AU625534B2 - Interleukin-1 receptors - Google Patents

Description

OPI DATE 14/06/89 APPLN. ID- 29015 89 S PCF wo AOJP DATE 20/07/89 BE PC J S /03926 INTERNATIONAL APPLICATION ruoDLolrnL unL-.r in r:i- r. N M 1 I'1 tr- II) (51) International Patent Classification 4 (11) International Publication Number: WO 89/ 04838 C07K 13/00, 3/28, A61K 37/02 Al A61K 39/395, C12P 21/00 (43) International Publication Date: 1 June 1989 (01.06.89) C12N 15/00, C07H 19/00 (21) International Application Number: PCT/US88/03926 (74) Agent: HALLQUIST, Scott, Immunex Corporation, 51 University Street, Seattle, WA 98101 (US).

(22) International Filing Date: 4 November 1988 (04.11.88) (81) Designated States: AU, BR, DK, FI, KR, NO.

(31) Priority Application Numbers: 125,627 160,550 258,756 Published With international search report.

(32) Priority Dates: 25 November 1987 (25.11.87) Before the expiration of the time limit for amending the February 1988 (25.02.88) claims and to be republished in the event of the receipt 13 October 1988 (13.10.88) of amendments.

(33) Priority Country:

US

(71) Applicant: IMMUNEX CORPORATION [US/US]; 51 University Street, Seattle, WA 98101 (US).

(72) Inventors: DOWER, Steven, K. 2620 East Lake Sammamish Parkway Northeast, Redmond, WA 98052 MARCH, Carl, J. 8133 8th Southwest, Seattle, WA 98106 SIMS, John, E. 5037 16th Avenue Northeast, Seattle, WA 98105 URDAL, David, L. 6826 55th Avenue Northeast, Seattle, WA 98115

(US).

(54) Title: INTERLEUKIN-1 RECEPTORS Murine (ApoLI)-- (EspI) (AvoI) l Human

I

I-(StuI) AvI-- Acc I Hind- Pvul-- Bam HI- Pvul Nco I NsiI AyuI -0.8 tuI BomHI -1.2 HindM -1.6 vu I S Asp718 -BglI -2.4 -PstI .2.8 (57) Abstract Mammalian Interleukin-! receptor proteins (IL-IRs), DNAs and expression vectors encoding mammalian IL-IRs, and processes for producing mammalian IL-IRs as products of cell culture, including recombinant systems, are disclosed.

V-10 89/04838 PC T/US88/03926 1

TITLE

Interleukin-1 Receptors BACKGROUND OF THE INVENTION The present invention relates generally to cytokine receptors, and more specifically, to Interleukin-1 receptors.

Interleukin-la and Interleukin-10 (IL-la and IL-10) are distantly related polypeptide hormones which play a central role in the regulation of immune and inflammatory responses. These two proteins were originally both classified as IL-1, based on a shared lymphocyte activation factor (LAF) activity, and a common major cellular source, activated macrophages. As information has accumulated from studies using purified natural and recombinant IL-1 molecules, it has become clear that IL-la and IL-10 each mediate most, if not all, of the wide range of activities previously ascribed to IL-1. The basis for this nearly identical spectrum of biological activities is thought to be a single class of plasma membrane IL-1 receptors which bind both IL-la and IL-1S.

A few preliminary reports concerning the existence of an IL-1 plasma membrane receptor have been published. To date, structural characterization of the Interleukin-1 receptor has been limited to estimates of the molecular weight of this protein by gel filtration, by SDS-PAGE analysis of covalent complexes formed by chemical crosslinking between the receptor and 125I-IL-1 molecules, and by immunoprecipitation of labeled surface proteins.

Dower et al. Exp. Med. 162:501, 1985), and Dover et al.

(Proc. Natl. Acad. Sci. USA 83:1060, 1986), describe chemical crosslinking studies indicating an apparent 79.5 kilodalton (kDa) plasma membrane protein on LBRM-33-1A5 murine T lymphoma cells and a 78 kDa surface protein on a murine fibroblast cell line which bound 1 25 I-labeled human Interleukin-10. Kilian et al. Immunol.

136:4509, 1986) reported that murine 12s l-IL-la binding to murine thymoma cells could be blocked by human IL-la and IL-~1. Dower et al.

(Nature 324:266, 1986) reported binding competition studies indicating that IL-la and IL-10 bound to the same cell surface receptors on t+ i ii;: i C-I~-X~I1 I- 'I- WO 89/04838 PCT/US88/0392 2 LBRM-33-1A5 cells, human dermal fibroblasts, murine BALB-3T3 cells, and ARH77, a human B lymphoblastoid cell line. The receptors in the different cell lineages exhibited similar but not identical binding characteristics. The IL-1 receptors on porcine synovial fibroblasts (Bird et al., Nature 324:263, 1986) and human dermal fibroblasts (Chin et al., J. Exp. Med. 165:70, 1987) have been shown to yield a major species in the size range Mr 97,000-100,000 when crosslinked to labeled IL-1, suggesting that a protein of Mr 80,000 was responsible for binding IL-1. In contrast, IL-1 receptors characterized in this fashion on human B cells (Matsushima et al., J. Immunol. 136:4496, 1986) displayed an apparent molecular weight of 60,000.

Bron and MacDonald, FEBS Letters 219:365 (1987), disclose immunoprecipitation of murine IL-1 receptor from surface-labeled EL-4 cells using a rabbit polyclonal antiserum directed to IL-1. This work indicated that the murine receptor is a glycoprotein having an apparent molecular weight of approximately 82,000 daltons.

Radiolabeled IL-1 has been used in chemical crosslinking studies and for the detection of receptor in detergent extracts of cells. The results of these experiments, noted above, suggest that a protein of Mr 60,000 or 80,000 is responsible for binding IL-1. The crosslinking of radiolabeled IL-1 to cells has also led to the occasional detection of proteins distinct from the major species of Mr 80,000, suggesting that the IL-1 binding molecule may exist in the membrane as part of a multi-subunit receptor complex.

In order to study the structure and biological characteristics of IL-1 receptors and the role played by IL-1 receptors in the responses of various cell populations to IL-1 stimulation, or to use IL-1 receptors effectively in therapy, diagnosis, or assay, homogeneous compositions of IL-1 receptor are needed. Such compositions are theoretically available via purification of solubilized receptors expressed by cultured cells, or by cloning and expression of genes encoding the receptors. However, prior to the present invention, several obstacles prevented these goals from being achieved.

Even in cell lines known to express detectable levels of IL-1 WO 89/04838 PCT/US88/03926 3 receptor, the IL-1 receptor is present as a very minor component of total cellular proteins. Moreover, no cell lines were known that expressed high levels of IL-1 receptors constitutively and continuously. For example, the murine EL-4 6.1 cell line expresses detectable levels of IL-1 receptor, but the level of II-i receptor expression tends to decay with time, which greatly complicates the process of obtaining sufficient quantities of receptor to provide a useful starting material for purification. Thus, a method of continuously selecting cells for acceptable levels of IL-1 receptor expression, employing fluorescence-activated "ell sorting (FACS), was devised.

Additional problems are inherent in attempting to clone mammalian genes encoding IL-1 receptor. Even if a protein composition of sufficient purity can be obtained to permit N-terminal protein sequencing, the degeneracy of the genetic code typically does not permit one to define a suitable probe without considerable additional experimentation. Many iterative attempts may be required to define a probe having the requisite specificity to identify a hybridizing sequence in a cDNA library. To circumvent this problem, a novel direct receptor expression cloning technique was devised to avoid the need for repetitive screening using different probes of unkn'mvn specificity. This technique, which has never before been employed, allows direct visualization of receptor expression following i transfection of a mammalian cell line with a high expression vector containing a cDNA clone encoding the receptor.

Purified IL-1 receptor compositions will be useful in diagnostic assays for IL-1 or IL-1 receptor, and also in raising antibodies to IL-1 receptor for use in diagnosis or therapy. In addition, purified IL-1 receptor compositions may be used directly in therapy to bind or scavenge IL-1, thereby providing a means for regulating the immune or inflammatory activities of this cytokine.

SUMMARY OF THE INVENTION The present invention provides DNA sequences consisting essentially of a single open reading frame nucleotide sequence WO 89/04838 PCT/US88/039 2 6 4 encoding a mammalian Interleukin-1 receptor (IL-1R) or subunit thereof. Preferably, such DNA sequences are selected from the group consisting of cDNA clones having a nucleotide sequence derived from the coding region of a native IL-1R gene; DNA sequences capable of hybridization to the cDNA clones of under moderately stringent conditions and which encode biologically active IL-1R molecules; and DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in and and which encode biologically active IL-1R molecules. The present invention also provides recombinant expression vectors comprising the DNA sequences defined above, recombinant IL-1R molecules produced using the r:ecombinant expression vectors, and processes for producing the recoribinant IL-1R molecules utilizing the expression vectors.

The present invention also provides substantially homogeneous protein compositions comprising murine or human IL-1 receptor. The murine molecule is a glycoprotein having a molecular weight of about 82,000 daltons by SDS-PAGE, a binding affinity for human IL-la of from 3-6 x 109 M-1, and the N-terminal amino acid sequence L E I D V C TE Y PN IVLF LS VNEI DIRK.

In another aspect, the present invention provides a process for purifying IL-1 receptor, comprising applying a sample comprising IL-1 receptor to an affinity matrix comprising an IL-1 molecule bound to an insoluble support, and eluting bound IL-1 receptor from the affinity matrix. The partially purified IL-1 receptor can be further purified by application to a lectin affinity column and subsequently eluting the IL-1 receptor from the lectin affinity column. The partially purified IL-1 receptor can then be treated by reversed phase high performance liquid chromatography, and eluted as a single peak of absorbance at 280 nanometers which, when analyzed by SDS-PAGE and silver staining, appeared as a single band. As noted above, the native murine IL-1 receptor had an apparent molecular weight of approximately 82,000 daltons as estimated by SDS-PAGE.

The present invention also provides compositions for use in therapy, diagnosis, assay of IL-1 receptor, or in raising antibodies to IL-1 receptors, comprising effective quantities of soluble native ia Iu SWO 89/04838 PCT/US88/03926 or recombinant receptor proteins prepared according to the foregoing processes. Such soluble recombinant receptor molecules include truncated proteins wherein regions of the receptor molecule not required for IL-1 binding have been deleted. These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.

BRIEF DESCRIPTION OF THE DRAVINGS Figure 1 is a restriction map of cDNA constructs comprising the coding regions of the murine and human IL-1R genes. The murine fragment, isolated from EL-4 6.1 C10 cells and present as an insert in clone GEMBL78, has been deposited with the American Type Culture Collection under deposit accession number ATCC 67563.

Figure 2 is a schematic illustration of the mammalian high expression plasmid pDC201, which is described in greater detail in Example 6.

Figures 3A-3C provide a graphical comparison of the IL-1 binding characteristics of natural and recombinant IL-1 receptors.

Figure 3A compares direct binding of 1 25 I-IL-la to cells expressing native IL-1 receptor (EL4 6.1 C10) or recombinant receptor (COS-IL-1R); Figure 3B shows the data from Figure 3A replotted in the Scatchard coordinate system. Figure 3C indicates competition for 12sI-IL-la binding by unlabeled IL-la and IL-10. In Figure 3, C indicates the concentration of IL-1 added to the binding incubation (molar); r indicates molecules of IL-1 bound per cell.

DETAILED DESCRIPTION OF THE INVENTION IL-la and IL-10 apparently regulate the metabolism of cells through a common plasma membrane receptor protein. IL-1 receptor from detergent solutions of EL-4 6.1 C10 cells has been stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 cells can be bound to and specifically eluted from IL-la coupled to 1 I I .I I I I 1 II% I I I 111 0 Z '4 WO 89/04838 PCT/US88/03926 6 Sepharose or other suitable affinity chromatography supports.

Purification by the foregoing process resulted in the identification by silver staining of polyacrylamide gels of a protein of M r 82,000 daltons that was present in'fractions exhibiting IL-1 binding activity. Experiments in which the cell surface proteins of EL-4 cells were radiolabeled and 125I labeled receptor was purified by affinity chromatography suggested that the M r 82,000 protein was expressed on the plasma membrane. N-glycanase treatment of this material showed that 21-35Z of the total M r (82,000) of the receptor was N-linked carbohydrate.

In order to define the chemical properties of the IL-1 receptor, a simple, reproducible and quantitative assay system was devised for the detection of IL-1 receptor in detergent solutions.

With this assay, receptor purification can be followed, and changes in receptor binding activity in response to chemical modification of the receptor can be easily monitored.

Binding Assay for IL-1 Receptor Recombinant human IL-10 and IL-1a can be prepared by expression in E. coli and purification to homogeneity as described by Kronheim et al. (Bio/Technology 4:1078, 1986). Recombinant human IL-la is preferably expressed as a polypeptide composed of the C-terminal 157 residues of IL-lc, which corresponds to the Mr 17,500 form of the protein released by activated macrophages. The purified protein is stored at -70°C in phosphate buffered saline as a stock solution of 3 mg/ml. 10 ul (30 Vg) aliquots of the stock solution are labeled with sodium (1251) iodide by a modified chloramine-T method described by Dower et al. (Nature 324:266, 1986) and Segal et al. (J.

Immunol. 118:1338, 1977). In this procedure, 10 ug rIL-la (0.57 nmol) in 10 ul phosphate (0.05 M) buffered saline (0.15 M) pF 7.2 (PBS) are added to 2.5 mCi (1.0 nmol) of sodium iodide in 25 Ul of 0.05 M sodium phosphate pH 7.0. The reaction is initiated by addition of 30 ul of 1.4 x 10 4 M chloramine-T (4.2 nmol; Sigma Chemical Co., St. Louis, MO, USA). After 30 minutes on ice the reaction mixture is fractionated by gel filtration on a 1 mL bed volume Biogel P6 WO 89/04838 PCT/US88/03926 7 (Bio-Rad, Richmond, CA, USA) column. Routinely, 40-50% of 1251 is incorporated into protein.

25 I-IL-la can be purified by gel filtration or other suitable methods and immediately diluted to a working stock solution of 3 x 10-' M in Roswell Park Memorial Institute (RPMI) 1640 medium comprising 1% bovine serum albumin (BSA), 0.1X sodium azide, 20 mM Hepes pH 7.4 (binding medium), to avoid radiolysis. Such dilute solutions can be stored for up to one month without detectable loss of receptor binding activity. The specific activity is routinely in the range 1-3 x 1015 cpm/mmole (ca 1 atom of iodine per IL-la molecule). Typically, the labeled protein is initially (prior to dilution) 100X active as determined by its capacity to elicit IL-2 production from EL-4 6.1 C10 cells. Further, 100% of the 125I cpm can be precipitated by trichloroacetic acid and >95X can be absorbed by IL-1 receptor bearing cells.

EL-4 6.1 C10 cells are propagated in suspension culture as described by MacDonald et al., J. Immunol. 135:3964 (1985). An IL-1 receptor negative variant line of EL-4 cells, EL-4 (ATCC TIB 39), is grown in an identical fashion. Cells are monitored on a weekly basis for IL-1 receptor expression by 12 5 I-IL-la binding.

To maintain relatively high levels of receptor expression, cells can be sorted using fluorescence-activated cell sorting (FACS) and fluorescein-conjugated recombinant IL-l. Fluorescein-conjugated rIL-la (FITC IL-la) is prepared by reacting 2.9 nanomoles protein with 100 nanomoles of fluorescein isothiocyanate (Research Organics, Cleveland, Ohio) in a total volume of 70 ul of borate (0.02 M) buffered saline (0.15 M) pH 8.5 for two hours at 37 0 C. Protein is separated from unconjugated dye by gel filtration on a 1 ml bed volume P6 column, as described by Dower et al. Exp. Med. 162:501, 1985).

Using an EPICS C flow cytometer (Coulter Instruments; 488 nM argon laser line. 300 MV, gain 20, PMT voltage 1700), cells providing the highest level fluorescence signal the top 1.0% or 0.1X, as desired) are collected and used to establish cell cultures for receptor expression.

For extractions, cells harvested from culture by I -_ric i5.1 WO 804838 pCTUS8103926 8 centrifugation are washed once with binding medium and sedimented at 2000 x g for 10 min to form a packed pellet (ca 8 x 108 cells/ml). To the pellet is added an equal volume of PBS containing 1% Triton X-100 and a cocktail of protease inhibitors (2 mM phenylmethylsulphonyl fluoride, 1 pM pepstatin, 1 um leupeptin, and 2 mM 0-phenanthroline).

The cells are mixed with the extraction buffer by vigorous vor'-xing and the mixture incubated on ice for 15 minutes; at the end of this time the mixture is centrifuged at 11,000 x g for 30 minutes at 8°C to remove nuclei and other debris. The supernatant is mada 0.02% v/v in sodium azide and stored either at 8°C or -70°C, with no loss in IL-1 receptor activity detected for periods of up to six months at either temperature.

For solid phase binding assays, uiiless otherwise indicated, 1 ul (4 x 105 cell equivalents) aliquots of extract are placed on dry BA85/21 nitrocellulose membranes (Schleicher Schuell, Keene, NH) and the membranes kept at room temperature until dry. Dry membranes can be stored at room temperature until use. Under these conditions, receptor binding actvity remains stable for up to two months. Prior to use, membranes are reconstituted by incubating for 30 minutes in Tris (0.05 M) buffered saline (0.15 M) pH 7.5 containing 3% w/v BSA to block nonspecific binding sites, washed twice with PBS (20 ml per filter), once with binding medium and cut while wet into 0.9 x 0.9 cm squares with the IL-1 receptor extract at the center. The squares are placed in 24 well trays (Costar, Cambridge, MA) and covered with 200 ul of binding medium containing 12 5 I-IL-la or 12 5 I-IL-la and unlabeled inhibitors. Trays are then placed on a nutator and incubated in a refrigerator (8 0 C) for two hours. At the end of this time a 60 ul aliquot can be taken from each well for determination of unbound 12 5 I-rIL-lc. Subsequently, the remaining solution is aspirated and discarded and the nitrocellulose filters washed by adding and aspirating sequentially 1 ml of binding medium and three times 1 ml of PBS to each well. The nitrocellulose squares are then removed and dried on filter paper. Subsequently, they are either placed on Kodak X-omat AR film for twelve hours at -70 0 C, or placed in 12 x 75 cm glass tubes and counted on a gamma counter.

WO 89/04838 PCT/US88/03926 9 Brief Description of Tables Table 1 depicts the cDNA sequence of clone GEMBL78.

Nucleisides are numbered from the beginning of the fragment. The CTG codoit specifying the leucine residue constituting the N-terminus is underlined at position 282, and the TAG terminator codon which ends the open reading frame is underlined at position 1953.

Tables 2A-2C depict the cDNA sequence and derived amino acid sequence of the coding region of the cDNA shown in Table 1. In Tables 2A-2C, nucleotides and amino acids are numbered from the leucine residue representing the N-terminus of the mature protein.

The alternative initiator methionines, N-terminus, and 21 amino acid putative transmembrane region of the murine IL-1 receptor are underlined.

Table 3 depicts a cDNA sequence which includes the complete coding region of the human IL-1R gene. Nucleotides are numbered from the beginning of a fragment, designated R3A, which includes the N-terminus and a short sequence of 5' nontranslated DNA. The CTG codon specifying the leucine residue constituting the N-terminus is underlined at position 135, and the TAG terminator codon which ends the open reading frame is underlined at position 1791.

Tables 4A-4C depict the cDNA sequence and derived amino acid sequence of the coding region of a cDNA encoding human IL-1 receptor.

In Tables 4A-4C, nucleotides and amino acids are numbered from the leucine residue (underlined) representing the N-terminus of the mature protein. The 20-amino acid transmembrane region is also underlined.

Table 5 is a comparison of the derived amino acid sequences of the murine and human IL-1 receptors. The transmembrane regions of each protein are underlined, and conserved cysteine residues are indicated by asterisks. Potential N-linked glycosylation sites are indicated by triangles adjacent to asparagit\e residues.

Definitions "Interleukin-1 receptor" and "IL-1R" refer to proteins which are capable of binding Interleukin-1 (IL-1) molecules and, in their native configuration as mammalian plasma membrane proteins, presumably WO 89/04838 PCT/US88/03926 play a role in transducing the signal provided by IL-1 to a cell. As used herein, the term includes analogs of native proteins with IL-1-binding or signal transducing activity. Specifically included are truncated or soluble forms of the IL-1 receptor protein not having a cytoplasmic and transmembrane region. The predicted molecular weight of the murine protein corresponding to the sequence of the mature protein depicted in Tables 2A-2B is 64,597 daltons, while the predicted weight of the precursor is 66,697 daltons. Both of these estimates are exclusive of any glycosylation. The predicted molecular weight of the human protein corresponding to the sequence of the mature protein depicted in Tables 4A-4C is 63,486 daltons, while the predicted weight of the precursor is 65,402 daltons.

"Substantially identical" and "substantially similar," when used to define amino acid sequences, mean that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences. For purposes C£ the present invention, amino acid sequences having greater than percent similarity are considered to be substantially similar, and amino acid sequences having greater than 80 percent similarity are considered to be substantially identical. In defining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered substantially similar to a reference nucleic acid sequence, and all nucleic acid sequences capable of encoding substantially identical amino acid sequences are considered substantially identical to a reference sequence. For purposes of determining similarity, truncation or internal deletions of the reference sequence should be disregarded. Sequences having lesser degrees of similarity, comparable biological activity, and equivalent expression characteristics are considered to be equivalents. For purposes of the present invention, a "subunit" of an IL-1R is deemed to constitute an amino acid sequence of at least 20 amino acids.

"Recombinant," as used herein, means that a protein is WO 89/04838 PCT/US88/03926 11 derived from recombinant microbial or mammalian) expression systems. "Microbial" refers to recombinant proteins made in bacterial or fungal yeast) expression systems. As a product, S"recombinant microbial" defines a protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Protein expressed in most bacterial cultures, E.

coli, will be free of glycan; protein expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.

"Biologically active," as used throughout the specification as a characteristic of IL-1 receptors, means either that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of binding at least 0.01 nmoles IL-1 per nanomole IL-1 receptor or IL-1 receptor analog, or, in the alternative, shares sufficient amino acid sequence similarity to be capable of transmitting an IL-1 stimulus to a cell, for example, as a component of a hybrid receptor construct.

Preferably, biologically active IL-1 receptors within the scope of the present invention are capable of binding greater than 0.1 nanomoles IL-1 per nanomole receptor, and most preferably, greater than -anomoles IL-1 per nanomole receptor.

"DNA sequence" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methoda, for example, using a cloning vector.

Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. However, it will be evident that genomic DNA containing the relevant sequences could also be used. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

I L _i J WO 89/04838 PCT/US88/03926, 12 "Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. DNA sequences encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.

"Recombinant expression vector" refers to a plasmid comprising a transcriptional unit comprising an assembly of a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, a structural or coding sequence which is transcribed into mRNA and translated into protein, and appropriate transcription and translation initiation and termination sequences. Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.

Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

"Recombinant microbial expression system" means a substantially homogeneous monoculture of suitable host microorganisms, for example, bacteria such as E. coli or yeast such as S. cerevisiae, which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid. Generally, cells constituting the system are the progeny of a single ancestral transformant.

Recombinant expression systems as defined herein will express heterologous protein upon induction of the regulatory elements linked to the DNA sequence or synthetic gene to be expressed.

Isolation of cDNAs Encoding IL-1 Receptors In order to secure the murine coding sequence, a DNA sequence encoding murine IL-1R (mIL-1R) was isolated from a cDNA library prepared by reverse transcription of polyadenylated RNA isolated from the murine cell line EL-4 6.1 C10. The library was screened by direct /t WO 89/04838 PCT/US88/03926 13 expression of pooled cDNA fragments in monkey COS-7 cells using a mammalian expression vector (pDC201) that uses regulatory sequences derived from SV40 and Adenovirus 2. Transfectants expressing biologically active IL-1R vere identified by incubating transfected COS-7 cells with medium containing 12 5 I-IL-lo, washing the cells to remove unbound labeled IL-lo, and contacting the cell monolayers vith X-ray film to detect concentrations of IL-la binding. Transfectants detected in this manner appear as dark foci against a relatively light background.

Using this approach, approximately 150,000 cDNAs were screened in pools of approximately 350 cDNAs until assay of one transfectant pool indicated positive foci of IL-la binding. A frozen stock of bacteria from this positive pool was grown in culture and plated to provide individual colonies, which were screened until a single clone (clone 78) was identified which directed synthesis of a surface protein with detectable IL-1 binding activity. This clone was isolated, and its insert sequenced to determine the sequence of the murine cDNA set forth in Table 1. The initiator methionine for the full-length translation product of the native murine gene is one of two alternative methionine residues found at positions -19 and -16 of Table 2A. The first amino acid residue of the mature receptor protein was deduced by comparison to an N-terminal amino acid sequence obtained from highly purified preparations of IL-1R derived from EL-4 6.1 C10 cells. This residue is a leucine residue shown at position i of Table 2A. The 1671 nucleotide coding region corresponding to the mature protein encodes 576 amino acids, including 15 cysteine residues and a 21-amino acid putative transmembrane region. Located N-terminal to the transmembrane region are 7 potential N-glycosylation sites. A cloning vector comprising the full-length murine cDNA, designated GEMBL78, has been deposited with the American Type Culture Collection, Rockville, MD, USA (ATCC) under accession number 67563. The deposit was made under the conditions of the Budapest Treaty.

A probe was constructed from the murine sequence and used to screen human cDNA libraries prepared from cultures of a human T-cell clone grown in the presence of OKT3 antibody and IL-2. cDNA clones g _J4 14 which hybridized to the murine probe were then isolated and sequenced.

Using a fragment derived from human cDNA clones, a 1707 nucleotide human coding sequence was obtained and sequenced. The nucleotide sequence of the human cDNA, including 5' and 3' nontranslated sequences, is shown in Table 3. The nucleotide sequence of the human open reading frame and derived amino acid sequence of the human protein is set forth in Tables 4A-4C. This sequence comprises 569 amino acids (including a 17 amino acid signal peptide), including 16 cysteine residues, 13 of which are conserved between the murine and human genes. In addition, the human sequence includes six potential N-glycosylation sites, of which 5 are conserved between murine and human. The amino acid sequence of Tables 4A-4C is numbered from a leucine residue considered to be the likely N-terminus on the basis of comparison to the murine protein. The putative transmembrane region of the human gene is 20 amino acids in length. The sequences of the presumed intracellular portions of the murine and human genes are highly conserved; the extracellular and transmembrane regions are somewhat less conserved, except for the location of cysteines presumably involved in intramolecular disulfide bonding and certain N-glycosylation sites. The derived amino acid sequences of the human and murine genes are compared in Table The murine and human genes encode integral membrane proteins including intracellular regions having no apparent homology with any known protein sequence and extracellular portions which appear to be organized into domains similar to those of members of the immunoglobulin gene superfamily. Immunoglobulin-like domains typically possess only minimal amino acid similarity but share a common threedimensional structure consisting of two O-sheets held together by a disulfide bond. The cysteine residues involved in formation of this disulfide bond, as well as a few other critical residues, are highly conserved and occur in the same relative position in almost all members of the family. Members of the immunoglobulin superfamily include not only immunoglobulin constant and variable regions but also a number of other cell surface molecules, many of which are involved in cell-cell interactions.

WO 89/04838 PCT/US88/03926 Like most mammalian genes, mammalian IL-1Rs are presumably encoded by multi-exon genes. Alternative mRNA constructs which can be attributed to different mRNA splicing events following transcription, and which share large regions of identity or similarity with the cDNAs claimed herein, are considered to be within the scope of the present invention.

In its nucleic acid embodiments, the present invention provides DNA sequences encoding mammalian IL-1Rs. Examples of mammalian IL-1Rs include primate IL-1R, human IL-1R, murine, canine, feline, bovine, ovine, equine and porcine IL-1Rs. IL-1R DNAs are preferably provided in a form which is capable of being expressed in a recombinant transcriptional unit under the control of mammalian, microbial, or viral transcriptional or translational control elements.

For example, a sequence to be expressed in a microorganism will contain no intrors. In preferred aspects, the DNA sequences comprise at least one, but optionally more than one sequence component derived from a cDNA sequence or copy thereof. Such sequences may be linked or flanked by DNA sequences prepared by assembly of synthetic oligonucleotides. However, synthetic genes assembled exclusively from oligonucleotides could be constructed using the sequence information provided herein. Exemplary sequences include those substantially identical to the nucleotide sequences depicted in Tables 2A-2C.

Alternatively, the coding sequences may include codons encoding one or more additional amino acids located at the N-terminus, for example, an N-terminal ATG codon specifying methionine linked in reading frame with the nucleotide sequence. Due to code degeneracy, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence; exemplary DNA embodiments are those corresponding to the sequence cf nucleotides 1-1671 of Tables 2A-2C, and nucleotides 1-1656 of Tables 4A-4C. Other embodiments include sequences capable of hybridizing to the sequence of Tables 2A-2C or 4A-4C under moderately stringent conditions (50 0 C, 2 x SSC) and other sequences degenerate to those described above which encode biologically active IL-1R polypeptides.

The present invention also provides expression vectors for i 'Vi

.I

WO 89/04838 PCT/US88/03926 16 producing useful quantities of purified IL-1R. The vectors can comprise synthetic or cDNA-derived DNA fragments encoding mammalian IL-1Rs or bioequivalent homologues operably linked to regulatory elements derived from mammalian, b'cterial, yeast, bacteriophage, or viral genes. Useful regulatory elements are described in greater detail below. Following transformation, transfection or infection of appropriate cell lines, such vectors can be induced to express recombinant protein.

Mammalian IL-IRs can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.

Cell-free translation systems could also be employed to produce mammalian IL-1R using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant disclosure of which is hereby incorporated by reference.

Various mammalian cell culture systems can be employed to express recombinant protein. Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing an appropriate vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. Additional details regarding the use of a mammalian high expression vector to produce a recombinant mammalian IL-1R are provided in Examples 4 and 6, below. Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, i WO 89/04838 PCT/fS88/03926 17 1983).

A useful system for stable high level expression of mammalian receptor cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Molecular Immunol. 23:935, 1986).

Yeast systems, preferably employing Saccharomyces species such as S. cerevisiae, can also be employed for expression of the recombinant proteins of this invention. Yeast of other genera, for example, Pichia or Kluyveromyces, have also been employed as production strains for recombinant proteins.

Generally, useful yeast vectors will include origins of replication and selectable markers permitting transformation of both yeast and E. coli, the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed yeast gene to induce transcription of a downstream structural sequence. Such promoters can be derived from yeast transcriptional units encoding highly expressed genes such as 3-phosphoglycerate kinase (PGK), o-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate reading frame with translation initiation and termination sequences, and, preferably, a leader sequence capable of directing secretion of translated protein into the extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide Asp--Tyr-Lys- (Asp) 4 -Lys) or other sequence imparting desired characteristics, stabilization or simplified purification of expressed recombinant product.

Useful yeast vectors can be assembled using DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) and yeast DNA sequences including a glucose-repressible alcohol dehydrogenase 2 (ADH2) promoter. The ADH2 promoter has been described by Russell et al. Biol. Chem.

258:2674, 1982), and Beier et al. (Nature 300:724, 1982). Such vectors may also include a yeast TRP1 gene as a selectable marker and the yeast 2 u origin of replication. A yeast leader sequence, for

J

WO 89/04838 PCT/US88/03926 18 example, the a-factor leader which directs secretion of heterologous proteins from a yeast host, can be inserted between the promoter and the structural gene to be expressed (see Kurjan et al., U.S. Patent 4,546,082; Kurjan et al., Cell 30:933 (1982); and Bitter et al., Proc.

Natl. Acad. Sci. USA 81:5330, 1984). The leader sequence may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes.

Suitable yeast transformation protocols are known to those skilled in the art; an exemplary technique is described by Hinnen et al. (Proc. Natl. Acad. Sci. USA 75:1929, 1978), selecting for Trp transformants in a selective medium consisting of 0.67X yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ug/ml adenine and 20 ug/ml uracil.

Host strains transformed by vectors comprising the ADH2 promoter may be grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ug/ml adenine and 80 ug/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4 0 C prior to further purification.

Useful expression vectors for bacterial use are constructed by inserting a DNA sequence encoding mammalian IL-1R together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure amplification within the host.' Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

Expression vectors are conveniently constructed by cleavage of cDNA clones at sites close to the codon encoding the N-terminal residue of the mature protein. Synthetic oligonucleotides can then be used to "add back" any deleted sections of the coding region and to provide a linking sequence for ligation of the coding fragment in appropriate reading frame in the expression vector, and optionally a F4 WO 89/04838 PCT/US88/03926 codon specifying an initiator methionine.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMI (Promega Biotec, Madison, VI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

A particularly useful bacterial expression system employs the phage X PL promoter and c1857 thermolabile repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the X PL promoter include plasmid pHUB2, resident in E.

coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082). Other useful promoters for expression in E. coli include the T7 RNA polymerase promoter described by Studier et al. (J.

Mol. Biol. 189: 113, 1986), the lacZ promoter described by Lauer (J.

Mol. Appl. Genet. 1:139-147, 1981) and available as ATCC 37121, and the tac promoter described by Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, p 412) and available as ATCC 37138.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed by appropriate means temperature shift or chemical induction) and cells cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Cells are grown, for example, in a 10 liter fermenter employing conditions of maximum aeration and vigorous agitation. An antifoaming agent (Antifoam A) is preferably employed.

Cultures are grown at 30 0 C in the superinduction medium disclosed by Mott et al. (Proc. Natl. Acad. Sci. USA 82:88, 1985), alternatively including antibiotics, derepressed at a cell density corresponding to

A

60 o 0.4-0.5 by elevating the temperature to 420C, and harvested

.IS

WO 89/04838 PCT/US88/03926 from 2-20, preferably 3-6, hours after the upward temperature shift.

The cell mass is initially concentrated by filtration or other means, then centrifuged at 10,000 x g for 10 minutes at 4°C followed by rapidly freezing the cell pellet.

Preferably, purified mammalian IL-IRs or bioequivalent homologues are prepared by culturing suitable host/vector systems to express the recombinant translation products of the synthetic genes of the present invention, which are then purified from culture media.

An alternative process for producing purified IL-1R involves purification from cell culture supernatants or extracts. In this approach, a cell line which elaborates useful quantities of the protein is employed. Supernatants from such cell lines can be optionally concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix as previously described. For example, a suitable affinity matrix can comprise an IL-1 or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.

Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.

Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e silica gel having pendant methyl or other aliphatic groups, can be employed to further purify an IL-1R composition. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size i; i: 1 i I I i i v- Irr L -ijl I I I I WO 89/04838 PCT/US88/03926 21 exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

Microbial cells employed in expression of recombinant mammalian IL-1R can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express mammalian IL-1R as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J.

Chromatog. 296:171, 1984). This reference describes two sequential, reversed-phase HPLC steps for purification of recombinant human GM-CSF on a preparative HPLC column.

In its various embodiments, the present invention provides substantially homogeneous recombinant mammalian IL-1R polypeptides free of contaminating endogenous materials, with or without associated native-pattern glycosylation. The native murine IL-1R molecule is recovered from cell culture extracts as a glycoprotein having an apparent molecular weight by SDS-PAGE of about 82 kilodaltons (kD).

IL-1Rs expressed in mammalian expression systems, COS-7 cells, may be similar or slightly different in molecular weight and glycosylation pattern to the native molecules, depending upon the expression system. Expression of IL-1R DNAs in bacteria such as E.

coli provides non-glycosylated molecules having an apparent molecular weight of about 60 kD by SDS-PAGE under nonreducing conditions.

Recombinant IL-1R proteins within the scope of the present invention also include N-terminal methionyl murine and human IL-1Rs.

Additional embodiments include soluble trrn:ated versions wherein certain regions, for example, the transmembrane region and intracellular domains, are deleted, providing a molecule having an IL-l-binding domain only. Also contemplated are mammalian IL-1Rs expressed as fusion proteins with a polypeptide leader comprising the sequence Asp-Tyr-Lys-(Asp 4 )-Lys, or with other suitable peptide oprotein sequences employed as aids to expression in microorganisms or purification of microbially-expressed proteins.

i WO 89/04838 PCT/US88/0392e 22 Bioequivalent homologues of the proteins of this invention include various analogs, for example, truncated versions of IL-1Rs wherein terminal or internal residues or sequences not needed for biological activity are deleted. Other analogs contemplated herein are those in which one or more cysteine residues have been deleted or replaced with other amino acids, for example, neutral amino acids.

Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present, or modification of the protein sequence to eliminate one or more N-linked glycosylation sites.

As used herein, "mutant amino acid sequence" refers to a polypeptide encoded by a nucleotide sequence intentionally made variant from a native sequence. "Mutant protein" or "analog" means a protein comprising a mutant amino acid sequence. "Native sequence" refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein. The terms "KEX2 protease recognition site" and "N-glycosylation site" are defined below. The term "inactivate," as used in defining particular aspects of the present invention, means to alter a selected KEX2 protease recognition site to retard or prevent cleavage by the KEX2 protease of Saccharomyces cerevisiae, or to alter an N-glycosylation site to preclude covalent bonding of oligosaccharide moieties to particular amino acid residues by the cell.

Site-specific mutagenesis procedures can be employed to inactivate KEX2 protease processing sites by deleting, adding, or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conver-ton of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites. The resulting analogs are less susceptible to cleavage by the KEX2 protease at locations other than the yeast m-factor leader sequence, where cleavage upon secretion is intended.

Many secreted proteins acquire covalently attached i $i

II

WO 89/04838 PCT/US88/03926 23 carbohydrate units following translation, frequently in the form of oligosaccharide units linked to asparagine side chains by N-glycosidic bonds. Both the structure and number of oligosaccharide units attached to a particular secreted protein can be highly variable, resulting in a wide range of apparent molecular masses attributable to a single glycoprotein. mI -i.R is a glycoprotein of this type.

Attempts to express glycoproteins in recombinant systems can be complicated by the heterogeneity attributable to this variable carbohydrate component. For example, purified mixtures of recombinant glycoproteins such as human or murine granulocyte-macrophage colony stimulating factor (GM-CSF) can consist of from 0 to 50% carbohydrate by weight. Miyajima et al. (EMBO Journal 5:1193, 1986) reported expression of a recombinant murine GM-CSF in which N-glycosylation sites had been mutated to preclude glycosylation and reduce heterogeneity of the yeast-expressed product.

The presence of variable quantities of associated carbohydrate in recombinant glycoproteins complicates purification procedures, thereby reducing yield. In addition, should the glycoprotein be employed as a therapeutic agent, a possibility exists that recipients will develop immune reactions to the yeast carbohydrate moieties, requiring therapy ro be discontinued. For these reasons, biologically active, homogeneous analogs of immunoregulatory glycoproteins having reduced carbohydrate may be desirable f r therapeutic use.

tional mutant analogs of msmmiian IL-1Rs having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques as described below. These analog proteins tan be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems. N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-A'-Z, vhere A' is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate. Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or x. WO 89/04838 PCT/US88/03926 24 inserting a non-Z amino acid between A' and Z, or an amino acid other than Asn between Asn and Preferably, substitutions are made conservatively; the most preferred substitute amino acids are those having physicochemical characteristics resembling those of the residue to be replaced. Similarly, when a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion upon biological activity should be considered.

In addition to the particular analogs described above, numerous DNA constructions including all or part of the nucleotide sequences depicted in Tables 2A-2C or 4A-4C, in conjunction with oligonucleotide cassettes comprising additional useful restriction sites, can be prepared as a matter of convenience. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. By way of example, Valder et al.

(Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (Biotechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U. S.

Patent No. 4,518,584 disclose suitable techniques, and are incorporated by reference herein.

In one embodiment of the present invention, the amino acid sequence of IL-1R is linked to a yeast o-factor leader sequence via an N-terminal fusion construct comprising a nucleotide encoding the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK). The latter sequence is highly antigenic and provides an epitope reversibly bound by specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at the residua immediately following the Asp-Lys pairing. Fusion proteins capped 3 l 1: with this peptide may also be resistant to intracellular degradation in E.coli, An alternative construction is Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Glu-Ile-Gly-Arg, which provides a Factor X recognition site immediately downstream from the enterokinase site.

Other forms of IL-1 receptor have recently been reported. Matsushima et al., J.

Immunol. 136:4496 (1986) disclose the existence of an IL-1 receptor protein which has a molecular weight of about 60 kDa. This 60 kDa IL-1 receptor protein is commonly referred to as type II 11.i receptor. In contrast, the present patent application relates to an entirely different IL-1 receptor protein which, in its fulllength mature form, has a molecular weight of about 79.5 kDa. The IL-1 receptor protein of the present invention is commonly referred to as type I IL-1 receptor.

Recent studies have confirmed that these two forms of IL-1 receptor are the products of entirely different genes. See, e.g. Bomsztyk et al, Proc. Natl. Acad. Sci.

USA 86: 8034, 1989, and McMahan et al., EMBO J. 10: 2821, 1991.

The following examples are offered by way of illustration, and not by way of *limitation.

EXAMPLES

Example 1 Preparation of IL-1 a Affinity Matrix and Affinity Purification of Receptor from Surface Labeled EL-4 6.1 C10 Cells Cell surface proteins on EL-4 6.1 C10 cells were radiolabeled with '25I by the tglucose oxidase-lactoperoxidase method disclosed by Cosman et al. (Molecular Immunol.23935, 1986). Labeled cells were pelleted by centrifugation, washed three times with PBS, and extracted with PBS containing 1% Triton X-100 and the 25 cocktail of protease inhibitors described in the assay protocol detailed above. The Triton X-100 extract was spun for 10 minutes in an Eppendorf microcentrifuge and the supernatant was stored at -70 C.

Recombinant IL-1 a was coupled to cyanogen bromide activated Sepharose 4B (Pharmacia, Piscataway, NJ, USA) or to Affigel-10 (Bio-Rad, Richmond, CA, USA) according to the manufacturer's suggestions. For example, to a solution of IL-1 a (1.64 mg/ml in 9.5 ml PBS), 3 ml were added of swollen, acid-washed, CNBR-activated Sepharose. The solution was rocked P a of IL- aAgbditioo9.29 a015A891en5 2 WO 89/04838 PCT/US88/03926 26 RIPA buffer (0.05 M Tris-HCl pH 7.5, 0.15 M NaCi, 1% NP40, 1% sodium deoxycholate, 0.1Z SDS), and PBS containing 0.1X Triton X-100 and mM ATP. Small columns (200 ul) were prepared in disposable polypropylene holders (Bio-Rad, Richmond, CA, USA) and washed with PBS containing 1% Triton X-100, Aliquots of 100 ul of 1'2I-labeled extract were applied to a column, which was then washed with PBS containing 1% Triton X-100, RIPA buffer, PBS containing 0.1 Triton X-100 and 10 mM ATP, and PBS with 1% Triton X-100.

The IL-1 receptor on murine T cells is a robust structure capable of binding 1 25 I-IL-la in Triton X-100 detergent solutions. To be able to recover receptor from such an affinity matrix, a mild elution procedure is necessary. Mild acid treatment can cause rapid dissociation of preformed IL-l/IL-1 receptor complexes. Based upon this observation, pH 3.0 glycine HC1 buffer containing 0.1% Triton X-100 were used to elute receptor from the IL-la affinity columns, which was collected in 0.05 ml fractions. The presence of receptor in the fractions was detected by dot blot as described above, using 125I-labeled IL-la.

Analysis by SDS-PAGE proceeded as follows. To 50 ul of each column fraction was added 50 ul of 2 x SDS sample buffer (0.125 M Tris HC1 pH 6.8, 4% SDS, 20% glycerol, 10X 2-mercaptoethanol). The solution was placed in a boilng water bath for three minutes and aliquots of 40 ul were applied to the sample well of a polyacrylamide gel which was set up and run according to the method of Laemmli (Nature 227:680, 1970). Gels were fixed and stained using 0.25% Coomassie brilliant blue in 25% isopropanol, 10% acetic acid), destained in 25% isopropanol, 10% acetic acid, treated with Enhance (New England Nuclear, Boston, MA, USA), dried and exposed to Kodak X-omat AR film at -70oC. Molecular weight markers, labeled with 14C, were obtained from New England Nuclear, and included: cytochrome C (Mr 12,300), lactoglobulin A (Mr 18,367), carbonic anhydrase (Mr 31,000), ovalbumin (Mr 46,000), bovine serum albumin (Mr 69,000), phosphorylase B (Mr 97,400) and myosin (Mr 200,000). Alternatively, fractions having receptor activity were analyzed by SDS polyacrylamide gel electrophoresis followed by silver staining as previously described by

I

NWO 89/04838 PCT/US88/03926 27 Urdal et al. (Proc. Natl. Acad. Sci. USA 81:6481, 1984).

Dot blot analysis of fractions eluted from the IL-la affinity matrix showed that IL-1 binding activity was detected in fractions that were collected after pH 3.0 glycine buffer was applied to the column. Fractions that scored positive in this assay, when analyzed by SDS-PAGE, revealed that a protein of Mr 82,000 could be detected upon developing the gel with silver stain. To determine which of the proteins detected by silver stain were expressed on the cell surface, EL-4 6.1 cells were surface labeled with 125I by the lactoperoxidas glucose oxidase procedure. Radiolabeled cells were then extracted with PBS containing X1 Triton X-100 and aliquots of the detergent extract applied to an IL-la affinity matrix. Fractions that were collected from this column, following application to the column of pH glycine buffer, contained a radiolabeled protein of Mr 82,000.

Example 2 Comparison of Properties of Cellular IL-1 Receptor and IL-1 Receptor Isolated from Cell Extracts In a preliminary experiment, the binding properties of the IL-1 receptor were compared in intact EL-4 6.1 C10 cells and after extraction from cells. 3.8 x 108 EL-4 6.1 C10 cells were divided into two equal aliquots, one of which was extracted as described above.

The remaining cells were resuspended at 3.8 x 107 cells/ml and used for direct binding studies. Extract was adsorbed to nitrocellulose and used for solid phase binding studies employing vArious concentrations of 12'I-IL-la with or without unlabeled IL-1. After washing and drying, the nitrocellulose filters were first counted for bound 12 "I-IL-la and subsequently pl :ed on film for autoradiography.

Nonspecific background was measured in the presence of 5.7 x 10 M unlabeled rIL-10. The data obtained shoved that 5 I-IL-la was bound to the extract on nitrocellulose in an IL-1 concentration-dependent fashion, and that the 1 25 I-IL-la was specifically bound to the region of the blot where extract is present. Further, binding could be extensively blocked by inclusion of unlabeled IL-la in the incubation i c WO 89/04838 pCT/US88/03926.

28 mixture.

The comparison further indicated that not only were the levels of receptor the same in both instances, but that the receptors after adsorption to nitrocellulose exhibited an affinity for ligand which was indistinguishable from that of the receptor in intact cells.

No significant difference between the number of receptors detected on intact cells and those detected following detergent extraction was found. This is consistent with the view that the majority of the receptors were present on the external face of the plasma membrane in intact cells.

To measure the specificity of binding of IL-1 receptors on nitrocellulose filters, two ul of EL-4 6.1 C10 extract were applied to nitrocellulose filters, dried, blocked and assayed as described above.

The following proteins were tested for their capacity to inhibit 25 I-IL-la binding: human rIL-la (7.62 x 10- 7 human rIL-11 (7.62 x 7 human IL-2 (8.9 x 10- 7 murine IL-3 (7.5 x 10- 4

M),

murine-GM-CSF (7.5 x 10- 7 recombinant murine IL-4 (5 x 10- 9

M),

human epidermal growth factor 3 ug/ml, fibroblast growth factor 1 ug/ml, rat submandibular gland nerve growth factor (2 ug/ml), bovine insulin (1 x 10- 7 human luteinizing hormone (1 ug/ml), human growth hormone (1.7 x 10- 7 thyroid stimulating hormone (1 ug/ml), and follicle stimulating hormone (1 ug/ml). All incubations were done with 1.9 x 10-10 M 125 I-IL-la.

This experiment demonstrated that extracted receptor retains the same specificity as that previously demonstrated for intact cells.

As found with intact cells, only IL-la and IL-1 produced any significant inhibition of 12 5I-IL-la binding. The data showed that unlabeled IL-la and IL-10 produced >90X inhibition of 125I-IL-la binding, while no significant blockade was observed with any of the other hormones.

To determine whether receptor in detergent solution would bind IL-1 with an affinity equal to that of receptor in cell membranes, or adsorbed to nitrocellulose, a third experiment was performed in which the nitrocellulose dot blot binding assar was used to test the capacity of an EL-4 6.1 C10 extract in Triton X-100 i I WO 89/04838 PCT/US88/03926 29 solution to inhibit binding of 12s 5 -IL-la to the solid phase. EL-4 6.1 C10 extracts were adsorbed to nitrocellulose, dried, blocked and incubated with mixture of 12 5 I-IL-la and extracts containing receptors in detergent solution.

The concentration of receptor in the solution phase was estimated from a saturation binding curve to 1 pl aliquots blotted on nitrocellulose, allowing receptors/pl to be calculated and hence IL-1 receptor concentration The extract was diluted through PBS Triton X-100 solution Triton) to keep the detergent concentration constant. The inhibition curve shoved that in solution, the receptor bound to 12 5 I-IL-lc with a K, (4.5 0.5 x 109 M- 1 that is the same as that of receptor on the solid phase or in membranes.

Further, the close fit between the theoretical curve, which is based on a simple competitive inhibition model, and the data was consistent with the hypothesis that a single type of IL-1 binding protein was present in the membrane extract.

In order to examine the integrity of the receptor as a function of the concentration of total EL-4 6.1 C10 membrane proteins, a fourth experiment was done. Mixtures of EL-4 6.1 C10 extract in various proportions ranging from 10 to 100X were made either with an extract from cells not expressing the IL-1 receptor, EL-4 cells, or with PBS Triton X-100 Each mixture was analyzed for receptor concentration, and affinity of '15I-IL-la binding by quantitative dot blot binding. Receptor concentration decreased linearly with the percentage of EL-4 6.1 C10 extract present, whether membrane protein concentration was maintained at a constant level or not. In both series of mixtures the affinity of the receptor for 12 sI-IL-la remained constant. These data are consistent with one of two hypotheses, either the receptor binding function is contained within a single polypeptide chain or, if the functional receptor requires two or more subunits for IL-1 binding, these are sufficiently tightly associated that dilution through detergent does not separate them.

1 WO 89/04838 PCT/US88/03926 Example 3 Purification of IL-1 Receptor to Homogeneity and Determination of N-terminal Sequence 300-500 liters of EL-4 6.1 C10 cells were grown to saturation under the conditions previously described, harvested, and extracted with PBS-1X Triton X-100. The detergent extract was applied to an IL-la affinity column and the column washed as previously described.

Fractions containing IL-1 receptor were detected by the 12 5 I-IL-la dot blot procedure following elution of the column with 0.1 M glycine HC1 pH 3.0 containing 0.1% Triton X-100. Aliquots of the fractions were analyzed by SDS polyacrylamide gel electrophoresis.

This partially purified IL-1 receptor composition prepared by affinity chromatography on Affigel-IL-la was adjusted to contain the following buffer composition: 10 mM Tris-HCl, pH 8, 250 mM NaC1, mM MgCl 2 0.5 mM Mncl 2 0.5 mM CaCI 2 and 0.01 X Triton X-100 (VGA buffer). The IL-1 receptor composition was then applied to a 1 ml column of wheat germ agglutinin (VGA) bound to Sepharose CL-6B, equilibrated with VGA buffer. Following application of the IL-1 receptor composition, the VGA column was washed with 20 ml of VGA buffer followed by 10 mM Tris HC1, pH 8, 0.01% Triton X-100.

The IL-1 receptor protein was eluted from the VGA column with 10 mM Tris-HCl, pH 8, 0.5 M N-acetylglucosamine, and 0.01% Triton X-100. The presence of biologically active IL-1 receptor was detected by the 12 5 I-IL-la dot blot procedure. The fractions were also analyzed by SDS polyacrylamide gel electrophoresis followed by silver staining.

Material eluting from the VGA column was applied to a C8 RP-HPLC column. The C8 RP-HPLC column (Brownlee Labs RP-300, 1 mm X mm) was previously equilibrated with 0.1% trifluoroacetic acid (TFA) in HPLC grade H20, at a flow rate of 50 ul/min. Following application of the IL-1 receptor containing material, the C 8 RP-HPLC column was washed with 0.1% TFA in H 2 0 at 50 ul/min until the absorbance at 280 nm returned to baseline. The IL-1 receptor protein was eluted from the column by running linear gradient of 0.1% (vlv) I 7' WO 89/04838 PCT/US88/03926 31 TFA in acetonitrile from 0-100X at a rate of 1% per minute. Aliquots of the fractions were analyzed by SDS polyacrylamide gel electrophoresis. The IL-1 receptor protein was found to consist of a single band on an SDS polyacrylamide gel migrating with a molecular weight of 82,000.

The purified IL-1 receptor protein was analyzed by Edman degradation using an Applied Biosystems Model 470A protein sequencer.

The protein (150 picomoles) was not modified before analysis. The results of the N-terminal protein sequence analysis of the IL-1 receptor indicated the folloviw.,: sequence of amino acid residues:

NH

2 -Leu-Glu-Ile-Asp-Val-Cys-Thr-Glu-Tyr-Pro-Asn-Gln-Ile-Val- Leu-Phe-Leu-Ser-Val-Asn-Glu-Ile-Asp-Ile-Arg-Lys.

This protein sequence saz found to be unique when compared to the March 17, 1987 release of the Protein Sequence Database of the Protein Identification Resource of the National Biomedical Research Foundation. This release of the database contained 4,253 sequences consisting of 1,029,056 residues.

Example 4 Isolation of cDNA Encoding Murine IL-1R by Direct Expression of Active Protein in COS-7 Cells A cDNA library was constructed by reverse transcription of polyadenylated mRNA isolated from total RNA extracted from EL-4 6.1 cells by a procedure similar to that of Chirgvin et al. (Biochem.

18:5294, 1979). Briefly, the cells were lysed in a guanidinium isothiocyanate solution, and the lysate layered over a pad of CsC1 and centrifuged until the RNA had pelleted. The RNA pellet was resuspended and further purified by protease digestion, organic extraction and alcohol precipitation. Poly A* RNA vas isolated by oligo dT cellulose chromatography and double-stranded cDNA was prepared by a method similar to that of Gubler and Hoffman (Gene 25:263, 1983). Briefly, the RNA was copied into cDNA by reverse transcriptase using either oligo dT or random oligonucleotides as primer. The cDNA was made double-stranded by incubation with E. coli DNA polymerase I and RNase H, and the ends made flush by further WO 89/04838 PCT/US88/03926 32 incubation with T 4 DNA polymerase. The blunt-ended cDNA was ligated into SmaI-cut dephosphorylated pDC201 vector DNA.

The eukaryotic high expression vector pDC201 was assembled from SV40, adenovirus 2, and pBR322 DNA comprising, in sequence: (1) an SV40 fragment containing the origin of replication, early and late promoters, and enhancer; an adenovirus 2 fragment containing the major late promoter, the first exon and part of the first intron of the tripartite late leader; a synthetic sequence comprising a HindIII site, a splice acceptor site, the second and third exons of the adenovirus 2 tripartite leader and a multiple cloning site including a SmaI site; additional SV40 sequences containing early and late polyadenylation sites; adenovirus 2 sequences including the virus-associated RNA genes; and pBR322 elements for replication in E. coli.

The resulting EL-4 6.1 C10 cDNA library in pDC201 was used to transform E. coli strain DH5o, and recombinants were plated to provide approximately 350 colonies per plate and sufficient plates to provide approximately 25,000 total colonies per screen. Colonies were scraped from each plate, pooled, and plasmid DNA prepared from each pool. The pooled DNA was then used to transfect a sub-confluent layer of monkey COS-7 cells using DEAE-dextran followed by chloroquine treatment, as described by Luthman et al. (Nucleic Acids Res. 11:1295, 1983) and McCutchan et al. Natl. Cancer Inst. 41:351, 1986). The cells were then grown in culture for three days to permit transient expression of the inserted sequences. After three days, cell culture supernatants were discarded and the cell monolayers in each plate assayed for IL-1 binding as follows. Three ml of RPMI medium containing 3 x 1O-'oM 125I-IL-la was added to each plate and the plates incubated for 2 hours at 8 0 C. This medium was then discarded, and each plate was washed with 10 ml RPMI 1640 medium [containing no labeled IL-l1a]. The edges of each plate were then broken off, leaving a flat disk which was contacted with X-ray film for 72 hours at -70 0 C using an intensifying screen. IL-1 binding activity was visualized on the exposed films as a dark focus against a relatively uniform background.

After approximately 150,000 recombinants from the library had i 1 i WO 89/04838 PCT/US88/03926 33 been screened in this manner, one transfectant pool was observed to provide IL-1 binding foci which were clearly apparent against the background exposure.

A frozen stock of bacteria from the positive pool was then used to obtain plates of approximately 350 colonies. Replicas of these plates were made on nitrocellulose filters, and the plates were then scraped and plasmid DNA prepared and transfected as described above to identify a positive plate. Bacteria from individual colonies from the nitrocellulose replicas of this plate were grown in 2 ml cultures, which were used to obtain plasmid DNA, which was transfected into COS-7 cells as described above. In this manner, a single clone, clone 78, was isolated which was capable of inducing expression of IL-1R in COS cells. The insert was subcloned into a plasmid derived from pBR322 (GEMBL) and sequenced by conventional techniques. The sequence is set forth in Table 1.

Example Isolation of Human cDNA Clones Which Hybridize to Murine IL-1 Receptor Probe DNAs A cDNA polynucleotide probe was prepared from the 2356 base pair (bp) fragment of clone 78 (see Example 4) by nick-translation using DNA polymerase I. The method employed was substantially similar to that disclosed by Maniatis et al. (supra, p. 109).

A cDNA library was constructed by reverse transcription of polyadenylated mRNA isolated from total RNA extracted from the cultured cells of a human T-cell line designated clone 22, described by Acres et al. Immunol. 138:2132, 1987). These cells were cultured in RPMI 1640 medium plus 10% fetal bovine serum as described by Acres et al. (supra), in the presence of 10 ng/ml 0KT3 antibody and 10 ng/ml human IL-2. The cDNA was rendered double-stranded using DNA polymerase I, blunt-ended with T4 DNA polymerase, methylated with EcoRI methylase to protect EcoRI cleavage sites within the cDNA, and ligated to EcoRI linkers. The resulting constructs were digested with EcoRI to remove all but one copy of the linkers at each end of the cDNA, and ligated to EcoRI-cut and dephosphorylated arms of

F

1.

WO 89/04838 PCT/US88/03926.

34 bacteriophage Xgt1O (Huynh et al., DNA Cloning: A Practical Approach, Glover, ed., IRL Press, pp. 49-78). The ligated DNA was packaged into phage particles using a commercially available kit (Stratagene Cloning Systems, San Diego, CA, USA 92121) to generate a library of recombinants. Recombinants were plated on E. coli strain C600(hfl-) and screened by standard plaque hybridization techniques under conditions of moderate stringency (50 0 C, 6 x SSC).

Following several rounds of screening, nine clones were isolated from the library which hybridized to the cDNA probe. The clones were plaque purified and used to prepare bacteriophage DNA which was digested with EcoRI. The digests were electrophoresed on an agarose gel, blotted onto nylon filters, and retested for hybridization. The clones were digested with EcoRI followed by preparative agarose gel electrophoresis, then subcloned into an EcoRI-cut derivative (pGEMBL) of the standard cloning vector pBR322 containing a polylinker having a unique EcoRI site, a BamHl site and numerous other unique restriction sites. An exemplary vector of this type is described by Dente et al. (Nucleic Acids Research 11:1645, 1983).

Restriction mapping and sequencing of a 4.8 kb human IL-1R clone indicated that the clone included a sequence encoding 518 amino acids which exhibited 80% amino acid sequence identity to the corresponding murine sequence in the extracellular, or N-terminal region distal to the transmembrane region, 63% identity in the transmembrane region, and 87% identity in the cytoplasmic, or C-terminal region. In addition, several cysteine residues and most N-linked glycosylation sites between the mouse and human sequences were conserved. A 440 bp EcoRI-NsiI fragment derived from the portion of the human IL-1R clone was "P-labeled by nick-translation as described above and used to screen a cDNA library produced by randomly-priming clone 22 mRNA prepared as described above. 23 clones which hybridized to the probe were isolated and analyzed by restriction mapping. Sequencing of one of these clones provided the sequence information corresponding to the remaining N-terminal 34 amino acids of the human protein. The coding and deduced amino acid

I

WO 89/04838 PCT/US88/03926 sequence of the complete coding region of human IL-1R is shown in Tables 4A-4C.

Example 6 Expression of Recombinant IL-1 Receptor Using a High-Efficiency Mammalian Expression System The mammalian expression plasmid pDC201, depicted in Figure 2, is designed to express cDNA sequences inserted at its multiple cloning site (MCS) when transfected into mammalian cells. Referring now to Figure 2, pDC201 includes the following components: (hatched box) contains SV40 sequences from coordinates 5171-270 including the origin of replication, enhancer sequences and early and late promoters. The fragment is oriented so that the direction of transcription from the early promoter is as shown by the arrow.

Ad-MLP (open box) contains adenovirus-2 sequences from coordinates 5779-6231 including the major late promoter, the first exon and part of the intron between the first and second exons of the tripartite leader. TPL (stippled box) contains a synthetic DNA sequence specifying adenovirus-2 sequences 7056-7172, 9634-9693 (containing the acceptor splice site of the second exon of the tripartite leader, the second exon and part of the third exon of the tripartite leader) and a multiple cloning site (MCS) containing sites for KpnI, SmaI, and BglII. pA (hatched box) contains SV40 sequences from 4127-4100 and 2770-2533 that include the polyadenylation and termination signals for early transcription. VA (solid box) contains adenovirus-2 sequences from 10226-11555 that include the virus-associated RNA genes (VAI and VAII). The solid lines are derived from pBR322 and represent (starting after the pA sequences and proceeding clockwise) coordinates 29-23, 651-185 (at which point the VA sequences are inserted), 29-1, 4363-2486, and 1094-375. pDC201 is a derivative of pMLSV, previously described by Cosman et al., Molec. Immunol. 23:935 (1986).

To express recombinant IL-1 receptor, COS cells were grown and transfected as described by Cosman et al., supra, with the plasmid DNA from a 1.5 ml culture of E. coli transformed with pDC201 having an IL-1R cDNA insert (clone 78). After 72 hours of culture 'Wr_ i,_ WO 89/04838 PCT/US88/03926.

36 cells were harvested by washing once with 10 ml of PBS and then treating for 20 minutes at 37 0 C with an EDTA solution (sodium phosphate 0.05 M, sodium chloride 0.15 M, EDTA 0.005 M, pH 7.4) followed by scraping. For comparisons, COS cells were transfected with a pDC201 control vector containing no insert, and EL-4 6.1 cells and EL-4 M cells (an IL-1 receptor-negative variant of EL-4 cells) were grown and harvested as described by McDonald et al., J.

Immunol. 135:3964 (1985).

At saturating DNA concentrations, the transfected COS cell monolayer contained an average of 45,000 sites per cell. Since the parental COS cells expressed only about 500 receptors per cell, it can be calculated that more than 98X of all IL-1 receptors in the transfected population were recombinant. Flow cytometry using FITC-IL-la revealed that only 4.2% of the cells stained brightly; therefore, each of these transfected COS cells contained about 1.1 x 106 IL-1 binding sites.

The plasma membrane proteins of EL-4 6.1 C10 cells and of COS cells transfected with vector DNA con aining cDNA encoding the IL-1 receptor (clone 78) were labeled with 125I as described in Example 1, above. Cells were subsequently extracted with PBS containing 1% Triton X-100 and a cocktail of protease inhibitors (2 mM phenylmethyl sulphonyl fluoride, 1 mM pepstatin, 1 mM leupeptin, and 2 mM 0-phenanthroline). Detergent extracts were subjected to affinity chromatography as described in Example 1 on Affigel-1O (Biorad, Richmond, CA) to which recombinant human IL-la had been coupled.

12SI-labeled receptor was then eluted with sample buffer (0.0625 M Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol) and analyzed by SDS polyacrylamide gel electrophoresis on a 10X gel. Gels were then subjected to autoradiography. The recombinant IL-1 receptor purified by affinity chromatography on IL-la columns migrated with a relative mobility of about 80,000 on SDS polyacrylamide gels, comparable to the mobility displayed by IL-I receptor purified in the same manner from EL-4 6.1 C10 cells.

The DNA from clone 78, when transfected into COS cells, led to expression of IL-1 binding activity which was virtually identical SWO 89/04838 PCT/US88/03926 37 to that displayed by EL-4 6.1 C10 cells, as shown in Figures 3A-3C.

For binding assays, COS cells were resuspended at 1.7 x 106 cells/ml with EL-4 M (1.5 x 107 cells/ml) cells as carriers. EL-4 M and EL-4 6.1 C10 were resuspended at 1.5 x 107 cells/ml. All cell suspensions were made and binding assays done in RPMI 1640/10X BSA/0.1X sodium azide/20 mM HEPES pH 7.4. Binding incubations with 12 5 I-IL-x or 12"I-IL-1 and unlabeled IL-la and IL-10 were done as described elsewhere in the specification. 12 5 I-IL-la bound to the transfected COS cells with a K, of 3.0 0.2 x 109 M- 1 (Figure 3B).

The K, for the native receptor on EL-4 6.1 C10 cells was 4.3 3 x 109

M-

1 All of the binding was to recombinant Freptors (see Figure 3A); the parental COS cell population did not bind detectable 12 5 I-IL-la in this experiment.

In a cold competition experiment, free 12 5 I-IL-la concentration was 7.72 0.13 x 10-10 M. On the transfected COS cells the maximal binding was 2.98 0.3 x 104 molecules/cell (no inhibition) and the background (measured in the presence of 6 x 10 7

M

unlabeled IL-la) was 921 60 molecules/cell (100% inhibition). On the EL-4 6.1 C10 cells maximal binding was 1.33 0.02 x 104 molecules/cell and background (see above) was 47 2 molecules/cell.

Binding of 125 I-IL-lo, both to the transfected COS cells and to EL-4 6.1 C10 cells, could be competed completely by an excess of either unlabeled IL-la or unlabeled IL-1 (Figure 3C). The inhibition constants for IL-la and for IL-11 were very similar with each cell type (Figure 3C).

Example 7 Preparation of Monoclonal Antibodies to IL-1R Preparations of purified recombinant IL-1R, for example, human IL-1R, or transfected COS cells expressing high levels of IL-1R are employed to generate monoclonal antibodies against IL-1R using conventional techniques, for example, those disclosed in U. S. Patent 4,411,993. Such antibodies are likely to be useful in interfering Swith IL-1 binding to IL-1 receptors, for example, in ameliorating i i WO 89/04838 PCT/US88/03926 38 toxic or other undesired effects of IL-1.

To immunize mice, IL-1R immunogen is emulsified in complete Freund's adjuvant and injected in amounts ranging from 10-100 ug subcutaneously into Balb/c mice. Ten to twelve days later, the immunized animals are boosted with additional immunogen emulsified in incomplete Freund's adjuvant and periodically boosted thereafter on a weekly to biweekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay, ELISA (enzyme-linked immunosorbent assay), or inhibition of binding of 25 I-IL-la to extracts of EL-4 6.1 cells (as desribed above). Other assay procedures are also suitable.

Following detection of an appropriate antibody titer, positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and fused to the murine myeloma cell line NS1. Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a HAT selective medium (hypoxanthine, aminopterin, and thymidine) to inhibit proliferation of non-fused cells, nyeloma hybrids, and spleen cell hybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivity with IL-1R, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochemistry 8:871 (1971) and in U. S.

Patent 4,703,004. Positive clones are then injected into the peritoneal cavities of syngeneic Balb/c mice to produce ascites containing high concentrations mg/ml) of anti-IL-1R monoclonal antibody. The resulting monoclonal antibody can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography, and/or affinity chromatography based on binding of antibody to Protein A of Staphylococcus aureus.

Example 8 Expression of IL-1R in Yeast For expression of human or murine IL-1R in yeast, a yeast expression vector derived from pIXY120 is constructed as follows.

i, i

J

WO 89/04838 PCT/US88/03926 39 pIXY120 is identical to pYaouGM (ATCC 53157), except that it contains no cDNA insert and includes a polylinker/multiple cloning site with an NcoI site. This vector includes DNA sequences from the following sources: a large SphI (nucleotide 562) to EcoRI (nucleotide 4361) fragment excised from plasmid pBR322 (ATCC 37017), including the origin of replication and the ampicillin resistance marker for selection in E. coli; S. cerevisiae DNA including the TRP-1 marker, 2P origin of replication, ADH2 promoter; and DNA encoding an 85 amino acid signal peptide derived from the gene encoding the secreted peptide a-factor (See Kurjan et al., U.S. Patent 4,546,082).

An Asp718 restriction site was introduced at position 237 in the o-factor signal peptide to facilitate fusion to heterologous genes.

This was achieved by changing the thymidine residue at nucleotide 241 to a cytosine residue by oligonucleotide-directed in vitro mutagenesis as described by Craik, Biotechniques:12 (1985). A synthetic oligonucleotide containing multiple cloning sites and having the following sequence was inserted from the Asp718 site at amino acid 79 near the 3' end of the a-factor signal peptide to a Spel site in the 2p sequence: Asp718 StuI Ncol BamHI

GTACCTTTGGATAAAAGAGACTACAAGGACGACGATGACAAGAGGCCTCCATGGAT...

GAAACCTATTTTCTGATGTTCCTGCTGCTACTGTTCTCCGCAGGTACCTA...

[+----Polylinker-- SmaI Spel

CCCCCGGGACA

GGGGGCCCTGTGATC

Polylinker---41 pBC120 also varies from pYoHuGM by the presence of a 514 bp DNA fragment derived from the single-standed phage fl containing the origin of replication and intergenic region, which has been inserted at the Nrul site in the pBR322 sequence. The presence of an fl origin of replication permits generation of single-stranded DNA copies of the vector when transformed into appropriate strains of E. coli and superinfected with bacteriophage fl, which facilitates DNA sequencing of the vector and provides a basis for in vitro mutagenesis. To WO 89/04838 PCT/US88/03926 insert a cDNA, pIXY120 is digested with Asp718 which cleaves near the 3' end of the a-factor leader peptide (nucleotide 237) and, for example, Neol which cleaves in the polylinker. The large vector fragment is then purified and ligated to a DNA fragment encoding the protein to be expressed.

To create a secretion vector for expressing human IL-1R, a cDNA fragment including the complete open reading frame encoding hIL-1R is cleaved with an appropriate restriction endonuclease proximal to the N-terminus of the mature protein. An oligonucleotide or oligonucleotides are then synthesized which are capable of ligation to the 5' and 3' ends of the hIL-1R fragment, regenerating any codons deleted in isolating the fragment, and also providing cohesive termini for ligation to pIXY120 to provide a coding sequence located in frame with respect to an intact a-factor leader sequence.

The resulting expression vectors are then purified and employed to transform a diploid yeast strain of S. cerevisiae (XV2181) by standard techniques, such as those disclosed in EPA 0165654, selecting for tryptophan prototrophs. The resulting transformants are cultured for expression of an hIL-1R protein as a secreted or extracted product. Cultures to be assayed for hIL-1R expression are grown in 20-50 ml of YPD medium yeast extract, 2% peptone, 1X glucose) at 37 0 C to a cell density of 1-5 x 108 cells/ml. To separate cells from medium, cells are removed by centrifugation and the medium filtered through a 0.45 p cellulose acetate filter prior to assay.

Supernatants produced by the transformed yeast strain, or extracts prepared from disrupted yeast cells, are assayed for the presence of hIL-1R using binding assays as described above.

Example 9 Construction, Expression and Purification of Truncated Recombinant Murine IL-1 Receptor A truncated version of the IL-1 receptor protein was produced using an expression system compatible with the HELA-EBNA1 cell line, which constitutively expresses Epstein-Barr virus nuclear antigen driven from the CMV immediate-early enhancer promoter. The expression ,WO 89/04838 PCT/US88/03926 41 vector used was termed HAV-EO, a derivative of pDC201 which contains the Epstein-Barr virus origin and allows high level expression in the HELA-EBNA cell line. HAV-EO is derived from pDC201 by replacement of the adenovirus major late promoter with synthetic sequences from HIV-1 extending from the cap site of the viral mRNA, using the SV-40 early promoter to drive expression of the HIV-1 tat gene.

The eip.S~&i~n construct for the soluble truncated IL-1 receptor was generated in a series of steps. The entire coding region of the receptor and part of the 5' untranslated region were removed from the original IL-1 receptor clone 78 by digestion with Asp 718 and NdeI. This fragment, containing no 3' untranslated sequences, was cloned into HAV-EO, to generate HAV-EO-FL9. A variant of this plasmid, containing a translational stop codon immediately following the codon for proline 316 and lacking all the coding sequence 3' to this, was subsequently constructed by standard methods and termed

HAV-EO-MEXT.

HAV-EO-MEXT vector DNA was introduced into HELA-EBNA cells by a modified polybrene transfection as disclosed by Kawei and Nishizawa (Mol. Cell Biol. 4:1172, 1984). 1.5 x 106 cells were seeded into ml DMEM 10X FCS, in a 10 cm tissue culture dish. Cells were incubated at 37 0 C, 10X CO, for 16 hours. The media was then removed and 3 ml of serum-free DMEN containing 10 ug/ml DNA and 30 ug/ml polybrene (Sigma) were added. Dishes were then incubated at 37eC/10%

CO

2 for a further six hours, st which time the DNA mix was removed and ca-lls were glycerol shocked by addition of 3 ml serum-free DMEM glycerol for one minute. Glycerol was removed, and the cells were washed twice with medium. Ten ml of DMEM 10% FCS were then added, and the cells were incubated at 37*/10X CO for 18 hours.

Transfected cells were then removed with trypsin and split in a ratio of 1:9 into T175 cm 2 flasks (to give approximately confluence) containing 25 ml DMEM 1% FCS. Supernatants containing transiently expressed soluble murine IL-1 receptor were harvested every 24 hours for up to ten days.

IL-la binding activity in the medium was measured by inhibition of 25 II-Io to EL4 6.1 C10 cells as described by Mosley et I WO 89/04838 PCT/US88/03926 42 al. Biol. Chem. 262:2941, 1987) with the exception that labeled IL-la (2 x 10- 11 50 ul was first incubated with the test sample pl) for two hours at 8 0 C, prior to addition of cells (2.5 x 106 cells, ul). Each test sample was assayed at six dilutions (X3) and the inhibition dose response curve used to assess the relative inhibitory titer.

Soluble IL-1 receptor was purified from culture supernataits as'described for natural receptor by Urdal et al. Biol. Chem.

263:280, 1988). Culture supernatants were passed over a 1 ml bed volume IL-la column, the column vas washed with PBS and eluted with 0.1 M glycine-HCl. Acid eluate fractions were immediately neutralized ana subsequently tested for IL-1 binding activity using the radioreceptor inhibition assay. SDS-polyacrylamide gel electrophoresis of the material eluted by tLh acid treatment showed that it contained two bands of Mr 60,000 and 54,000. N-glycanase treatment of this material indicated that the size heterogeneity is due to differences in N-linked glycosylation between the two species.

Soluble IL-1 receptor retains full IL-1 binding activity.

i tWO 89/04838 PMVJS88/03926 43 TABLE 1: cDNA SEQUENCE OF IL-lR CLONE IN GEMBL78 1 51 101 151 201 251 301 351 401 451 501 551 601 651, 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 5' -TGGGTCGTCT

CCAGTAATCA

TGACTTGAGG

CAAACGTTGT

TGCCCCGA-4C,

GCTCATTTGT

CAGAATATCC

ATTCGCAAGT

TTGGTACAAG

GGATTCATCA

GACTCAGGAT

AACTAAAGTA

GCACACAGC

CTTGTGTGCC

CGAGGTCCAG

GCTTCTTCGG

CACAGAGGGG

ATATCCGGTC

GGGACAGACC

CCAGGATCAA

CCTTGTCTAC

TTCTAGCTGA

TACACACTCA

TCGCTATCCG

CGCATGTGCA

GGGGGCTTTA

TAAAGTCTTC

GTTTTCTTCC

CTTTATCCCA

TGTTTTAAA

TGTTCATTTA

ACTMATGAAA

AGATATGGGA

CCATATACAA

TTGGAGAAAA

TAAGCAGAAA

CACAGTCTGC

GCCCAACGGA

TGTGCGGGAC

GCTAGCATGG

ATAAGAGGCT

AACCTGTGCA

AACAGGACTT

TGCTGCTCCG

AAAGCGTCAG

GAGTCTCACA

GTCCAAGAAG

CCCCAC-31

GACTAGAAGT

TTTGGAGGCA

AGGCAGTTTT

CTCGGGGAGA

CGTGAACMAC

CTCATGGTGC

AAATCAGATC

GTCCTCTTAC

MATGACAGCA

GCAGAATGAA

ATTACTATTG

ACCGTAACTG

CACCTTCCCA

CTTATGTGAG

TGGTATAAGA

AGCAAAAGAT

ACTATATATG

ACACGAGTAA

TGTTA'ZCCTG

TGATACAACT

TGGAAGTGGA

AGACTATCAA

TTACAACACT

TTTATCTGTG

GTTAATATAC

TCATCCTCAC

AAGGTTGACA

TTCAAAAGCT

AGACCCTGGG

CTGTTGCCTG

TGGAAGGGAT

ATGTAAAGAA

GGCTTCAGCT

TGCTCTCATC

TCCAAGACTA

CACGGAGTCA

AAAGACCAGG

GATCACCATT

ACTAAGGAGA

CAAAAGTGGG

GCAGCTGGGC

GTCCCTTGTT

GGTGGCCAGG

GGATGM~GA

GAAATGGCCA

GTGGGATTGT

TGGCrCCCCA

GAGCTGTCTG

AAGCAAACTG

CGTTTTAACA

AATGTCGCTG

ACAAATGGAG

CTCTGCTGTC

GTTTTGTTTT

TCCMAATAAA

AGACCCCCAT

CATCTTTGGT

TATAGTAAGA

TGTTAGA(

3

AA

CAGCGGCTCC

TTATTTTAAA

ACTGTAAACC

AAACTGTTGG

CCGTATGTCC

TACAATTTAT

AGCCCTCGGA

GATCTGCAAC

ATGGATCAGA

TTTGTGGAAC

TAACATTTCA

TTGTTAAGAA

CCAGTCCCTG

GGCTACAATT

TAGTGCTTTG

TCAGATGGAA

AGAGGGGTCC

AGGTCTTGGA

GACTATGTTG

AAGCAGGAGG

GGCTGGGCCA

CAGGAAGGAA

TGAGAAAATG

TTTGCTGGTC

TTCTGGAAAA

GTCTAAACAC

AACTGCCGGC

CAGGCCAAGA

TGTGCCTCCC

CCAGATCACC

ACCGC1TCAGA

CTAACAGTCG

CATGTGTGGA

GGCTGTCTGA

AAGTATAAGT

TCATTCTTGT

TAAGTAATGC

GCCAGTGTTT

GATGTCATCA

AATATGAAAG

GCTGGAGATT

TATCTGTAAA

ATGCACGGCG

ATCAGCGGAC

TTGTACCTGC

ACCAACTT

TGACCCTGGC

ACATTGCCGG

GATGAAAATA

TCTGCTTCTT

TGAGGAATGT

TATACGTTCC

CACAATAGAT

ATGAGACGAT

GTCACGGGCC

AATTGAATG

ATCCTTCAAC

GAAGTTAAAA

CACAAATATT

ACTTCAAGAA

GTATGCTGTG

GTACAGGGAC

AGACATACGA

TTCTCAGACT

GGGACAGTTT

GAGAAGATAC

CTGATTATCA

GTCATCTGAA

TTAAAATCGT

CCAGATTCTA

AGGAGACTTT

ACTTAAGATA

CGCTTACTAA

AGCAACACAC

ACTTCGGAAT

&GTMAAACAG

TGGAACTGGA

GAGCCATGGT

AGGCAGTGAA

TGGTTTAATT

GGACACTTTG

GCGGGTGAGG

GCACGCCAGC

TGTCCTGGGC

ATTTGCTCAG

GAGTTCCCAG

TGCTACTGGG

GACGTATGTA

TGAAATTGAT

ACACCATAAT

CGGGACTCCA

CAAGGTGGAG

ACTGCCTCAA

TTGTGTTACA

GGATGGAAGT

ATGAGTTACC

GACAACGTGA

GGCTGAAGAG

GGGGGAAGCA

GAAAACAAGA

CGAAGCTGAC

AGTTCTCAGA

AATGATCCAT

CAAAAGAAAA

GCCAGTTTTA

TT'TGAGTCGG

TTACCTCATC

TGTGCATCTA

TCCTGCTCTG

TGCCTATATT

TAGATACTTT

GGATACAAGC

CATCGAGGTT

TTCTAGTGAG

GAGCAAATAG

CCTGCTTGAG

TTCAGTTCAT

CAAGAAAGAC

CCAGATGCCA

CCCrGGATCC

TTACCACTCG

ATCTCCCATC

TCACGAACCA

TTGGGAAGAG

TG~TCAGGGA

CTGGGTGTAG

AGATTCTGTG

GGGGGTCGCT

TTTACTGATA

y

I

I

WO 89/04838 PCT/US88/03926.

TABLE 2A: Sequence of Codihg Region of Hurine IL-1 Receptor Gene

-ATG

Met

COT

Pro

CAG

Gin

TOT

Cys

TAC

Tyr

AGO

Arg

GTG

Val

TAO

Tyr

CT

Pro

CAC

His

TTT

Phe

MAC

Asn

AMA

Lys

GAO

Asp 000 Pro GAG MAT Oiu Asn CTG OTO Leu Leu ATC OTT Ile Val COT OTT Pro Leu MOG MT Lys Asn ATT OAT Ile His GAG GAO Giu Asp TOO OTO Cys Leu 000 TTO Gly Leu ATT 000 Ile Ala AA OAT Lys Asp TOT AMA Cys Lys OAT A Asp Lys TAT ATA Tyr Ile GTC ACA Val Thr

ATO

Met

TOO

Ser

TTO

Leu

ACT

Thr

GAO

Asp

CAG

Gin

TOA

Ser

AMA

Lys

TOT

Cys Gly

GMA

Oiu

OCT

Pro

CTG

Leu

TOO

Cys

CGA

Arg

AAA

Lys

OTO

Leu

TTT

Phe

OCA

Pro

AGO

Ser

CAG

Gin

GGA

Gly

AOT

Thr

TAO

Tyr

OAT

Asp

MAT

Asn o'ro Leu

TTG

Leu

COT

Ar

GTA

Val

OTA

Leu

ATT

Ile

TOT

Ser

AMA

Lys

ACC

Thr

GMA

Giu

TAO

Tyr

OTA

Val

ACA

Thr

AGT

Ser

OTO

Leu

GAC

Asp

OTA

Val

ATO

Met

M.T

Asn

CAC

His

ATA

Ile

OTT

Leu

TOT

Cys

OTA

Val

CC

Ala

OTO

Vai

COO

Pro

MOC

Asn

GTO

Val

ACG

Thr

ATO

Ile

ATA

Ile

ACT

Thr

ACC

Thr

TOO

Cys

GAO

Glu

OTG

Vai

GOT

Ala TTc Phe

ACA

Thr

OTA

Val

OTO

Val

TTC

Phe

CT

Pro

GTC

Val

AGO

Ser

GMA

Giu

GAT

Asp

ACC

Thr

GAO

Asp

GTA

Val

AGA

Arg

TTA

Leu

OCA

Pro

TAT

Tyr

CAG

Gin

TTC

Phe

GAG

Giu

GG

Gly

OAT

Asp

OTC

Leu

TAT

Tyr Ile

ATA

Ile

CG

Arg

OOT

Pro

MAC

Asn

GAG

Oiu

CAG

Gin

OTG

Val

TOO

Trp

TTC

Phe 33 11 78 26 123 41 168 56 213 71 258 86 303 101 348 116 393 131 438 146 483 161 528 176 573 191 618 206

AGA

Arg

CMA

Gin

MAC

Asn WO 89/04838 PCJ7/US88/03926 TABLE 2B:,Sequence of Coding Region of Murine IL-i Receptor Gene AGG GAC AGA CCT GTT ATC CTG AGC CCT CGG MAT GAG ACG ATC GMA 663 Arg Asp Arg Pro Val Ile Leu Ser Pro Arg Asn Giu Thr Ile Glu 221 GCT GAC CCA GGA TCA ATG ATA CAA GIG ATC TGC MAC GTC ACG GGC 708 Ala Asp Pro Gly Ser Met Ile Gin Leu Ile Cys Asn Val Thr Gly 236 GAG TIC TCA GAC CTT GTC TAC TGG MAG TGG MAT GGA TCA GMA ATT 753 Gin Phe Ser Asp Leu Val Tyr Trp Lys Trp Asn Gly Scr Glu le 251 GMA TGG MAT GAT CCA TTT CTA GCT GMA GAC TAT CMA TTT GTG GMA 798 Giu Trp Asn Asp Pro Phe Leu Ala Giu Asp Tyr Gin Phe Val Giu 266 CAT CCT TCA ACC AAA AGA AAA TAC ACA CTC ATT ACA ACA CIT MAC 843 His Pro Ser Thr Lys Arg Lys Tyr Thr Leu Ile Thr Thr Leu Asn 281 ATI TCA GMA GTT MAA AGC CAG TIT TAT CGC TAT CCG TTT ATC TGT 888 le Ser Giu Val Lys Ser Gin Phe Tyr Arg Tyr Pro Phe Ile Cys 296 GTI GTT MAG MAC ACA MAT All ITT GAG TCG GCG CAT GTG CAG TTA 933 Val Val Lys Asn Thr Asn le Phe Giu Scr Ala His Val Gin Leu 311 ATA TAG CCA GTC CCI GAG TIC MAG MT TAC GIG ATC GGG GGC III 978 le Tyr Pro Val Pro Asp Phe Lys Asn Tyr Leu le Giy Gly Phe 326 ATC ATC CIC ACG GCT ACA ATT GTA TGC TGT GTG TGC ATO TAT AAA 1023 le le Leu Thr Ala Thr le Vai Cys Cys Val Cys le Tyr Lys 341 GTC TIC MAG GTT GAC ATA GTG CIT TGG TAC AGG GAG TCC TGC TCT 1068 Val Phe Lys Val Asp Ile Val Leu Trp Tyr Arg Asp Ser Cys Ser 356 GGT TTT CIT CCI ICA AMA GCT ICA GAT GGA MAG ACA T.C GAT GCC 1113 Giy Phe Leu Pro Ser Lys Ala Ser Asp Gly Lys Thr Tyr Asp Aia 371 TAT ATT CIT TAT CCC MAG ACC CIG GGA GAG GGG TCC TIC TCA GAC. 1158 Tyr Ile Leu Tyr Pro Lys Thr Leu Gly Giu Gly Ser Phe Ser Asp 386 TTA GAT ACT TTI GTT TIT AMA CTG TTG CCT GAG GTC TTG GAG GGA 1203 Leu Asp Thr Phe Val. Phe Lys Leu Leu Pro Giu Val Leu Giu Gly 401 CAG TTT GGA TAC MAG GIG TIC All TAT GGA AGG GAT GAC TAT GTT 1248 Gin Phe Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val 416 GGA GMA GAT ACC AIC GAG GTl ACT MAT GAA MT GTA MAG MAA AGC 1293 Gly Giu Asp Thr le Giu Val Thr Asn Giu Asn Val Lys Lys Ser 431 ft WO 89/04838 PCT/US88/03926t TABLE 2C: Seauence of Codinff Reffion of Murine IL-1 ReceDtor Gene

AGO

Arg

TGG

Trp

CTC

Leu

ATC

le

CAG

Gin

CCA

Pro

ATG

Met

ACC

Thr

ACA

Thr

ATC

le

TCA

Ser

OGA

Gly

GAG

Giu

GTC

Val

MAG

Lys

CGG

Arg

GTG

Val

CTC

Leu Ile

TCT

Ser

ATT

le

AAA

Lys

ATT

Ile

ACC

Thr

AGA

Arg

CGG

Arg

GC

Gly CTA GTG Leu Vai GMA GAG Giu Giu AMA ATC Lys le ATO CCA Met Pro TGC TG Cys Trp AGG TTC Arg Phe TCA CCA Ser Pro GAC ACT Asp Thr TAG-3' End

AGA

Arg

CMA

Gin

GTC

Vai

GAT

Asp

TCA

Ser

TOO

Trp

TTG

Leu

MAG

Lys

GGA

Gly

ATA

le

GAG

Giu

CAG

Gin

TTT

Phe

TTA

Leu

CAC

His

CTG

Leu

GC

Gly

TAC

Tyr

TTG

Leu

TTC

Phe

CMA

Gin

AGA

Arg

COC

Arg

CO

Pro

TTC

Phe

MAT

Asn

GAG

Giu

ATT

le

GMA

Oiu

TAC

Tyr

TTA

Leu

GCA

Ala

AGC

Ser

OCT

Aia

A

Lys

MAG

Lys

AGA

Arg

CAG

Gin

CTA

Leu

GCA

Ala 1338 446 1383 461 1428 476 1473 491 1518 506 1563 521 1608 536 1653 551 1671 557 WO 89/04938 WO 8904838PCT/US88/ 03926 TABLE 3: eDN SEQUENCE OF HUMAN IL-iR CONSTRUCT 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 5' -AGACGCACCC

TTGGTAAAAG

ACTTATTTGT

GCAAGGMACG

GATGTTCGTC

TTGGTATAAA

GGATTCATCA

GATTCAGGAC

AATTtAAAATA

ATGCACAAGC

CTTGTGTGCC

TAAATTACAG

ACTTTAGTCG

CATAGAGGGA

ATATCCTATT

CCACAAGGCC

TTGGGATCCC

CATTGCTTAC

TGCTAGGGGA

AGTACCCTCA

TAAACATCCA

CATATATCCA

GGTATATGTG

TAAAATCTTC

ATTTTCTCCC

CTGTATCCAA

TGTGTTTAAA

TGTTCAT1TTA

ATTAMTGAAA

AGAAACATCA

CCATGTATAA

CTGGAGAAAA

TAAGCAGAAA

CACAGTCTGC

GTCCAGCGAC

TAAGGAGAAA

AAGTTGCCAA

AGGTTATGCC

TCCTTTATCC

TGACGTCAAT

CTTTAAAAA

GCACTTGG

ACCAGCCCAG

TGAGCTAGGC

GAGGCAGGAG

GTTTGGGCCA

AAAAAGGGCA

CATGGAGACA

TACAGATGGC

TTGTCTGGCG

GGTGCTGCAG

TTCTTGGAGA

TCTGAAGATG

ACAAGGCCTT

TTCATAGCTC

TGAAGAAAAA

CCTGTCCTCT

GATGACAGCA

ACACAAAGAG

ATTACTATTG

AGTGCAAAAT

CATATTTAAG

CTTATATGGA

TGGTATAAGG

AGTCAAAGAT

ACTATACTTG

ACCCGGGTAA

TGTGATTGTG

AGATACAATT

TGGAAGTGGA

AGACTATTAC

TCACAGTGCT

TTTACCTGTT

GTTAATATAT

TCACGTTGAC

AAGATTGACA

AATAAAAGCT

AGACTGTTGG

GTCTTGCCTG

TGGAAGGGAT

ACGTAAAGAA

GGCrTCAGC

TGCTCTTGTT

TCCAAG~aCTA

CATGGGGCTA

AAAGACAAGG

GGTCACCTTC

CrGCAAAGAG

GAGTTCTTTA

TCATGCTGAC

CTGAGGTCAC

AGCAGCCCAG

GGCAGTAGGC

AGGCTGAAGT

CCAACATGGC

ATGGTGGCAC

AATTGCTTGA

CTGCACTCTA

ATAAATGCCC

GCGAACTAGA

TTAGTTAAGT

ACCCTGTAGA

GCAAAGTGAG

ACTTTCCATC

GTGGACTCCC

CTCCAAGAAG

TACTGATTTC

ATAATTTTAG

TAACCCAAAT

AGACACCTGT

AAACTTTGGT

CGTGGTAAGA

TTGTGGAGAA

CAGAAACTAC

GTTTTTAAA

ATTGCAAACC

AGGCTCATCG

TCATGCATCC

TAGAATTTAT

AGCCCAGCTA

GATCTGTAAT

ATGGGTCAGT

AGTGTGGAAA

TAATATATCG

TTGCCAAGAA

CCAGTCACTA

AGTCATAATT

TTGTGCTTTG

TCAGATGGAA

GGAAGGGTCT

AGGTCTTGGA

GACTACGTTG

AAGCAGAAGA

GGCTGGGTGG

CAGGATGGAA

TGAGAAAATG

TCCGCTGGTC

TTCTGGAAGA

ATCTAAACAC

AGGCTCACGT

GGTGCCTCCT

TTGCAGAGTT

CTGGAATCAG

GGCACTTCAG

CCGGTGTGGT

GGGTGGATCA

AAAACCCCAT

ACGCCTGTAA

ACCGGGGAGA

GCCTGGCAAC

TCTCTGAATG

AGAAAGGGCA

CATCCACAGC

GTCACTGACC

ACTGACACCT

TGCTTGTATT

TCCTGAGAAG

AATATGAAAG

TTCTCTGGAG

TGTCATCTGC

GAACACAAAG

ATCTACAGAA

TTGTTCCTGC

AATTCATCTT

TGAGCCTAAC

CCGTTGCAGG

AATGAAAATA

TCTACTTCTT

TGATGAATGT

TACACATACT

TACTCTAGAG

ATGAGACAAT

GTCACCGGCC

AATTGATGAA

ATCCTGCAAA

GAAATTGAAA

TACACATGGT

ATTTCCAGAA

GTGTGTTCTG

GTACAGGGAT

AGACCTATGA

ACCTCTGACT

AAAACAGTGT

GGGAAGACAT

CTGATTATCA

TTCATCTGAA

TTAAAGTTGT

CCAGAATCGA

AGGGGACTT

ATGTCAGGTA

CAGTTACrGT GCCTCrCGGG

GTCTTATGGC

CATGGAATGT

ATTATTAAGG

AGTAGAGGGC

GGCTCACGCC

CCAGAGGTCA

CTCTACTAAA

TCCCAGCTAC

CGGAGGTTGC

AGAGCAAGAC

TTTGAACTGC

AGAACCAAAT

CCAAGGGCGG

CTGGAGCGGC

CACrGAGGAA

TTCCATACAC

CTGGGACCCC

TGTTACTCAG

GCTGATAAAT

AAATGAAATT

GCACTATAAC

CAAGCCTCCA

TAAGGTGGAG

ACTGCCTCAG

TTATGTTATA

AGACGGAGGA

ATGAGTTACC

GACAATATAC

GGCTGAAAAG

TGGGCAAGCA

GAAAACAAAC

GGAAGTAGAC

AGTTGAGTGA

GATGACCCAG

CAAAAGAAGG

GTAGATTTTA

ATAGATGCAG

GCACATGATT

TTTCATCTA

TCCTGCTATG

CGCATATATA

GTGATATTTT

GGATATAAGC

TGTTGAGGTC

TTTAGTCAG

GAGCAAATAG

CCTGCTTGAG

TTAAATTCAT

ACACAGGGAC

CCACATGCCA

CACCAGCCAC

TAGCATGGAG

GTTGCAGGCC

AACrATATCA

GAATAAGCCA

TTGGGAAGAT

TATAATCCCA

GGAGTTCGAG

AATACAAAAA

ACCTGAGGCT

AGTGAGCCGA

TCCGTCTCAA

CAAGAAAAGG

AGCCACCGTC

CGGCTATGCC

TCTCCTGAGA

GGGAGACATA

ATCCCCAGCC-31 WO 89/04838 WO 8904838PCr/US88/03926 TABLE 4A~ Senuen~e of Coding Region of Human IL-1 Receptor Gene TABLE 4A: Seauence ATG AAA GIG TTA C Met

TCT

Ser

ATT

Ile

CT

Leu

GAC

Asp

CMA

Gin

TCA

Ser

AGA

Arg

TGT

Cys

GGA

Gly

GMA

Glu

CCT

Pro

CTC

Leu

TGT

Cys

CGG

Arg

CCI

Pro Lys Val TCT CIG Ser Leu TTA GIG Leu Val MAC CCA Asn Pro AGC MAG Ser Lys CAC AMA His Lys GGA CAT Gly His ATl AMA Ile Lys TAT MAT Tyr Asn GAC GGA Asp Gly MAT MAT Asn Asn CIA CT Leu Leu ATC GIG le Val CAT GCA His Ala GTA ATA Val Ile GIG AT Val Ile Leu

GAG

Giu

TCA

Ser

MAT

Asn

ACA

Ihr

GAG

Giu

TAC

Tyr

ATA

Ile

GCA

Ala

GGA

Gly Leu

GCT

Ala

ICT

Ser

GMA

Giu

CCT

Pro

A

Lys

TAT

Tyr

AGT

Ser

CMA

Gin

CT

Leu

AGA

Arg

GAT

Asp

OCA

Ala

CAC

His

GTA

Val

CII

Leu

TGC

Cys

GCA

Ala

GCC

Ala

GIG

Val

CCI

Pro

MAT

Asn

GIG

Val

ACA

Ihr All le

CCA

Pro

CII

Leu

AAA

Lys

MIT

Asn

AMA

Lys

ICI

Ser

TGG

Trp

GIG

Val

AMA

Lys

ATA

le

TGC

Cys

AAA

Lys AlA Ile

GCI

Ala

TAC

Tyr

ACT

Ihr

GCI

Ala

ATT

Ile

IGC

Cys

GMA

Giu

GGC

Gly

ACA

Ihr

III

Phe

GTA

Val

III

Phe Ill Phe

CCI

Pro

ITA

Leu

CAC

His

GMA

Glu

TTG

Leu

CIA

Leu

GMA

Giu

GTT

Val

AGA

Arg

GIG

Val

MAG

Lys

TAT

Tyr

CAG

Gin

TT

Phe

MAG

Lys

GGC

Gly

GAG

Giu

CAA

Gin

CCT

Pro

MAT

Asn

GAG

Giu

CAG

Gin

AIG

Met

TGG

Trp

AGT

Ser

CAT

His

MAG

Lys

GMA

Glu AlA Ile

CGT

Arg

GTT

Val

ACI

Ihr

GCC

Ala

GCI

Ala

ICA

Ser

MIT

Asn

A

Lys

GAG

Giu

TAT

Tyr

GGA

Gly

AGA

Arg

CMA

cGIn

MAC

Asn

ATG

Met

CIA

Leu

GMA

Giu ICC AGG Ser Arg MAG GIG Lys Val ICI TAC Ser Tyr GAG CCI Giu Pro CIA CCC Leu Pro Ill "IT Phe Phe MAG GAT Lys Asp GIC AMA Val Lys GGG MAC Gly Asn TAT CCI Tyr Pro AAA CCC Lys Pro GAA GIA Giu Val ICT CCI Cys Pro MAA GAT Lys Asp All CAT le His GAG GAT Giu Asp IGO CIC Cys Leu MAC ITA Asn Leu GTl GCA Val Ala AA MI Lys Asn TGC AMA Cys Lys GAl AGG Asp Arg TAT ACT Tyr Ihr AT? ACC le Thr ACA AGG Thr Arg GAC TIG Asp Leu

CIG

Leu

AMA

Lys AlT Ile

ATA

le MAT GAG ACA Asn Giu Ihr I WO 89/04838 PCr/US88/03926 TABLE 4B: Sequence of Coding Region of Human IL-1 Receptor Gene

GGA

Gly

GAC

As p

GAC

Asp

AAC

Asn

ATT

le

MAT

Asn

GTC

Val

ACA

Thr TCC CAG ATA CMA TTG ATC TGT MAT GTC ACC GGC CAG TTG AGT Gin

GCT

Ala

GTG

Val

AGA

Arg

AGT

Ser

CAT

His

MAT

Asn

ATA

le le Gin TAC TGG Tyr Trp CTA GGG Lou Gly AGG AGT Arg Ser AGA TTT Arg Phe GGT ATA Giy Ile TTC CAG Phe Gin ATT GTG le Val Leu Ile MAG TGG Lys Trp GMA GAC Giu Asp ACC CTC Thr Leu TAT AAA Tyr Lys GAT GCA Asp Aia MAG CAC Lys His TGT TCT Cys Ser Cys

MAT

Asn

TAT

Tyr

ATC

le

CAT

His

GCA

Ala

ATG

Met Asn

GGG

Giy

TAC

Tyr

ACA

Thr

CCA

Pro

TAT

Tyr

ATT

le Val

TCA

Ser

AGT

Ser

GTG

Val

TTT

Phe

ATC

le

GGT

Giv Gly

ATT

le

GMA

Glu

MAT

Asn

TGT

Cys

TTA

Lou

TGT

Cvs Gin

GAT

Asp

MAT

Asn

ATA

le

TTT

Phe

ATA

Ile

GTC

Val Lou

GMA

Glu

CCT

Pro

TCG

Ser

CC

Aia

TAT

Tyr

ACC

Thr ATT GAC ATT GTG CTT TGC TAC Ile Asp Ile Val Lou Trp Tyr CCA ATA AAA GCT TCA GAT CCA Pro le Lys Ala Ser Asp Giy TAT CCA MAG ACT GTT GGG GMA Tyr Pro Lys Thr Val Gly Giu TTT GTG TTT AMA GTC TTG CCT Phe Vai Phe Lys Val Leu Pro TAT MAG CTG TTC ATT TAT GGA Tyr Lys Lou Phe Ile Tyr Gly ATT GTT GAG GTC ATT MAT GMA Ile Val Giu Val Ile Asn Giu ATT ATC ATT TTA GTC AGA GMA Ile Ile Ile Lou Val Arg Ciu GGT TCA TCT GMA GAG CMA ATA Giy Ser Ser Ciu Giu Gin le CAT GGA Afl AMA CTT GTC CTC Asp Cly Ile Lys Val Val Leu GTT TTC ATC TAT AMA Val Phe Ile Tyr Lys AGG GAT TCC TOC TAT Arg Asp Ser Cys Tyr MAG ACC TAT GAC GCA Lys Thr Tyr Asp Ala GGG TCT ACC TCT GAC Gly Ser Thr SerA~p GAG GTC TIC GMA AMA Ciu Vai Leu Giu Lys AGG CAT GAC TAC CT Arg Asp Asp Tyr Val MAC GIA MAG AMA AGC Asn Val Lys Lys Ser ACA TCA GGC TTC AC Thr Ser Giy Pho. Ser CC ATG TAT MAT C Ala Met Tyr Asn Ala CTT GAG CTG GAG AMA Lou Giu Leu Giu Lys

MAG

Lys

C

Lou

CTG

Lou

ATT

le

GA

Gly

GAC

Asp

CTG

Lou

CCI

Gly

CAG

Gin

GAC

Asp 804 268 849 283 894 298 939 313 984 328 1029 343 1074 358 1I19 373 1164 388 1209 403 1254 418 1299 433 1344 448 1389 463 1434 478 ATC, CMA le Gin WO 89/04838 WO 9/0838PCT/US88/03926 of Codinff Remion of Human IL-1 ReceDtor Gene TABLE 4C: Seauence

TAT

Tyr

GGG

Gly

GCA

Ala

CAG

Gin

ACT

Thr

ATG

Met

CGC

Arg

AGG

Arg

TCA

Ser

AAA

Lys

CAG

Gin

CCA

Pro

ATG

Met

TCA

Ser

CTC

Leu

A

Lys

CAG

Gin

CCA

Pro

CCA

Pro

GGG

Gly 1479 493 1524 508 1569 523 1614 538 1656 552 k- l__L- 0 00 Table 5: Comparison of Human and Nurine IL-1 Receptor Amino Acid Sequences h NKVLLRLICFIA-LLISSLEADKCKEREEKIILVSSANEIDVRPCPLNPNE-HKG-TITYKDDSKTPVSTEASRIHQHKEKLFVPAK lii! II I 1111 I 11111111 I1 1111111 il1I111I I Iliii mn MKVLLGLICLNtVPLL--SLEIDVTEYPNIVLFLSVNEIDIRKCPLTPNKH-GDTIIYKNDSKTPISADRDSRIHQNEHLFVPAK h VEDSGHYYCVVRNSSYCLRIKISAKFVENEPNLCYNAQAIFKI(LPVAGDGGLVCPYMEFFKNENNELPKLQWYKDCKPLLLDN4IHFSGV 11111 III Jill11 II 1 11 1111 1iii 1 l11 ii1 11111 11 111111 liii 11111111 1I1I m VEDSGYYYCIVRNSTYCLKTKVTVTVLENDPGLCYSTOATFPQRLRIAGDGSLVCPYVSYFKDENNELPEVOWKCKPLLLDNVSFFGV h KDRLIVMNVAEKHRGNTCJ1ASYTYLGKYPITRVIEFITLEENKPTRPVIVSPANETMEVDLGSOIOLICNTQ--!DAYVKV4GSVI 1111 1 1 I I ,IIIIII 111111 III III liii 1I 111 111 111I11 11l~l~ II 111 l~ll m KDKLLVRNVAEEHRGDYICRMSYTFRGKYPVTRVIFITIDENKRDRPVILSPRHETIEADPGSNILICNVTGQFSDLVYVNGSEI h DEDDPVLGEDYYSVENPANKRRSTLITVLNISEIESRFYKJIPFTCFAKNTHGIDAAYIQLIYPV'rNFOKHIGICVTLTVIIVCSVFIYK 1I 1 111 1I 1 11 1111 11111 1 11 II 1 111 1 111111 1 11 11 111 1 111 iEINDPFLAEDYQFVEHPSTKRKYTLITTLNISEVKSQFYRYPFICVVKNTNIFESAHVQLIYPVPDFKNYLIGGFIILTATIVCCVCIYK At* h IFKIDIVLYRDSCYDFLPIKASDGKTYDAYILYPKTVGEGSTSDCDIFVFKVLPEVLEKCGYKLFIYGRDDYVGEDIVEVINENVKKS II Ilt1111l1l 111I~fI~IIII 1111 11 1111111 H1 I~IIIIII 11 1111111 mn VFKVDIVLVYRDSC3GFLPSKASDGKTYDAYILYPKTLGEGSFSDLDTFVFKLLPEVLEQFGYKLFIYRDDYVEDTIEVNENVKKS h RRLIIILVRETSGFSLGGSSEEIAMYNALVQDGIKV'JLLELEKIQDYEKNPESIKFIKKH AKTRFVKNVRYH mn RRLIIILVRDGGFSVLGQSSEEOIAIYNALIEGIKIVLLELEKIDYEKPDSIFIKOKGVICVSGDFERPQSAKTRFKLRY h NPVQRRSPSSKHQLLSPA----tKEKLREHVPLG 0 00 m HPAQRRSPLSKHRLLTLDPVRDTKEI(LPAATHLPLG

L

Claims (1)

1. 52- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An isolated DNA sequence encoding a mammalian Type I Interleukin-1 receptor (Type I IL-1R) protein, Type I IL-1R analog protein, or soluble truncated Type I IL-1R protein which exhibits IL-1 binding activity. 2. A DNA sequence according to claim 1, selected from the group consisting of: cDNA clones having the nucleotide sequence of contiguous coding regions of a native mammalian IL-1R gene; DNA sequences capable of hybridisation to the clones of under moderately stringent conditions and which encode biologically active IL-1R molecules; and DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in and and which encode biologically active IL-1R molecules. 3. A DNA sequence according to claim 2, consisting essentially of a synthetic gene encoding a mammalian IL-1R which is capable of being expressed in a recombinant transcriptional unit comprising inducible regulatory elements derived from a microbial or viral operon. 4. A DNA sequence according to claim 2, which encodes a murine IL-1R, murine IL-1R analog protein, or soluble truncated murine IL-1R protein which 25 exhibits IL-1 binding activity. A DNA sequence according to claim 2, which encodes a human IL-1R, human IL-1R analog protein, or soluble truncated human IL-1R protein which exhibits IL-1 binding activity. 6. A recombinant expression vector comprising a DNA sequence according to any one of claims 1-6. 9204isnigbdaL009.29015*89JeS2 vy" *'pf MImbfrtSBJct IL -53- 7. A process for preparing a mammalian Type I IL-1R, human Type I IL-1R analog protein, or solub'-: truncated human Type I IL-1R protein which exhibits IL-1 binding activity, comprising culturing a suitable host cell comprising a vector according to claim 6 under conditions promoting expression. 8. A population of eukaryotic cells which express more than 30,000 surface type I IL-1 receptors per cell. 9. A population of eukaryotic cells according to claim 8, which express more than 45,000 surface type I IL-1 receptors per cell. A homogenous biologically active mammalian type I IL-1 receptor protein. 11. A homogenous protein according to claim 10 consisting essentially of a mammalian type I IL-1R, mammalian type I IL-1R analog protein, or soluble truncated mammalian type I IL-1R protein which exhibits IL-1 binding activity. 12. A homogenous protein according to claim 11, consisting essentally of a murine type I IL-1R, murine type I IL-1R analog protein, or soluble truncated murine type I IL-1R protein which exhibits IL-1 binding activity. i 13. A homogenous protein according to claim 11, consisting essentially of a human type I IL-1R, human type I IL-1R analog protein, or soluble truncated 25 human type I IL-1R protein which exhibits IL-1 binding activity. 14. A protein comprising a soluble truncated murine type I IL-1R protein produced by recombinant cell culture which exhibits IL-1 binding activity and comprises an amino acid sequence which is substantially identical to the extracellular domain of the amino acid sequence of residues 1-316, 1-317, 1- 318 or 1-319 in Tables 3A-3C. 9215ImgbdaL009.29015-89.et,53 -L -54- A protein comprising a soluble truncated human IL-1R protein procuced by recombinant cell culture which exhibits IL-1 binding activity and comprises an amino acid sequence which is substantially identical to the extracellular domain of the amino acid sequence of residues 1-316 or 1-319 in Tables 16. A method for regulating immune or inflammatory responses in a mammal comprising administering to said mammal a regulating effective amount of an IL-1 receptor according to any one of claims 10 to 17. The method according to claim 16 wherein the IL-1 receptor is human IL-1 receptor and the mammal to be treated is a human. 18. A pharmaceutical composition for regulating immune or inflammatory 15 responses in mammals, comprising an effective amount of a human type I IL-I receptor or biologically active human type I IL-1 receptor analog or subunit in admixture with a suitable diluent or carrier. 19. The pharmaceutical composition according to claim 18 in a form for parental administration. 20, A process for purifying a mammalian type I IL-1 receptor, comprising: applying a sample comprising mammalian type I IL-1 receptor to an affinity matrix comprising a type I IL-1 or antibody having specificity 25 to the type I IL-1 receptor molecule bound to an insoluble support; and eluting the type I IL-1 receptor from the affinity matrix. 21. The process according to claim 20 wherein the antibody is a monoclonal antibody. "9205.n. 009 -89.iet 9" t *SinibdlOl390Sl589.cti4 P i 22. A process according to claim 20 or 21, further comprising steps of: applying the partially purified murine type I IL-1 receptor to a lectin affinity column obtained following step of claim eluting the murine type I IL-1 receptor from the lectin column; and treating the partially purified murine type I IL-1 receptor by reversed phase high performance liquid chromatography and eluting therefrom murine type I IL-1 receptor as a single peak of absorbance at 280 nanometres which, when analysed by SDS-PAGE and silver staining, appears as a single band. 23. A process according to claim 20 or 21, wherein the IL-1 molecule is recombinant human type I IL- a. 15 24. A method for detecting IL-1 or IL-1 receptor molecules or the interaction thereof, comprising contacting a known quantity of type I IL-1 receptor according to any one of claims 10 to 15 with IL-1 for a time and under conditions sufficient for said type I IL-1 receptor to bind to IL-1 and then measuring the quantity of unbound IL-1 or type I 11-1 receptor. 25. Antibodies immunoreactive with type I IL-1 receptor. DATED this 15th day of April, 1992. IMMUNEX CORPORATION By Its Patent Attorneys DAVIES COLLISON 92G415.gbdaLOO9,M915-Z9Je55