DK175810B1 - DNA coding for mammalian interleukin-1 receptor - used for obtaining protein and antibodies for diagnosis and therapy involving immune or inflammatory activities - Google Patents

Abstract

The following are claimed (a) a DNA sequence comprising a single open reading frame nucleotide sequence encoding mammalian interleukin-1 receptor (Ih-1R) or a subunit of it, (b) a recombinant expression vector comprising the DNA sequence, (c) a population of eukaryotic cells which express more than 30,000 surface Ih-1 receptors per cell, (d) a homogeneous biologically active mammalian IL-1R compsn., (e) antibodies immunoreactive with IL-1R.

Description

DK 175810 B1

The present invention relates to interleukin-1 receptor (IL-1R).

Interleukin-1a and interleukin-1 β (IL-1 no. On II -ΐβ) are distantly hesleanterle polypeptide hormones that play a central role in the regulation of immune and inflammatory responses. These two proteins were originally both classified as IL-1 5 on the basis of a common lymphocyte activation factor (LAF) activity and a common main cell source, activated macrophages. The pooled data from studies using purified natural and recombinant IL-1 molecules have made it clear that IL-1a and IL-1 β each mediate most, if not all, of the broad spectrum of activities. previously attributed to IL-1. The basis for this almost identical spectrum of biological activities is thought to be a single class of plasma membrane IL-1 receptors that bind both IL-1 α and IL-1 β.

A few preliminary reports have previously been published regarding the existence of an IL-1 plasma membrane receptor. Heretofore, structural characterization of the interleukin-1 receptor has been limited to determinations 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 125 I-IL-1 molecules, and by immunoprecipitation of labeled surface proteins.

Dover et al. (j. Exp. Med. 162: 501,1985) and Dover et al. (Proc. Natl. A-cad. Sci. USA 83: 1060,1986) describes chemical crosslinking studies showing 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 that bound 1251-labeled human interleukin-1 β. Kilian et al. (J. Immunol. 136: 4509, 1986) reported that murine 125 I-IL-1α, which binds to murine thymoma cells, could be blocked with human IL-1α and IL-β. Dover et al. (Nature 324: 266,1986) rappor-! 25 competitive binding studies showing that IL-1 α and IL-1 β bound to the same cell surface receptors on LBRM-33-1 A5 cells, human dermal fibroblasts, murine BALB-3T3 cells, and ARH77, a human B lymphoblastoid cell line. The receptors in the different cell lines exhibited similar but not identical binding characteristics. The IL-1 receptors on porcine synovial fibroblasts (Bird 30 et al., Nature 324: 263,1986) and human dermal fibroblasts (Chin et al., J. Exp. Med.

165: 70,1987) gives a major species in the size range Mr 97,000-100,000 when cross-linked with the label IL-7, suggesting that a protein with Mr 80,000 was responsible for binding IL-1.1 as opposed to IL-1 receptors , which was characterized in this way on human B cells (Matsushima et al., J. Immunol.

136: 4496, 1986), an apparent molecular weight of 60,000.

I DK 175810 B1 I

I I

In Bron and MacDonald, FEBS Letters 219: 365 (1987) describe immune I

precipitation of murine IL-1 receptor from surface-labeled EL-4 cells during use

In view of a polyclonal rabbit antiserum directed against IL-1. This work shows that the murine

In receptor, a glycoprotein with an apparent molecular weight of approx. 82,000 daltons. IN

I 5 The radiolabelled IL-1 has been used in chemical crosslinking sub- I

In searches and for the detection of receptor in detergent extracts of cells. The results of I

these experiments above suggest that a protein with Mr 60,000 or 80,000 I

You are responsible for the binding of IL-1. The cross-linking of radiolabeled IL-1 to cells also has

I led to the occasional detection of proteins that differ from the main species with I

In 10 Mr 80,000, which suggests that the IL-1 binding molecule may be present in membranes.

as part of a multi-subunit receptor complex. IN

I To study the structure and biological properties of IL-1 re- I

I receptors and the role that IL-1 receptors play in the responses of different cell populations

I ions to IL-1 stimulation, or the effective use of IL-1 receptors for tea- I

15 rapi, diagnosis or analysis, homogeneous IL-1 receptor composition is required

ger. Such compositions are theoretically available via purification of solubilization- I

In prepared receptors expressed by cultured cells or by cloning and expression of I

In genes that encode the receptors. So far, however, several obstacles have stood in the way

In order to achieve these goals. IN

20 Even in cell lines known to express detectable levels of IL-1 receptor

The IL-1 receptor is present as a very small component of the total cell pro- I

In pots. Furthermore, no cell lines were known that constitutively and continuously expressed I

In high levels of IL-1 receptors. For example, the murine EL-4 expresses 6.1 cell-I

nie detectable levels of IL-1 receptor, but the level of IL-1 receptor expression has I

I 25 tends to degrade over time, which greatly complicates the work met that I

obtain sufficient amounts of receptor to provide a useful release

gang material for purification. Thus, a method for I

continuous selection of cells to obtain acceptable levels of IL-1 receptor

repression using fluorescence activated cell sorting (FACS). IN

I 30 Additional problems arise when trying to clone mammalian genes, I

In which encodes IL-1 receptor. Although a protein composition with I can be obtained

In sufficient purity to allow N-terminal protein sequencing, allow dough- I

the generation of the genetic code typically does not mean that a suitable probe is defined without I-

taking further attempts. Defining a probe I may require many iterative attempts

35 with the necessary specificity to identify a hybridizing sequence in a cDNA-I

library. To circumvent this problem, a novel direct receptor was devised

DK 175810 B1 3 expression cloning technique to avoid the need for repeated screening using various probes of unknown specificity. This technique, which has never been used before, allows direct visualization of receptor expression after transfection of a mammalian cell line with a high expression vector containing a 5 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 for obtaining antibodies to IL-1 receptor for use in diagnosis and therapy. In addition, purified IL-1 receptor compositions can be used directly in therapy to bind or remove IL-1, thereby providing a method of regulating the immune activities or the inflammatory activities of this cytokine.

The present invention provides substantially homogeneous protein compositions comprising human IL-1 receptor.

The present invention also provides compositions for use in the therapy of IL-1 receptor or for the provision of antibodies to IL-1 receptors comprising effective amounts of soluble native or recombinant receptor proteins prepared by a suitable method. A suitable method of raising IL-1 receptor comprises adding a sample comprising IL-1 receptor to an affinity matrix comprising an IL-1 molecule bound to an insoluble carrier and eluting bound IL-1 receptor from the affinity matrix. The partially purified IL-1 receptor can be further purified by transfer to a lectin affinity column and subsequent elution of the IL-1 receptor from the lectin affinity column. The partially purified IL-1 receptor can then be treated by reverse phase high pressure liquid chromatography and eluted as a single absorbance peak at 280 nm, which when analyzed by SDS-PAGE! 25 and silver staining, appeared as a single band. As stated above, the native murine IL-1 receptor had an apparent molecular weight of approx. 82,000 daltons as determined by SDS-PAGE. Suitable DNA sequences for carrying out the present method consist essentially of a single open reading frame nucleotide sequence encoding a mammalian interleukin-1 receptor (IL-1R) or a subunit thereof. Such DNA sequences are preferably selected from (a) cDNA clones having a nucleotide sequence derived from the coding region of a native IL-1R gene, (b) DNA sequences capable of hybridizing to the cDNA clones of (a) under moderately severe conditions and encoding biologically active IL-1R molecules, and (c) DNA sequences degenerate as a result of the genetic code of the DNA sequences defined in (a) and (b) and encoding biologically active IL-1R molecules. Suitable recombinant expression vectors include the DNA sequences defined above,

I DK 175810 B1 I

I 4 I

In recombinant IL-1R molecules prepared using the recombinant ex- I

In pressure vectors, as well as methods for producing the recombinant IL-1 R I

In molecules using the expression vectors. IN

The soluble recombinant receptor molecules comprise truncated proteins in which om- I

In 5 regions of the receptor molecule that are not required for IL-1 binding, · I

I ladt. These and other aspects of the invention will become apparent from the following details

In the description and the drawing. H

In FIG. 1 is a restriction map of cDNA constructs comprising code-H

In the ranges of the murine and human IL-1R genes. The murine fragment, isolated I

I 10 from EL-4 6.1 C10 cells and present as an insert in clone GEMBL78, have been deposited.

I neret at the American Type Culture Collection under serial number ATCC 67563. I

In FIG. 2 is a schematic representation of the highly expressive mammalian I

onsplasmid pDC301, which is described in more detail in Example 6.

In FIG. 3A-3C is a graphical comparison of IL-1 binding I

In the properties of natural and recombinant IL-1 receptors. FIG. 3A compares I

In direct binding of 125 I-IL-1α to cells expressing native IL-1 receptor (EL4 6.1 C10) I

I or recombinant receptor (COS-IL-1R), fig. 3B shows the data of FIG. 3A plotted in I

In the Scatchard coordinate system. FIG. 3C shows competitive 125 I-IL-1α binding between H

In unlabeled IL-1 α and IL-1 li. In FIG. 3 indicates the C concentration of IL-1 added to binding incu- I

In the bation (molar), r indicates molecules of IL-1 bound per cell. IN

In IL-1 α and IL-1 R apparently regulate the metabolism of cells through an I

In common plasma membrane receptor protein. IL-1 receptor from detergent solutions of EL-4 I

In 6.1 C10 cells have been stably adsorbed to nitrocellulose while fully maintaining I

In IL-1 binding activity. This assay system was used to monitor purification I

I 25 of the IL-1 receptor and to study the effects of several chemical modi- I

In fixations on the receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 I

In cells can be bound to and specifically eluted from IL-1 coupled to Sepharose or other self-H

the affinity chromatography carriers. IN

Purification by the previous procedure resulted in

30 silver staining of polyacrylamide gels in identification of a protein of Mr 82,000 daltons, I

In which were present in fractions showing IL-1 binding activity. Experiments at which cells

liver surface proteins of EL-4 cells were radiolabeled and 125 I-labeled receptor was purified

In by affinity chromatography, it was assumed that the protein with Mr 82,000 was expressed on

In the plasma membrane. N-glycanase treatment of this material showed that 21-35% of the I

In 35 total Mr (82,000) of the receptor was N-linked carbohydrate. IN

I i I

11 I DK 175810 B1

To define the chemical properties of the IL-1 receptor, a simple, reproducible and quantitative assay system for the detection of IL-1 receptor in detergent solutions was devised. With this assay, the receptor purification can be followed and changes in I

the receptor binding activity in response to chemical modification of the receptor can easily I

5 are monitored. IN

Binding kinase saw for IL-1 receptor I

Recombinant human IL-1β and IL-1α can be prepared by expression in E.I.

coli and purification to homogeneity as described by Kronheim et al. (Bio / Technology I

4: 1078, 1986). Recombinant human IL-1α is preferably expressed as a polypeptide I

composed of the C-terminal 157 residues of IL-1α corresponding to Mr 17,500 the form of I

the protein released by activated macrophages. The purified protein is stored at - I

70 ° C in phosphate buffered saline as a stock solution of 3 mg / ml. 10 μΙ (30 pg) portion- I

The stock solution is labeled with sodium (125 L) iodide at a modified chlorine level.

15 min T-method described by Dover et al. (Nature 324: 266, 1986) and Segal et al. (J. Im- 1

munol. 118: 1338, 1977). In this procedure, 10 μg rlL-1a (0.57 nmol) is added to 10 μl I

phosphate (0.05 M) buffered saline (0.15 M), pH 7.2, (PBS) to 2.5 mCi (1.0 nmol) sodium iodine

did in 25 μΙ 0.05 M sodium phosphate, pH 7.0. The reaction is started by adding 30 μΙ I

1.4 x 10 -4 M chloramine-T (4.2 nmol, Sigma Chemical Co., St. Louis, MO, USA). After I

For 30 minutes on ice, the reaction mixture is fractionated by gel filtration on a Biogel P6 I

(Bio-Rad, Richmond, CA, USA) column with 1 ml bed volume. Routinely incor- I

porate 40-50% 125l in protein. IN

125 I-IL-1α can be purified by gel filtration or other suitable methods and by I

the same is diluted to a working stock solution of 3 x 10'® M in Roswell Park Me- I

Mortal Institute (RPMI) 1640 medium comprising 1% w / v bovine serum albumin I

(BSA), 0.1 w / v% sodium azide, 20 mM Hepes, pH 7.4 (binding medium) to

avoid radiolysis. Such diluted solutions can be stored for up to 1 month without:

detectable loss of receptor binding activity. The specific activity is routinely in I

range 1-3 x 1015 cpm / mmol (about 1 atom of iodine per IL-1α molecule). Typically, it is

30 chain protein initially (before dilution) 100% active as determined by its I

ability to induce IL-2 production from EL-4 6.1 C10 cells. In addition, 100% of I

125 I cpm is precipitated by trichloroacetic acid and> 95% can be absorbed by IL-1 I

receptor-bearing cells. IN

EL-4 6.1 C10 cells are propagated in suspension culture as described by I

MacDonald et al., J. Immunol. 135: 3964 (1985). An IL-1 receptor negative variant line of I

SDK 175810 B1 I

the. Cells are monitored in

iceptor expression can I

sorting (FACS) and fluo- I

and rLL-1a (FITC IL-1a)

sceinisothiocyanate (Re- I

70 μΙ borate (0.02 M) buffered I

; illes from ukonj ugeet color- I

m as described by Dover I

t EPICS C flow cytometer I

strength 20, PMT- I

ieste fluorescensslgnalni- I

s for the establishment of cell culture- I

ifugering is harvested from cultu- I

OO x g for 10 minutes to dan- I

ttes same volume PBS I

inhibitors (2 mM phenyl- I

mM O-phenanthroline). Cel- I

g and the mixture is incubated

11,000 x g for 30 minutes at I

0.02 w / v% in na- I

loss of IL-1 I can be detected

rerne. IN

ice binding assays 1 μΙ (4 x I

nitrocellulose membranes I

ice at room temperature until I

literature until use. Under I

up to two months. Before use - I

buffered saline (0.15 M), pH I

g of nonspecific bin- I

ml pr. filter), once with I

m squares with IL-1 re- I

ids bakker (Costar, Cam- I

aging 125I-IL-1 α or 12S-I

ist on a nutator and incube- I

DK 175810 B1 7 refrigerate (8 ° C) for two hours. A 60 μ. Aliquot can then be taken from each well to determine unbound 125l-rLL-1a. The remaining solution is then aspirated and discarded, and the nitrocellulose filters are washed by adding and aspirating 1 ml of binding medium and three times 1 ml of PBS to each well, respectively. The nitrocellulo-5 squares are then removed and dried on filter paper. Then they are either placed on Kodak X-omat AR film for twelve hours at -70 ° C, or they are placed in 12 x 75 cm glass tubes and counted on a gamma counter.

Table 1 shows the cDNA sequence of clone GEMBL78. Nucleotides are numbered from the beginning of the fragment. The CTG codon specifying the leucine residue, which constitutes N-terminals, is underlined at position 282, and the TAG terminator codon terminating the open reading frame is underlined at position 1953.

Tables 2A-2C show the cDNA sequence and the deduced amino acid sequence for 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, the N-terminus and the putative 21 amino acid transmembrane region of the murine IL-1 receptor are underlined.

Table 3 shows a cDNA sequence comprising the complete coding region of the human IL-1R gene. Nucleotides are numbered from the beginning of a fragment called R3A, which comprises the N-terminus and a short sequence 20 of 5 'untranslated DNA. The CTG codon specifying the leucine residue constituting the N-terminus is underlined at position 135 and the TAG terminator codon terminating the open reading frame is underlined at position 1791.

Tables 4A-4C show the cDNA sequence and the deduced amino acid sequence of the coding region of a cDNA-encoding human IL-1 receptor. In Tables 25A-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 amino acid sequences derived from murine and human IL-1 receptors. The transmembrane regions of each protein are underlined and conserved cysteine residues are shown with an asterisk. Potential N-linked glycosylation sites are shown with triangles next to asparagine residues.

Definitions "Interleukin-1 receptor" and "IL-IR" denote proteins that are capable of binding interleukin-1 (IL-1) molecules and in their native configuration as mammals. Plasma membrane proteins are thought to play a role in the metabolism of IL-1.

I DK 175810 B1 I

I 8 I

I brought signal to a cell. As used herein, the term analog I includes

I to native proteins with IL-1 binding or signaling activity. Specifically about- I

In the form are truncated or soluble forms of the IL-1 receptor protein which do not have an I

In cytoplasmic or transmembrane region. The predicted molecular weight of the murine I

I 5 protein corresponding to the sequence of the mature protein shown in Tables 2A-2B is I

In 64,597 daltons, while the predicted molecular weight of the precursor is 66,697 daltons. IN

Both of these estimates are exclusive of glycosylation. The predicted molecular weight of the hu- I

In mane protein corresponding to the sequence of the mature protein I shown in Tables 4A-4C

I is 63,486 daltons, while the predicted molecular weight of the precursor is 65,402 daltons. IN

I 10 "Substantially identical" and "substantially equal" means when express- I

ne is used to define amino acid sequences that a given sequence, e.g. and mutant- I

In sequence, differs from a reference sequence by one or more substitutions, except I

In parts or additives the net effect of which does not result in an adverse functional difference

H between reference sequence and referred sequence. In connection with the present I

According to the invention, amino acid sequences with more than 30% similarity are considered to be in the I

substantially similar to each other and amino acid sequences with more than 80% similarity are considered I

to be substantially identical. For the definition of nucleic acid sequences, I

all of the nucleic acid sequences referred to which are capable of encoding amino acid sequences

sequences substantially equal to each other to be substantially equal to an I

20 reference nucleic acid sequence, and all nucleic acid sequences capable of

code essentially identical amino acid sequences are considered to be in the I

H essentially identical to a reference sequence. In determining equality, I must

ignores abbreviations or internal omissions of the reference sequence. Sequences I

with a lesser degree of similarity, comparable biological activity and equivalent ex- I

25 pressure characteristics are considered to be equivalents. In connection with the present- I

According to the present invention, a "subunit" of an IL-1R is considered to constitute an amino acid

As used herein, "recombinant" means that a protein hereto- I

H stems from recombinant (eg microbial or mammalian) expression systems. "Mikrobi- I

30 el "refers to recombinant proteins produced in bacterial or fungal (eg yeast) I

H expression systems. As a product, "recombinant microbially" defines a protein, I

which is essentially free of native endogenous substances and not accompanied by associ- I

native glycosylation. Protein expressed in most bacterial cultures, e.g. E. coli, will be I

glycan free; protein expressed in yeast may have a glycosylation pattern different from I

DK 175810 B1 9

As used herein as a characterization of IL-1 receptors, "biologically active" either means that a given molecule has sufficient amino frequency in common with the embodiments of the invention to be able to bind at least 0.01 nmol of IL-1 1 pr. nmol IL-1 receptor or IL-1 receptor analog, or alternatively has a sufficient amino acid sequence in common to be able to transmit an IL-1 stimulus to a cell, e.g. as a component of a hybrid receptor construct. Preferably, biologically active IL-1 receptors of the invention are capable of binding more than 0.1 nmol IL-1 per nmol receptor, and most preferably more than 0.5 nmol IL-1 per nmol 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 which has been isolated at least once in the substantially pure form, i.e. . free of contaminating endogenous materials and in an amount or concentration that allows identification, manipulation and recovery of the sequence and its nucleotide sequence components by standard biochemical methods, e.g. using a cloning vector. Such sequences are preferably provided in the form of an open reading frame that is not interrupted by internal untranslated sequences, or introns that are typically present in eukaryotic genes. However, it will be apparent that genomic DNA containing the relevant sequences may also be used. Sequences of untranslated DNA 20 may be present 5 'or 3' from the open reading frame, where these sequences do not interfere with manipulation or expression of the coding regions.

"Nucleotide sequences" refers to a heteropolymer of deoxyrbonucleotides. DNA sequences encoding the proteins provided in accordance with the invention are assembled by cDNA fragments and short oligonucleotide linkers or by a series of oligonucleotides to obtain a synthetic gene which can be expressed in a recombinant transcriptional unit.

"Recombinant expression vector" refers to a plasmid comprising a transcriptional unit comprising a collection of (1) one or more genetic elements having a regulatory role in gene expression, e.g. promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences. Structural elements for use in yeast expression systems preferably comprise a leader sequence that enables extracellular secretion of translated protein by a host cell. Alternatively, recombinant protein, when expressed without a leader or transport sequence, may comprise an N-terminal methionine residue. This residue may

I DK 175810 B1 I

I 10 I

I is then cleaved from the expressed recombinant protein to provide a final protein

In duct. IN

In "Recombinant microbial expression system", one in the I

substantially homogeneous monoculture of suitable host microorganisms, e.g. bacteria so- I

I 5 as E. coli or yeast such as S. cerevisiae, which has a stable in chromosomal DNA inte- I

In Greater Recombinant Transcription! entity or carries the transcriptional entity as I

In a component of a plasmid contained therein. In general, cells that make up the system are of- I

I come from a single parent transformant. Recombinant expression systems such as I

I defined herein will express heterologous protein by induction of the regulatory I

In 10 latoric elements linked to the DNA sequence or the synthetic gene which I

must be expressed. IN

In Isolation of cDNAs. encoding 1L-1 receptors I

To obtain the murine coding sequence, a DNA-I was isolated

In 15 sequence encoding murine IL-1R (mlL-1R) from a cDNA library prepared in

by reverse transcription of polyadenylated RNA isolated from the murine cell line EL-4 I

I 6.1 C10. The library is screened by direct expression of combined cDNA-I

In fragments of COS-7 monkey cells using a mammalian expression vector I

(pDC201) using regulatory sequences derived from SV40 and Adenovirus I

20 2. Transfectants expressing biologically active IL-1R were identified by incubation I

of transfected COS-7 cells with medium containing 125 I-IL-1α, washing the cells to I

removal of unbound labeled IL-1α and by contacting the cell monolayers with I

X-ray film to detect concentrations of IL-1cx binding. In this way you proved

transfectants appear as dark places against a relatively light background. IN

H 25 Using this technique, approx. 150,000 cDNAs in I

pools of approx. 350 cDNAs until assay of a transfectant pool showed positive IL-1 a bin- I

dingssteder. A frozen bacterial stock from this positive pool was grown in I

culture and plated to provide individual colonies screened until I

H a single clone (clone 78) was identified which controlled the synthesis of a surface pro-

30 tein with detectable IL-1 binding activity. This clone was isolated and its insertion I

sequenced to determine the sequence of the murine I listed in Table 1

H cDNA. The initiator methionine for the unabridged translation product of the native murine I

H gene is one of two alternative methionine residues found at positions -19 and -16 in I

H Table 2 A. The first amino acid residue of the mature receptor protein was deduced by I

35 comparison with an N-terminal amino acid sequence obtained from highly purified IL-1R I

H preparations derived from EL-4 6.1 C10 cells. This residue is a leucine residue shown at I

DK 175810 B.1 11 position 1 in table 2A. The 1671 nucleotide coding region, corresponding to the mature protein, encodes 576 amino acids, including 15 cysteine residues and a putative 21 amino acid trans membrane region. Localized N-terminus to the transmembrane region are 7 potential N-glycosylation sites. A cloning vector comprising the unabridged murine cDNA, called GEMBL78, has been deposited with the American Type Culture Collection.

Rockville, MD, USA under serial number ATCC 67563. The deposit was made under the terms of the Budapest Convention.

A probe was constructed from the murine sequence and used to screen human cDNA libraries prepared from cultures of a human T-10 cell clone grown in the presence of OKT3 antibody and IL-2. Next, cDNA clones that hybridized to the murine probe were 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' untranslated sequences, is shown in Table 3. The nucleotide sequence of the 15 human open reading frame and the deduced amino acid sequence of the human protein are listed in Tables 4A-4C. This sequence comprises 569 amino acids (including a signal peptide of 17 amino acids), including 16 cysteine residues, 13 of which are conserved between the murine and human genes. The human sequence further comprises 6 potential N-glycosylation sites, 5 of which are conserved between murine and human. The amino acid sequence in Tables 4A-4C is numbered from a leucine residue that is considered to be the probable N-terminus based on comparisons with the murine protein. The putative transmembrane region of the human gene is 20 amino acids in length. The sequences of the putative intracellular portions of the murine and human genes are highly conserved (87%), the extracellular regions (78%) and transmembrane-25. the n areas (63%) are retained slightly less except for the localization of cysteines presumably involved in intramolecular disulfide bonding and certain N-glycosylation sites. The amino acid sequences derived from the human and murine genes are compared in Table 5.

The murine and human genes encoding integrated membrane proteins comprise intracellular regions without any apparent homology to any known protein sequence and extracellular moieties that appear to be organized in regions similar to those of the members of the immunoglobulin gene superfamily. Immunoglobulin-like regions typically have only minimal amino acidity, but have a common three-dimensional structure consisting of two li-surfaces held together by a disulfide bond. The cysteine residue involved in the formation of this disulfide bond as well as some other critical residues is highly conserved and occurs in the same relative position in almost all

I DK 175810 B1 I

I i

In members of the family. Members of the immunoglobulin superfamily include not only con- I

constant and variable immunoglobulin regions, but also a number of other cell surface mo- I

In lesions, many of which are involved in cell-cell interactions. IN

Like most mammalian genes, mammalian IL-1Rs are presumably encoded by I

I 5 multi-exon genes. Alternative mRNA constructs attributable to different I

In mRNA splice cases after transcription and which have large identity or similarity I

In regions common to the cDNAs of the invention, are considered to fall within the scope of I-I

In the framework of the fine ice. IN

In its nucleic acid embodiments, the present invention provides

In 10 parts DNA sequences encoding mammalian IL-1 Rs. Examples of mammalian IL-I

1 Rs include primate IL-1R, human IL-1R, murine, canine, feline, bovine, ovine, equine and I

In porcine IL-1 R. IL-1 R DNAs are preferably provided in a form which can be expressed in an I

In recombinant transcriptional unit under the control of mammalian, microbial or viral I

In transcriptional or translational controls. For example, a sequence, I

In 15 to be expressed in a microorganism, do not contain intranes. In preferred aspects I

The DNA sequences comprise at least one, but optionally more than one sequence component

derivatives derived from a cDNA sequence or a copy thereof. Such sequences may be I

I bound to or flanked by DNA sequences prepared by assembling synthetic oligo- I

nucleotides. Synthetic genes assembled exclusively from oligonucleotides could I

20 can be constructed using the se- I described herein

gender information. Examples of sequences include those which are essentially I

In identical to the nucleotide sequences shown in Tables 2A-2C. Alternatively, code- I

The sequences comprise codons encoding one or more additional amino acids, I

which is located at the N-terminal, e.g. an N-terminal ATG codon specifying I

25 methionine bound in reading frame with the nucleotide sequence. P.g.a. code degeneration can I

there are considerable variations in nucleotide sequences encoding the same I

amino acid sequence; examples of DNA embodiments are those corresponding to I

the sequence of nucleotides 1-1671 in Tables 2A-2C and nucleotides 1-1656 in Table I

Bells 4A-4C. Other embodiments include sequences that can hybridize to I

The sequence in Tables 2A-2C or 4A-4C under moderately severe conditions (50 ° C, 2 L

x SSG), and other sequences degenerate into those described above that encode biological I

active IL-1 R polypeptides. IN

The present invention also provides expression vectors for I

H preparation of purified IL-1R in useful amounts. The vectors may comprise synthetic or I

35 cDNA-derived DNA fragments encoding mammalian IL-1 Rs or bioequivalent I

H homologues operatively linked to regulatory elements derived from mammalian, bak- I

DK 175810 B1 13 terial, viral, yeast or bacteriophage genes. Useful regulatory elements are described in more detail below. Following transformation, transfection, or infection of Dassende cell lines, such vectors can be induced to express recombinant protein.

Mammalian IL-1Rs can be expressed in mammalian cells, yeast, bacteria or other cells under the control of appropriate promoters. Cell-free translation systems could also be used to produce mammalian IL-1R using RNAs derived from the DNA constructs of the invention. Suitable cloning and expression vectors for use with bacterial, mammalian, fungal and yeast cell hosts are described by Pouvels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New 10 York, 1985), the relevant parts of the description being incorporated herein by reference.

Various mammalian cell culture systems can be used 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, 1985), and other cell lines which may express an appropriate vector, e.g. C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors may include non-transcribed elements such as a replication region, a suitable promoter and enhancer and other 5 'and 3' flanking non-transcribed sequences and 5- or 3 'untranslated sequences such as required ribosome binding sites, a polyadenylation site, splice 20 donor and acceptor sites as well as termination sequences. From the viral SV40 genome-derived DNA sequences, e.g. SV40 origin, early promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements required for expression of a heterologous DNA sequence. Further details regarding the use of a mammalian high expression vector for the production of a recombinant mammalian IL-1R are given in Examples 4 and 6 below. Examples of vectors can be constructed as described by Okayama and Berg (Mol. Cell. Biol. 3: 280, 1983).

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

Yeast systems that preferably use Saccharomyces species such as S.

, cerevisiae, can also be used to express the recombinant proteins of the invention. Yeasts of other genera, e.g. Pichia, or Kluyveromyces, have also been used as production strains for recombinant proteins.

In general, useful yeast vectors will include replication regions and selectable markers that allow transformation of both yeast and E. coli, e.g. ampicillin resistance

I DK 175810 B1 I

I 14 I

In networks from E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a yeast I

I with high expression to induce transcription of a downstream structural see * I

kvens. Such promoters may be derived from transcriptional yeasts encoding I

In genes with high expression such as i.a. 3-phosphoglycerate kinase (PGK), α-factor, acid I

In 5 phosphatase or heat shock proteins. The heterologous structural sequence is assembled in I

an appropriate reading frame with translation initiation and termination sequences and I

I preferably a leader sequence that can control secretion of translated protein in the ex- I

In tracellular medium. Optionally, the heterologous sequence may encode a fusion protein, I

containing an N-terminal identification peptide (eg Asp-Tyr-Lys- (Asp) 4-Lys) or an I

In 10 second sequence resulting in the desired characteristics, e.g. stabilization or pre- I

In simple purification of expressed recombinant product. IN

Useful yeast vectors can be assembled using DNA sequences from I

pBR322 for selection and replication in E. coli (Ampr gene and replication region) and I

In yeast DNA sequences containing a glucose repressible alcohol dehydrogenase 2 I

I 15 (ADH2) promoter. The ADH2 promoter has been described by Russell et al. (J. Biol. I

I | Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). Such vectors can I

also include a yeast TRP1 gene as a selectable marker and yeast 2p replication I

I | the area. A yeast leader sequence, e.g. the α-factor leader that controls the secretion of heterologous I

I I proteins from a yeast host can be inserted between the promoter and the structural gene that I

I 1 20 to be expressed (see Kurjan et al., U.S. Patent 4,546,082, Kurjan et al., Cell 30: 933 I

I (1982), and Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330, 1984). The leader sequence I

may be modified to contain near its 3 'end one or more useful restriction sites I

to facilitate the fusion of the leader sequence with foreign genes. IN

H Suitable yeast transformation protocols are skilled in the art. Et ek- I

25 samples of a technique are described by Hinne et al. (Proc. Natl, Acad. Sci. USA 75: 1929, I

1978), in which Trp + transformants are selected in a selective medium consisting of I

0.67% gemitrogen base, 0.5% casamino acids, 2% glucose, 10 pg / ml adenine and 20 l

pg / ml uracil. IN

Host strains transformed with vectors comprising ADH2-I

Promoter, can be grown for expression in an enriched medium consisting of 1% yeast extract

extract, 2% peptone and 1% glucose supplemented with 80 pg / ml adenine and 80 pg / ml uracil. IN

Derepression of the ADH2 promoter occurs when the glucose of the medium is depleted. Raw I

yeast supernatants are harvested by filtration and kept at 4 ° C before further purification. IN

Useful expression vectors for bacterial use are built up by ind- I

Addition of a DNA sequence encoding mammalian IL-1R together with suitable trans- I

lation initiation and termination signals in operational read phase with a functional pro- I

DK 175810 B1 15 engine. The vector will comprise one or more phenotypically selectable markers and a replication region to ensure amplification in the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species of the genera Pseudomonas, Streptomyces and Staphylococcus, although other hosts may also be used if desired.

Expression vectors are conveniently constructed by cleaving cDNA clones at sites near the codon encoding the N-terminal residue of the mature protein. Synthetic oligonucleotides can then be used to "reverse" omitted portions of the coding region and to provide a binding sequence for ligation | '10 of the coding fragment in the appropriate reading frame of the expression vector and optionally a codon specifying an initiator methionine.

As a representative but non-limiting example, useful expression vectors for bacterial use may comprise a selectable marker and a bacterial replication region derived from commercially available plasmids comprising 15 genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercial vectors include e.g. pKK223-3 (Pharmacia Fine Chemicals, Uppsala,

Sweden9 and pGEM1 (Promega Biotec, Madison, Wl, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

A particularly useful bacterial expression system uses phage λ PL

the promoter and the thermolabile cl857 repressor. Plasmid vectors available from the American Type Culture Collection which include derivatives of the λ PL promoter include plasmid pHUB2 present in E. coli strain JMB9 (ATCC 37092) and pPLc28 present in E. coli RR1 (ATCC 53082) . Other useful promoters for expression in E. coli include that of Studier et al. (J. Mol. Biol. 189: 113, 1986) described T7 RNA polymerase promoter, the lacZ promoter described by Lauer (J. Mol. Appl. Genet. 1: 139-147, 1981). available as ATCC 37121, and that of Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, p.

412) described tac promoter available as ATCC 37138.

After transforming a suitable host strain and culturing the host strain to an appropriate cell density, the selected promoter is subjected to derepression by appropriate means (e.g., temperature change or chemical induction), and cells are cultured for an additional period of time. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Cells are grown e.g. in a 101 fermentation vessel using maximum aeration and vigorous stirring. An antifoam is preferably used.

I DK 175810 B1 I

I I

In formative (Antifoam A). Cultures are grown at 30 ° C in that of Mott et al. (Proc. Natl. I

I Acad. Sci. USA 82:88, 1985) described superinduction medium which alternatively re- I

I fatter antibiotics, exposed to derepression at a cell density corresponding to A600 = I

0.4-0.5 by raising the temperature to 42 ° C and harvesting from 2-20, preferably 3-6, hours I

In 5 after the temperature increase. The cell mass is initially concentrated by filtration

Somehow and then centrifuged at 10,000 x g for 10 minutes at 4 ° C ef-I

I followed by rapid freezing of the cell pellet. IN

In Purified mammal I L-1Rs or bioequivalent homologues are prepared

I preferably by culturing suitable host / vector systems for expression of the recombi 'I

In nant translation products of the synthetic genes of the invention, which then I

You are purified from cultural media. IN

An alternative method of making purified 11-1R comprises op- I

In purification from cell culture supernatants or extracts. By this method an- I

In turns a cell line that future useful amounts of the protein. Superfoods from I

15 such cell lines may optionally be concentrated using a commercial I

In available protein concentration filter, e.g. an Amicon or Millipore Pellicon ultrafil- I

tration unit. After the concentration step, the concentrate can be placed on a suitable I

In purification matrix as previously described. For example, a suitable affinity matrix I

comprise an IL-1 or lectin or antibody molecule bound to a suitable carrier. IN

B 20 Alternatively, an anion exchange resin may be used, e.g. a matrix or substrate with I

diethylaminoethyl (DEAE) side groups. The matrices may be acrylamide, agarose, dex- I

cod liver oil, cellulose or other types commonly used for protein purification. Alter- I

Alternatively, a cation exchange step may be used. Suitable cation exchangers include various I

B insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl- I

In 25 groups is preferred. IN

B Finally, for further purification of an IL-1R composition, I- I

B, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps are performed during

B use of hydrophobic RP-HPLC media, e.g. silica gel with methyl or other aliphatic I

B side groups. Some or all of the previous purification steps may be in various combinations

B ions are also used to provide a homogeneous recombinant protein. IN

B In bacterial culture, recombinant protein produced is usually isolated by I

B initial extraction from cell pellets followed by one or more concentrations, I

B desalination, aqueous ion exchange or size exclusion chromatography steps. Finally I

B, high pressure liquid chromatography (HPLC) can be performed as the final purification step. Microbial I

B 35 cells used to express recombinant mammalian IL-1R can be disrupted by an I

17 DK 175810 B1 any suitable method, including alternating freezing / thawing, sonication, mechanical breaking or the use of cell disintegrants.

Fermentation of yeast that expresses mammalian IL-IR as a secreted protein makes purification much simpler. Secreted recombinant protein derived from a large scale fermentation can be purified by methods analogous to those of Urdal et al. (J. Chromatog. 296: 171, 1964), described. This reference describes two sequential, reverse 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 that are free of contaminating endogenous materials, with or without associated glycosylation of the native pattern. The native murine IL-IR molecule is recovered from cell culture extracts as a glycoprotein with an apparent molecular weight of approx. 82 kD determined by SDS-PAGE. In mammalian expression systems, IL-1Rs expressed e.g. COS-7 cells, may have a molecular weight and a glycosylation pattern that are similar or slightly different from those of the native molecules depending on the expression system. Expression of IL-1R DNAs in bacteria such as E. coli provides non-glycosylated molecules with an apparent molecular weight of approx. 60 kD as determined by SDS-PAGE under non-reducing conditions.

Recombinant IL-1R proteins of the invention also include N-terminal methionyl murine and human IL-1R; Additional embodiments include soluble abbreviated versions in which certain ranges, e.g. the transmembrane region and the intracellular regions are omitted, thereby providing a molecule having only one IL-1 binding region. Mammalian IL-1 Rs expressed as fusion proteins with a polypeptide leader comprising the sequence Asp-Tyr-Lys- (Asp4) -Lys, or with other suitable peptide or protein sequences used as aids for expression in microorganisms or purification of microbially expressed proteins is also included.

Bioequivalent homologues of the proteins of the invention include various analogs, e.g. abbreviated versions of IL-1 Rs in which terminal or internal residues or sequences not necessary for biological activity are omitted. Other analogues are those in which one or more cysteine residues have been omitted or replaced with other amino acids, e.g. neutral amino acids. Other mutagenesis methods include modification of adjacent dibasic amino acid residues to increase 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.

I DK 175810 B1 I

I 18 I

As used herein, "mutant amino acid sequence" refers to I

In a polypeptide encoded by a nucleotide sequence deliberately differentiated from I

In a native sequence. "Mutant protein" or "analog" means a protein comprising an I

In mutant amino acid sequence. "Native sequence" refers to an amino acid or nucleic acid I

In 5 sequence which is identical to a wild type or native form of a gene or protein. IN

I The terms "KEX2 protease recognition site" and "N-glycosylation site" are defined in

In below. As used to define particular aspects of the invention, ud- I

In the pressure "inactivation", change of a selected KEX2 protease recognition site to I

retard or prevent cleavage by the KEX2 protease of Saccharomyces cerevisiae, I

I 10 or alteration of an N-glycosylation site to exclude covalent binding of oligosac- I

charid moieties for special amino acid residues at the cell. IN

Site-specific mutagenesis procedures can be used to inactivate I

In KEX2 protease treatment sites by omission, addition or replacement of residues I

I to change Arg-Arg, Arg-Light and Light-Arg pairs to eliminate the occurrence of these I

15 adjacent alkaline residues. Light-Light pairs are significantly less prone to KEX2-I

In cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a con- I

In servative and preferred method of inactivating KEX2 sites. The result- I

the analogues are less likely to be cleaved with KEX2 protease elsewhere I

I than in yeast the α-factor leader sequence, where cleavage by secretion is intentional. : I

I 20 Many secreted proteins assume covalently bound carbohydrate moieties in I

after translation, frequently in the form of oligosaccharide units which by N-glycoside bonds I

is bound to asparagine side chains. Both the structure and the number of oligosaccharide units

In that, which is bound to a given secreted protein can vary considerably, resulting in I

in many different apparent molecular masses attributable to a single gly- I

H 25 coprotein. mlL-1R is a glycoprotein of this type. Attempts to express glycoprotei- I

recombinant systems can be complicated by the heterogeneity attributable to I

this variable carbohydrate component. For example, purified mixtures of re- I

combining glycoproteins such as human or murine granulocyte macrophage colonies

stimulating factor (GM-CSF) consist of 0-50% by weight of carbohydrate. Miyajima et al. : I

30 (EMBO Journal 5: 1195,1986) reported expression of a recombinant murine GM-I

CSF in which N-glycosylation sites had been mutated to exclude glycosylation and I

reduce the heterogeneity of the yeast expressed product. IN

The presence of variable amounts of associated carbohydrate in re- I

combined glycoproteins complicate the purification procedures, whereby the yield re- I

35 duceres. In addition, if the glycoprotein is used as a therapeutic agent, in I

possibility that the recipient will develop immune reactions to yeast carbohydrate de-: I

I 'I

In DK 175810 B1 lene, which requires that the therapy be interrupted. For these reasons, biologically active, homogeneous analogs of immunoc regulatory glycoproteins with reduced carbon hydratinH content may be desirable for therapeutic use.

Functional mutant analogs of mammalian IL-1Rs with inactivated N-5 glycosylation sites can be prepared by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques as described below. These analogous proteins can be produced in good yield in a homogeneous, carbohydrate-reduced form using yeast expression systems. N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-A1-Z, where A1 is an amino acid 10 except Pro and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for the covalent bonding of carbohydrate. Such a site can be eliminated by substituting Asn or the residue Z with another amino acid, omitting Asn or Z, or inserting a non-Z amino acid between A1 and Z, or a non-Asn amino acid between Asn and A1. Preferably, substitutions are performed in a conservative manner, i.e. the most preferred substitution amino acids are those whose physicochemical properties are similar to those of the residue to be replaced. Similarly, the potential effect of the deletion or inclusion on the biological activity should be considered when applying an exclusion or insertion strategy.

In addition to the particular analogs described above, numerous DNA constructs can be conveniently prepared comprising all or part of the nucleotide sequences shown in Tables 2A-2C or 4A-4C, together with oligonucleotide cassettes comprising additional useful restriction sites. Mutations can be introduced at particular sites by synthesizing oligonucleotides containing a mutant sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide an altered gene with particular codons that have been altered according to the required substitution, deletion, or insertion. As Examples 30, Walder 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 suitable techniques, which are incorporated herein by reference.

In one embodiment of the invention, the amino acid sequence of IL-1R is bound to a yeast α-factor leader sequence via an N-terminal fusion construct comprising a nucleotide encoding the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Asp- Light (DYKDDDDK).

I DK 175810 B1 I

I 20 I

The latter sequence is highly antigenic and provides an epitope that binds re- I

I versible of specific monoclonal antibody, enabling a rapid assay and an easy I

In purification of expressed recombinant protein. This sequence is also specifically cleaved by bovine I.

mucosal enterokinase by the residue immediately following the Asp-Lys pair. Fusi- I

I 5 onsproteins masked with this peptide may also be resistant to I

In intracellular degradation in E. coli. An alternative construction is Asp-Tyr-Lys-Asp-Asp-I

In the Asp-Asp-Lys-Glu-lle-Gly-Arg which provides a factor X recognition site immediately

I bare downstream of enterokinase siteL I

The invention is further described in the following examples, which are to serve I

I 10 for lighting, but not for limiting the invention. IN

In Example 1 I

Preparation of IL-1 α affinity matrix and affinity purification of surface I receptor

I labeled EL-4 6.1 C10 cells I

15 Cell transfer proteins on EL-4 6.1 C10 cells were radiolabeled with 125 L

by that of Cosman et al. (Molecular Immunol. 23: 935,1986) described glucoseoxt- I

In the dase lactoperoxidase method. Labeled cells were pelleted by centrifugation, wash- I

three times with PBS and extracted with PBS containing 1% Triton X-100 and I

In the cocktail of protease inhibitory I described in the assay protocol specified above

20 rer. The Triton X-100 extract was centrifuged for 10 minutes in an Eppendorf microcentric

joint and the supernatant was stored at -70 ° C. IN

Recombinant IL-1α was bound to cyanogen bromide-activated Sepharose I.

4B (Pharmacia, Piscataway, NJ, USA) or to Affigel-10 (Bio-Rad, Richmond, CA, I

USA) according to the manufacturer's proposal. For example, a solution of IL-1α (1.64 L) was added

25 mg / ml in 9.5 ml PBS) 3 ml swollen, acid-washed, CNBR-activated Sepharose. Resolution- I

The gene was shaken overnight at 4 ° C and a portion of the supernatant was tested for protein I

by a fluorescamine protein assay as described by Udenfriend et al. (Science I

178: 871,1972) using BSA as a standard. 98% of the protein had become I

bound to the gel, which suggested that the column had a final charge of 5.1 mg IL-1a I

H 30 pr. ml gel. 300 μl of 1 M glycine ethyl ester (Sigma Chemical Co., St. Louis, MO, USA) sat- I

to the slurry to block unreacted sites on the gel. IN

The gel was washed thoroughly with 0.1 M glycine buffer, pH 3.0, containing I

0.1% Triton X-100, PBS containing 0.1% Triton X-100, RIPA buffer (0.05 M Tris-I

HCl, pH 7.5, 0.15 M NaCl, 1% NP40.1% sodium deoxycholate, 0.1% SDS) and PBS in

Containing 0.1% Triton X-100 and 10 mM ATP. Small columns (200 μΙ) were prepared in I

disposable polypropylene holders (Bio-Rad, Richmond, CA, USA) and washed I

I DK 175810 B1: with PBS containing 1% Triton X-100. Portions of 100 μΙ 125l-labeled extract! was transferred to a column which was then washed with PBS containing 1% Triton X

I 100, RIPA buffer, PBS containing 0.1% Triton X-100 and 10 mM ATP, and PBS with I

i I

j 1% Triton X-100. IN

! ' The IL-1 receptor on murine T cells is a robust structure that can bind I

in 125 I-IL-1α in Triton X-100 detergent solutions. In order to extract receptor from an I

; such an affinity matrix requires a mild elution procedure. Mild acid treatment- I

ling can result in rapid dissociation of preformed IL-1 cx / IL-1 receptor complexes. IN

Based on this observation, pH 3.0 glycine HCl buffer containing 0.1% I was used

Triton X-100 for eluting receptor from the IL-1 α affinity columns, which receptor op- I

was collected in 0.05 ml fractions. The presence of receptor in the fractions was detected by I

dot blot as described above using 125 I-labeled IL-1 α. IN

Analysis by SDS-PAGE procedure as follows. To 50 μΙ of each colon- I

nefraction was added 50 μΙ 2 x SDS sample buffer (0.125 M Tris HCl, pH 6.8.4% SDS, 20% I

Glycerol, 10% 2-mercaptoethanol). The solution was placed in a boiling water bath for 3 l

j minutes and 40 μ portion aliquots were placed in a sample well with 10% polyacrylamide gel, I

which test device was set up and run according to that of Laemmli (Nature 227: 680, I

1970). Gels were fixed and stained using I

0.25% Coomassie brilliant blue in 25% isopropanol and 10% acetic acid, decolorized in I

25 25% isopropanol and 10% acetic acid, treated with Enhance (New England Nu- I

clear, Boston, MA, USA), dried and exposed to Kodak X-omat AR film by - I

70 ° C. Molecular weight markers, labeled 14C, were obtained from New England Nuclear I

and included cytochrome C (Mr 12,300), lactoglobulin A (Mr 18,367), carboxylic acid

drase (Mr 31,000), ovalbuman (Mr 46,000), bovine serum albumin (M2 69,000), phos-. IN

Phorylase B (M2 97,400) and myosin (Mr 200,000). Alternatively, fractions I were analyzed

with receptor activity by SDS-polyacrylamide gel electrophoresis followed by silver staining I

as previously described by Urdal et al. (Proc. Natl. Acad. Sci. USA 81: 6481, 1984). IN

Dot-blot analysis of fractions eluted from the IL-1 α affinity matrix showed that I

IL-1 binding activity was detected in fractions collected after pH 3.0 glycine-I

30 buffers had been added to the column. Fractions that gave positive results in this assay

revealed by analysis by SDS-PAGE that a protein with Mr 82,000 could be detected in

by developing the gel with silver color. To determine which of them by silver- I

staining detected proteins expressed on the cell surface were surface-labeled El- I

4 6.1 cells with 125 I by the lactoperoxidase glucose oxidase procedure. Radiolabeled I

35 cells were then extracted with PBS containing 1% Triton X-100, and aliquots of I

the detergent extract was added to an IL-1 α affinity matrix. Fractions collected from I

SDK 175810 B1

> ldt et radio- I

isolated from I

ne of IL-1 re- I

c 10B EL-4 6.1! IN

ss as be- I

r / ml and an- I

irocellulose and I

Hige koncen- I

taltes nitro- I

dr autoradio- I

IL-1IJ. The op- I

on an IL-1 I

an area of I

skning bloke- I

saueme var de I

ulose expelled I

i i intact cell- I

shown on inakte I

jrens with the I

surface of plas- I

filters, dried, I

ier tested I

10-7 M), hu- I

; 10-4 M), mu- I

and epidermal I

ilnerve- I

ormon (1 I

jshormon (1 I

In DK 175810 B1 1 μς / ml) and follicle stimulation hormone (1 pg / ml). All incubations were performed at 1.9 x 10-10 M 125 I-IL-1α.

This experiment showed that extracted receptor retains the same specificity as previously demonstrated for intact cells. As was found to be the case with intact 5 cells, only IL-1 α and IL-1 β produced significant inhibition of 125 I-IL-1 α binding. The data showed that unlabeled IL-1α and IL-1β produced> 90% inhibition of 125 I-IL-1cc binding, while no significant blockade of any of the other hormones was observed.

To determine whether receptor in detergent solution binds IL-1 with an affinity similar to that of receptor in cell membranes or adsorbed to nitrocellulose, a third experiment was performed in which a nitrocellulose dot-blot binding assay was used for testing the ability of EL-4 6.1 C10 extract in Triton X-100 solution to inhibit binding of 125 I-IL-1a to the solid phase. EL-4 6.1 C10 extracts were adsorbed to nitrocellulose, dried, blocked and incubated with a mixture of 12S1-IL-1α and extracts containing receptors in detergent solution.

The concentration of receptor in the solution phase was determined from a saturation binding curve to 1 μl aliquots blotted onto nitrocellulose, which made it possible to calculate receptors / μΙ and thus IL-1 receptor concentration (M). The extract was diluted through PBS Triton X-100 solution (0.5% Triton) to keep the detergent concentration constant. The inhibition curve showed that the receptor in solution bound to 125 I-IL-1a 20 with a Ka (4.5 ± 0.5 x 109 M-1), which corresponds to that of the receptor on the solid phase or in membranes. Furthermore, the good agreement between the theoretical curve, which is based on a single 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.

To investigate the integrity of the receptor as a function of the concentration of total EL-4 6.1 C10 membrane proteins, a fourth experiment was performed. Mixtures of EL-4 6.1 C10 extract in various ratios ranging from 10 to 100% were prepared either with an extract from cells not expressing the IL-1 receptor, EL-4 (M) cells, or with PBS Triton X-100 ( 0.5%). Each mixture was assayed for receptor concentration and affinity of 125 I-IL-1α binding was assayed by quantitative dot-blot binding. Receptor concentration decreased linearly by the percentage of the EL-4 6.1 C10 extract present, whether or not membrane protein concentration was maintained at a constant level. In both mixing series, the affinity of the 125 I-IL-1 α receptor remained constant. These data are consistent with one of two hypotheses, either the receptor binding function is contained in a single polypeptide chain or if the functional re- I DK 175810 B1

I 24 I

ceptor requires two or more subunits for IL-1 binding, the chains are sufficiently dense I

In connection with that dilution via detergent does not separate them. IN

In Example 3 I

I 5 Purification of IL-1 receptor to homogeneity and determination of N-terminal sequence I

300-500 I EL-4 6.1 C10 cells were grown to saturation during the previous I

described conditions, were harvested and extracted with PBS * 1% Triton X-100. It is in

the gene extract was transferred to an IL-1α affinity column and the column was washed as I

previously described. Fractions containing IL-1 receptor were detected at 125 I-IL-1α dot-I

In just the procedure followed by eluting the column with 0.1 M glycine HCl, pH 3.0, containing

In holding 0.1% Triton X-100. Aliquots of the fractions were analyzed by SDS-po-: I

lyacrylamide gel electrophoresis. IN

This prepared by affinity chromatography on Affigel-IL-1a, partially I

purified IL-1 receptor composition was adjusted to contain the following I

In buffer composition: 10 mM Tris-HCl, pH 8, 250 mM NaCl, 0.5 mM MgCl 2, 0.5 mM I

In MnCl 2, 0.5 mM CaCl 2 and 0.01% (v / v) Triton X-100 (WGA buffer). IL-1 receptor- I

the composition was then transferred to a 1 ml column of wheat germ glutinin I.

(WGA) bound to Sepharose C1-6B, equilibrated with WGA buffer. After the transfer of I

The IL-1 receptor composition was washed in the WGA column with 20 ml of WGA buffer I

In 20 followed by 10 mM Tris HCl, pH 8.0.01% (v / v) Triton X-100. IL-1 receptor protein I

H was eluted from the WGA column with 10 mM Tris-HCl, pH 8, 0.5 M N-acetylglucosamine I

and 0.01% (v / v) Triton X-100. The presence of biologically active IL-1 receptor on- I

by the 125 I-IL-1α dot-blot procedure. Fractions were also analyzed by SDS-I

polyacryphamide gel electrophoresis followed by silver staining. IN

Material eluting from the WGA column was transferred to a C8 RP-I

HPLC column. The C8 RP-HPLC column (Brownlee Labs RP-300.1 mm x 50 mm) was I

previously equilibrated with 0.1% (v / v) trifluoroacetic acid (TFA) in H 2 O of HPLC-I

quality at a flow rate of 50 pl / minute. After transfer of the IL-1 receptor

dry material, the C8 RP-HPLC column was washed with 0.1% (v / v) TFA in H

30 at 50 μΙ / minute until the absorbance at 280 nm returned to baseline. IL-1 re- I

The H receptor protein was eluted from the column by running a linear gradient of 0.1% I

(v / v) TFA in acetonitrile from 0-100% at a rate of 1% / minute. Portions of frak- I

the ions were analyzed by SDS-polyacrylamide gel electrophoresis. It turned out that IL-1 I

the receptor protein consisted of a single band on an SDS-polyarylamide gel that migrated I

H 35 with a molecular weight of 82,000. IN

DK 175810 B1 25

The purified IL-1 receptor protein was analyzed by Edmann 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 show the following sequence of 5 amino acid residues: NH2-Leu-Glu-lle-Asp-Val-Cys-Thr-Glu-Tyr-Pro-Asn-Gln-lle-Val-Leu -Phe-Leu-Ser-Val-Asn-Glu-lle-Asp-lle-Arg-Lys.

This protein sequence was found to be unique when compared to the information available on March 17, 1987 in the Protein Identification Resource National Biomedical Research Foundation's Protein Sequence Database. The available information in 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 15 COS-7 cells

A cDNA library was constructed by reverse transcription of polyadenylated mRNA isolated from total RNA extracted from EL-4 6.1 C10 cells by a method similar to that of Chirgwin et al. described (Biochem. 18: 5294,1979). Briefly, the cells were lysed in a guanidinium isothiocyanate solution, and the lysate was deposited over a pad 20 of CsCl and centrifuged until the RNA was pelleted. The RNA pellet was resuspended and further purified by protease degradation, organic extraction and alcohol precipitation. Poly A + RNA was isolated by oligo dT cellulose chromatography, and double-stranded cDNA was prepared by a method similar to that described by Gubier and Hoffman (Gene 25: 263,1983). Briefly, the RNA was copied to cDNA 25 by reverse transcriptase using either oligo dT or random oligonucleotides as primer. The cDNA was double-stranded by incubation with E. coli DNA polymerase I and RNase H, and the ends were blunt-ended by further incubation with T4 DNA polymerase. The blunt-ended cDNA was ligated into Narrow-cut dephosphorylated pDC201 vector DNA.

The eukaryotic high expression vector pDC201 was assembled from SV40, adenovirus 2 and pBR322 DNA, which sequentially comprised (1) an SV40 fragment containing the replication region, early and late promoters and enhancer, (2) an adenovirus 2 fragment containing the late main promoter, the first exon and a portion of the first intran of the three-part late leader, (3) a synthetic sequence comprising a HindIII site, a splice acceptor site, second and third exons of the three-part adenovirus 2 leader, and a multiple cloning site comprising a SmaI site, (4) further

DK 175810 B1 I

26 I

SV40 sequences containing early and late potyadenylation sites, (5) adenovirus I

2 sequences comprising the virus-associated RNA genes, and (6) pBR322 elements for I

replication in E. coli. IN

The resulting EL-4 6.1 C10 cDNA library in pDC201 was used for | IN

Transformation of E. coli strain DH5α, and recombinants were plated to provide

approx. 350 colonies per. plate and sufficient plates to provide approx. IN

25,000 total colonies per. screen. Colonies were scraped from each plate, combined, and I

from each pool plasmid DNA was prepared. The combined DNA was then used

next for transfection of subconfluent layers of monkey COS-7 cells using I

DEAE-dextran followed by chlorquin treatment as described by Luthman et al. (Nu- H

cleic Acids Res. 11: 1295, 1983) and McCutchan et al. (J: Natl. Cancer Inst. 41: 351, I

1986). The cells were then cultured for three days to allow transient ex- I

pressure of the inserted sequences. After three days, the cell culture superman- I was discarded

and the cell monolayers in each plate were analyzed for IL-1 binding as follows. 3 ml H

15 RPMI medium containing 3 x 10-10M 125 I-IL-1a was added to each plate and the plates I

incubated for 2 hours at 8 ° C. This medium was then discarded and each plate was removed

was done with 10 ml of RPMI 1640 medium (not containing labeled IL-1a). The edges of I

each plate was then broken off, leaving a flat disc which was brought into contact with

X-ray film for 72 hours at -70 ° C using an amplifying screen. IL-1 I

20 binding activity became visible on the developed films as a dark spot against a relative I

uniform background. IN

After screening approx. 150,000 recombinants from the library on this H

Thus, it was observed that a transfectant pool provided IL-1 binding sites which in

were clearly visible against the background. IN

A frozen stock of bacteria from the positive pool was then used

to obtain plates with approx. 350 colonies. Replicas of these plates were made on I

nitrocellulose filters, after which the plates were scraped and plasmid DNA was prepared and I

was transfected as described above to identify a positive plate. Bacteria from I

individual colonies from the nitrocellulose replicas of this plate were grown in 2 ml of culture medium.

30 rers used to obtain plasmid DNA which was transfected into COS-7 cells as H

described above. In this way, a single clone, clone 78, which was capable I was isolated

to induce expression of 11-1R in COS cells. The insert was subcloned into a place- I

mid derived from pBR322 (GEMBL) and sequenced by conventional techniques. IN

The sequence is shown in Table 1.

27 DK 175810 B1 \

Example 5 j i

Isolation of human cDNA clones. which hybridizes 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-5 polymerase I. The method used was substantially similar to that of Maniatis et al.

(above, p. 109) described.

A cDNA library was constructed by reverse transcription of polyadenylated mRNA isolated from total RNA extracted from the cultured cells by a human T cell line called clone 22, described by Acres et al. (J. Immunol. 138: 2132, 1987). These cells were grown in RPM11640 medium plus 10% fetal calf serum as described by Acres et al. (above), in the presence of 10 ng / ml OKT3 antibody and 10 ng / ml human IL-2. The cDN was double-stranded using DNA polymerase I, blunt-ended with T4 DNA polymerase, methylated with EcoRI methylase to protect the EcoRI cleavage sites in the cDNA, and ligated to EcoRI linkers. The resulting constructs were digested with EcoRI to remove the copies of the links at each end of the cDNA except one, and ligated to EcoRI-cut and dephosphorylated arms of bacteriophage Agt10 (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 Synstems, San Diego, CA, USA 92121) to form a library of recombinants. The recombinants were plated on E. coli strain C600 (hf1-) and screened by standard plaque hybridization techniques under moderately severe conditions (50 ° C, 6 x SSC).

After several rounds of screening, nine clones were isolated from the library that hybridized to the cDNA probe. The clones were plaque purified and used to prepare 25 bacteriophage DNA, which was digested with EcoRI. The degradation products were electrophoresed on an agarose gel, blotted onto nylon filters and tested again 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, which contained a polylinker with a unique EcoRI site, a BamH1 site and numerous other single sites. An example of a 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 showed that the clone comprised a sequence encoding 518 amino acids that showed 80% amino acid sequence identity with the corresponding murine sequence in the extracellular or N-terminal region. distal to the transmembrane region, 63% identity in

DK 175810 B1 I

28 I

transmembrane region and 87% identity in the cytoplasmic or C-terminal region. i m

IN

In addition, several cysteine residues and most N-linked glycosylation sites were between the I

murine and human sequences. A 440 bp EcoRI-Nsil fragment derived from the 5 part of the I

human IL-IR clone 32P was labeled by nick translation as described above and I

5 was used to screen a cDNA library prepared by random priming of I

clone 22 mRNA prepared as described above. 23 clones that hybridized to pro- I

bones, were isolated and analyzed by restriction mapping. Sequencing of I

one of these clones provided the sequence information corresponding to the remaining N-I

terminal 34 amino acids of the human protein. The coding and the deduced amino acid- I

10 sequences of the complete coding region of human IL-1R are shown in Tables 4A-4C. IN

Example 6 I

Expression of recombinant IL-1 receptor using a highly efficient mammalian I

expression svtem I

The mammalian expression plasmid pDC201, depicted in FIG. 2, is designed I

to express cDNA sequences inserted at its multiple cloning site (MCS) at I

transfection into mammalian cells. Referring now to FIG. 2, pDC201 comprises the following I

components: SV40 (shaded field) contains SV40 sequences from coordinates 5171- I

270 including the replication region, amplifier sequences, and early and late promoters. IN

The fragment is oriented so that the direction of transcription from the early promoter is H

as shown by the arrow. Ad-MLP (unshaded field) contains adenovirus-2 sequences from I

coordinates 5779-6231 including the late main promoter, the first exon and part I

of the intron between the first and the second exon of the three-part conductor. TPL (dashed I

field) contains a synthetic DNA sequence specifying adenovirus-2 sequences H

25 7056-7172, 9634-9693 (containing the acceptor splice site of the second exon of I

the three-part conductor, the second exon and part of the third exon of the three-part conductor)

and a multiple cloning site (MCS) containing sites for Kpnl, Smal and Bglll. pA I

(shaded box) contains SV40 sequences from 4127-4100 and 2770-2533 that include I

the polyadenylation and the early transcription termination signals. VA (filled in field) I

30 contains adenovirus-2 sequences from 10226-11555 comprising the virus associate I

the RNA genes (VAI and VAII). The solid lines are derived from pBR322 and repre- I

centers (starting after the pA sequences and clockwise) coordinates 29-23, I

651-185 (at which point the VA sequences are inserted), 29-1,4363-2486 and 1094- I

375. pDC201 is a derivative of pMLSV previously described by Cosman et al., Mo- I

35 lec. Immunol. 23: 935 (1986). IN

DK 175810 B1 29

To express recombinant IH receptor, COS cells were cultured 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, which has an IL-1R cDNA insertion (clone 78). After 72 hours of culture, cells were harvested by washing once with 10 ml of 5 PBS and subsequent treatment with an EDTA solution (sodium phosphate 0.05 M, | sodium chloride 0.15 M, EDTA 0.005 M, pH 7.4) for 20 minutes. at 37 ° C followed by scraping. In comparison, COS cells were transfected with a pDC201 control vector containing no insert and EL-4 6.1 C10 cells and EL-4 M cells (an 11-1 receptor negative variant of EL-4 cells) were cultured 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 well. cell. Since the parental COS cells expressed only approx. 500 receptors per cell, it can be estimated that more than 98% of all IL-1 receptors in the transfected population were recombinant. Flow cytometry using FITC-IL-1a revealed that only 4.2% of the cells were stained clear, so that each of these transfected COS cells contained approx. 1.1 x 106 IL-1 binding sites.

Plasma membrane proteins of EL-4 6.1 C10 cells and of COS cells transfected with vector DNA containing cDNA encoding the IL-1 receptor (clone 78) were labeled with 125 I as described in Example 1 above. Cells were then extracted with PBS containing 1% Triton X-100 and a cocktail of protease inhibitors (2 mM phenylmethylsulphonyl fluoride, 1 mM pepstatin, 1 mM leupeptin and 2 mM O-phenanthroline). Detergent extracts were subjected to affinity chromatography as described in Example 1 of Affigel-10 (Biorad, Richmond, CA) to which recombinant human IL-1α 125 I-labeled receptor had been bound, then eluted with sample buffer 25 (0.0625 M Tris-HCl, pH 6). , 8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol) and analyzed by SDS-polyacrylamide gel electrophoresis on a 10% gel. Gels were then subjected to autoradiography. The recombinant IL-1 receptor purified by affinity chromatography on IL-1α columns migrated with a relative mobility of approx. 80,000 on SDS-polyacrylamide gels, which was comparable to the mobility exhibited by the 30 similarly from EL-4 6.1 C10 cells purified IL-1 receptor.

The DNA from clone 78, when transfected into COS cells, led to expression of IL-1 binding activity that was virtually identical to that exhibited by EL-4 6.1 C10 cells, as shown in Figs. 3A-3C.

For binding assays, COS cells were resuspended at 1.7 x 10 6 cells / ml to 35 EL-4 M (1.5 x 10 7 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 and binding assays were performed in

I DK 175810 B1 I

H

I I

In RPM11640 / 10% BSA / 0.1% sodium azide / 20 mM HEPES, pH 7.4. Binding incubations I

I with 125 I-IL-1α or 125 I-IL-1 β and unlabeled IL-1α and IL-1 β were performed as described in I

Elsewhere in the description. 125 I-IL-1α bound to the transfected COS cells with a Ka I

I at 3.0 ± 0.2 x 109 M-1 (Fig. 3B). The Ka for the native receptor on EL-4 6.1 C10 cells I

5 was 4.3 ± 3 x 109 M-1. All bonds were to recombinant receptors (see Fig. 3A), the I

parental COS cell population did not detectably bind 125I-IL-1 α in this experiment. IN

In a cold comparative experiment, the concentration of free 1251-1 L-1a I

I 7.72 ± 0.13 x 10-10 M. On the transfected COS cells, the maximum binding was I

2.98 ± 0.3 x 104 molecules / cell (no inhibition), and the background (measured in the presence of 6 L

In 10 x 10-7 M unlabeled IL-1α) was 921 ± 60 molecules / cell (100% inhibition). On EL-4 6.1 I

In the C10 cells, the maximum binding was 1.33 ± 0.02 x 104 molecules / cell, and

Basically (see above) was 47 + 2 molecules / cell. Binding of 125 I-IL-1α, both to the I

transfected COS cells and to EL-4 6.1 C10 cells, could be completely outcompeted I

I of an excess of either unlabeled IL-1 α or unlabeled IL-1 β (Fig. 3C). Inhibition con- I

The 15 stants for IL-1 α and for IL-1 β were very similar for each cell type (Fig. 3C). IN

Example 7 I

I Preparation of monoclonal antibodies to IL-1 R I

In Preparations of purified recombinant IL-1R, e.g. human IL-1R, or transfice- I

In 20 ready COS cells expressing high levels of IL-1R are used to form

noclonal antibodies to IL-1R using conventional techniques, e.g. de i I

U.S. Pat. No. 4,411,993. Such antibodies are likely to be used

In delusion to affect IL-1 binding to IL-1 receptors, e.g. to improve toxic or I

In other adverse effects of IL-1.

For immunization of mice, the IL-1 R immunogen is emulsified in complete Freund's I

I! adjuvant and injected into Baib / c mice in amounts from 10-100 pg. 10 to 2 days later I

In, the immunized animals were boosted with additional immunogen emulsified in incomplete H

In Freund's adjuvant and is then periodically boosted according to a weekly or weekly im-

In munitions form. Serum samples are taken periodically by retroorbital vein loading or

In 30 tail tip excision for testing by dot-blot assay, ELISA test (enzyme-linked immune H

In sorbent assay) or inhibition of binding of 1251-IL-loc to extracts of EL-4 6.1 C10 I

In cells (as described above). Other assay methods are also suitable. After I

Upon detection of an appropriate antibody titer, an intravenous injection of antigen into saline-H is administered

In water for positive animals. 3 to 4 days later the animals are sacrificed, splenocytes are harvested and fused

In 35 res to the murine myeloma cell line NS1. By this method, hy- I

In bridoma cell lines, plated in multiple microtiter plates in a HAT-selective medium (hyx DK 175810 B1 31 poxanthin, aminopterin and thymidine) to inhibit proliferation of non-fused cells, mveloma hybrids and spleen cell hybrids.

Hybridoma clones thus generated can be screened by an ELISA assay for reactivity with IL-1R, e.g. by adapting the techniques described by Engvall et al., Immunochmeistry 8: 871 5 (1971) and in U.S. Patent No. 4,703,004. Positive clones are then injected into the peritoneal cavities of syngeneic Balb / c mice to produce ascites containing high concentrations (> 1 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 15 pXY120 is constructed as follows. plXY120 is identical to pYo-HuGM (ATCC 53157) except that it contains no cDNA insert and comprises a polylinker / multiple cloning site with an NcoI site. This vector comprises DNA sequences from the following sources: (1) a large SphI (nucleotide 562) to EcoRI (nucleotide 4361) fragment excised from plasmid pBR322 (ATCC 37017), including the replication region and the ampicillin resistance marker for selection in E. coli, (2) S. cerevisiae DNA comprising the TRP-1 marker, the 2 μm replication region, the ADH2 promoter, and (3) DNA encoding an 85 amino acid signal peptide derived from the gene encoding the secreted peptide-α factor (see Kurjan et al., U.S. Patent 4,546,082). An Asp718 restriction site was introduced at position 237 of the α-factor signal peptide to facilitate fusion to heterologous genes. This was accomplished by changing the thymidine residue at nucleotide 241 to a cytosine residue by oligonucleotide-directed in vitro mutagenesis as described by Craik, Bio-techniques: 12 (1985). A synthetic oligonucleotide containing multiple cloning sites and having the following sequences was inserted from the Asp718 site at amino acid 79 near the 3-terminus of the α-factor signal peptide to a SpeI site in the 2 μm sequence:

I DK 175810 B1 I

I 11

Asp718 Stul Ncol BamHI I

GTACCTTTGGATAAAAGAGACTACAAGGACGACGATGACAAGAGGCCTCCATGGAT ...

GAAACCTATTTTCTCTGATGTTCCTGCTGGTACTGTTCTCCGGAGGTACCTA ...

j <—— Polylinker— I

I Narrow Games I

... CCCCCGGGACA

... GGGGGCCCTGTGATC

I —Polylinker —- »| i I

IN

pBC120 also differs from pYoHuGM in the presence of a 514 bp DNA

I 5 fragment derived from the single-stranded phage f1 containing the replication region and j I

In the intergenic region that has been inserted at the Nru 1 site in the pBR322 sequence. IN

The presence of an f1 replication area allows the creation of single lines

DNA copies of the vector by transformation into appropriate strains of E. coli and superin- I

infection with bacteriophage f1, which facilitates DNA sequencing of the vector and

I 10 provides a basis for in vitro mutagenesis. To insert a cDNA, I is broken down

In pIXY 120 with Asp718 cleaving near the 3 'end of the α-factor leader peptide (nucleotide

I 237), and e.g. Ncol that splits in the polylinker. The large vector fragment is then purified

I and ligated to a DNA fragment encoding the protein to be expressed. H

To create a secretion vector for expression of human IL-1R I

15, a cDNA fragment is digested which contains the complete open reading frame contained in I

I encodes hIL-1 R, with an appropriate restriction endonuclease proximal to the N-terminus of I

the mature protein. Then one or more oligonucleotides capable of synthesis I are synthesized

I to ligate to the 5 'and 3' ends of the hIL-1R fragment, any codons being H

I omitted by isolating the fragment, regenerates and also provides cohesive I

In 20 terminals for Siging to pXY120 to provide a coding sequence which is located in

In there in frame with respect to an intact α-factor leader sequence. IN

I The resulting expression vectors are then purified and used for

In transformation of a diploid yeast strain of S. cerevisiae (XV2181) by standard technique, I

I nods, such as those described in EPA 0165654, selecting on the basis of tryp- I

I 25 tophan prototropher. The resulting transformants are grown for expression of a hIL-I

In 1R protein as a secreted or extracted product. Cultures to be analyzed for I

In hlL-1R expression, grown in 20-50 ml YPD medium (1% yeast extract, 2% peptone, 1% I

In glucose) at 37 ° C to a cell density of 1-5 x 108 cells / ml. For separation of cells from I

In medium, cells are removed by centrifugation and the medium is filtered through a 0.45 μm cellulose acetate filter before analysis. Supernatants prepared from the transformed yeast strain, or extracts prepared from disintegrated 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 An truncated version of the IL-1 receptor protein was prepared using an expression system compatible with the HELA-EBNA1 cell line that constitutively expresses the Epstein-Barr virus nucleus antigen driven by CMV-10 immediately. amplifies the promoter. The expression vector used was designated HAV-EO, a derivative of pDC201 that contains Epstein-Barr virus origin and allows a high level of expression in the HELA-EBNA cell line. HAV-EO is derived from pDC201 by replacing the late adenovirus major promoter with synthetic sequences from HIV-1 extending from the cap site of the viral mRNA, using the early SV40 promoter to drive expression of HIV 1 tat gene.

The expression construct for the soluble truncated IL-1 receptor was generated stepwise. The entire coding region of the receptor and a portion of the untranslated 5 'region was removed from the original IL-1 receptor clone 78 by digestion with Asp 718 and NdeI. This fragment, which did not contain untranslated 3 'sequences, was cloned into HAV-EO to give HAV-EO-FL9. A variant of this plasmid, which contained a translational stop codon immediately after the codon for proline 316 and which lacked the entire coding sequence 3 'for it, was then constructed by standard methods and called HAV-EO-MEXT.

HAV-EO-MEXT vector DNA was introduced into HELA-EBNA cells by a modified polybrene transfection as described by Kavai and Nishizava (Mol. Cell Biol.

4: 1172, 1984). 1.5 x 10 6 cells were seeded in 10 ml DMEM + 10% FCS in a 10 cm tissue culture dish, Cells were incubated at 37 ° C, 10% CO 2 for 16 hours. The media was then removed and 3 ml of serum-free DMEM containing 10 pg / ml DNA and 30 pg / ml polybrene (Sigma) was added. The dishes were then incubated at 37 ° C / 10% CO 2 for an additional 6 hours, at which time the DNA mixture was removed and the cells were subjected to glycerol shock by adding 3 ml of serum-free DMEM + 25% glycerol (v / v) for 1 minute. . Glycerol was removed and the cells were washed twice with medium. Then, 10 ml of DMEM + 10% FCS was added and the cells were incubated at 37 ° C / 10% CO 2 for 18 hours.

Transfected cells were then removed with trypsin and divided into a ratio of 1: 9 in T175 cm 2 flasks (to obtain about 10% confluence) containing 25 ml

DK 175810 B1 I

34

DMEM + 1% FCS. Supernatants containing temporarily expressed soluble murine IL-I

1 receptor was harvested every 24 hours for up to 10 days. IN

IL-1α binding activity in the medium was measured by inhibition of 125IL-1α to EL4 I

6.1 C10 cells as described by Mosley et al. (J. Biol. Chem. 267: 2941, 1987) with I

5 exception, that the label IL-1a (2x10-11, 50 μl) was first incubated with test sample I

(50 μΙ) for two hours at 8 ° C before adding cells (2.5 x 10e cells, 50 μΙ). Each test sample

was analyzed in 6 dilutions (X3) and the inhibitory dose-response curve was used for H

determination of the relative inhibitory titer. H

Soluble IL-1 receptor was purified from culture supernatants such as H

10 described for natural receptor by Urdal et al. (J. Biol. Chem. 263: 280, 1988). Kultursu- I

pematants were passed over a 1 ml bed volume IL-1 oc column, the column was washed with H

PBS and eluted with 0.1 M glycine-HCl. Acid eluate fractions were neutralized with the I

same and then tested for IL-1 binding activity using radiore- I

receptor inhibition assay. SDS-polyacrylamide gel electrophoresis of the acid-treated I

15 ling eluted material showed that it contained two bands with Mr 60,000 and 54,000. N- I

glycanase treatment of this material showed that the size heterogeneity is due to H

differences in N-linked glycosylation between the two species. Soluble IL-1 receptor maintained- I

there full IL-1 binding activity. H

DK 175810 B1 35

Table 1: cDNA sequence of IL-1R clone in GEMBI78 1 5 '-TGGGTCGTCT GACTAGAAGT GAGCTGTCTG ΤΠΑΤΤΓΤΤΓΓΓ artrr.iy ^ hnr

51 CCAGTAATCA TTTGGAGGCA AAGCAAACTG TAAGTAATGC TGTCCTGGGC

101 TGACTTCAGG AGGCAGITTT CGTTTTAACA GCCAGTGTTT ATTTGCTCAG

151 CAAACGTTGT CTCGGGGAGA AATGTCGCTG GATGTCATCA GAGTTCCCAG

201 TGCCCCGAAC CGTGAACAAC ACAAATGGAG AATATGAAAG TGCTACTGGG

251 GCTCATTTGT CTCATGCTGC CTCTCCTGTC CCTCCACATT CACCTATGTA

301 CAGAATATCC AAATCAGATC GTTTTGXTTT TATCTGTAAA TGAAATTGAT

351 ATTCGCAAGT GTCCTCTTAC TCCAAATAAA ATGCACGGCG ACACCATAAT

401 TTGGTACAAG AATGACAGCA AGACCCCCAT ATCAGCGGAC CGGGACTCCA

451 GGATTCATCA GCAGAATGAA CATCTTTGGT TTGTACCTGC CAAGGTGGAG

501 GACTCAGGAT ATTACTATTG TATAGTAAGA AACTCAACTT ACTGCCTCAA

551 AACTAAAGTA ACCGTAACTG TGTTAGAGAA TGACCCTGGC TTGTGTTACA

601 GCACACAGGC CACCTTCCCA CÅGCGGCTCC ACATTGCCGG GGATGGAAGT

651 CTTGTGTGCC CTTATGTGAG TTATTTTAAA GATGAAAATA ATGAGTTACC

701 CGAGGTCCAG TGGTATAAGA ACTGTAAACC TCTGCTTCTT GACAACGTGA

751 GCTTCTTCGG AGTAAAAGAT AAACTGTTGG TGAGGAATGT GGCTGAAGAG

801 CACAGAGGGG ACTATATATG CCGTATGTCC TATACGTTCC GGGGGAAGCA

851 ATATCCGGTC ACACGAGTAA TACAATTTAT CACAATAGAT GAAAACAAGA

901 GGGACAGACC TGTTATCGTG AGCCCTCGGA ATGAGACGAT CGAAGCTGAC

951 CCAGGATCAA TGATACAACT GATCTGCAAC GTCACGGGCC AGTTCTCAGA

1001 CCTTGTCTAC TGGAAGTGGA ATGGATCAGA AATTGAATGG AATGATCCAT

1051 TTCTAGCTGA AGACTATCAA TTTGTGGAAC ATCCTTCAAC CAAAAGAAAA

1101 TACACACTCA TTACAACACT TAACATTTCA GAAGTTAAAA GCCAGTTTTA

1151 TCGCTATCCG TTTATCTGTG TTGTTAAGAA CACAAATATT TTTGAGTCGG

1201 CGCATGTGCA GTTAATATAC CCAGTCCCTG ACTTCAAGAA TTACCTCATC

1251 GGGGGCTTTA TCATCCTCAC GGCTACAATT GTATGCTGTG TGTGCATCTA

1301 TAAAGTCTTC AAGGTTGACA TAGTGCTTTG GTACAGGGAC TCCTGCTCTG

1351 GTTTTCTTCC TTCAAAAGCT TCAGATGGAA AGACATACGA TGCCTATATT

1401 CTTTATCCCA AGACCCTGGG AGAGCGGTCC TTCTCAGACT TAGATACTTT

1451 TGTTTTTAAA CTGTTGCCTG AGGTCTTGGA GGGACAGTTT GGATACAAGC

1501 TGTTCATTTA TGGAACGGAT GACTATGTTG GAGAAGATAC CATCGAGGTT

1551 ACTAATGAAA ATGTAAAGAA AAGCAGGAGG CTGATTATCA TTCTAGTGAG

1601 AGATATGGGA GGCTTCAGCT GGCTGGGCCA CTCATCTGAA GAGCAAATAG

1651 CCATATACAA TGCTCTCATC CAGGAAGGAA TTAAAATCGT CCTGCTTGAG

1701 TTGGAGAAAA TCCAAGACTA TGAGAAAATG CCAGATTCTA TTCAGTTCAT

1751 TAAGCAGAAA CACGGAGTCA TTTGCTGGTC AGGAGACTTT CAAGAAAGAC

1801 CACAGTCTCC AAAGACCAGG TTCTGGAAAA ACTTAAGATA CCAGATGCCA

1851 GCCCAACGGA GATCACCATT GTCTAAACAC CGCTTACTAA CCCTGGATCC

1901 TGTGCGGGAC ACTAAGGAGA AACTGCCGGC AGCAACACAC TTACCACTCG

1951 GCTAGCATGG CAAAAGTGGG CAGGCCAAGA ACTTCGGAAT ATCTCCCATC

2001 ATAAGAGGCT GCAGCTGGGC TGTGCCTCCC AGTAAAACAG TCACGAACCA

2051 AACCTGTGCA GTCCCTTGTT CCAGATCACC TGGAACTGGA TTGGGAAGAG

2101 AACAGGACTT GGTGGCCAGG ACCGCTCAGA GAGCCATGGT TGCTCAGGGA

2151 TGCTGCTCCG GGATGCTTCA CTAACAGTCG AGGCAGTGAA CTGGGTGTAG

2201 AAAGCGTCAG GAAATGGCCA CATGTnrnr ^ A TGGTTTAATT AGATTCTGTG

2251 GAGTCTCACA GTGGGATTGT GGCTGTCTGA GGAGACTTTG GGGGGTCGCT

2301 GTCCAAGAAG TGGCTCCCCA AAGTATAAGT GCGGGTGAGG TTTACTGATA

! 2351 CCCCAC-3 '

I DK 175810 B1 I

I 36 I

In Table 2A: Sequence of coding region of murine IL-1 receptor I

I 5'-ATC GAG AAT ATG AAA GTG CTA CTC CGC CTC ATT TGT CTC ATG GTG -15 I

I Met Clu Asn Hct Lys Tel leu Leu Gly Leu Ile Cys Leu Net Val I

I CCT CTG CTG TCG CTG GAG ATT GAC CTA TCT ACA CAA TAT CCA AAT 33 I

I Pro Leu Leu Ser Leu Clu Ile Asp Val Gys Thr Clu Tyr Pro Asn 11 I

I CAG ATC CTT TTC TTT TTA TCT CTA AAT CAA ATT CAT ATT CCC AAC 78 I

I Gin Ile Val Leu Phe Leu Ser Val Asn Glu Ile Asp Ile Arg Lys 26, I

TGT CCT CTT AGT CCA AAT AAA ATG GAC GGC GAC ACC ATA ATT TCG 123 I

I Cys Pro Leu Thr Fro Asn Lys Met Bis Gly Asp Thr Ile Ile Trp 41 I

I TAC AAG AAT CAC ACC AAC ACC CCC ATA TCA CCC GAC CGC GAC TCC 168 I

I Tyr Lys Asn Asp Ser Lys Thr Pro Ile Ser Ala Asp Arg Asp Ser 36 I

AGG ATT CAT CAG CAG AAT GAA CAT CTT TGG ITT CTA CCT GCC AAG 213 I

I Arg Ile Bis Gin Gin Asn Glu Bis Leu Trp Fhe Val Pro Ala Lys 71 I

I GTG GAG GAC TCA GGA TAT TAC TAT TGT ATA GTA AGA AAC TCA ACT 258 I

Val Glu Asp Ser Gly Tyr Tyr Tyr Cys Ile Val Arg Asn Ser Thr 86 I

TAC TGC CTC AAA ACT AAA CTA ACC CTA ACT CTC TTA GAG AAT GAC 303 I

Tyr Cys Leu Lys Thr Lys Val Thr Val Thr Val Leu Glu Asn Asp 101 I

CCT GCC TTG TGT TAC AGC ACA CAG GCC ACC TTC CCA CAG CGG CTC 348 I

Pro Gly Leu Cys Tyr Ser Thr Gin Ala Thr Phe Pro Gin Arg Leu 116 I

I CAC ATT GCC GGG GAT GGA AGT CTT CTG TCC CCT TAT GTG AGT TAT 393 I

Bis Ile Ala Gly Asp Gly Ser Leu Val Cys Pro Tyr Val Ser Tyr 131 I

I ITT AAA GAT GAA AAT AAT CAG TTA CCC GAG CTC CAG TGG TAT AAG 438 I

Phe Lys Asp Glu Asn Asn Glu Leu Pro Glu Val Gin Trp Tyr Lys 146 I

I AAC TCT AAA CCT CTC CTT CTT CT AAC CTG ACC TTC TTC GGA GTA 4B3 I

Asn Cys Lys Pro Leu Leu Leu Asp Asn Vel Ser Phe Phe Gly Val 161 I

I AAA GAT AAA CTG TTG GTG AGG AAT GTG CCT GAA GAG CAG AGA GGG 528 I

Lys Asp Lys Leu Leu Val Arg Asn Val Ala Glu Glu Bis Arg Gly 176 I

GAC TAT ATA TGC CCT ATG TCC TAT AOG TTC CGG GGG AAG CAA TAT 573 I

Asp Tyr Ile Cys Arg Net Ser Tyr Thr Phe Arg Gly Lys Gin Tyr 191 I

CCC CTC ACA CCA GTA ATA CAA TTT ATC ACA ATA GAT GAA AAC AAG 618 I

Pro Val Thr Arg Val Ile Gin Phe Ile Thr Ile Asp Glu Asn Lys 206 I

I DK 175810 B1

Table 2B: Sequence of codinino region of murine IL-1 receptor ACO GAC AGA CCT GTT KTC CTC AGC OCT OGG AAT GAG ACG ATC GAA 663

Arg Asp Arg Pro Val Ile Len Ser Pro Arg Asn Glu Thr Ile Glu 221

CCT GAC CCA CCA TCA ATG ATA CAA CTG ATC tCC AAC CTC ACC CCC 70S

Ale Asp Pro Gly Ser Met Ile Gin leu Ile Cys Asn Tel Thr Gly 236 CAG TTC TCA GAC CTT CTC TAC TGC AAG TGC AAT GGA TCA GAA ATT 753

Cln Phe Ser Asp Leu Tal Tyr Trp Lys Trp Asn Gly Ser Glu Ile 231 GAA TGC AAT GAT CCA TTT CTA CCT GAA GAC TAT CAA ITT CTC GAA 79Θ

Glu Trp Asn Asp Pro Phe Leu Ala Glu Asp lyr Gin Phe Vel Glu 266 CAT CCT TCA ACC AAA AGA AAA TAC ACA CTC ATT ACA ACA CTT AAC 843

Bl «Pro Set Thr Lys Arg Lys Tyr Thr Leu Ile Thr Thr Leu Asn 281

ATT TCA GAA CTT AAA AGC CAG TTT TAT CGC TAT CCG TTT ATC TGT 88B

Ile Ser Glu Val Lys Ser Gin Phe Tyr Arg Tyr Pro Phe Ile Cys 296 GTT GTT AAG AAC ACA AAT ATT TTT GAG TCC G CG CAT CTG CAG TTA 933

Val Val Lys Asn Thr Asn Ile Phe Glu Ser Ala Bis Val Gin Leu 311 ATA TAC CCA CTC CCT GAC TTC AAC AAT TAC CTC ATC GGG CGC TTT 978

Ile Tyr Pro Val Pro Asp Phe Lys Asn Tyr leu Ile Gly Gly Phe 326 ATC ATC CTC ACG CCT ACA ATT GTA TGC TGT CTG TCC ATC TAT AAA 1023

Ile Ile Leu Thr Ala Thr Ile Val Cys Cys Val Cys Ile Tyr Lys 341 CTC TTC AAG GTT GAC ATA CTG CTT TGC TAC ACG GAC TCC TGC TCT 1068

Val Phe Lys Val Asp Ile Vel Leu Trp Tyr Arg Asp Ser Cys Ser 336 CCT ITT CTT CCT TCA AAA CCT TCA CAT CCA AAC ACA TAC CAT CCC 1113

Gly Phe Leu Pro Ser Lys Ale Ser Asp Asp Gly Lys Thr Tyr Asp Ala 371 TAT ATT CTT TAT CCC AAG ACC CTG GGA GAG GGG TCC TTC TCA GAC 1138

Tyr Ile Leu Tyr Pro Lys Thr Leu Gly Glu Gly Ser Phe Ser Asp 386 TTA GAT ACT TTT CTT TTT AAA CTC TTG CCT CAG CTC TTC CAG GGA 1203

Leu Asp Thr Phe Val Phe Lys Leu leu Pro Glu Val Leu Glu Gly 401 CAG TTT GGA TAC AAG CTG TTC ATT TAT GGA AGG GAT CAC TAT GTT 1248

Cln Phe Gly Tyr Lys leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val 416 CCA GAA GAT ACC ATC GAG GTT ACT AAT GAA AAT GTA AAG AAA AGC 1293

Gly Glu Asp Thr Ile Glu Val Thr Asn Glu Asn Val Lys Lys Ser 431

I DK 175810 B1 I

I 38 I

In Table 2C: Sequence of codinino region of murine IL-1 receptor gene I

AGG AGG CTG ATT ATC ATT CTA GTG AGA GAT ATG GGA GGC TTC AGC 1338 I

I Arg Arg Leu Ile Ile Ile Leu Val Arg Asp Het Gly Gly Phe Ser 446 I

I TGG CTG GGC CAG TCA TCT GAA GAG CAA ATA GCC ATA TAC AAT GCT 1383 I

I Trp Leu Gly Gin Ser Ser Glu Glu Gin Ile Ala Ile tyr Asn Ale 461 I

I CTC ATC CAG GAA GGA ATT AAA ATC GTC CTG CTT GAG TTG GAG AAA 1428 I

I Leu Ile Gin Glu Gly Ile Lys Ile Val Leu Leu Glu Leu Glu Lys 476

ATC CAA GAC TAT GAG AAA ATG CCA GAT TCT ATT CAG TTC ATT AAG 1473 I

I Ile Gin Asp Tyr Glu Lys Het Pro Asp Ser Ile Gin Phe Ile Lys 491 I

I CAG AAA CAC GGA GTC ATT TGC TGG TCA GGA GAC TTT CAA GAA AGA 151B I

Gin Lys Bis Gly Val Ile Cys Trp Ser Gly Asp Phe Gin Glu Arg 506 I

I CCA CAG TCT GCA AAG ACC AGG TTC TGG AAA AAC TTA AGA TAC CAG 1563 I

Pro Gin Ser Ala Lys Thr Arg Phe Trp Lys Asn Leu Arg Tyr Gin 521 I

H ATG CCA GCC CAA CGG AGA TCA CCA TTG TCT AAA CAC CGC TTA CTA 1608 I

Net Pro Ala Gin Arg Arg Ser Pro Leu Ser Lys His Arg Leu Leu 536 I

ACC CTG GAT CCT GTG CGG GAC ACT AAG GAG AAA CTG CCG GCA GCA 1653 I

Thr Leu Asp Pro Val Arg Asp Thr Lys Glu Lys Leu Pro Ala Ala 551 I

I ACA CAC TTA CCA CT C GGC TAG-3 '1671 I

Thr His Leu Pro Leu Gly End 557 I

DK B1 175 810 39 1.5 * -AGACGCACCC TCTCAAOATG CTCCACTCCC TCCTCACAAC CTCGCACCCC 51 TTGCTAAAAG ACAAGGCCTT CTCCAAGAAG AATATCAAAC TCTTACTCAG 101 ACTTATTTGT TTCATAGCTC TACTGATTTC TTCTCTGGAG GCTGATAAAT 151 6GAAGGAAGG TGAAGAAAAA ATAATTTTAG TGTCATCTGC AAATGAAATT 201 GATGTTCGTC CCTCTCCTCT TAACCCAAAT GAACACAAAG GCACTATAAC 251 TTGGTATAAA GATGACAGCA AGACACCTGT ATCTACAGAA CAAGCCTCCA 301 GGATTCATCA ACACAAAGAG AAACTTTGGT TTGTTCCTCC TAAGGTGGAG 351 GATTCAGGAC ATTACTATTC CGTGGTAAGA AATTCATCTT ACTCCCTCAC A01 aattaaaata ACTGCAAAAT ttctggagaa tcagcctaac ttatgttata A51 atgcacaagc catatttaag cagaaactac ccgttgcagg agacggagga

301 CTTGTCTCCC CTTATATGCA CTTTTTTAAA AATGAAAATA ATCAGTTACC 551 TAAATTACAG TGGTATAAGC ATTGCAAACC TCTACTTCTT CACAATATAC 601 ACTTTAGTGG ACTCAAAGAT AGCCTCATOG TGATGAATGT GGCTGAAAAG 651 CATAGAGGGA ACTATACTTG TCATGCATCC TACACATACT TGGGCAAGCA 701 ATATCCTATT ACCCGGGTAA TAGAATTTAT TACTCTAGAG GAAAACAAAC 751 CCACAAGGCC TGTGATTGTG AGCCCAGCTA ATGAGACAAT GGAAGTACAC B01 TTGGGATCCC AGATACAATT GATCTCTAAT GTCACCGGCC AGTTGAGTGA 851 CATTGCTTAC TGGAACTCGA ATGGGTCAGT AATTGATGAA GATGACCCAG 901 TGCTAGGGGA AGACTATTAC AGTGTCGAAA ATCCTGCAAA CAAAAGAAGG 951 AGTACCCTCA TCACACTGCT TAATATATCG CAAATTGAAA CTAGATTTTA 1001 TAAACATCCA TTTACCTGTT TTGCCAAGAA TACACATGGT ATAGATGCAG 1051 CATATATCCA CTTAATATAT CCAGTCACTA ATTTCCAGAA GCACATGATT 1101 GGTATATCTG TCACGTTGAC AGTCATAATT GTGTGTTCTG TTTTCATCTA 1151 TAAAATCTTC AACATTGACA TTCTCCTTTG CTACAGGGAT TCCTGCTATG 1201 ATTTTCTCCC AATAAAAGCT TCAGATGGAA AGACCTATGA CGCATATATA 1251 CTGTATCCAA AGACTCTTGG GGAAGCGTCT ACCTCTGACT GTGATATTTT 1301 TGTGTTTAAA GTCTTGCCTG AGGTCTTGGA AAAACAGTGT G GATATAAGC 1351 TCTTCATTTA TGGAACGGAT GACTACCTTG GGGAACACAT TGTTGAGGTC 1401 ATTAATGAAA ACGTAAAGAA AAGCAGAAGA CTGATTATCA TTTTAGTCAG 1451 ACAAACATCA GGCTTCAGCT CGCTGGCTGG TTCATCTCAA GAGCAAATAC 1501 CCATGIATAA TGCTCTIGTT CAGGATGGAA TTAAAGTTGT CCTGCTTGAG 1551 CTGGAGAAAA TCCAAGACTA TGAGAAAATG CCAGAATCGA TTAAATTCAT 1601 TAAGCAGAAA CATGGGGCTA TCCGCTGGTC AGGGGACTTT ACACAGGGAC 1651 CACAGTCTGC AAAGACAAGG TTCTGCAAGA ATGTCAGGTA CCACATGCCA 1701 Gtccaccgac GGTCACCTTC ATCTAAACAC CACTTACTCT CACCAGCCAC 1751 TAAGCAGAAA CTGCAAAGAG AGGCTCACGT GCCTCTCCGG TAGCATGGAG 1801 AAGTTGCCAA GAGTTCTTTA GCTGCCTCCT CTCTTATGGC GTTCCAGGCC 1851 AGGTTATGCC TCATGCTGAC TTGCAGAGTT CATGGAATGT AACTATATCA 1901 TCCTTTATCC CTGAGGTCAC CTGCAATCAG ATTATTAAGG GAATAAGCCA 1951 TGACGTCAAT AGCAGCCCAG GGCACTTCAG AGTAGAGGGC TTGGGAAGAT 2001 CTTTTAAAAA GGCAGTAGGC CCGGTGTGGT GGCTCACGCC TATAATCCCA 2051 GCACTTTGGG AGGCTGAAGT GGCTGCATCA CCAGAGGTCA GGACTTCGAG 2101 ACGACCCCAG CCAACATGGC AAAACCCCAT CTCTACTAAA AATACAAAAA 2151 TGAGCTAGGC ATGGTGGCAC ACC CCTGTAA TCCCAGCTAC ACCTGACGCT 2201 CAGGCAGGAG AATTCCTTCA ACCCGGGAGA CGGAGCTTGC AGTGAGCCGA 2301 GTTTGGGCCA CTGCACTCTA GCCTGGCAAC AGAGCAAGAC TCCCTCTCAA 2351 AAAAAGCGCA ATAAATGCCC TCTCTGAATG TTTGAACTGC CAAGAAAAGG 2401 CATGGAGACA GCGAACTAGA AGAAAGGGCA AGAAGGAAAT AGCCACCGTC 2451 TACACATGGC TTACTTAAGT CATCCACAGC CCAAGGCCGG CGGCTATGCC 2501 TTGTCIGGGG ACCCTGTACA CTCACTGACC CTGGAGCGGC TCTCCTGAGA 2551 GGTGCTGCAG CCAAAGTGAG ACTGACACCT CACTGAGGAA CGGAGACATA

2601 TTCTTGGAGA ACTTTCCATC TGCTTGTATT TTCCATACAC A7CCCCAGCC-3 '

Table 3: cDNA sequence of human IL-1R construct

40 I

DK 175810 B1 I

Table 4A: Sequence of codninase region of human IL-1 receptor gene I

ATG AAA GTC TTA CTC ACA CIT ATT TGT TTC ATA CCT CTA CTG ATT -9 I

Het Lys Val Leu Leu Arg Leu Ile Cys Phe Ile Ala Lev Lev Ile -3 I

TCT TCT CTG GAG CCT CAT AAA TCC AAG GAA CCT CAA GAA AAA ATA 39 I

Ser Ser bro Glu A1 & Asp Lys Cys Lys Clu Arg Clu Clu Lys Ile 13 H

ATT TTA CTC TCA TCT GCA AAT CAA ATT GAT GTT CCT CCC TGT CCT 84

Ile Leu Val Ser Ser Ala Asn Glu Ile Asp Val Arg Pro Cys Pro 28 ^ I

CTT AAC CCA AAT GAA CAC AAA GGC ACT ATA ACT TGG TAT AAA GAT 129 I

Leu Aan Pro Asn Glu His Lys Gly Thr Ile Thr Trp Tyr Lys Asp 43 I

GAC AGC AAG ACA CCT CTA TCT ACA GAA CAA GCC TCC ACG ATT CAT 174 I

Asp Ser Lys Thr Pro Val Ser Thr Glu Gin Ala Ser Arg Ile His 58 I

CAA CAC AAA GAG AAA CTT TGG TTT CTT CCT GCT AAG CTG GAG GAT 219 I

Gin His Lys Glu Lys Leu Trp Phe Val Pro Ala Lys Val Glu Asp 73

TCA GGA CAT TAC TAT TCC GTG CTA AGA AAT TCA TCT TAC TGC CTC 264 I

Ser Gly His Tyr Tyr Cys Val Val Arg Asn Ser Ser Tyr Cys Leu 88 H

AGA ATT AAA ATA AGT GCA AAA TTT CTC GAC AAT GAG CCT AAC TTA 309 I

Arg Ile Lys Ile Ser Ala Lys Phe Val Glu Asn Glu Pro Asn Leu 103 I

TGT TAT AAT GCA CAA GCC ATA TTT AAG CAG AAA CTA CCC GTT GCA 354 I

Cys Tyr Asn Ala Gin Ala Ile Phe Lys Gin Lys Leu Pro Val Ala 118 I

GGA GAC GGA GGA GTA CTT GTG TGC CCT TAT ATG GAG TTT TTT AAA AAT 399 I

Gly Asp Gly Gly Gly Leu Val Cys Pro Tyr Het Glu Phe Phe Lys Asn 133 I

CAA AAT AAT GAG TTA CCT AAA TTA CAC TGG TAT AAG GAT TCC AAA 444 I

Clu Asn Asn Glu Leu Pro Lys Leu Gin Trp Tyr Lys Asp Cys Lys 148

CCT CTA CTT CTT CAC AAT ATA CAC ITT AGT GGA CTC AAA GAT AGG 489 I

Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly Vel Lys Asp Arg 183 I

CTC ATC CTG ATG AAT GTG GCT GAA AAG CAT AGA GGG AAC TAT ACT 534 I

Leu Ile Val Het Asn Val Ala Glu Lys Bis Arg Gly Asn Tyr Thr 178 I

TGT CAT CCA TCC TAC ACA TAC TTC GGC AAG CAA TAT CCT ATT ACC 579 I

Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gin Tyr Pro Ile Thr 193 I

CCC CTA ATA CAA TTT ATT ACT CTA GAG GAA AAC AAA CCC ACA AGG 624 I

Arg Val Ile Glu Phe Ile Thr Leu Glu Glu Asn Lys Pro Thr Arg. 208 I

CCT GTG ATT GTG AGC CCA GCT AAT GAG ACA ATG GAA CTA GAC TTG 669 I

Pro Val Ile Val Ser Pro Ala Asn Glu Thr Het Glu Val Asp Leu 223 I

DK 175810 B1 I

Table 4B: Sequence of coding region of human IL-1 receptor gene I

CCA TCC CAC ATA CAA TTG ATC TCT AAT CTC ACC CCC CAC TTG ACT 714 Gly Ser Cln Ile Gin Leu Ile Cys Asn Val Thr Cly Cln Leu Ser 238 GAC ATT GCT TAC TGG AAC TOG AAT GGG TCA GTA ATT CAT CAA CAT 739

Asp Ile Aln Tyr Trp Lys Trp Asn Cly Ser Val Ile Asp Glu Asp 253 CAC CCA CTC CTA GGG CAA CAC TAT TAC ACT CTC CAA AAT CCT GCA 804

Asp Pro Tal Leu Gly Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala 268 AAC AAA AGA AGG AGT ACC CTC ATC ACA CTC CTT AAT ATA TCG GAA 869

Asn Lys Arg Arg Ser Thr Leu Ile Thr Val Leu Asn Ile Ser Glu 283 ATT GAA ACT AGA TTT TAT AAA CAT CCA ΤΤΓ ACC TCT TTT CCC AAG 894

Ile Clu Ser Arg Phe Tyr Lys His Pro Phe Thr Cys Phe Ala Lys 298 AAT ACA CAT GCT ATA GAT CCA CCA TAT ATC CAC TTA ATA TAT CCA 939

Asn Thr His Gly Ile Asp Ala Ala lyr Ile Gin Leu Ile Tyr Pro 313 I CTC ACT AAT TIC CAC AAG CAC ATC ATT CCT ATA TGT CTC ACG TTG 984

Val Thr Asn Phe Gin Lys Bis Met Ile Gly Ile Cys Val Thr Leu 328 ACA CTC ATA ATT GTG TGT TCT CTT TTC ATC TAT AAA ATC TTC AAC 1029

Thr Vel Ile Ile Val Cys Ser Val Phe Ile Tyr Lys Ile Phe Lys 343 ATT GAC ATT CTC CTT TGG TAC AGG GAT TCC TCC TAT CAT TTT CTC 1074

Ile Asp Ile Val Leu Trp Tyr Arg Asp Ser Cys Tyr Asp Phe Leu 358 CCA ATA AAA GCT TCA GAT CGA AAG ACC TAT GAC GCA TAT ATA CTG 1119

Pro Ile Lys Ala Ser Asp Gly Lys Thr Tyr Asp Ala Tyr Ile Leu 373 TAT'CCA AAG ACT CTT GGG GAA GGG TCT ACC TCT GAC TCT GAT ATT 1164 Tyr Pro Lys Thr Val Gly Glu Gly Ser Thr Ser Asp Cys Asp Ile 388 TTT CTG TTT AAA CTC TTG CCT GAG CTC TTG GAA AAA CAG TGT GGA 1209

Phe Vel Pha Lys Val Leu Pro Glu Val Leu Glu Lys Gin Cys Gly 403 TAT AAG CTG TTC ATT TAT GGA AGG GAT GAC TAC GTT GGG GAA GAC 1254

Tyr Lys Leu Phe Ile Tyr Cly Arg Asp Asp Tyr Vel Gly Glu Asp 418 ATT CTT GAG CTC ATT AAT GAA AAC CTA AAG AAA AGC AGA AGA CTG 1299

Ile Val Glu Val Ile Asn Glu Asn Val Lys Lys Ser Arg Arg Leu 433 ATT ATC ATT TTA CTC AGA GAA ACA TCA GGC TTC ACC TGG CTG CCT 1344

Ile Ile Ile Ile Leu Val Arg Glu Thr Ser Gly Phe Ser Trp Leu Gly 448 GCT TCA TCT CAA GAG CAA ATA GCC ATG TAT AAT GCT CTT CTT CAG 1389

Cly Ser Ser Glu Clu Gin Ile Ala Het Tyr Asn Ala Leu Val Gin 463 CAT CGA ATT AAA CTT CTC CTG CTT CAG CTG CAC AAA ATC CAA GAC 1434

Asp Gly Ile Lys Val Val Leu Leu Glu Leu Clu Lys Ile Gin Asp 478

DK 175810 B1 I

IN

Table 4C: Sequence of codinase regions of the human IL-1 receptor I

I TAT GAG AAA ATG CCA C AA TCG ATT AAA TTC ATT AAG CAG AAA CAT 1479 I

I Tyr Glu Lys Net Pro Glu Ser Ile Lys Phe Ile Lys Gin Lys Bis 493 I

CGG CCT ATC CGC TGG TCA GGG GAC ΤΤΓ ACA CAG GCA CCA CAG TCT 1524 I

Gly Ala Ile Arg Trp Ser Gly Asp Phe Thr Gin Gly Pro Gin Ser 508 I

I GCA AAG ACA AGG TTC TGG AAG AAT GTC AGC TAC CAC ATG CCA GTC 1569 I

Ala Lys Thr Arg Phe Trp Lys Asn Val Arg Tyr Bis Net Pro Val 523 I

I CAG CGA CGG TCA CCT TCA TCT AAA CAC CAG TTA CTG TCA CCA GCC 1614 I

I Gin Arg Arg Ser Pro Ser Ser Lys Bis Gin Leu Leu Ser Pro Ala 538 I

I ACT AAG GAG AAA CTG CAA AGA GAG GCT CAC GTG CCT CTC GGG TAG 1656 I

Thr Lys Glu Lys Leu Gin Arg Glu Ala Bis Val Pro Leu Gly End 552 I

I I

I 'I

Η I

43 DK 175810 B1

Table 5: Comparison of human oo murine 1L-1 receptor amino acid sequence in f 1 = 1 ΙξΙ j-1 1 | -μ | Lj I-j ij fif * l = j * pa j: j 1-1 1 = 1 | J ||| j: | | il - | = | |: | jfj | ξ | 1 I | = | , | = | | z ^

U Sil Sil “111 · | J III

in

Claims (7)

44 I DK 175810 B1 I A homogeneous biologically active soluble human interleukin-1 receptor (IL-1R) composition, wherein said human IL-1R has an amino acid sequence more than 80% homologous to the amino acid sequence shown in Table 4A-4C. H or to that portion of the sequence consisting of amino acids 1-319 shown in said Figures H and wherein said IL-1R does not have a cytoplasmic or transmembrane region. IN An isolated IL-1 receptor comprising the amino acid sequence consisting of amino acids 1-319 of Table 4A-4C. IN An isolated soluble IL-1 receptor comprising a soluble I form of the polypeptide of Table 4A-4C, wherein the soluble IL-1 receptor has IL-1 I binding activity and wherein said IL-1R does not have a cytoplasmic or trans In 15 membrane region. IN A soluble human IL-1 receptor for medical use, wherein the receptor has an amino acid sequence that is more than 80% homologous to the amino acid sequence shown in Tables 4A-4C or to that portion of the amino acid sequence. 1-319 shown in said figures and wherein said IL-1R does not have a cytoplasmic or transmembrane region. H Use of a soluble human IL-1 receptor for the manufacture of a medicament for regulating immune or inflammatory responses, wherein the receptor has an amino acid sequence that is more than 80% homologous to the amino acid H sequence shown in Table 4A-4C or to that part of the sequence consisting of amino acids H 1-319 shown in said figures and wherein said IL-1R does not have a cytoplasmic H or transmembrane region. H Pharmaceutical composition suitable for parenteral administration to a human patient for regulating immune or inflammatory responses, H CHARACTERIZED in that it comprises an effective amount of a soluble human IL-1 I receptor according to Table 4A- 4C in admixture with a suitable diluent or carrier. IN Antibody that is immunoreactive with an IL-1 receptor having H having amino acids 1-552 in Table 4A-4C. IN