IE83679B1 - Type II interleukin-1 receptors - Google Patents

Description

The present invention relates generally to cytokine receptors, and more specifically, to Interleukin-1 receptors. ‘_ Interleukin- la (IL-la) and Interleukin-1B and (IL-13) are distantly related polypeptide hormones which play a central role in the regulation of immune and inflammatory responses. These two proteins act on a variety of cell types and have multiple biological activities. The diversity of biological activity ascribed to lL- la and IL- 113 is mediated by specific plasma membrane receptors which bind both IL-la and IL-13.

Due to the wide range of biological activities mediated by IL-la and IL-15 it was originally believed that the IL-1 receptors should be highly conserved in a variety of species and expressed on a large variety of cells.

Structural characterization by ligand affinity cross-linking techniques has demonstrated that, despite their significant divergence in sequence, IL-10: and IL-13 bind to the same cell surface receptor molecule on T cells and fibroblasts (Dower et al., Nature (London) 324:266, 1986; Bird et al., Nature (London) 3242263, 1986; Dower et al., Proc.

Natl. Acad. Sci. USA 83:1060, 1986). The IL-1 receptor on murine and human T cells has been identified by CDNA expression cloning and N—terrninal sequence analysis as an integral membrane glycoprotein that binds IL—la and IL-13 and has a molecular weight of 80,000 kDa (Sims et al., Science 24I:585, 1988; Sims et al., Proc. Natl. Acad. Sci. USA 86:8946, 1989).

It is now clear, however, that this 80 kDa IL-1 receptor protein does not mediate all the diverse biological effects of IL-1. Subsequent affinity cross-linking studies indicate that IL-1 receptors on the Epstein Barr virus (EBV)-transformed human B cell lines VDS-O and 3B6, the EBV-positive Burkitt's lymphoma cell line Raji, and the murine pre-B cell line 70Z/3, have a molecular weight of 60,000 to 68,000 kDa (Matsushima et al., J.

Immunol. I,36:4496, 1986; Bensimon et al., J. Immunol. 14212290, 1989; Bensimon et al., J. Immunol. I43:1168, 1989; Horuk et al., J. Biol. Chem. 262: 16275, 1987; Chizzonite et al., Proc. Natl. Acad. Sci. USA 86:8029, 1989; Bomsztyk et al., Proc. Natl.

Acad. Sci. USA 8628034, 1989). Moreover, comparison of the biochemical properties and kinetic analysis of the IL-1 receptor in the Raji B cell line with EL-4 murine T lymphoma cell line showed that Raji cells had lower binding affinity but much higher receptor density per cell than a subclone of EL-4 T cells (Horuk eta1., J. Biol. Chem. 262:1627S, 1987).

Raji cells also failed to internalize IL-1 and demonstrated altered receptor binding affinities with IL-1 analogs. (Horuk et al., J. Biol. Chem. 262216275, 1987). These data suggest that the IL-1 receptors expressed on B cells (referred to herein as type II IL-1 receptors) are different from IL-l receptors detected on T cells and other cell types (referred to herein as type I IL-1 receptors).

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

First, no cell lines have previously been known to express high levels of type II IL- 1R constitutively and continuously, and cell lines known to express type H IL-IR did so only in low numbers (500 to 2,000 receptors/cell) which impeded efforts to purify receptors in amounts sufficient for obtaining amino acid sequence infomiation or generating monoclonal antibodies. The low numbers of receptors has also precluded any practical translation assay-based method of cloning.

Second, the significant differences in DNA sequence between type I ll.-IR and type II IL-lR has precluded cross-hybridization using a murine type IL-IR cDNA (Bomsztyk et al., Proc. Natl. Acad. Sci. USA 8628034, 1989, and Chizzonite et al., Proc. Natl. Acad.

Sci. USA 86:8029, 1989).

Third, even if a protein composition of sufficient purity could be obtained to permit N-terminal protein sequencing, the degeneracy of the genetic code may not permit one to define a suitable probe without considerable additional experimentation. Many iterative attempts may be required to define a probe having the requisite specificity to identify a hybridizing sequence in a cDNA library. Although direct expression cloning techniques avoid the need for repetitive screening using different probes of unknown specificity and have been useful in cloning other receptors (e.g., type I IL-1R), they are not sufficiently sensitive to be suitable for using in identifying type II IL-IR clones from cDNA libraries derived from cells expressing low numbers of type II IL-1R.

Thus, efforts to purify the type II IL-1R or to clone or express genes encoding type H IL-lR have been significantly impeded by lack of purified receptor, a suitable source of receptor mRNA, and by a sufficiently sensitive cloning technique.

The present invention provides purified and homogeneous type II IL-IR proteins and isolated DNA sequences encoding type H IL-IR proteins, in particular, human type II II.-IR, or analogs thereof. Preferably, such DNA sequences are selected from the group consisting of (a) cDNA clones having a nucleotide sequence derived from the coding region of a native type II IL-1R gene, such as clone 75; (b) DNA sequences capable of hybridization to the cDNA clones of (a) under moderately stringent conditions and which encode biologically active IL-1R molecules; and (c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode biologically active IL-1R molecules. The present invention also provides recombinant expression vectors comprising the DNA sequences defined above, recombinant type IIIL-1R molecules produced using the recombinant expression vectors, and processes for producing the recombinant type II IL-IR molecules utilizing the expression-vectors.

The present invention also provides substantially purified and homogeneous proteins and protein compositions comprising type II IL-IR.

The present invention also provides compositions for use in therapy, diagnosis, assay of type II IL-IR, or in raising antibodies to type II IL-IR, comprising effective quantities of soluble native or recombinant receptor proteins prepared according to the foregoing processes. ' These and other aspects of the present invention will become evident upon reference to the following detailed description.

FIG. 1 is a schematic diagram of the expression plasmid pDC406. cDNA molecules inserted at the Sal site are transcribed and translated using regulatory elements derived from HIV and adenovirus. pDC406 contains origins of replication derived from SV40, Epstein-Ban’ virus and pBR322.

FIG. 2 is a schematic diagram of the human and murine type II IL-1 receptors and the various human and murine clones used to determine the sequences. Thin lines represent untranslated regions, while the coding region is depicted by a box. The sections encoding the signal peptide are filled in; the transmembrane regions are cross-hatched; and the cytoplasmic portions are stippled. Potential N-linked glycosylation sites are marked by inverted triangles. The predicted immunoglobulin-like disulfide bonds are also indicated by dashes connecting two sulfide molecules (S-----S).

FIG. 3 compares the amino acid sequences of the human and murine type H IL-1 receptors (as deduced from the cDNA clones) with the amino acid sequences of the human and murine type I IL-1 receptors (Sims et al., Proc. Natl. Acad. Sci. USA 86:8946, 1989; Sims et al., Science 241 :585, 1988) and the amino acid sequences of the ST2 cellular gene (T orninaga, FEBS Lett. 258:301, 1989) and the B15R open reading frame of vaccinia virus (Smith and Chan, J. Gen. Virology 72:51l, 1991). Numbering begins with the initiating methionine. The predicted position of the signal peptide cleavage in each sequences was determined according to the method described by von Heijne, Nucl. Acids. Res. 14:4683, 1986, and is indicated by a gap between the putative signal peptide and the main body of the protein. The predicated transmembrane and cytoplasmic regions for the type II IL-1 receptors are shown on the bottom line, and are separated from one another by a gap.

Residues conserved in all four lL—l receptor sequences are presented in bold.

Residues conserved in type II receptors that are also found in one of the other sequences are shaded; residues conserved in type I IL-l receptors that are found in one of the other sequences are boxed. Cysteine residues involved in forrnin g the disulfide bonds characteristic of the immunoglobulin fold are marked with solid dots, while the extra two pairs of cysteines found in the type I IL-1 receptor and in some of the other sequences are indicated by stars. The approximate boundaries of domains 1, 2 and 3 are indicated above the lines. The predicted signal peptide cleavage in the type II IL-1 receptors follow Ala13, resulting in an unusually short signal peptide and an N-terminal extension of 12 (human) or 23 (mouse) amino acids beyond the point corresponding to the mature N-terrninus of the human or mouse type I IL-1 receptor. Other less favored but still acceptable sites of cleavage in the murine type H IL-1 receptor are after Thrl5 or Pro17. This sequence alignment was made by hand and does not represent an objectively optimized alignment of the sequences. The nucleotide and amino acid sequences of the full length and soluble human and murine type II IL-1 receptor cDNAs are also set forth in the Sequence Listing herein.

FIG. 4 shows an autoradiograph of an SDS/PAGE gel with crosslinked IL-1 receptors. Cells expressing IL-1 ‘receptors were cross-linked to 1251-IL-1 in thd absence or presence of the cognate unlabeled IL-1 competitor, extracted, electrophoresed and autoradiographed as described in Example 6. Recombinant receptors were expressed transiently in CV1/EBNA cells. The cell lines used for cross-linking to natural receptors were KB (ATCC CCL 1717) (for human type I IL-1R) , CB23 (for human type II ll.-1R), EL4 (ATCC TIB 39) (for murine type I IL-1R), and 702/3 (AT CC TIB 158) (for murine type 11 IL-1R).

Definitions "IL-1" refers collectively to IL-10: and IL-15.

"Type II Interleukin-1 receptor" and "type II IL-1R" refer to proteins which are capable of binding Interleukin-1 (IL-1) molecules and, in their native configuration as mammalian plasma membrane proteins, play a role in transducing the signal provided by IL-1 to a cell. The mature full-length human type II IL-1R is a glycoprotein having an apparent molecular weight of approximately 60-68 kDa. Specific examples of type 11 IL- lR proteins are shown in SEQ ID N021 and SEQ ID NO:l2. As used herein, the above terms include analogs or subunits of native type II IL-1R proteins with IL-l-binding or signal transducing activity. Specifically included are truncated or soluble forms of type II IL-IR protein, as defined below. In the absence of any species designation, type II IL-1R refers generically to mammalian type II IL-1R, which includes, but is not limited to, human, murine, and bovine type II IL-IR. Similarly, in the absence of any specific designation for deletion mutants, the term type II IL-1R means all forms of type II IL-IR, including mutants and analogs which possess type II IL-IR biological activity.

"Interleukin-1 Receptor" or "lL-lR" refers collectively to type I IL-1 receptor and type II IL-1 receptor.

"Soluble type II IL-1R" as used in the context of the present invention refer to proteins, or substantially equivalent analogs, which are substantially similar to all or part of the extracellular region of a native type II IL-IR, and are secreted by the cell but retain the ability to bind IL-1 or inhibit IL-1 signal transduction activity via cell surface bound IL-IR proteins. Soluble type II IL-lR proteins may also include part” of the transmembrane region, provided that the soluble type II IL-1R protein is capable of being secreted from the cell. Specific examples of soluble type II IL-1R proteins include proteins having the sequence of amino acids 1-330 or amino acids 1-333 of SEQ ID NO:l and amino acids 1- 342 and amino acids 1-345 of SEQ ID NO:l2. Inhibition of IL-1 signal transduction activity can be determined using primary cells or cells lines which express an endogenous IL-IR and which are biologically responsive to IL-1 or which, when transfected with recombinant IL-1R DNAs, are biologically responsive to IL-1. The cells are then contacted with IL-1 and the resulting metabolic effects examined. If an effect results which is attributable to the action of the ligand, then the recombinant receptor has signal transduction activity. Exemplary procedures for determining whether a polypeptide has signal transduction activity are disclosed by Idzerda et al., J. Exp. Med. 171:86l (1990); Curtis et al., Proc. Natl. Acad. Sci. USA 86:3045 (1989); Prywes et al., EMBO J. 522179 (1986) and Chou et al., J. Biol. Chem. 26221842 (1987).

The term "isolated" or "purified", as used in the context of this specification to define the purity of type 11 IL-1R protein or protein compositions, means that the protein or protein composition is substantially free of other proteins of natural or endogenous origin and contains less than about 1% by mass of protein contaminants residual of production processes. Such compositions, however, can contain other proteins added as stabilizers, carriers, excipients or ccrtherapeutics. Type II IL-IR is "isolated" if it is detectable as a single protein band in a polyacrylarnide gel by silver staining.

The term "substantially similar," when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the type II IL-1R protein as may be determined, for example, in a type II IL-1R binding assays, such as is described in Example 5 below. Alternatively, nucleic acid subunits and analogs are "substantially similar" to the specific DNA sequences disclosed herein if: (a) the DNA sequence is derived from the coding region of SEQ ID N021 or; SEQ ID NO: 12; (b) the DNA sequence is capable of hybridization to DNA sequences of (a) under moderately stringent conditions (25% formamide, 42'C, 2xSSC) or alternatively under more stringent conditions (50% formamide, 50°C, 2xSSC or 50% formamide, 42°C, 2xSSC) and which encode biologically active ll.-1R molecules; or DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) or (b) and which encode biologically active IL-1R molecules.

"Recombinant," as used herein, means that a protein is derived from recombinant (e.g., microbial or mammalian) expression systems. "Microbial'f refers to recombinant proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, "recombinant microbial" defines a protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Protein expressed in most bacterial cultures, e.g., E. call, will be free of glycan; protein expressed in yeast may have a glycosylation pattem different from that expressed in mammalian cells.

“Biologically active," as used throughout the specification as a characteristic of type II ll.-IR, means either that a particular molecule shares sufficient amino acid sequence similarity with SEQ ID NO:2 or SEQ ID NO:l3 to be capable of binding detectable quantities of IL-1, preferably at least 0.01 nmoles IL-1 per nanomole type II IL-1R, or, in the alternative, shares sufficient amino acid sequence similarity to be capable‘ of transmitting an IL-1 stimulus to a cell, for example, as a component of a hybrid receptor construct.

More preferably, biologically active type II IL-IR within the scope of the present invention is capable of binding greater than 0.1 nanomoles IL-1 per nanomole receptor, and most preferably, greater than 0.5 nanomoles IL-1 per nanomole receptor.

"DNA sequence" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. However, it will be evident that genomic DNA containing the relevant sequences could also be used. Sequences of non—u'anslated DNA may be present 5' or 3‘ from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. DNA sequences encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.

"Recombinant expression vector" refers to a plasmid comprising a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Altematiyely, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terrninal inethionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

"Recombinant microbial expression system" means a substantially homogeneous monoculture of suitable host microorganisms, for example, bacteria such as E. coli or yeast such as S. cerevisiae, which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid. Generally, cells constituting the system are the progeny of a single ancestral transforrnant. Recombinant expression systems as defined herein will express heterologous protein upon induction of the regulatory elements linked to the DNA sequence or synthetic gene to be expressed.

Tyx H H.-lR Proteins and Analogs The present invention provides isolated recombinant mammalian type II H.-IR polypeptides. The type H H.-1R proteins of the present invention include, by way of example, primate, human, murine, canine, feline, bovine, ovine, equine, caprine and porcine type H H.-1R. Type H IL-1R can be obtained by cross species hybridization, using a single stranded cDNA derived from the human or murine type II H..—lR DNA sequence, for example, human clone 75, as a hybridization probe to isolate type H H.-IR cDNAs from mammalian cDNA libraries. 1R is presumably encoded by multi-exon genes. Alternative mRN A constructs which can be attributed to different mRNA splicing events following transcription, and which share large regions of identity or similarity with the cDNAs claimed herein, are considered to be Like most mammalian genes, mammalian type 11 IL- within the scope of the present invention. DNA sequences which encode 11.-IR-H, possibly in the form of alternate splicing arrangements, can be isolated from the following cells and tissues: B lymphoblastoid lines (such as CB23, CB33, Raji, RPMI1788, ARH77), resting and especially activated peripheral blood T cells, monocytes, the monocytic cell line THPI, neutrophils, bone marrow, placenta, endothelial cells, keratinocytes (especially activated), and HepG2 cells.

Derivatives of type H H.-IR within the scope of the invention also include various structural forms of the primary protein which retain biological activity. Due to the presence of iortizable amino and carboxyl groups, for example, a type H H.-1R protein may be in the form of acidic or basic salts, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction.

The primary amino acid structure may be modified by; forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants.

Covalent derivatives are prepared by linking particular functional groups to type H H.-1R amino acid side chains or at the N- or C-termini. Other derivatives of type II IL-1R within the scope of this invention include covalent or aggregative conjugates of type H H.-1R or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated peptide may be a a signal (or leader) polypeptide sequence at the N -terrninal region of the protein which co- translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast a-factor leader). Type H H.-1R protein fusions can comprise peptides added to facilitate purification or identification of type II H.-1R (e.g., poly-His). The amino acid sequence of type 11 H.- lR can also be linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hopp et al., Bio/Technology 6:l204,1988.) The latter sequence is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing. Fusion proteins capped with this peptide may also be resistant to intracellular degradation in E. coli.

Type H IL-1R derivatives may also be used as immunogens, reagents in receptor- based immunoassays, or as binding agents for affinity purification procedures of H.-l or other binding ligands. Type H H.-lR derivatives may also be obtained by cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N—hydroxysuccinimide, at cysteine and lysine residues. Type H H.-1R proteins may also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen brornide-activated, bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking). Once bound to a substrate, type II IL-1R may be used to selectively bind (for purposes of assay or purification) anti-type II IL-1R antibodies or H.-1.

The present invention also includes type H H.-lR with or without associated native- , pattern glycosylation. Type H IL-1R expressed in yeast or mammalian expression systems, e.g., COS-7 cells, may be similar or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system.

Expression of type H IL-1R DNAs in bacteria such as E. coli provides non-glycosylated molecules. Functional mutant analogs of mammalian type H IL-1R having inactivated N- glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site- A. specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems.

N - glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn- A1-Z, where A1 is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate.

Examples of N-glycosylation sites in human type H H.-1R are amino acids 66-68, 72-74, 112-114, 219-221, and 277-279 in SEQ ID NO:l. Such sites can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between A1 and Z, or an amino acid other than Asn between Asn and A 1.

Type H H.-1R derivatives may also be obtained by mutations of type H H.-IR or its subunits. A type H H.-1R mutant, as referred to herein, is a polypeptide homologous to type H IL-1R but which has an amino acid sequence different from native type H H.-1R because of a deletion, insertion or substitution.

Bioequivalent analogs of type II IL-1R proteins may be constructed by, for example, malcing various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfrde bridges upon renaturation. Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in‘ which KEX2 protease activity is ‘present.

Generally, substitutions should be made conservatively; i.e., the most preferred substitute amino acids are those having physiochemical characteristics resembling those of the residue to be replaced. Similarly, when a deletion or insertion strategy is adopted, the potential effect of the deletion or insenion on biological activity should be considered. Substantially similar polypeptide sequences, as defined above, generally comprise a like numberof amino acids sequences, although C~termina1 truncations for the purpose of constructing soluble type II IL-lRs will contain fewer amino acid sequences. In order to preserve the biological activity of type II IL-lRs, deletions and substitutions willpreferably result in homologous or conservatively substituted sequences, meaning that a given residue is replaced by a biologically similar residue. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Moreover, particular amino acid differences between human, murine and other mammalian type II IL-lRs is suggestive of additional conservative substitutions that may be made without altering the essential biological characteristics of type II IL-lR.

Subunits of type II IL-IR may be constructed by deleting terrriinal or internal residues or sequences. The present invention contemplates, for example, C terminal deletions which result in soluble type H IL-1R constructs corresponding to all or part of the exuacellular region of type II IL-1R. The resulting protein preferably retains its ability to bind IL-1. Particularly preferred sequences include those in which the transmembrane region and intracellular domain of type II II_.- lR are deleted or substituted with hydrophilic residues to facilitate secretion of the receptor into the cell culture medium. Soluble type II IL-1R proteins may also include part of the transmembrane region, provided that the soluble type II IL-1R protein is capable of being secreted from the cell. For example, soluble human type II IL-lR may comprise the sequence of amino acids l-333 or amino acids 1-330 of SEQ ID NO:l and amino acids 1-345 and amino acids 1-342 of SEQ ID NO:l2. Alternatively, soluble type II IL-lR proteins may be derived by deleting the C- terminal region of a type II IL-lR within the extracellular region which are not necessary for IL-1 binding. For example, C-terminal deletions may be made to proteins having the sequence of SEQ ID NO:1 and SEQ ID NO:l2 following amino acids 313 and 325, respectively. These amino acids are cysteines which are believed to be necessary to maintain the tertiary structure of the type II IL-1R molecule and pennit binding of the type II IL-1R molecule to IL-1. Soluble type II IL-1R constructs are constructed by deleting the 3'-terminal region of a DNA encoding the type II IL-1R and then inserting and expressing the DNA in appropriate expression vectors. Exemplary methods of constructing such soluble proteins are described in Examples 2 and 4. The resulting soluble type II IL-1R proteins are then assayed for the ability to bind IL-1, as described in Example 5. Both the DNA sequences encoding such soluble type II IL-lRs and the biologically active soluble type 11 IL-IR proteins resulting from such constructions are contemplated to be within the scope of the present invention.

Mutations in nucleotide sequences constructed for expression of analog type 11 IL- lR must, of course, preserve the reading frame phase of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRN A structures such as loops or hairpins which would adversely affect translation of the receptor mRNA. Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed type II IL-1R mutants screened for the desired activity.

Not all mutations in the nucleotide sequence which encodes type II IL-IR will be expressed in the final product, for example, nucleotide substitutions may be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA (see EPA 75,444A, incorporated herein by reference), or to provide codons that are more ' readily translated by the selected host, e.g., the well-known E. coli preference codons for E. coli expression.

Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotlde-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 422133, 1986); Bauer et 211. (Gene 37:73, 1985); Craik (Br’oTechnz'ques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. ,518,584 and 4,737,462 disclose suitable techniques, and are incorporated by reference herein.

Both monovalent forms and polyvalent forms of type II IL-1R are useful in the compositions and methods of this invention. Polyvalent forms possess multiple type H IL- 1R binding sites for IL-1 ligand. For example, a bivalent soluble type II IL-1R may consist of two tandem repeats of the extracellular region of type 11 IL-1R, separated by a linker region. Alternate polyvalent forms may also be constructed, for example, by chemically coupling type II IL-1R to any clinically acceptable carrier molecule, a polymer selected from the group consisting of Ficoll, polyethylene glycol or dextran using conventional coupling techniques. Alternatively, type II IL-1R may be chemically coupled to biotin, and the biotin-type I1 IL-1R conjugate then allowed to bind to avidin, resulting in tetravalent avidin/biotin/type II IL-1R molecules. Type H IL-1R may also be covalently coupledto dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugate precipitated with anti—DNP or anti-TN?-IgM, to form decarneric conjugates with a valency of 10 for type II IL-IR binding sites. ‘ A recombinant chimeric antibody molecule may also be produced having type II IL- lR sequences substituted for the variable domains of either or both of the immunoglubulin molecule heavy and light chains and having unmodified constant region domains. For example, chimeric type II IL-1R/IgG1 may be produced from two chimeric genes -- a type II IL-1R/human K light chain chimera (type II IL-1R/Cg) and a type II IL-1R/human 71 heavy chain chimera (type II IL-1R/CH). Following transcription and translation of the two chimeric genes, the gene products assemble into a single chimeric antibody molecule having type II IL-1R displayed bivalently. Such polyvalent forrrts of type II IL-1R may have enhanced binding affinity for IL-1 ligand. Additional details relating to the construction of suchlchimeric antibody molecules are disclosed in W0 89/09622 and 5 EP 315062.

Expression of Recombinant Tvoe ll IL-1R The present invention provides recombinant expression vectors to amplify or express DNA encoding type II IL-1R. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding mammalian type II IL-1R or bioequivalent analogs operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes.

A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulatory elements may include an operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites. The ability to replicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants may additionally be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading frame.

Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final product . DNA sequences encoding mammalian type II IL-1Rs which are to be expressed in a microorganism will preferably contain no introns that could prematurely terminate transcription of DNA into mRNA; however, premature termination of transcription may be desirable, for example, where it would result in mutants having advantageous C-terrninal truncations, for example, deletion of a transmembrane region to yield a soluble receptor not bound to the cell membrane. Due to code degeneracy, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence. Other embodiments include sequences capable of hybridizing to clone 75 under moderately stringent conditions (50'C, 2x SSC) and other sequences hybiidizing or degenerate to those which encode biologically active type II IL-1R polypeptides.

Recombinant type H IL-1R DNA is expressed or amplified in a recombinant expression system comprising a substantially homogeneous monoculture of suitable host microorganisms, for example, bacteria such as E. coli or yeast such as S. cerevisiae, which have stably integrated (by transfomtation or transfection) a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid. Generally, cells constituting the system are the progeny of a single ancestral transforrnant. Recombinant expression systems as defined herein will express heterologous protein upon induction of the regulatory elements linked to the DNA sequence or synthetic gene to be expressed Transformed host cells are cells which have been transformed or transfected with type 11 IL-1R vectors constructed using recombinant DNA techniques. Transformed host cells ordinarily express type II IL-1R, but host cells transformed for purposes of cloning or amplifying type II 1L- 1R DNA do not need to express type H IL-1R. Expressed type H IL- 1R will be deposited in the cell membrane or secreted into the culture supernatant, depending on the type II IL-IR DNA selected. Suitable host cells for expression of mammalian type II [L-IR include prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E . coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed to produce mammalian type II IL-1R using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al.

(Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant disclosure of which is hereby incorporated by reference.

Prokaryotic expression hosts may be used for expression of type II IL-lR that do not require extensive proteolytic and disulfrde processing. Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host.

Suitable prokaryotic hosts for transformation include E . coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, S treptomyces, and S taphyolococcus, although others may also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223—3 (Pharrnacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. E . coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2295, 1977). pBR322 contains genes for arnpicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.

Promoters commonly used in recombinant microbial expression vectors include the [3—lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275 :6l5, 1978; and Goeddel et al., Nature 2812544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 824057, 1980; and EPA 36,776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful bacterial expression system employs the phage 3. PL promoter and cI857ts thermolabile repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the 1. PL promoter include plasmid pHUB2, resident in E. coli strain J MB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).

Recombinant type II IL-1R proteins may also be expressed in yeast hosts, preferably from the Saccharomyces species, such as S. cerevisiae. Yeast of other genera, such as Pichia or K Iuyveromyces may also be employed. Yeast vectors will generally contain an origin of replication from the 2p yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA encoding type 11 IL-1R, sequences for polyadenylation and transcription termination and a selection gene. Preferably, yeast vectors will include an origin of replication and selectable marker permitting transformation of both yeast and E. coli, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 or URA3 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, and a promoter derived from a highly expressed yeast gene to induce transcription of a structural sequence downstream. The presence of the TRP1 or URA3‘ lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan or uracil.

Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255 :2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehydephosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofmctokinase, glucose phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucolcinase. Suitable vectors and promoters for use in yeast expression are further described in R. I-Iitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pUCl8 for selection and replication in E. colt’ (Amp? gene and origin of replication) and yeast DNA sequences including a glucose-repressible ADH2 promoter and a-factor secretion leader.

The ADH2 promoter has been described by Russell et al. (J. Biol. Chem. 25812674, 1982) and Beier et al. (Nature 3002724, 1982). The yeast ot-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed. See, e.g., Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proc.

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

Suitable yeast transformation protocols are known to those of skill in the art; an exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75 :1929, , selecting for Trp+ transforrnants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 pg/ml adenine and 20 pg/ml uracil or URA+ transformants in medium consisting of 0.67% YNB, with amino acids and bases as described by Sherman et al., Laboratory Course Manual for Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1986.

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

Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells is particularly preferred because such proteins are generally correctly folded, appropriately modified and completely functional. Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:l75, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines.

Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other ‘ or 3' flanking nomranscribed sequences, and 5‘ or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV4O viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV4O viral origin of replication (Fiers et al., Nature 273:1 13, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind 3 site toward the Bgll site located in the viral origin of replication is included. Further, mammalian genomic type II IL-1R promoter, control and/or signal sequences may be utilized, provided such control sequences are compatible with the host cell chosen. Additional details regarding the use of a mammalian high expression vector to produce a recombinant mammalian type II IL-1R are provided in Examples 2 below.

Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983).

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

In preferred aspects of the present invention, recombinant expression vectors comprising type II IL-IR cDNAs are stably integrated into a host cell's DNA. Elevated levels of expression product is achieved by selecting for cell lines having amplified numbers of vector DNA. Cell lines having amplified numbers of vector DNA are selected, for example, by transforming a host cell with a vector comprising a DNA sequence which encodes an enzyme which is inhibited by a known drug. The vector may also comprise a DNA sequence which encodes a desired protein. Alternatively, the host cell may be co- transformed with a second vector which comprises the DNA sequence which encodes the desired protein. The transformed or co-transforrned host cells are then cultured in increasing concentrations of the known drug, thereby selecting for drug-resistant cells.

Such drug-resistant cells survive in increased concentrations of the toxic drug by over- production of the enzyme which is inhibited by the drug, frequently as a result of amplification of the gene encoding the enzyme. Where drug resistance is caused by an increase in the copy number of the vector DNA encoding the inhibitable enzyme, there is a concomitant co-arnplification of the vector DNA encoding the desired protein (e.g., type H IL-1R) in the host cell's DNA. , A preferred system for such co-amplification uses the gene for dihydrofolate reductase (DHFR), which can be inhibited by the drug methotrexate (MTX). To achieve co-amplification, a host cell which lacks an active gene encoding DHFR is either transformed with a vector which comprises DNA sequence encoding DHFR and a desired protein, or is co-transfomred with a vector comprising a DNA sequence encoding DHFR and a vector comprising a DNA sequence encoding the desired protein. The transformed or co-transforrned host cells are cultured in media containing increasing levels of MTX, and those cells lines which survive are selected.

A particularly preferred co-amplification system uses the gene for glutamine synthetase (GS), which is responsible for the synthesis of glutamine from glutamate and ammonia using the hydrolysis of ATP to ADP and phosphate to drive the reaction. GS is subject to inhibition by a variety of inhibitors, for example methionine sulphoximine (MSX). Thus, type II IL-1R can be expressed in high concentrations by co-amplifying cells transformed with a vector comprising the DNA sequence for GS and a desired protein, or co-transformed with a vector comprising a DNA sequence encoding GS and a vector comprising a DNA sequence encoding the desired protein, culturing the host cells in media containing increasing levels of MSX and selecting for surviving cells. The GS co- amplification system, appropriate recombinant expression vectors and cells lines, are described in the following PCT applications: WO 87/04462, WO 89/01036, W0 89/ 10404 and WO 86/05807.

Recombinant proteins are preferably expressed by co-amplification of ‘DHFR or GS in a mammalian host cell, such as Chinese Hamster Ovary (CHO) cells, or alternatively in a murine_ myeloma cell line, such as SP2/0-Ag14 or N80 or a rat myeloma cell line, such as YB2/3.0-Ag20, disclosed in PCT applications WO/89/ 10404 and WO 86/05807.

A preferred eukaryotic vector for expression of type II IL-lR DNA is disclosed below in Example 2. This vector, referred to as pDC406, was derived from the mammalian high expression vector pDC201 and contains regulatory sequences from SV40, HIV and EBV.

Purification of Recombinant Ty}; II H.-1R Purified mammalian type II IL-1Rs or analogs are prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts.

For example, supematants from systems which secrete recombinant soluble type II IL-1R protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration- unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix can comprise an IL-1 or lectin or antibody molecule bound to a suitable support. Alternatively, an anioh exchange resin can be employed, for example, a mauix or substrate having pendant diethylarninoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.

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

Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of recombinant mammalian type II IL-IR can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express soluble mammalian type 11 IL-lR as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large- scale fermentation can be purified by methods analogous to those disclosed by Urdal et al.

(J. C hromatog. 2962171, 1984). This reference describes two sequential, reversed-phase HPLC steps for purification of recombinant human GM-CSF on a preparative HPLC column.

Human type II IL-lR synthesized in recombinant culture is characterized by the presence of non-human cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover human type II IL-IR from the culture. These components ordinarily will be of yeast, prokaryotic or non-human higher eukaryotic origin and preferably are present in innocuous contaminant quantities, on the order of less than about 1 percent by weight. Further, recombinant cell culture enables the production of type II IL-1R free of proteins which may be normally associated with type II IL-1R as it is found in nature in its species of origin, e.g. in cells, cell exudates or body fluids.

Therapeutic Administration of Recombinant figlgblg Type II IL-1R The present invention provides methods of using therapeutic compositions comprising an effective amount of soluble type II IL-1R proteins and a suitable diluent and carrier, and methods for suppressing IL-'1-dependent immune responses in humans comprising administering an effective amount of soluble type II IL-1R protein.

For therapeutic use, purified soluble type II IL-1R protein is administered to a patient, preferably a human, for treatment in a manner appropriate to the indication. Thus, for example, soluble type II IL-1R protein compositions can be administered by bolus injection, continuous infusion, sustained release from implants, or other suitable technique.

Typically, a soluble type H IL-1R therapeutic agent will be administered in the form of a composition comprising purified protein in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the type II IL-1R with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with conspecific serum albumin are exemplary appropriate diluents. Preferably, product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Appropriate dosages can be determined in trials; generally, shuIL-1R dosages of from about 1 ng/kg/day to about 10 mg/kg/day, and more preferably from about 500 pg/kg/day to about 5 mg/kg/day, are expected to induce a biological effect.

Because IL-IR-I and type II IL-1R proteins both bind to IL-1, soluble type H IL- 1R proteins are expected to have similar, if not identical, therapeutic activities. For example, soluble human type II IL-1R can be administered, for example, for the purpose of suppressing immune responses in a human. A variety of diseases or conditions are caused by an immune response to alloantigen, including allograft rejection and graft-versus-host reaction. In alloantigen-induced immune responses, shuIL-IR suppresses lymphoproliferation and inflammation which result upon activation of T cells. shull-lR can therefore be used to effectively suppress alloantigen-induced immune responses in the clinical treatment of, for example, rejection of allog-rafts (such as skin, kidney, and heart transplants), and graft-versus-host reactions in patients who have received bone marrow transplants. ‘ Soluble human type II lL-lR can also be used in clinical treatment of autoimmune dysfunctions, such as rheumatoid arthritis, diabetes and multiple sclerosis, which are dependent upon the activation of T cells against antigens not recognized as being indigenous to the host.

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

I EXAMBLES * Example 1 Isolation of CDNA Encoding Human Type II IL-IR by Direct Ex ression of Active Protein in V-1 BNA-l Cells A. Radiolabeling of rIL-la. Recombinant human IL-15 was prepared by expression in E. coli and purification to homogeneity as described by Kronheim et al.

(Bio/Technology 421078, 1986). The IL-1 [5 was labeled with di-iodo (1251) Bolton-Hunter reagent (New England Nuclear, Glenolden, PA). Ten micrograms (0.57 nmol) of protein in 10 uL of phosphate (0.015 mol/L)-buffered saline (PBS; 0.15 mol/L), pH 7.2, was mixed with 10 uL of sodium borate (0.1 mol/L)-buffered saline (0.15 mol/L), pH 8.5, and reacted with 1 mCi (0.23 nmol) of Bolton-Hunter reagent according to the manufacturer's instructions for 12 hours at 8'C. Subsequently, 30 uL of 2% gelatin and 5 uL of 1 mol/L glycine ethyl ester were added, and the protein was separated from unreacted Bolton- r 21 Hunter reagent on a 1 mL bed volume Biogel” P6 column (Bio-Rad Laboratories, Richmond, CA). Routinely, 50% to 60% incorporation of label was observed.

Radioiodination yielded specific acdvities in the range of 1 x 1015 to 5 x 1015 cpm/mmol-1 (0.4 to 2 atoms I per molecule protein), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single labeled polypeptide of 17.5 kD, consistant with previously reported values for IL-1. The labeled protein was greater than 98% TCA precipitable, indicating that the 1351 was covalently bound to protein.

B. Qgnstrucjgn and Screening Qf QB23 QDNA librgg. A CB23 library was constructed and screened by direct expression of pooled cDNA clones in the monkey kidney cell line CV-1/EBNA-1 (which was derived by transfection of the CV-1 cell line with the gene encoding EBNA-1, as described below) using a mammalian expression vector (pDC406) that includes regulatory sequences from SV40, human immunodeficiency vinrs (HIV), and Epstein-Barr virus (EBV). The CV-1/EBNA-1 cell line constitutively expresses EBV nuclear antigen-1 driven from the human cytomegalovirus (CMV) immediate-early enhancer/promoter and therefore allows the episomal replication of r expression vectors such as pDC406 that contain the EBV origin of replication. The expression vector used was pDC406, a derivative of HAV-E0, described by Dower et al., J. Immunol. I42:4314, 1989), which is in turn a derivative of pDC201 and allows high A level expression in the CV-1/EBNA-1 cell line. pDC406 differs from HAV-E0 (Dower et al., supra) by the deletion of the intron present in the adenovirus 2 tripartite leader sequence in HAV-E0 (see description of pDC303 below).

The CB23 cDNA library was constructed by reverse transcription of poly(A)+ mRNA isolated from total RNA extracted from the human B cell lymphoblastoid line CB23 (Benjamin & Dower, Blood 75:2017, 1990) substantially as described by Ausubel et al., eds., Current Protocols in Mole'cuIar Biology, Vol. 1, 1987. The CB23 cell line is an EBV-transformed cord blood (CB) lymphocyte cell line, which was derived by using the methods described by Benjamin et al., Proc. Natl. Acad. Sci. USA 8123547, 1984.

Poly(A)+ mRNA was isolated by oligo dT cellulose chromatography and double-stranded cDNA was made substantially as described by Gubler and Hoffman,Gene 252263, 1983.

Briefly, the po1y(A)‘* mRNA was converted to an RNA-CDNA hybrid with reverse transcriptase using random hexanucleotides as a primer. The RNA-CDNA hybrid was then converted into double-stranded cDNA using RNAase H in combination with DNA polymerase I. The resulting double stranded cDNA was blunt-ended with T4 DNA polymerase. The following two unldnased oligonucleotides were annealed and blunt end ligated with DNA ligase to the ends of the resulting blunt-ended CDNA as described by Haymerle, et al., Nucleaic Acids Research, 14: 8615, 1986. sso ID NO:3 s'- rcc ACT GGA ACG AGA cca ccr GCT -3" GA CCT TGC TCT GCT GGA CGA -5' SEQ ID NO:4 3'- In this case only the 24-mer oligo will ligate onto the cDNA. The non-ligated oligos were removed by gel filtration chromatography at 68‘C, leaving 24. nucleotide non-self- complementary overhangs on the CDNA. The same procedure was used to convert the 5' ends of Sall-cut mammalian expression vector pDC406 to 24 nucleotide overhangs complementary to those added to the CDNA. Optimal proportions of adaptored vector and CDNA were ligated in the presence of T4 polynucleotideldnase. Dialyzed ligation mixtures were electroporated into E. coli strain DH5a. Approximately 3.9 x 105 clones were generated and plated in pools of approximately 3,0()0. A sample of each pool was used to prepare frozen glycerol stocks and a sample was used to obtain a pool of plasmid DNA. A The pooled DNA was then used to transfect a sub-confluent layer of monkey CV- /EBNA-‘I cells using DEAE-dextran followed by chloroquine treatment, similar to that - described by Luthman et al., Nucl. Acids Res. 1 1:1295 (1983) and McCutchan et al., J.

Natl. Cancer Inst. 412351 (1986). CV -1/EBNA-1 cells were derived as follows. The CV- 1/EBNA-1 cell line constitutively expresses EBV nuclear antigen-l driven from the CMV immediate-early enhancer/promoter. The African Green Monkey kidney cell line, CV-1 (ATCC CCL 70, was cotransfected with 5 pg of pSV2gpt (Mulligan & Berg, Proc. Natl.

Acad. Sci. USA 7822072, 1981) and 25 ug of pDC303/EBNA-I using a calcium phosphate coprecipitation technique (Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley, New York, 1987). pDC303/EBNA-1 was constructed from pDC302 (Mosley et al., Cell 59:335, 1989) in two steps. First, the intron present in the adenovirus tripartite leader sqquence was deleted by replacing a Pvull to Seal fragment spanning the intron with the following synthetic oligonucleotide pair to create plasmid pDC303: SEQ ID NO:5 5‘-CTGTTGGGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGT-3' SEQ ID N0:6 3'-GACAACCCGAGCGCCAACTCCTGTTTGAGAAGCGCCAGAAAGGTCA-5' Second, a Hindlll-AhaII restriction fragment encoding Epstein-Barr virus nuclear antigen I (EBNA-1), and consisting essentially of EBV coordinates 107,932 to 109,894 (Baer et al., Nature 310:207, 1984), was then inserted into the multiple cloning site of pDC303 to create the plasmid pDC303/EBNA-1. The transfected cells were grown in the presence of hypoxanthine, aminopterin, thymidine, xanthine, and mycophenolic acid according to standard methods (Ausubel et al., supra; Mulligan & Berg, supra) to select for the cells that had stably incorporated the transfected plasmids. The resulting drug resistant colonies were isolated and expanded individually into cell lines for analysis. The cell lines were screened for the expression of functional EBNA-1. One cell line, clone 68, was found to express EBNA-l using this assay, and was designated CV-1/EBNA-1.

In order to transfect the CV-1/EBNA-1 cells with the CDNA library, the cells were maintained in complete medium (Dulbecco's modified Eagle's media (DMEM) containing % (v/V) fetal calf serum (FCS), 50 U/ml penicillin, 50 U/ml streptomycin, 2 mM L- glutamine) and were plated at a density of 2 x 105 cells/well in either 6 well dishes (Falcon) or single well chambered slides (Lab-Tek). Both dishes and slides were pretreated with 1 ml human fibronectin (10 ug/ml in PBS) for 30 minutes followed by I wash with PBS.

Media was removed from the adherent cell layer and replaced with 1.5 ml complete medium containing 66.6 M chloroquine sulfate. 0.2 rnls of DNA solution (2 ug DNA, 0.5 mg/ml DEAE-dextran in complete medium containing chloroquine) was then added to the cells and incubated for 5 hours. Following the incubation, the media was removed and the cells shocked by addition of complete medium containing 10% DMSO for 2.5 to 20 minutes followed by replacement of the solution with fresh complete medium. The cells were grown in culture to permit transient expression of the inserted sequences. These conditions led to an 80% transfection frequency in surviving CV-1/EBNA-1 cells.

After 48 to 72 hours, transfected monolayers of CV-1/EBNA cells were assayed for expression of IL-1 binding proteins by binding radioiodinated IL-1B prepared as described above by slide autoradiography. Transfected CV-1/EBNA-1 cells were washed once with binding medium (RPMI medium 1640 containing 25 mg/ml bovine serum albumin (BSA), 2 mg/ml sodium azide, 20 mM HEPES, pH 7.2, and 50 mg/ml nonfat dry milk (NFDM)) and incubated for 2 hours at 4'C with 1 ml binding medium + NFDM containing 3 x 109 M 1251-IL-lfi. After incubation, cells in the chambered slides were washed three times with binding buffer + NFDM, followed by 2 washes with PBS, pH 7.3, to remove unbound -IL-1B. The cells were fixed by incubating for 30 minutes at room temperature in 10% glutaraldehyde in PBS, pH 7.3, washed twice in PBS, and air dried. The slides were dipped in Kodak GTNB-2 photographic emulsion (6x dilution in water) and exposed in the dark for 48 hours to 7 days at 4'C in a light proof box. The slides were then developed for approximately 5 minutes in Kodak D19 developer (40 g/500 ml water), rinsed in water and fixed in Agfa G433C fixer. The slides were individually examined with a microscope at -40x magnification and positive cells expressing type II IL-1R were identified by the presence of autoradiogiaphic silver grains against a light background.

Cells in the 6 well plates were washed once with binding buffer + NFDM followed by 3 washings with PBS, pH 7.3, to remove unbound ‘Z51-IL-1B. The bound cells were then trypsinized to remove them from the plate and bound 1251-11..-15 were counted on a beta counter.

Using the slide autoradiography approach, approximately 250,000 cDNAs were screened in pools of approximately 3,000 cDNAs until assay of one transfectant pool showed multiple cells clearly positive for IL-15 binding. This pool was then partitioned into pools of 500 and again screened by slide autoradiography and a positive pool was identified. This pool was further partitioned into pools of 75, plated in 6-well plates and screenedlby plate binding assays analyzed by quantitation of bound 1251-IL-113. The cells were scraped off the plates and counted to determine which pool of 75 was positive.

Individual colonies from this pool of 75 were screened until a single clone (clone 75) was identified which directed synthesis of a surface protein with detectable IL-15 binding activity. This clone was isolated, and its insert was sequenced to determine the sequence of the human type II ll.-1R cDNA clone 75. The pDC406 cloning vector containing the human type II IL-1R cDNA clone 75, designated pHuIL-IR-II 75, was deposited with the American Type Culture Collection, Rockville, MD, USA (ATCC) on June 5, 1990 under accession number CRL 10478. The Sequence Listing setting forth the nucleotide (SEQ ID Nozl) and predicted amino acid sequences of c1oneI75 (SEQ ID Nozl and SEQ ID NO:2) and associated information appears at the end of the specification immediately prior to the claims.

A cDNA encoding a soluble human type II IL-1R (having the sequence of amino acids_ 333 of SEQ ID N021) was constructed by polymerasefchain reaction (PCR) amplification using the full length type II IL-IR cDNA clone 75 (SEQ ID NO:1) in the vector pDC406 as a template. The following 5' oligonucleotide primer (SEQ ID NO:7) and 3' oligonucleotide primer (SEQ ID NO:8) were first constructed: SEQ ID NO: 7 5 ' -GCGTCGACCTAGTGACGCTCATACAAATC-3 ' SEQ ID NO: 8 5 ' -GCGCGGCCGCIQAGGAGGAGGCTTCCTTGACTG-3' <-NotI->End\119l \1172 The 5' primer corresponds to nucleotides 31-51 from the untranslated region of human type II I1.-1R clone 75 (SEQ ID NO:l) with a 5' add—on of a Sall restriction site; this nucleotide sequence is capable of annealing to the (-) strand complementary to nucleotides 31-51 of human clone 75. The 3' primer is complementary to nucleotides 1191-1172 (which includes anti-sense nucleotides encoding 3 amino acids of human type II IL-1R clone 75 (SEQ ID NO:1) and has a 5‘ add—on of a Not] restriction site and a stop codon.

The following PCR reagents were added to a 1.5 ml Eppendorf micmfuge tube: 10 pl of 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3 at 25°C, 15 mM MgCl2, and 1 mg/ml gelatin) (Perkin-Elmer Cetus, Norwalk, CN), 10 pl of a 2mM solution containing each dNTP (2 mM dATP, 2 mM dC'I'P, 2 mM dGTP and 2 mM d'l'I'P), 2.5 units (0.5 pl of standard 5000 units/ml solution) of Taq DNA polymerase (Perkin-Elmer Cetus), 50 ng of template DNA and 5 pl of a 20 pM solution of each of the above oligonucleotide primers and 74.5 pl water to a final volume of 100 pl. The final mixture was then overlaid with 100 pl paraffin oil. PCR was carried out using a DNA thermal cycler (Ericomp, San Diego, CA) by initially denaturing the template at 94° for 90 seconds, reannealing at 55° for 75 seconds and extending the cDNA at 72° for 150 seconds. PCR was carried out for an additional 20 cycles of amplification using a step program (denaturation at 94°, 25 sec; annealing at 55°, 45 sec; extension at 72°, 150 sec.), followed » by a 5 minute extension at 72°.

The sample was removed from the paraffin oil and DNA extracted by phenol- chloroform extraction and spun column chromatography over G-50 (Boehringer Mannheim). A 10 pl aliquot of the extracted DNA was separated by electrophoresis on 1% SeaKem"‘ agarose (FMC Biol-’roducts, Rockland, ME) and stained with ethidium bromide to confirm that the DNA fragment size was consistent with the predicted product pl of the PCR-amplified CDNA products were then digested with Sall -and NotI restriction enzymes using standard procedures. The Sall/Notl restriction fragment was then separated on a 1.2% Seaplaquem low gelling temperature (LGT) agarose, and the band representing the fragment was isolated. The fragment was ligated into the pDC406 vector by a standard "in gel" ligation method, and the vector was transfected into CV1- EBNA cells and expressed as described above in Example 1.

Example 3 Isolation of cDNAs Encoding Murine Type ll IL-1R Murine type II IL-IR cDNAs were isolated from a cDNA library made from the murine pre-B cell line 7OZ/3 (ATCC 'I'IB 158), by cross species hybridization with a human Type H IL-1R probe. A CDNA library was constructed in a 7. phage vector using Xgt10 arms and packaged in vitro (Gigapaclt®, Stratagene, San Diego) according to the manufacturer's instructions. A double-stranded human Type I] IL-lR probe was produced by excising an approximately 1.35 kb Sall restriction fragment of the human type II IL-1R clone 75 and 32?-labelling the cDNA using random primers (Boehringer-Mannheim). The murine cDNA library was amplified once and a total of 5x105 plaques were screened with the human probe in 35% formamide (5xSSC, 42°C). Several murine type II IL-1R CDNA clones (including clone D) were isolated; however, none of the clones appeared to be full- length. Nucleotide sequence infomration obtained from the partial clones was used to clone a full-length murine type II IL-lR CDNA as follows.

A full-length CDNA clone encoding murine type II IL-1R was isolated by the method of Rapid Amplification of CDNA Ends (RACE) described by Frohman et al., Proc.

Natl. Acad. Sci. USA 85 :8998, 1988, using RNA from the murine pre-B cell line 702/3.

Briefly, the RACE method uses PCR to amplify copies of a region of cDNA between a known point in the cDNA transcript (determined from nucleotide ‘sequence obtained as described above) and the 3' end. An adaptor-primer having a sequence containing 17 dT base pairs and an adaptor sequence containing three endonuclease recognition sites (to place convenient restriction sites at the 3' end of the cDNA) is used to reverse transcribe a population of mRNA and produce (-) strand cDNA. A primer complementary to a known stretch of sequence in the 5‘ untranslated region of the murine type II IL— 1R clone 2 cDNA, described above, and oriented in the 3' direction is annealed with the (-) strand cDNA and extended to generate a complementary (+) strand cDNA. The resulting double-strand cDNA is amplified by PCR using primers that anneal to the natural 5'-end and synthetic 3'- end po1y(A) tail. Details of the RACE procedure are as follows. _ The following PCR oligonucleotide primers (d(T)17 adaptor-primer, 5' amplification primer and 3' amplification primer, respectively) were first constructed: ' -CTGCAGGCGGCCGCGGATCC (T) 17-3 ‘ <-NotI-> SEQ ID NO:9 '-GCGTCGACGGCAAGAAGCAGCAAGGTBC-3‘ \15 \34 SEQ ID NO:10 '-CTGCAGGCGGCCGCGGATCC-3' <-NotI-> SEQ ID NO:l1 Briefly, the d(T)17 adapter-primer (SEQ ID N029) contains nucleotide sequence anneals to the poly(A)+ region of a population mRNA transcripts and is used to generated (-) su'and cDNA reverse transcripts from mRNA; it also contains endonuclease restriction sites for Pstl, NotI and Baml-ll to be introduced into the DNA being amplified by PCR. The 5' amplification primer (SEQ ID NO:10) corresponds to nucleotides 15-34 from the 5' untranslated region of murine type II IL-1R clone 7&2 with a 5‘ add-on of a Sall restriction site; this nucleotide sequence anneals to the (-) strand CDNA generated by reverse transcription with the d(T)17 adaptor-primer and is extended to generate (+) strand cDNA.

The 3' primer (SEQ ID NO:1 1) anneals to the (+) strand DNA having the above endonuclease restriction sites and is extended to generate a double-stranded full-length cDNA encoding murine type II IL-IR, which can then be amplified by a standard PCR reaction. Details of the PCR procedme are as follows.

Poly(A)+ mRNA was isolated by oligo dT cellulose chromatography from total RNA extracted from 70Z/3 cells using standard methods described by Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY, 1982) and reverse transcribed as follows". Approximately 1 pg of poly(A)+ mRNA in 16.5 pl of water was heated at 68°C for 3 minutes and then quenched on ice, and added to 2 pl of 10X RTC buffer (500 mM Tris-HCI, pH 8.7 at 22°C, 60 mM MgCl2, 400 mM KCl, mM DTT, each dNTP at 10 mM), 10 units of RNasin (Promega Biotech), 0.5 pg of d(T)17-adapter primer and 10 units of AMV reverse transcriptase (Life Sciences) in a total volume of 20 pl, and incubated for a period of 2 hours at 42°C to reverse transcribe the mRNA and synthesize a pool of CDNA. The reaction mixture was diluted to 1 ml with TE buffer (10 mM 'l‘ris—HCl, pH 7.5, 1 mM EDTA) and stored at 4°C overnight.

Approximately 1 or 5 pl of the CDNA pool was combined with 5 pl of a 20 pM solution of the 5' amplification primer, containing sequence corresponding to the sequence of nucleotides 15-34 of murine type II IL-1R" clone 12, 5 pl of a 20 pM solution of the 3' amplification primer, 10 pl of 10X PCR buffer (500 mM KCl, 100 mM Tris—HCl (pH 8.4, °C), 14 mM MgCl2, and 1 mg/ml gelatin), 4 pl of 5mM each dNTP (containing 5 mM dATP, 5mM dCTP, 5mM dGT'P and 5mM d'I'I'P), 2.5 units (0.5 pl of standard 5000 units/ml solution) of Taq DNA polymerase (Perkin-Elmer Cetus Instruments), diluted to a volume of 100 pl. The final mixture was then overlaid with 100 pl paraffin oil. PCR was carried out using a DNA thermal cycler (Perkin-Elmer/Cetus) by initially denaturing the template at 94° for 90 seconds, reannealing at 64° for 75 seconds and extending the cDNA at 72° for 150 seconds. PCR was carried out for an additional 25 cycles of amplification using the following step program (denaturation at 94° for 25 sec; annealing at 55° for 45 sec; extension at 72° for 150 sec.), followed by a 7 minute final extension at 72°.

The sample was removed from the paraffin oil and DNA extracted by phenol- chloroform extraction and spun column chromatography over G-50 (Boehringer Mannheim). A 10 pl aliquot of the extracted DNA was separated by electrophoresis on 1% SeaKem"“ agarose (FMC BioProducts, Rockland, ME) and stained with ethidium bromide to confirm that the DNA fragment size was consistent with the predicted product. The gel was then blotted and probed with a 5' 6l0bp EcoRI fragment of murine type H H.-1R clone 12 from above to confirm that the band contained DNA encoding murine type II IL-1R.

The PCR-amplified cDNA products were then concentrated by centrifugation in an Eppendorf microfuge at full speed for 20 min., followed by ethanol precipitation in 1/10 volume sodium acetate (3 M) and 2.5 volume ethanol. 30 pl of the concentrate was digested with Sall and NotI restriction enzymes using standard procedures. The Sall/Not] restriction fragment was then separated on a 1.2% LGT agarose gel, and the band representing the fragment was isolated. The restriction fragments were then purified from the agarose using GeneClean"“ (Bio-101, La Jolla, CA).

The resulting purified restriction fragment was ligated into the pDC406 vector, which was then transfected into CV1-EBNA cells and expressed as described above in Example 1.

The Sequence Listing setting forth the nucleotide (SEQ ID No:12) and predicted amino acid sequences (SEQ ID No:12 and SEQ ID NO:13) and associated information appears at the end of the specification immediately prior to the claims.

Example 4 Qonstruction and Expression of cDNAs Encoding Murine Soluble Typ_e II IL-1R A cDNA encoding soluble murine type II IL-1R (having the sequence of amino acids 345 of SEQ ID NO:l2) was constructed by PCR amplification 702./3 poly(A)+ mRNA as a template and the following procedure as described for the full length clone encoding murine type II IL-lR. The following PCR oligonucleotide primers (d(T)17 adaptor-primer, 5‘ amplification primer and 3' amplification primer, respectively) were constructed: < -NOtI -> '-GCGTCGACGGCAAGAAGCAGCAAGGTAC-3‘ \15 \34 SEQ ID NO:10 SEQ ID NO:14 5'-GCGCGGCCGCCTAGGAAGAGACTTCTTTGACTGTGG-3' <--Notl-—>EndSerSerValGluLysVal'I‘hrThr The d('I')17 adaptor-primer and 5‘ amplification primer are identical with SEQ ID NO:9 and SEQ ID NO:10, described in Example 5. The 3' end of SEQ ID NO:l2 is complementary to nucleotides 1145-1166 of SEQ ID NO:12 and has a 5' add-on of a Notl restriction site and a stop codon.

A pool of cDNA was synthesized from poly(A)+ mRNA using the d(T)17 adaptor- primer as described in Example 3. To a 1.5 ml Eppendorf microfuge tube was added approximately 1 pl of the cDNA pool, 5 |.lI of a 20 ttM solution of the 5' amplification primer, 5 pl of a 20 |.tM solution of the 3' amplification primer, 10 IJL of 10X PCR buffer (500 mM KC1, 100 mM Tris-HCl (pH 8.4 at 20°C), 14 mM MgCl2, and 1 mg/ml gelatin), 4 [.11 of 5mM each of dNTP (containing 5 mM dATP, 5mM dCl'P, 5mM dGTP and 5mM dTI'P), 2.5 units (0.5 tr] of standard 5000 units/tnl solution) of Taq DNA polymerase (Perkin-Elmer Cetus Instruments), diluted with 75.4 pl water to a volume of 100 pl. The final mixture was then overlaid with 100 pl paraffin oil. PCR was carried out using a DNA thermal cycler (Ericomp) by initially denaturing the template at 94° for 90 seconds, reannealing at 55° for 75 seconds and extending the cDNA at 72° for 150 seconds. PCR was carried out for an additional 20 cycles of amplification using the following step program (denaturation at 94° for 25 sec; annealing at 55° for 45 sec; extension at 72° for 150 sec.), followed by a 7 minute final extension at 72°.

The sample was removed from the paraffin oil and DNA extracted by phenol- chloroform extraction and spun column chromatography over G-50 (Boehringer Mannheim). A 10 pl aliquot of the extracted DNA was separated by electrophoresis on 1% SeaKem“" agarose (FMC BioProducts, Rockland, ME) and stained with ethidium bromide to confirm that the DNA fragment size was consistent with the predicted product.

The PCR—amplified cDNA products were then concentrated by centrifugation in an Eppendorf microfuge at full speed for 20 min., followed by ethanol precipitation in 1/10 volume sodium acetate (3 M) and 2.5 volume ethanol. 50 pl was digested with SalI and NotI restriction enzymes using standard procedures. The Sall/Notl restriction fragment was then separated on a 1.2% Seaplaque LGT agarose gel, and the band representing the fragment was isolated. The restriction fragment was then purified from the isolated band using the following freeze/thaw method. The band from the gel was split into two 175 pl fragments and placed into two 1.5 ml Eppendorf microfuge tubes. 500 pl of isolation buffer (0.15 M NaCl, 10 mM Tris, pH 8.0, 1rnM EDTA) was added to each tube and the tubes heated to 68°C to melt the'ge1. The gels were then frozen on dry ice for 10 minutes, thawed at room temperature and centrifuged at 4°C for 30 minutes. Supernatants were then removed and placed in a new tube, suspended in 2 mL ethanol, and centrifuged at 4°C for an additional 30 minutes to form a DNA pellet The DNA pellet was washed with 70% ethanol, centrifuged for 5 minutes, removed from the tube and resuspended in 20 pl TE buffer.

The resulting purified restriction fragments were then ligated into the pDC406 vector. A sample of the ligation was transformed into DH5a and colonies were analyzed to check for correct plasmids. The vector was then transfected into COS—7 cells and expressed as described above in Example 1.

Example 5 Tym [I IL-IR Binding Stugfigs The binding inhibition constant of recombinant human type H IL—lR, expressed and purified as described in Example 1 above, was determined by inhibition binding assays in which varying concentrations of a competitor (IL-18 or IL-la) was incubated with a constant amount of radiolabeled IL-18 or IL-la and cells expressing the type II 1L-IR. The competitor binds to the receptor and prevents the radiolabeled ligand from binding to the receptor. Binding assays were performed by a phthalate oil separation method essentially as describe by Dower et al., J. Immunol. I32:751, 1984 and Park et al., J. Biol. Chem. 26l:4l77, 1986. Briefly, CV1/EBNA cells were incubated in six-well plates (Costar, Cambridge, MA) at 4°C for 2 hours with 1251-11.-1B in 1 ml binding medium (Roswell Park Memorial Institute (RPMI) 1640 medium containing 2% BSA, 20 mM Hepes buffer, and 0.2% sodium azide, pH 7.2). Sodium azide was included to inhibit internalization and degradation of 1251-ll.-1 by cells at 37°C. The plates were incubated on a gyratory shaker for 1 hour at 37°C. Replicate aliquots of the incubation mixture were then transferred to polyethylene centrifuge tubes containing a phthalate oil mixture comprising 1.5 parts dibutylphthalate, to 1 part bis(s-ethylhexyl)phthalate. Control tubes containing a 100X molar excess of unlabeled IL-13 were also included to determine non-specific binding. The cells with bound 1251-IL-1 were separated from unbound 1251-IL-1 by centrifugation for 5 minutes at l5,000X g in an Eppendorf Microfuge. The radioactivity associated with the cells was then determined on a gamma counter. This assay (using unlabeled human IL-1B as a competitor to inhibit binding of 1251-IL-15 to type II IL-1R) indicated that the full length human type II IL-1R exhibits biphasic binding to IL-18 with a K11 of approximately 19:8 x 109 and K12 of approximately O.2i0.002 x 109. Using unlabeled human IL-lb to inhibit binding of 1251-IL-la to type II IL-IR, the full length human type II IL-1R exhibited biphasic binding to IL-18 with a K11 of approximately 2.0il x 109 and K12 of approximately 0.0l3:t0.003 x 109.

The binding inhibition constant of the soluble human type II 11,- IR, expressed and purified as described in Example 2 above, is determined by a inhibition binding assay in which varying concentrations of an IL-113 competitor is incubated with a constant amount of radiolabeled I-IL-18 and CB23 cells (an Epstein Barr virus transformed cord blood B lymphocyte cell line) expressing the type H IL-IR. Binding assays were also performed by a phthalate oil separation method essentially as describe by Dower et al., J. Immunol.

I32:751, 1984 and Park et al., J. Biol. Chem. 26l:4l77, 1986. Briefly, COS-7 cells were transfected with the expression vector pDC406 containing a cDNA encoding the soluble human type II H.-1R described above. Supematants from the COS cells were harvested 3 days after transfection and serially diluted in binding medium (Roswell Park Memorial Institute (RPMI) 1640 medium containing 2% BSA, 20 mM Hepes buffer, and 0.2% sodium azide, pH 7.2) in 6 well plates to a volume of 50 pl/well. The supernatants were incubated with 50 ul of 9x1o10 M 1251-11.-15 plus 2.5x1o6 CB23 cells at 8°C for 2 hours with agitation. Duplicate 60 pl aliquots of the incubation mixture were then transferred to polyethylene centrifuge tubes containing a phthalate oil mixture comprising 1.5 parts dibutylphthalate, to 1 part bis(s-ethylhexyl)phthalate. A negative control tube containing 3x10‘5 M unlabeled IL-15 was also included to determine non-specific binding ( 100% inhibition) and a positive control tube containing 50 ml binding medium with only radiolabeled IL-15 was included to determine maximum binding. The cells with bound 1251-IL-1B were separated from unbound 1251-IL-15 by cenuifugation for 5 minutes at ,000 X g in an Eppendorf Microfuge. Supematants containing unbound 1251-IL-1B were discarded and the cells were carefully rinsed with ice-cold binding medium The cells were then incubated in 1 ml of trypsin—EDTAat 37°C for 15 minutes and cells were harvested. The radioactivity of the cells was then determined on a gamma counter. This inhibition binding assay (using soluble human type II IL-1R to inhibit binding of IL-15) indicated that the soluble human typeéll IL-1R has a K101" approximately 3.5 x 109 M'1.

Inhibition of IL-la binding by soluble human type II IL-IR using the same procedure indicated that soluble human type H IL-IR has a K1 of 1.4 x 103 M4.

Murine type II IL-1R exhibits biphasic binding to IL-1 5 with a K11 of 0.8 x 109 and a K12 of less then 0.01 x 109.

— Example 6 rm; 11 IL-IR Affinig; Qrgsslinking Studies ’ Affinity crosslinking studies were performed essentially as described by Park et al., Proc. Natl. Acad. Sci. USA 84:l669, 1987. Recombinant human IL-10: and IL-13 used in the assays were expressed, purified and labeled as described previously (Dower et al., J.

Exp. Med. 162:501, 1985; Dower et al., Nature 3242266, 1986). Recombinant human IL- I receptor antagonist (IL-lra) was cloned using the cDNA sequence published by Eisenberg et al., Nature 3432341, 1990, expressed by transient transfection in COS cells, and purified by affinity chromatography on a column of soluble human type I 11.-IR coupled to affigel, as described by Dower et al., J. Immunol. I43:43l4, 1989, and eluted at low pH.

Briefly, CV1/EBNA cells (4 x 107/ml) expressing recombinant type H IL-IR were incubated with 1251-IL-la or 1251-IL-lfi (1 nM) at 4°C in the presence and absence of 1 uM excess of unlabeled IL-1 as a specificity control for 2 hours. The cells were then washed and bis(sulfosuccinimidyl)suberate was added to a final concentration of 0.1 mg/ml. After min. at 25°C, the cells were washed and resuspended in 100 til of phosphate-buffered saline (PBS)/1% Triton containing 2 mM leupeptin, 2 mM o-phenanthroline, and 2 mM EGTA to prevent proteolysis. Aliquots of the extract supematants containing equal amounts (CPM) of 1251-IL-1 and equal volumes of the specificity controls, were analyzed by SDS/PAGE on a 10% gel using standard techniques. .

Figure 4 shows the results of affinity crosslinking studicsconducted as described above, using radiolabeled IL-la and IL-13, to compare the sizes of the recombinant murine and human type II IL-1 receptor pnpteins to their natural counterparts, and to natural and recombinant murine and human type I IL-1 receptors. In general, the sizes of the transiently-expressed recombinant receptors are similar to the natural receptors, although the recombinant proteins migrate slightly faster and as slightly broader bands, possibly as a result of differences in glycosylation patter when over-expressed in CV_l/EBNA cells. The results also indicate that the type II ll.-1 receptors are smaller than the type I IL-1 receptors.

One particular combination (natural human type I receptor with IL-1 13) failed to yield specific crosslinl-ting products. Since approximately equal amounts of label were loaded into each experimental lane, as indicated by the intensity of the freeligand bands at the ‘ bottom of the gels, this combination must cnosslink relatively poorly.

The lane showing natural human type II IL-1 receptor-bearing cells cross-linked with 1251-IL-la reveals a component in the size range (M,=lO0,000) of complexes with natural and recombinant type I receptors. No such complex can be detected in the lane containing recombinant type II IL-1 receptor, possibly as a result of low level expression of type I IL-1 receptors on the CB23 cells, since these cells contain trace amounts of type I IL- 1 receptor mRNA.

Example 7 .

Preparation gf Monglgnal Antimgies t_Q mg; IIILA-1R Preparations of purified recombinant type II IL-IR, for example, human type 11 ll..- lR, or transfected COS cells expressing high levels of type II IL-1R are employed to generate monoclonal antibodies against type II IL-lR using conventional techniques, for example, those disclosed in U.S. Patent 4,411,993. Such antibodies are likely to be useful in interfering with IL-1 binding to type II IL—lR, for example, in ameliorating toxic or other undesired effects of IL-1, or as components of diagnostic or research assays for IL-1 or soluble type II IL-IR.

To immunize mice, type II IL-IR immunogen is emulsified in complete Freund's adjuvant and injected in amounts ranging from 10-100 ug subcutaneously and interaperitoneally into Balb/c mice. Ten to twelve days later, theimmunized animals are boosted with additional immunogen emulsified in incomplete Freund's adjuvant and periodically boosted thereafter on a weekly to biweekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-up excision for testing by dot-blot assay (antibody sandwich) or ELISA (enzyme-linked immunosorbent assay), or receptor binding inhibition. Other assay procedures are also suitable. Following detection of an appropriate antibody titer, positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and fused to the murine myeloma cell line N81 or Ag8.653. I-lybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a HAT selective medium (hypoxanthine, arninopterin, and thymidine) to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivity with type II ll.-IR, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Patent 4,703,004. Positive clones are then injected into the peritoneal cavities of syn geneic Balb/c mice to produce ascites containing high concentrations (>1 mg/ml) of anti-type II IL-IR monoclonal antibody, or grown in flasks or roller bottles. 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 or protein G from Streptococci.

EELM SEQ ID NO:l and SEQ H) NO:2 show the nucleotide sequence and predicted amino acid sequence of human type II IL-lR. The mature peptide encoded by this sequence is defined by amino acids 1-385. The predicted signal peptide is defined by amino acids -13 through -1. The predicted transrnernbrane region is defined by amino acids 331-356.

SEQ ID N023 - SEQ ID NO:6 are various oligonucleotides used to clone the full- length human type H IL-IR.

SEQ ID NO:7 and SEQ ID N028 are oligonucleotide primers used to construct a soluble human type II IL-IR by polymerase chain reaction (PCR).

SEQ ID N029 - SEQ ID NO:11 are oligonucleotide primers used to clone a full- length and soluble murine type II IL-1Rs.

SEQ ID NO:l2 and SEQ ID NO:l3 show the nucleotide sequence and predicted amino acid sequence of the full-length murine type II IL-1R. The mature peptide encoded by this sequence is defined by amino acids 1-397. The predicted signal peptide is defined by amino acids -13 through -1. The predicted transmembrane region is defined by amino acids 343-368.

SEQ ID NO: 14 is an oligonucleotidc primer used to construct a soluble murine type II IL-1R.

TABLE 1 — SEQUENCE LISTINGS (1) GENERAL INFORMATION: (i) APPLICANT: Sims, John E.

Cosman, David J.

Lupton, Stephen D.

Mosley, Bruce A.

Dower, Steven K. (ii) TITLE OF INVENTION: Type II Interleukin-1 Receptors (iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSES: Imunex Corporation 1(8) STREET: 51 University Street (C) CITY: Seattle (D) STATE: WA (E) COUNTRY: USA (F) ZIP: 98101 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (D) COMPUTER: IBM PC Compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.24 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 16-MAY-1991 (C)'CLASSIFICATION: (vii) paxoa APPLICATION DATA: (A) APPLICATION NUMDBR: us 07/334,193 (B) FILING DATE: 05-JUN-1990 (viii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 07/573,576 (B) FILING DATE: 24-AUG-1990 (ix) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: O7/627,071 (B) FILING DATE: 13-DEC-1990 (x) ATTORNEY/AGENT INFORMATION: (A) NAME: Wight, Christopher L.

(B) REGISTRATION NUMBER: 31680 (C) REFERENCE/DOCKET NUMBER: 2003-C (xi) TELECOMUNICATION INFORMATION: (A) TELEPHONE: 2065570 (B) TELEFAX: 2060644 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1357 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (G) CELL TYPE: Human B cell lymphoblastoid (H) CELL LINE: CB23 (vii) IMMEDIATE SOURCE: (A) LIBRARY: CB23 CDNA (B) CLONE: pHuIL~1RII75 (ix) FEATURE: (A) NAME/KEY: cos (B) LOCATION: 154..1350 (D) OTHER INFORMATION: (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 193..l347 (D) OTHER INFORMATION: (ix) FEATURE: ‘ (A) NAME/KEY: sig_peptide (B) LOCATION: l54..192 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTGGAAAATA CATTCTGCTA CTCTTAAAAA CTAGTGACGC TCATACAAAT CAACAGAAAG AGCTTCTGAA GGAAGACTTT AAAGCTGCTT CTGCCACGTG CTGCTGGGTC TCAGTCCTCC ACTTCCCGTG TCCTCTGGAA GTTGTCAGGA GCA ATG TTG CGC TTG TAC GTG TTG ' Met Leu Arg Leu Tyr Val Leu -13 -10 GTA ATG GGA GTT TCT GCC TTC ACC CTT CAG CCT GCG GCA CAC ACA GGG Val Met Gly Val Ser Ala Phe Thr Leu Gln Pro Ala Ala His Thr Gly -S 1 5 10 GCT GCC AGA AGC TGC CGG TTT CGT GGG AGG CAT TAC AAG CGG GAG TTC Ala Ala Arg Ser Cys Arg Phe Arg Gly Arg His Tyr Lys Arg Glu Phe 20 25 AGG CTG GAA GGG GAG CCT GTA GCC CTG AGG TGC CCC CAG GTG CCC TAC Arg Leu Glu Gly Glu Pro Val Ala Leu Arg Cys Pro Gln Val Pro Tyr 35 . 40 TGG TTG TGG GCC TCT GTC AGC CCC CGC ATC AAC CTG ACA TGG CAT AAA Trp Leu Trp Ala Se: val Se: Pro Arg Ile Asn Leu Thr Trp His Lys 45 50 120 GCC Ala 75 GGC Gly TCC Ser ATC Ile TGC Cys CAA Gln 155 GAA Glu CAG Gln GCT Ala 235 ACC Thr CAC His CAG Gln GAC Asp 60 CAG Gln ACC Thr ATT Ile TCA Ser CCT Pro 140 AGT Ser GAT Asp CAA Gln GAA Glu 220 GGC Gly ATA Ile GAA Glu TCT Ser GAC Asp TAC Tyr GAG Glu TAC Tyr 125 GAC Asp TAC Tyr GTG Val GCT Ala TAC Tyr 205 GAG Glu CTG Leu ACA Thr GAG Glu TAT Tyr 285 GCT Ala GGT Gly GTC Val CTC Leu 110 CCG Pro CTG Leu AAG Lys AGG Arg GGC Gly 190 AAC Asn ACC Thr GGG Gly CCC Pro AGC Ser 270 TCA Ser AGG Arg GCT Ala TGC Cys 95 AGA Arg CAA Gln AGT Ser GAT Asp GGG Gly 175 TAT Tyr ATC Ile ATT Ile TCA Ser TTA Leu 255 GCC Ala GAA Glu ACG Thr CTG Leu ACT Thr GTT Val ATT Ile GAA Glu TCT Ser 160 ACC Thr TAC Tyr ACT~ CCT Pro AGA Arg 240 ACC Thr Tyr' AAT Asn GTC Val 65 TGG Trp ACT Thr TTT Phe TTA Leu TTC Phe 145 CTT Leu ACT Thr CGC Arg .Arg GTG Val 225 ACC Thr CCG Pro AAT ASH CCA Pro CTT Leu AGA Arg GAG Glu ACC Thr 130 ACC Thr CTT Leu CAC His TGT Cys AGT Ser 210 ATC Ile ACA Thr ATG Met GGA Gly GAG Glu 2 GGA GAA GAA GAG CTG Leu AAT Asn AAT Asn 115 TTG Leu CGT Arg TTG Leu TTA Leu GTC Val 195 ATT Ile ATT Ile ATC Ile CTG Leu GGC Gly 275 AAC Asn CCA Pro GCT Ala 100 ACA Thr TCA Ser GAC Asp GAT Asp CTC Leu 180 CTG Leu GAG Glu TCC Ser CCG Pro TGG Trp 260 CGC Arg TAC Tyr GCC Ala 85 GAT Asp ACC Thr Lys 165 GTA Val ACA Thr CTA Leu CCC Pro TGT Cys 245 TGG Trp GTG Val ATT Ile Glu 70 TTG Leu TAC Tyr GCT Ala TCT Ser ACT Thr 150 GAC Asp CAC His TTT Phe CGC Arg CTC Leu 230 AAG Lys ACG Thr ACC Thr GAA Glu ACA Thr CAG Gln TGT Cys TTC Phe GGG Gly 135 GAC Asp AAT ASH GAT Asp GCC Ala ATC Ile 215 AAG Lys GTG Val GCC Ala GAG Glu GTG Val 295 CGG Arg GAG Glu GAC Asp CTG Leu 120 GTA Val GTG Val GAG Glu GTG Val CAT His 200 AAG Lys ACC Thr TTT Phe AAT Asn GGG Gly 280 CCA Pro ATG Met GAC Asp Lys 105 TTA Leu AAG Lys GCC Ala 185 GAA Glu ATA Ile CTG Leu GAC Asp 265 TTG Leu TGG Trp TCT Ser 90 ATG Met TTC Phe GTA Val ATT Ile TTT Phe 170 GGC Gly TCA Ser GGA Gly 250 ACC Thr CGC Arg ATT Ile TTT Phe GTC Val 315 GCC Ala GCC Ala AGA Arg GAC Asp Met- -13.

Ala 100 GAT Asp 300 CAT His TCC Ser TTC Phe ACT Thr TTT Phe 380 INFORMATION FOR SEQ ID NO:2: CCT Pro TCC Ser TTG Leu GGA Gly 365 CAA Gln GTC Val ACC Thr ACG Thr GTT Val 350 TCC Ser ACA Thr CTG Leu TTC Phe 335 GCA Ala TAT Tyr AGA Arg AGT Ser 320 GGG Gly GAT Asp CCC Pro TYPE: (D) TOPOLOGY: linear GAG Glu 305 TTT Phe TGG Trp GGA Gly GGT Gly AAG Lys 385 GAT Asp CAG Gln ATA Ile CTG Leu 370 TTG Leu ACA Thr ATT Ile TGG Trp 355 ACT Thr CAC His CTA Leu GTG Val 340 ATG Met GTG Val AATAAAT (ii) MOLECULE TYPE: protein (xi) SEQUENCE Leu Arg Leu Tyr Lys Arg Gln Val Thr Trp Thr Arg Gln Glu Cys Asp DESCRIPTION: Lys 105 (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 398 amino acids amino acid SEQ ID ATG Met CGC Arg 325 CAC His CTA Leu GAT Asp 310 ACC Thr GCC Ala AGA Arg TGG Trp N0:2: Leu 110 TTT Phe ACA Thr CCA Pro CGG Arg CCT Pro 375 AAA Lys GTC Val CTT Leu TGC Cys 360 CAT His TGT GTT AAG Lys TCA Ser 345 AAA Lys CAT His GAA Glu 330 CTG Leu CAC His CAA Gln Asn 115 Pro 375 INFORMATION FOR SEQ ID NO:3: Ser Tyr Pro Gln Phe 380 (i) SEQUENCE CHARACTERISTICS: (A) (B) (C) (D) TYPE: STRANDEDNESS: single TOPOLOGY: linear LENGTH: 24 base pairs nucleic acid (ii) MOLECULE TYPE: DNA (genomic) A811 Ile Glu Ser Lys 385 (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCGACTGGAA CGAGACGACC TGCT 24 2) INFORMATION FOR SEQ ID N034: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: Y (xi) SEQUENCE DESCRIPTION: SEQ ID No:4: GACCTTGCTC TGCTGGACGA ‘ 2o (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTGTTGGGCT CGCGGTTGAG GACAAACTCT TCGCGGTCTT TCCAGT 46 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: Y (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GACAACCCGA GCGCCAACTC CTGTTTGAGA AGCGCCAGAA AGGTCA (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GCGTCGACCT AGTGACGCTC ATACAAATC (2) INFORMATION FOR SEQ ID NO:8:_ (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GCGCGGCCGC TCAGGAGGAG GCTTCCTTGA CTG (2) INFORMATION FOR SEQ ID N019: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CTGCAGGCGG CCGCGGATCC TTTTTTTTTT TTTTTTT 37 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GCGTCGACGG CAAGAAGCAG CAAGGTAC 28 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CTGCAGGCGG CCGCGGATCC 20 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1366 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (vi) ORIGINAL SOURCE: (A) ORGANISM: Mouse (vii) GTCGACGGCA AGAAGCAGCA AGGTACAAGA ATACACAGCT CCAGGCTCCA AGGGTCCTGT (ix) (ix) (ix) (x1) (H) CELL LINE: 7oz/3 IMMEDIATE SOURCE: (A) LIBRARY: 7oz/3 (B) CLONE: 12 FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 85..13l7 (D) OTHER INFORMATION: FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: lZ4..13l4 (D) OTHER INFORMATION: FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: B5..123 (D) OTHER INFORMATION: SEQUENCE DESCRIPTION: SEQ ID NO:12: GCGCTCAGGA AGTTGGTGCG GACA ATG TTC ATC TTG CTT GTG TTA GTA ACT Met Phe Ile Leu Leu Val Leu Val Th: TCT Ser TGT Cys GAA Glu 45 CTG Leu TGG Trp TTC Phe GTT Val GAA Glu CAG Gln CCT Pro AGT Ser ATC Ile ATT Ile AGA Arg 110 -13 -10 TTC ACC ACT CCA ACA Phe Thr Thr Pro Thr TCT Ser Ala Val Val His AAG Lys CCC Pro ACA Thr GTC Val TCG Ser GAG Glu ACA Thr CCC Pro ATT Ile TCC Ser TTG Leu 40 TCT Ser GAA Glu TTC Phe GAG Glu GGC Gly AGA Arg CGT Arg Phe Lys CCT Pro 55 CAC His TTG Leu GCA Ala TGC Cys CCC Pro AGG Arg 50 CTG Leu GTG Val GTT Val TTG Leu AGT Ser 70 ACC Thr TGG Trp TTT Phe CTG Leu TCC Ser CAT His AGT Ser 65 AAG Lys TGG Trp GTG Val AGG Arg ATG Met 85 GAG Glu CCA Pro AGA Arg 80 GAT Asp CCA Pro GGT Gly TCT Ser ACC Thr CAA Gln 100 GAC Asp GTG val CAG Gln GCA Ala CTG Leu 95 CCA Pro TCT Ser ATG Met GTG Val 120 GAG Glu CAA Gln CAC His TGT Cys 115 AAC ASH GCA Ala TCC Ser CAT His AGG Arg TCC Ser GAC Asp GGT Gly TAC Tyr 105 GAA Glu Gly GGA Gly CTG Leu GAC Asp TCT Ser AAC Asn 90 ATT Ile CTC Len GAC Asp GAA Glu ATC Ile TCT Ser 75 ATA Ile TGC Cys AAG Lys GTG GTG CAC ACA GGA AAG GTT AAC Asn GGT Gly TCC Ser 60 CAG Gln CTC Leu ACA Thr GTC Val TTT Phe 125 TCA Ser TTC Phe ATA Ile ACA Thr AGA Arg 205 AGG Arg GTG Val CTG Leu ACC Thr CCA Pro 285 GAT Asp GAG Glu TCT Ser TGG Trp AAG Lys GCT Ala ATC Ile CTC Leu CGC Arg 190 TGT Cys AAT ASH ATC Ile ATA Ile ATT Ile 270 AGA Arg GAA Glu GAT Asp CAG Gln AGC Ser 350 AAT Asn CTC Leu TCC Ser TTG Leu 175 CTA Leu GTT Val ATT Ile ATT Ile GTC Val 255 GTG Val GGC Gly AAC Asn CTG Leu TCA Ser 335 ATT Ile ACT Thr TCC Ser AGC Ser 160 GAT Asp TTG Leu ATG Met GAA Glu TCT Ser 240 CCG Pro TGG Trp CGT Arg TAT Tyr CAT His 320 CTC GCG Ala GAA Glu ACC Thr 145 AAC Asn ATA Ile ACA Thr CTC Leu 225 CCC Pro TGC Cys TGG Trp GTG Val GTG Val 305 ACA Thr CAT His CTG Leu GCA Ala 130 ACC Thr GCT Ala GGC Gly TCC Ser TTT Phe 210 CGG Arg CTG Leu TTG Leu ACC Thr 290 GAA Glu GAT Asp ACC Thr GCA Ala TCT Ser GGG Gly GAT Asp AAT Asn AAC Asn 195 ACC Thr GTC Val GAG Glu GTG Val GCT Ala 275 GAG Glu GTG Val TTT Phe ACA Thr CCT Pro 355 CTG Leu TTA Leu GGA Gly AAG Lys 180 ACG Thr TAC Tyr ACA Thr TTT Phe 260 GGG Gly TCG Ser GTC Val 340 CTG Leu CCT Pro CTA Leu AAG Lys 165 GAA Glu TCC Ser AAT Asn GGA Gly ATA Ile 245 CTG Leu AGC Ser CTA Leu CTG Leu TGT Cys 325 AAA Lys TCT Ser CAT His GTG Val 150 ATA Ile TTT Phe ATG Met GGC Gly GCA Ala 230 CCA Pro GGA Gly ACG Thr CAC His ATT Ile 310 GTT Val GAA Glu CTG Leu GTC Val 135 TGC Cys CAG Gln CTG Leu GAC Asp CAG Gln 215 ACC Thr GCA Ala ACT Thr TTT Phe CAC His 295 TTT Phe GCC Ala GTC Val ATC Ile TCC Ser CCT Pro TGG Trp AGT Ser GAT Asp 200 GAA Glu ACG Thr TCA Ser GGT Gly ATC Ile 280 CAG Gln GAT Asp TCG Ser ‘Ser ATC Ile 360 TAC Tyr GAC Asp TAT Tyr GCA Ala 185 GCA Ala TAC Tyr GAA Glu TTG Leu ACA Thr 265 TCG Ser TAC Tyr CCA Pro AAT ASH TCC Ser 345 TTG Leu TTG Leu CTG Leu AAG Lys 170 GGA Gly GGC Gly AAC Asn CCC Pro GGG Gly 250 TCT Ser GCT Ala TCA Ser GTC Val CCA Pro 330 ACG Thr GTT Val CAA Gln Lys 155 GGC Gly GAC Asp TAT Tyr ATC Ile ATC Ile 235 TCA Ser TCC Ser GCT Ala GAG Glu ACA Thr 315 CGG Arg TTC Phe GTG Val ATC Ile 140 GAA Glu GCC Ala CCC Pro TAC Tyr ACT Thr 220 CCT Pro AGA Arg AAC Asn TAC Tyr AAT Asn 300 AGG Arg AGT Ser TCC Ser GGG Gly GCA Ala 365 GGA Gly ATA Ile CTG Leu TGG ATG CGC AGA CGG TGT AAA CGC AGG GCT GGA AAG ACA TAT Trp Met Arg Arg Arg Cys Lys Arg Arg Ala Gly Lys Thr Tyr 370 375 380 ACC AAG CTA CGG ACT GAC AAC CAG GAC TTC CCT TCC AGC CCA Thr Lys Leu Arg Thr Asp Asn Gln Asp Phe Pro Ser Ser Pro 385 390 395 ATAAAGGAAA TGAAATAAAA AAAAAAAAAA AAAAAGGATC CGCGGCCGC INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 410 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: Lys 165 SEQ ID NO:l3: Ile Leu Leu Val Leu Val Thr Gly Val Ser Ala Phe Thr Thr -10 -5 1 Val Val His Thr Gly Lys Val Ser Glu Ser Pro Ile Thr Ser ‘ 10 15 Pro Thr Val His Gly Asp Asn Cys Gln Phe Arg Gly Arg Glu 30 35 Ser Glu Leu Arg Leu Glu Gly Glu Pro Val Val Leu Arg Cys 40 45 50 Ala Pro His Ser Asp Ile Ser Ser Ser Ser His Ser Phe Leu 55 . 60 65 Ser Lys Leu Asp Ser Ser Gln Leu Ile Pro Arg Asp Glu Pro 70 75 80 Trp Val Lys Gly Asn Ile Leu Trp Ile Leu Pro Ala Val Gln 90 95 Ser His Cys Ser Gly Thr Tyr Ile Cys Thr Phe Arg Asn Ala 115 Met Ser Val Glu Leu Lys Val Phe Lys Asn Thr Glu Ala Ser 120 125 130 His Val Ser Tyr Leu Gln Ile Ser Ala Leu Ser Thr Thr Gly 135 140 145 Val Cys Pro Asp Leu Lys Glu Phe Ile Ser Ser Asn Ala Asp 150 155 160 Ile Gln Trp Tyr Lys Gly Ala Ile Leu Leu Asp Lys Gly Asn 170 175 Phe 260 GCGCGGCCGC CTAGGAAGAG ACTTCTTTGA CTGTGG His His Gln 295 Leu Ile Phe Cys A3 Val Ile 360 Arg Arg Ala Gln 390 Ala Gly 185 Ala Gly Tyr Asn Glu Pro Leu Gly 250 Thr Ser 265 Ser Ala Tyr Ser Pro 330 Ser Thr Leu Val Gly Lys Pro Ser Ser 3 Pro Thr Arg Leu Arg 205 Thr 220 Pro Val Arg Leu Asn Thr Tyr Pro 285 Asn Asp 300 Val Thr Arg Glu 315 Ser Ser Ala 365 Tyr Gly Pro Asn INFORMATION FOR SEQ ID NO:l4: (ii) (iii) (iv) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear TYPE: MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: N ANTI-SENSE: N Cys Val Asn Ile Ile Ile Ile Val Ile 270 Arg Gly Glu Asn Gln 335 Ser 350 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l4: Tyr Val Glu Leu His Thr Ser Leu His Ile Ala Leu Trp Met Arg Thr Lys Leu Asp

Claims (1)

1. An isolated DNA sequence selected from the group consisting of: (a) cDNA clones having a nucleotide sequence encoding a polypeptide having an amino acid sequence of human type II IL-1 receptor as shown in