine zippers described above (Kostelny et al. 1992 J Immunol 148:1547-1553). Since these leucine zippers form predominately heterodimers, they may be used to drive formation of the heterodimers where desired. As for the chimeric proteins described using leucine zippers, these may also be tagged with metal chelates or an epitope. This tagged domain can be used for rapid purification by metal-chelate chromatography, and/or by antibodies, to allow for detection on western blots, immunoprecipitation, or activity depletion/blocking in bioassays.

In additional embodiments, heterodimers may be prepared using other immunoglobulin derived domains that drive the formation of dimers. Such domains include, for example, the heavy chains of IgG (Cg1 and Cg4), as well as the constant regions of kappa (.kappa.) and lambda (.lamda.) light chains of human immunoglobulins. The heterodimerization of C.gamma. with the light chain occurs between the CH1 domain of C.gamma. and the constant region of the light chain (C.sub.L), and is stabilized by covalent linking of the two domains via a single disulfide bridge. Accordingly, as described in Example 4, constructs may be prepared using these immunoglobulin domains. Alternatively, the immunoglobulin domains include domains that may be derived from T cell receptor components which drive dimerization.

In another embodiment of the invention, the sR.alpha.:.beta.1 heterodimers are prepared by expression as chimeric molecules utilizing flexible linker loops. A DNA construct encoding the chimeric protein is designed such that it expresses two soluble or extracellular domains fused together in tandem ("head to head") by a