IL-1 Receptor fusion proteins used as antagonists and methods of making and using

Abstract: The present invention provides a fusion polypeptide capable of binding Interleukin-1 (IL-1) to form a nonfunctional complex. It also provides a nucleic acid sequence encoding the fusion polypeptide and methods of making and uses for the fusion polypeptide.

Description: FIELD OF THE INVENTION The present invention relates to IL-1 receptor fusion proteins used as antagonists to IL-1. BACKGROUND OF THE INVENTION WO93/19777 provides fusion proteins comprising a tumour necrosis factor receptor (TNF-R) polypeptide and at least one additional polypeptide selected from an interleukin-1 (IL-1R) and a second TNF-R polypeptide. WO98/08969 provides soluble interleukin-1 receptor accessory molecule (IL-1R AcM) protein, which is a member of the Ig super family, along with isolated nucleic acid molecules encoding human IL-1R AcM, and vectors, host cells and recombinant methods for producing said protein. WO97/31010 provides a receptor protein designated 2F1, the amino acid sequence which is said to indicate that it is a member of the IL-1 receptor family. EP-A-0 460,846 provides type II interleukin-1 receptor proteins, DNAs and expression vectors encoding type IIL-R, and processes for producing type II IL-1R as products of recombinant cell culture. In addition, WO95/11303 provides a cytokine antagonist protein capable of binding a cytokine to form a nonfunctional complex, comprising the soluble α specificity determining component of the cytokine receptor, and an extracellular domain of a β-component of the cytokine receptor. SUMMARY OF THE INVENTION The invention provides an isolated nucleic acid molecule encoding a fusion polypeptide, which fusion polypeptide comprises the following fusion polypeptide components: (a) an Interleukin-1 (IL-1) binding portion of the extracellular domain of the specificity determining component of an IL-1 receptor; (b) an IL-1 binding portion of an extracellular domain of the signal transducing component of said IL-1 receptor; and (c) a multimerising component; which multimerising component (c) multimerises with a multimerising component (c) comprised in another of said fusion polypeptides to form a multimer of said fusion polypeptides; which multimer binds to and traps IL-1 and thus acts as an antagonist of IL-1. BRIEF DESCRIPTION OF THE FIGURES FIGURE 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 α component, followed by binding to β1, and then β2. The β 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 β components is signal transduction initiated, as schematized by the tyrosine phosphorylations (P) of the β components and the Jak/Tyk kinases. FIGURE 2A-2E: Nucleotide sequence encoding and deduced amino acid sequence of fusion polypeptide designated 569 which is capable of binding the cytokine IL-1 to form a nonfunctional complex. FIGURE 3: Shows that the trap 1SC569 (described in Figs. 2A-2E) is able to antagonize the effects of IL-1 and block the IL-6 production from MRC 5 cells upon treatment with IL-1. FIGURE 4: 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 µg/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 µg/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). DETAILED DESCRIPTION OF THE INVENTION An isolated nucleic acid molecule encoding a fusion polypeptide, which fusion polypeptide comprises the following fusion polypeptide components: (a) an Interleukin-1 (IL-1) binding portion of the extracellular domain of the specificity determining component of an IL-1 receptor; (b) an IL-1 binding portion of an extracellular domain of the signal transducing component of said IL-1 receptor; and (c) a multimerising component; which multimerising component (c) multimerises with a multimerising component (c) comprised in another of said fusion polypeptides to form a multimer of said fusion polypeptides; which multimer binds to and traps IL-1 and thus acts as an antagonist of IL-1. By "IL-1 binding portion" what is meant is the minimal portion of the extracellular domain necessary to bind IL-1. 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, J.F., 1990, PNAS 87: 6934-6938). Sequences encoding the extracellular domains of the binding component of the IL-1 receptor and of the signal transducing component of the IL-1 receptor may also be used to create the fusion polypeptide of the invention. Similarly, longer sequences encoding larger portions of the components of the IL-1 receptor may be used. However, it is contemplated that fragments smaller than the extracellular domain will function to bind IL-1 and therefore, the invention contemplates fusion polypeptides comprising the minimal portion of the extracellular domain necessary to bind IL-1 as the IL-1 binding portion. The invention comprises a "specificity determining component" of an IL-1 receptor and a "signal transducing component" of said IL-1 receptor. Regardless of the nomenclature used to designate a particular component or subunit of an IL-1 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. Examples of the receptor components that may be used to prepare cytokine antagonists according to the invention are set forth in Table 1. Cytokine Specificity determining Component Signal transducing Component Interleukin-1 (IL-1) Type I IL-1R (ref. 1) IL-1R AcP (refs. 1,2) Type II IL-1R (ref. 1) IL-1RI (ref. 2) IL-1RII (ref. 2) REFERENCES CITED IN TABLE 1 1. Greenfeder, et al., Journal of Biological Chemistry 270: 13757-13765 (1995) - See page 13757, col. 1, line 6 to col. 2, line 3 and col. 2, lines 10-12; page 13764, col. 2, last 3 lines and page 13765, col. 1, lines 1-7; 2. Wesche, et al., Journal of Biological Chemistry 272: 7727-7731 (1997) See page 7731, lines 20-26. 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 heterodirneric mixtures that would result if the first and second components were produced as separate molecules and then multimerized. For example, U.S. Patent No. 5,470,952 issued November 28, 1995 describes the production of heterodimeric proteins that function as CNTF or IL-6 antagonists. The heterodimers are purified from cell lines cotransfected with the appropriate alpha (α) and beta (β) 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, PCT International Application WO 96/11213 published 18 April 1996 entitled Dimeric IL-4 Inhibitors 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-2Rgamma 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. The cytokine bound by the fusion polypeptide is interleukin-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 of IgG, the heavy chain of IgG, and the light chain of IgG. 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(ΔC1). 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 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, Cell 29 :671-679); 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 third multimerizing component. The present invention also contemplates a vector which comprises the nucleic acid molecule of the invention as described herein. 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 which comprises 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 technology described herein may be applied to develop a cytokine trap for any cytokine that utilizes an α-component that confers specificity, as well as a β component which, when bound to the α-specificity component, has a higher affinity for the cytokine than either component alone. Accordingly, antagonists according to the invention are antagonists of interleukin-1 [Greenfeder, et al. J. Biol. Chem. 270:13757-13765 (1995); Guo, et al. J. Biol. Chem. 270:27562-27568 (1995)]. Pharmaceutical compositions for use according to the invention include the antagonists described above in a pharmacologically acceptable liquid, solid or semi-solid carrier, linked to a carrier or targeting molecule (e.g., antibody, hormone, growth factor, etc.) and/or incorporated into liposomes, microcapsules, and controlled release preparation (including antagonist expressing cells) prior to administration in vivo . For example, the pharmaceutical composition may comprise one or more of the antagonists in an aqueous solution, such as sterile water, saline, phosphate buffer or dextrose solution. Alternatively, the active agents may be comprised in a solid (e.g. wax) or semi-solid (e.g. gelatinous) formulation that may be implanted into a patient in need of such treatment. The administration route may be any mode of administration known in the art, including but not limited to intravenously, intrathecally, subcutaneously, by injection into involved tissue, intraarterially, intranasally; orally, or via an implanted device. Administration may result in the distribution of the active agent of the invention throughout the body or in a localized area. For example, in some conditions which involve distant regions of the nervous system, intravenous or intrathecal administration of agent may be desirable. In some situations, an implant containing active agent may be placed in or near the lesioned area. Suitable implants include, but are not limited to, gelfoam, wax, or microparticle-based implants. EXAMPLES CLONING OF FUSION POLYPEPTIDE COMPONENTS The extracellular domains of human cytokine receptors were obtained by standard PCR techniques using tissue cDNAs (CLONTECH), cloned into the expression vector, pMT21 (Genetics Institute, Inc.), and the sequences were sequenced by standard techniques using an ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). For the IL-1RAcP, nucleotides 1 through 1074 (corresponding to the amino acids 1-358) from the Genbank sequence, AB006357, were cloned. For the IL-1RI, nucleotides 55 through 999 (corresponding to the amino acids 19-333) from the Genbank sequence, X16896, were cloned. PRODUCTION OF FUSION POLYPEPTIDES (CYTOKINE TRAPS) Nucleotide sequences encoding cytokine traps were constructed from the individual cloned DNAs (described supra ) by standard cloning and PCR techniques. The sequences were constructed in frame such that the sequence encoding the first fusion polypeptide component was fused to the sequence encoding the second fusion polypeptide component followed by an Fc domain (hinge, CH2 and CH3 region of human IgG1) as the multimerizing component. Extra nucleotides were inserted in frame between sequences encoding the first and second fusion polypeptide components to add a linker region between the two components (See Figure 2A - Figure 2E - trap 569). For the IL-1 trap 569 (Figure 2A - to Figure 2E), the IL-1RAcP component is 5' followed by the IL-1RI component and then the Fc domain. The final constructs were cloned into the mammalian expression vector pCDNA3.1 (STRATAGENE). In the 569 sequence (Figure 2A - to Figure 2E), nucleotides 1-1074 encode the IL1RAcP component, nucleotides 1075 -1098 encode a linker region, nucleotides 1099-2043 encode the IL1RI component and nucleotides 2044-2730 encode the Fc domain. DNA constructs were either transiently transfected into COS cells or stably transfected into CHO cells by standard techniques well known to one of skill in the art. Supernatants were collected and purified by Protein A affinity chromatography and size exclusion chromatography by standard techniques. (See for example Harlow and Lane, Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). MRC5 BIOASSAY FOR IL1 TRAPS MRC5 human lung fibroblast cells respond to IL-1 by secreting IL-6 and thus were utilized to assay the ability of IL-1 traps to block the IL-1-dependent production of IL-6. IL1 Trap 1SC569 (Figure 2A - Figure 2E) was tested against IL-1-RI.Fc which is the extracellular domain of the IL-1 Type I receptor fused to an Fc domain. MRC5 cells are suspended at 1 x 10 5 cells per ml in medium and 0.1 ml of cells are plated (10,000 cells per well) into the wells of a 96 well tissue culture plate. Plates are incubated for 24 hours at 37°C in a humidified 5% CO 2 incubator. IL-1 trap and recombinant human IL-1 at varying doses are pre-incubated in a 96 well tissue culture dish and incubated for 2 hours at 37°C. 0.1 ml of this mixture is then added to the 96 well plate containing the MRC5 cells such that the final concentration of IL-1 Trap is 10nM and the final concentrations of the IL-1 ranges from 2.4 pM to 5nM. Control wells contain trap alone or nothing. Plates are then incubated at 37°C for 24 hours in a humidified 5% CO 2 incubator. Supernatant is collected and assayed for levels of IL-6 using R&D Systems Quantikine Immunoassay Kit according to the manufacturer's instructions. RESULTS Figure 30 shows that the trap 569 (Figure 2A - Figure 2E) is able to antagonize the effects of IL-1 and block the IL-6 production from MRC 5 cells upon treatment with IL-1. At a concentration of 10nM, the trap 569 is able to block the production of IL-6 up to an IL-1 concentration of 3nM. In contrast, the IL-1RI.Fc is a much poorer antagonist of IL-1. It is only able to block the effects of IL-1 up to about 10-20 pM. Thus, the trap 569 is approximately 100x better at blocking IL-1 than IL1RI.Fc. BLOCKING OF INJECTED IL-1 BY IL-1 TRAP IN VIVO IL-1 is a pro-inflammatory cytokine. Systemic administration of IL-1 has been shown to elicit acute responses in animals, including transient hyperglycemia, hypoinsulinemia, fever, anorexia, and increased serum levels of interleukin-6 (IL-6) (Reimers, 1998). Since mice are responsive to both murine and human IL-1, human IL-1 can be used and in vivo binding effects of human specific IL-1 antagonists can be evaluated. This acute mouse model was used to determine the ability of a human IL-1 trap to antagonize the in vivo effects of exogenously administered human IL-1. This provides a rapid indication of in vivo efficacy of the human IL-1 trap and can be used as an assay to help molecule selection. Experimental Design: Mice were given subcutaneous injections of human IL-1 (0.3 µg/kg). Twenty-four hours prior to human IL-1 injection, the animals were pre-treated with either vehicle or 150-fold molar excess of human IL-1 trap (0.54 mg/kg). Two hours prior to sacrifice (26 hrs), the mice were given a second injection of human IL-1 (0.3 µg/kg). Blood samples were collected at various time points and sera were assayed for IL-6 levels. RESULTS Exogenous administration of human IL-1 resulted a dramatic induction of serum IL-6 levels. At 150-fold molar excess, the human IL-1 trap completely blocked the IL-6 increase (Figure 4). Furthermore, the effects of the human IL-1 trap persisted for at least another 24 hours, preventing an IL-6 increase even when IL-1 was re-administered (Figure 4). Such long-lasting efficacy suggests that daily injection of an IL-1 trap may not be necessary for chronic applications. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims: An isolated nucleic acid molecule encoding a fusion polypeptide, which fusion polypeptide comprises the following fusion polypeptide components: (a) an Interleukin-1 (IL-1) binding portion of the extracellular domain of the specificity determining component of an IL-1 receptor; (b) an IL-1 binding portion of an extracellular domain of the signal transducing component of said IL-1 receptor; and (c) a multimerising component; which multimerising component (c) multimerises with a multimerising component (c) comprised in another of said fusion polypeptides to form a multimer of said fusion polypeptides; which multimer binds to and traps IL-1 and thus acts as an antagonist of IL-1. The isolated nucleic acid molecule of claim 1 wherein component (a) of said fusion polypeptide comprises an IL-1 binding portion of the extracellular domain of Type I IL-1R and component (b) comprises an IL-1 binding portion of an extracellular domain of IL-1R AcP. The isolated nucleic acid molecule of claim 1 wherein component (a) of said fusion polypeptide comprises an IL-1 binding portion of the extracellular domain of Type II IL-1R and component (b) comprises an IL-1 binding portion of an extracellular domain of IL-1R AcP. The isolated nucleic acid molecule of any one of the preceding claims wherein said multimer is a dimer. The isolated nucleic acid molecule of any one of the preceding claims, wherein the multimerizing component comprises an immunoglobulin-derived domain. The isolated nucleic acid molecule of claim 5, wherein the immunoglobulin-derived domain is selected from the Fc domain of IgG, the heavy chain of IgG, and the light chain of IgG. The isolated nucleic acid molecule of any one of the preceding claims, wherein the nucleotide sequence encoding component (a) is upstream of the nucleotide sequence encoding component (b). The nucleic acid molecule of any one of claims 1 to 6, wherein the nucleotide sequence encoding component (a) is downstream of the nucleotide sequence encoding component (b). A multimer comprising two or more fusion polypeptides encoded by the nucleic acid molecules of any one of the preceding claims, which multimer binds to and traps IL-1 and acts as an antagonist of IL-1. A multimer of claim 9 which is a dimer comprising two of said fusion polypeptides. A vector which comprises the nucleic acid molecule of any one of claims 1 to 8. An expression vector comprising a nucleic acid molecule of any one of claims 1 to 8, wherein the nucleic acid molecule is operatively linked to an expression control sequence. A host-vector system for the production of a fusion polypeptide, which comprises the expression vector of claim 12, in a host cell. The host-vector system of claim 13, wherein the host cell is a bacterial cell, yeast cell, insect cell, or mammalian cell. The host-vector system of claim 13, wherein the host cell is E. coli , a COS cell, a CHO cell, a 293 cell, a BHK cell or a NS0 cell. A method of producing fusion polypeptides which comprises growing cells of the host-vector system of claim 13, 14 or 15 under conditions permitting production of the fusion polypeptides and recovering the fusion polypeptides so produced. A method according to claim 16 further comprising allowing said fusion polypeptides to form multimers of fusion polypeptides as defined in claim 9. A method according to claim 17 wherein said multimers are dimers as defined in claim 10. A pharmaceutical composition comprising a multimer of claim 9 or a dimer of claim 10 in a pharmacologically acceptable liquid, solid or semi-solid carrier, linked to a carrier or targeting molecule and/or incorporated into liposomes, microcapsules or a controlled release preparation.

Inventors: STAHL NEIL, YANCOPOULOS GEORGE D, STAHL, NEIL, YANCOPOULOS, GEORGE D.
Applicants: REGENERON PHARMA, REGENERON PHARMACEUTICALS, INC.
Application year: 1999
Main classification: C12N15
IPC codes: C12N15, C07K14