NZ256293A - Method of treating tumour necrosis factor - mediated inflammation by use of tnf antagonist - Google Patents

Description

New Zealand No. 256293 International No. PCT/US93/08666 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 15.09.1992; Complete Specification Filed: 14.09.1993 Classification:(6) A61K38/19 Publication date: 24 June 1997 Journal No.: 1417 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Method of treating TNF-dependent inflammation using tumor necrosis factor antagonists Name, address and nationality of applicant(s) as in international application form: IMMUNEX CORPORATION, a Washington corporation of 51 University Street, Seattle, Washington 98101, United States of America New Zealand No. 256293 International No. PCT/US93/08666 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates; International filing date: Classification: Publication date: Journal No.: NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of inventioti: Method of treating TNF-dependent inflammation using tumor necrosis factor antagonists Name, address and nationality of applicant(s) as in international application form: IMMUNEX CORPORATION, 51 University Street, Seattle, Washington 98101, USA 6 2 9 3 1 TTTT.F.

Method of Treating TNF-Dependent Inflammation Using Tumor Necrosis Factor Antagonists . BACKGROUND OF THE INVENTION The present invention relates generally to cytokine receptors and more specifically to a method of using tumor necrosis factor antagonists to suppress TNF-dependent inflammatory diseases.

Tumor necrosis factor-a (TNFa, also known as cachectin) and tumor necrosis factor-p (TNFp, also known as lymphotoxin) are homologous mammalian endogenous secretory proteins capable of inducing a wide variety of effects on a large number of cell types. The great similarities in the structural and functional characteristics of these two cytokines have resulted in their collective description as "TNF." Complementary cDNA clones encoding TNFa (Pennica et al., Nature 312:724, 1984) and TNFp (Gray et al., Nature 312:121, 1984) have been isolated, permitting further structural ana biological characterization of TNF.

TNF proteins initiate their biological effect on cells by binding to specific TNF receptor (TNFR) proteins expressed on the plasma membrane of a TNF-responsive cell. Two distinct forms of TNFR are known to exist: Type I TNFR (TNFRI), having a molecular weight of approximately 75 kilodaltons, and Type II TNFR (TNFRII), having a molecular weight of approximately 55 kilodaltons. TNFRI and TNFRII each bind to both TNFa and TNFp. TNFRI and TNFRII have both been molecularly cloned (Smith et al., Science 248:1019,1990; Loetscher et al., Cell 61:351, 1990 and Schall et al., Cell 61:361, 1990), permitting recombinant expression and purification of soluble TNFR proteins.

Soluble TNF binding proteins from human urine have also been identified (Peetre et al., Eur. J. Haematol. 47:414, 1988; Seckinger et al., J. Exp. Med. 767:1511, 1988; Seckinger et al., J. Biol. Chem. 264:11966, 1989; UK Patent N.Z. PATENT OFFICE AliS 1996 i RECFIVT. I.) • 256295 2 Application, Publ. No. 2 218 101 A to Seckinger et al.; Engelmann et al., J. Biol.

Chem. 2(54:11974, 1989).

TNF antagonists, such as soluble TNFR and TNF binding proteins, bind to TNF and prevent TNF from binding to cell membrane bound TNF receptors. Such 5 proteins may therefore be useful to suppress biological activities caused by TNF.

The role of TNF in mediated inflammatory diseases and the in vivo biological . effects of such soluble TNFR and TNF binding protein proteins in suppressing such TNF-dependent inflammatory diseases have not been fully elucidated and potential therapeutic uses for TNF antagonists have yet to be identified.

SUMMARY OF THF. INVENTION The present invention provides a method of using TNF antagonists to suppress TNF-dependent inflammatory diseases. Specifically, the present invention provides a method of treating a human having arthritis comprising the step of administering a TNF antagonist, such as soluble human TNFR, to a human.

In particular, the invention provides the use, in the manufacture of a medicament, of a TNF antagonist selected from the group consisting of a soluble Type 1 TNF receptor, «nH a chimeric antibody comprising a TNF receptor and the constant domain of an immunoglobulin molecule as an active ingredient in a composition for the treatment of TNF-medicated inflammatory diseases.

The invention also provides the use, in the manufacture of a medicament of a soluble human TNFR as an active ingredient in a composition for treating arthritis in a mammal, wherein the amount to be given rangfes from substantially 0.1 mg/kg/week to substantially 100 mg/kg/week.

These and other aspects of the present invention will become evident upon reference to the following detailed description. > (followed by page 2a) 6 293 2a BRIEF DESCRIPTION OF THF. DRAWINGS FIGURE 1 shows the dimeric structure of the recombinant human TNFR/Fc fusion protein. The primaiy translation product of the plasmid coding for rhu TNFR/Fc is a single molecule of soluble TNFR linked to single chain of Fc derived from human IgGl. Following translation, but prior to secretion, this fusion molecule dimerizes via 3 cysteine residues in the Fc region to form dimeric rhu TNFR/Fc. Boxes denote structural domains of TNFR.

FIGURE 2 shows the construction of plasmid pCAVDHFR rhu TNFR/Fc. Abbreviations arc as follows: ADH2, yeast alcohol dehydrogenase gene and regulatory region; CMV, cytomegalovirus immediate early enhancer; TPL, adenovirus-2 tripartite leader, VA, adenovirus-2 virus-associated RNA genes I and II; DHFR, hamster dihydrofolate reductase gene.

FIGURES 3 and 4 are graphs showing the effect of intra-articular administration of recombinant human TNFR/Fc, monomelic TNFR, recombinant murine IL-1R and TNFR monomer combined with rmuIL-lR on antigen-induced arthritis in rats. The data indicate that TNFR/Fc, TNFR monomer, rmu IL-1R and TNFR combined with IL-1R suppress inflammation associated with antigen-induced arthritis.

PCT/ US93/08666 3 256253 FIGURE 5 shows the effect of intraperitoneal administration of recombinant human TNFR/Fc and PBS (vehicle control) on the development of collagen induced arthritis (CIA) in B10.RUI mice. TNFR/Fc significantly delayed the onset of CIA.

FIGURE 6 shows the effect of intraperitoneal administration of recombinant 5 human TNFR/Fc and PBS (vehicle control) on the development of collagen induced arthritis (CIA) in DBA/1 mice. TNFR/Fc significantly delayed the onset of CIA.

FIGURE 7 shows that administration of TNFR/Fc in mice reduced the arthritis index and the number oi' joints showing signs of arthritis.

As used herein, the terms "TNF receptor" and "TNFR" refer to proteins having amino acid sequences which are substantially similar to the native mammalian TNF receptor or TNF binding protein amino acid sequences, and which are capable of 15 binding TNF molecules and inhibiting TNF from binding to cell membrane bound TNFR. Two distinct types of TNFR are known to exist: Type I TNFR (TNFRI) and Type II TNFR (TNFRII). The mature full-length human TNFRI is a glycoprotein having a molecular weight of about 75-80 kilodaltons (kDa). The mature full-length human TNFRII is a glycoprotein having a molecular weight of about 55-60 kilodaltons 20 (kDa). The preferred TNFRs of the present invention are soluble forms of TNFRI and TNFRII, as well as soluble TNF binding proteins. Soluble TNFR molecules include, for example, analogs or subunits of native proteins having at least 20 amino acids and which exhibit at least some biological activity in common with TNFRI, TNFRII or TNF binding proteins. Soluble TNFR constructs are devoid of a transmembrane 25 region (and are secreted from the cell) but retain the ability to bind TNF. Various bioequivalent protein and amino acid analogs have an amino acid sequence corresponding to all or part of the extracellular region of a native TNFR, for example, huTNFRIA235, huTNFRIA185 and huTNFRIA163, or amino acid sequences substantially similar to the sequences of amino acids 1-163, amino acids 1-185, or 30 amino acids 1-235 of SEQ ID NO:l, and which are biologically active in that they bind to TNF ligand. Equivalent soluble TNFRs include polypeptides which vary from these sequences by one or more substitutions, deletions, or additions, and which retain the ability to bind TNF or inhibit TNF signal transduction activity via cell surface bound TNF receptor proteins, for example huTNFRIAx, wherein x is selected from the group 35 consisting of any one of amino acids 163-235 of SEQ ID NO: 1. Analogous deletions may be made to muTNFR. Inhibition of TNF signal transduction activity can be determined by transfecting cells with recombinant TNFR DNAs to obtain recombinant receptor expression. The cells are then contacted with TNF and the resulting metabolic PET ATT,ED DESCRIPTION OF THE INVENTION Definitions WO 94/06476 PCT/US93/08666 256253 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. 171:861 (1990); Curtis et al., Proc. Natl. Acad. Sci. USA 5 56:3045 (1989); Prywes et al., EMBO J. 5 :2179 (1986) and Chou et al., J. Biol. Chem. 262:1842 (1987). Alternatively, primary cells or cell lines which express an endogenous TNF receptor and have a detectable biological response to TNF could also be utilized.

The nomenclature for TNFR analogs as used herein follows the convention of 10 naming the protein (e.g., TNFR) preceded by either hu (for human) or mu (for murine) and followed by a A (to designate a deletion) and the number of the C-terminal amino acid. For example, huTNFRA235 refers to human TNFR having Asp2^5 as the C-terminal amino acid (i.e., a polypeptide having the sequence of amino acids 1-235 of SEQ ID NO:l). In the absence of any human or murine species designation, TNFR 15 refers generically to mammalian TNFR. Similarly, in the absence of any specific designation for deletion mutants, the term TNFR means all forms of TNFR, including mutants and analogs which possess TNFR biological activity.

The term "isolated" or "purified", as used in the context of this specification to define the purity of TNFR protein or protein compositions, means that the protein or 20 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 co-therapeutics. TNFR is isolated if it is detectable as a single protein band in a polyacrylamide gel by silver staining. 25 "Recombinant," as used herein, means that a protein is derived from recombinant (e.g., microbial or mammalian) expression systems. "Microbial" refers to recombinant proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, "recombinant microbial" defines a protein produced in a microbial expression system which is essentially free of native endogenous substances. Protein expressed in 30 most bacterial cultures, e.g., E. coli, will be free of glycan. Protein expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.

"Biologically active," as used throughout the specification as a characteristic of TNF receptors, means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable 35 of binding detectable quantities of TNF, transmitting a TNF stimulus to a cell, for example, as a component of a hybrid receptor construct, or cross-reacting with anti-TNFR antibodies raised against TNFR from natural (i.e., nonrecombinant) sources. Preferably, biologically active TNF receptors within the scope of the present invention WO 94/06476 PCT/US93/08666 256 2 5 3 are capable of binding greater than 0.1 nmoles TNF per nmole receptor, and most preferably, greater than 0.5 nmole TNF per nmole receptor in standard binding assays (see below).

Soluble TNF Antagonists and Analogs The present invention utilizes isolated and purified TNF antagonist polypeptides. The isolated and purified TNF antagonist polypeptides used in this invention are substantially free of other contaminating materials of natural or endogenous origin and contain less than about 1% by mass of protein contaminants 10 residual of production processes. The TNF antagonist polypeptides used in this invention are optionally without associated native-pattern glycosylation.

In preferred aspects of the present invention, the TNF antagonists are selected from the group consisting of soluble human TNFRI and TNFR II. The pCAV/NOT-TNFR vector, containing the human TNFRI cDNA clone 1, was used to express and 15 purify soluble human TNFRI. pCAV/NOT-TNFR has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA (Accession No. 68088) under the name pCAV/NOT-TNFR.

Like, most mammalian genes, mammalian TNF receptors are presumably encoded by multi-exon genes. Alternative mRNA constructs which can be attributed to 20 different mRNA splicing events following transcription, and which share large regions of identity or similarity with the cDNAs claimed herein may also be used.

Other mammalian TNFR cDNAs may be isolated by using an appropriate human TNFR DNA sequence as a probe for screening a particular mammalian cDNA library by cross-species hybridization. Mammalian TNFR used in the present invention 25 includes, by way of example, primate, human, murine, canine, feline, bovine, ovine, equine and porcine TNFR. Mammalian TNFRs can be obtained by cross species hybridization, using a single stranded cDNA derived from the human TNFR DNA sequence as a hybridization probe to isolate TNFR cDNAs from mammalian cDNA libraries.

Derivatives of TNFR which may be used in the present invention also include various structural forms of the primary protein which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, a TNFR 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. 35 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 TNFR WO 94/06476 PCT/US93/08666 256253 amino acid side chains or at the N- or C-termini. Other derivatives of TNFR include covalent or aggregative conjugates of TNFR 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 signal (or leader) polypeptide 5 sequence at the N-terminal 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). TNFR protein fusions can comprise peptides added to facilitate purification or k identification of TNFR (e.g., poly-His). The amino acid sequence of TNF receptor 10 can also be linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hopp et al., Bio/Technology 6:1204,1988.) 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 15 following the Asp-Lys pairing. Fusion proteins capped with this peptide may also be resistant to intracellular degradation in E. coli.

TNFR with or without associated native-pattern glycosylation may also be used. TNFR 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 20 the native molecules, depending upon the expression system. Expression of TNFR DNAs in bacteria such as E. coli provides non-glycosylated molecules. Functional mutant analogs of mammalian TNFR having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-25 carbohydrate form in good yield using yeast expression systems. N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-Aj-Z, where A] is any amino acid except Pro, and Z is Ser or Thr. In this sequence, Asn provides a side chain amino group for covalent attachment of carbohydrate. Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or 30 Z, or inserting a non-Z amino acid between Ai and Z, or an amino acid other than Asn between Asn and Aj.

TNFR derivatives may also be obtained by mutations of TNFR or its subunits. A TNFR mutant, as referred to herein, is a polypeptide homologous to TNFR but which has an amino acid sequence different from native TNFR because of a deletion, 35 insertion or substitution.

Bioequivalent analogs of TNFR proteins may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine WO 94/06476 PCT/US93/08666 256 2 5 3 residues can be deleted (e.g., Cys178) or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide 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 5 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 insertion on biological activity should be considered. Substantially similar polypeptide sequences, as defined above, 10 generally comprise a like number of amino acids sequences, although C-terminal truncations for the purpose of constructing soluble TNFRs will contain fewer amino acid sequences. In order to preserve the biological activity of TNFRs, deletions and substitutions will preferably result in homologous or conservatively substituted sequences, meaning that a given residue is replaced by a biologically similar residue. 15 Examples of conservative substitutions include substitution of one aliphatic residue for another, such as lie, 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 Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Moreover, particular 20 amino acid differences between human, murine and other mammalian TNFRs is suggestive of additional conservative substitutions that may be made without altering the essential biological characteristics of TNFR.

Subunits of TNFR may be constructed by deleting terminal or internal residues or sequences. Particularly preferred sequences include those in which the 25 transmembrane region and intracellular domain of TNFR are deleted or substituted with hydrophilic residues to facilitate secretion of the receptor into the cell culture medium. The resulting protein is referred to as a soluble TNFR molecule which retains its ability to bind TNF. A particularly preferred soluble TNFR construct is TNFRIA235 (the sequence of amino acids 1-235 of SEQ ID NO:l), which comprises the entire 30 extracellular region of TNFRI, terminating with Asp235 immediately adjacent the transmembrane region. Additional amino acids may be deleted from the transmembrane region while retaining TNF binding activity. For example, huTNFRIA183 which comprises the sequence of amino acids 1-183 of SEQ ID NO:l, and TNFR1A163 which comprises the sequence of amino acids 1-163 of SEQ ID NO:l, retain the ability 35 to bind TNF ligand. TNFRIA142, however, does not retain the ability to bind TNF ligand. This suggests that one or both of Cys157 and Cys163 is required for formation of an intramolecular disulfide bridge for the proper folding of TNFRI. Cys178, which was deleted without any apparent adverse effect on the ability of the soluble TNFRI to 256 2 5 3 bind TNF, does not appear to be essential for proper folding of TNFRI. Thus, any deletion C-terminal to Cys163 would be expected to result in a biologically active soluble TNFRI. The present invention contemplates use of such soluble TNFR constructs corresponding to all or pan of the extracellular region of TNFR terminating 5 with any amino acid after Cys163. Other C-terminal deletions, such as TNFRIA157, may be made as a matter of convenience by cutting TNFR cDNA with appropriate restriction enzymes and, if necessary, reconstructing specific sequences with synthetic oligonucleotide linkers. Soluble TNFR with N-terminal deletions may also be used in the present invention. For example, the N-terminus of TNFRI may begin with Leu1, 10 Pro2 or Ala3 without significantly affecting the ability of TNFRI to effectively act as a TNF antagonist. The resulting soluble TNFR constructs are then inserted and expressed in appropriate expression vectors and assayed for the ability to bind '.'rNF.

Mutations in nucleotide sequences constructed for expression of analog TNFP must, of course, preserve the reading frame phase of the coding sequences and 15 preferably will not create complementary regions that could hybridize to produce secondary mRNA 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 20 mutagenesis may be conducted at the target codon and the expressed TNFR mutants screened for the desired activity.

Not all mutations in the nucleotide sequence which encodes TNFR 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 25 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 30 of the native sequence. Following ligation, the resulting reconstructed sequence encodes an ana log having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according 'o the substitution, deletion, or insertion required. Exemplary methods of making the 35 alterations set forth above are disclosed by Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:13, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent WO 94/06476 PCT/US93/08666 258253 Nos. 4,518,584 and 4,737,462 disclose suitable techniques, and are incorporated by reference herein.

Both monovalent forms and polyvalent forms of TNFR may also be used in the present invention. Polyvalent forms possess multiple TNFR binding sites for TNF 5 ligand. For example, a bivalent soluble TNFR may consist of two tandem repeats of amino acids 1-235 of SEQ ID NO:l, separated by a linker region. Alternate polyvalent forms may also be constructed, for example, by chemically coupling TNFR to any clinically acceptable carrier molecule, a polymer selected from the group consisting of Ficoll, polyethylene glycol or dextran using conventional coupling techniques. 10 Alternatively, TNFR may be chemically coupled to biotin, and the biotin-TNFR conjugate then allowed to bind to avidin, resulting in tetravalent avidin/biotin/TNFR molecules. TNFR may also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugate precipitated with anti-DNP or anti-TNP-IgM, to form decameric conjugates with a valency of 10 for TNFR binding sites. 15 A recombinant chimeric antibody molecule may also be produced having TNFR sequences substituted for the variable domains of either or both of the immunoglobulin molecule heavy and light chains and having unmodified constant region domains. For example, chimeric TNFR/IgGi may be produced from two chimeric genes — a TNFR/human k light chain chimera (TNFR/Ck) and a TNFR/human yi heavy chain 20 chimera (TNFR/Cy„i). Following transcription and translation of the two chimeric genes, the gene products assemble into a single chimeric antibody molecule having TNFR displayed bivalently. Such polyvalent forms of TNFR may have enhanced binding affinity for TNF ligand. One specific example of a TNFR/Fc fusion protein is disclosed in SEQ ID NO:3 and SEQ ID NO:4. Additional details relating to the 25 construction of such chimeric antibody molecules are disclosed in WO 89/09622 and EP 315062.

Expression of Recombinant TNFR Recombinant expression vectors are preferably used to amplify or express 30 DNA encoding TNFR to obtain purified TNFR. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding mammalian TNFR 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 35 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 WO 94/06476 PCT/US93/08666 25 6 ^53 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 von^rred by an origin of replication, and a selection gene to facilitate recognition uf tr? nsformants may additionally be incorporated. DNA regions are 5 operably lirkfu 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 10 orsitioned 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 15 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 TNF receptors which are to be expressed in a microorganism will preferably contain no introns that could prematurely 20 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-terminal 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 25 acid sequence. Other embodiments include sequences capable of hybridizing to the sequences of the provided cDNA under moderately stringent conditions (50*C, 2x SSC) and other sequences hybridizing or degenerate to those which encode biologically active TNF receptor polypeptides.

Recombinant TNFR DNA is expressed or amplified in a recombinant 30 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 transformation 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 35 the system are the progeny of a single ancestral transfoimant. 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. 256 2 5 3 Transformed host cells are cells which have been transformed or transfected with TNFR vectors constructed using recombinant DNA techniques. Transformed host cells ordinarily express TNFR, but host cells transformed for purposes of cloning or amplifying TNFR DNA do not need to express TNFR. Expressed TNFR will be 5 deposited in the cell membrane or secreted into the culture supernatant, depending on the TNFR DNA selected. Suitable host cells for expression of mammalian TNFR 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 10 mammalian origin as described below. Cell-free translation systems could also be employed to produce mammalian TNFR 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 15 disclosure of which is hereby incorporated by reference.

Prokaryotic expression hosts may be used for expression of TNFR that do not require extensive proteolytic and disulfide 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 20 requirement, and an origin of replication recognized by the host to ensure amplification within the host. Suitable prokaiyotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphyolococcus, 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 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA). These 30 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 2:95, 1977). pBR322 contains genes for ampicillin 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 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 5:4057, 1980; and EPA 36,776) WO 94/06476 PCT/US93/08666 ^56253 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 X Pl promoter and cI857ts thermolabile repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of 5 the X Pl promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).

Recombinant TNFR proteins may also be expressed in yeast hosts, preferably from the Saccharomyces species, such as 5. cerevisiae. Yeast of other genera, such as ; ichia or Kluyveromyces may also be employed. Yeast vectors will generally contain 10 an origin of replication from the 2\i yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA encoding TNFR, 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 5. cerevisiae TRP1 or URA3 15 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. 20 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. 77:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-25 phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pUC18 30 for selection and replication in E. coli (Ampr 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. 255:2674, 1982) and Beier et al. {Nature 300:124,1982). The yeast a-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter 35 and the structural gene to be expressed. See, e.g., Kuijan et al., Cell 30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 57:5330, 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. 94/06476 PCT/ JS93/08666 13 2 5 8 2 5 3 Suitable yeast transformation protocols are known to those of skill in the art; an exemplary technique is describec by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp+ transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ng/ml adenine and 5 20 ng/ml uracil or URA+ tranformants 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 transformed by vectors comprising the ADH2 promoter may be 10 grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% or 4% glucose supplemented with 80 ng/ml adenine and 80 ng/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants 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 20 by Gluzman (Cell 25:175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, CI 27, 3T3, Chinese hamster ovaiy (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 5' or 3' flanking nontranscribed 25 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 30 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 SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other 35 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 SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided WO 94/06476 PCT/US93/08666 256 2 5 3 14 the approximately 250 bp sequence extending from the Hind 3 site 'oward the Bgl\ site located in the viral origin of replication is included. Further, mammalian genomic TNFR promoter, control and/or signal sequences may be utilized, provided such control sequences are compatible with the host cell chosen. Additional details regarding 5 the use of a mammalian high expression vector to produce a recombinant mammalian TNF receptor are provided in Examples 2 and 7 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 CI27 murine mammary epithelial cells can be constructed substantially as described 10 by Cosman et al. {Mol. Immunol. 25:935, 1986). - Recombinant expression vectors comprising TNFR cDNAs ai o 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 15 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-transformed host cells are then cultured in increasing concentrations of the known 20 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 '4~'e, 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-amplification of 25 the vector DNA encoding the desired protein (TNFR) m 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 30 a desired protein, or is co-transformed with a vector comprising a DNA sequence encoding DHFR and a vector comprising a DNA sequence encoding the desired protein. The transformed or co-transformed 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 35 synthetase (GS), which is responsible for the synthesis of 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, TNFR can be expressed in high concentrations by co-amplifying cells WO 94/06476 PCT/US93/Q8666 256 2 5 3 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 5 co-amplification system, appropriate recombinant expression vectors and cells lines, are described in the following PCT applications: WO 87/04462, WO 89/01036, WO 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 10 alternatively in a murine myeloma cell line, such as SP2/0-Agl4 or NS0 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 TNFR DNA is disclosed below in Example 1. This vector, referred to as pCAV/NOT, was derived from the 15 mammalian high expression vector pDC201 and contains regulatory sequences from SV40, adenovirus-2, and human cytomegalovirus.

Purification of Recombinant TNFR Purified mammalian TNF receptors or analogs are prepared by culturing 20 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, supernatants from systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein 25 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 a TNF or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant 30 diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.

Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a TNFR WO 94/06476 PCT/US93/08666 258253 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, 5 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 TNFR can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express mammalian TNFR as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:\7\, 1984). This reference describes two sequential, reversed-phase HPLC steps for purification of recombinant human GM-CSF on a preparative 15 HPLC column.

Human TNFR 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 TNFR from the culture. These components ordinarily will be of yeast, prokaryotic or non-human 20 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 TNFR free of proteins which may be normally associated with TNFR as it is found in nature in its species of origin, e.g. in cells, cell exudates or body fluids.

Therapeutic Administration of Recombinant Soluble TNFR The present invention provides methods of suppressing TNF-dependent inflammatory responses in humans comprising administering an effective amount of a TNF antagonist, such as TNFR, and a suitable diluent and carrier. 30 For therapeutic use, purified soluble TNFR protein is administered to a patient, preferably a human, for treatment of arthritis. Thus, for example, soluble TNFR protein compositions can be administered, for example, via intra-articular, intraperitoneal or subcutaneous routes by bolus injection, continuous infusion, sustained release from implants, or other suitable techniques. Typically, a soluble 35 TNFR 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 WO 94/06476 PCT/US93/08666 17 H 5 6 2 5 3 TNFR 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 5 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. In accordance with appropriate industry standards, preservatives may also be added, such as benzyl alcohol. The amount and frequency of administration will depend, of course, on such factors as the 10 nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth.

TNF antagonist proteins are administered to a mammal, preferably a human, for the purpose treating TNF-dependent inflammatory diseases, such as arthritis. For example, TNFRI proteins inhibit TNF-dependent arthritic responses. Because of the 15 primary roles IL-1 and IL-2 play in the production of TNF, combination therapy using TNFR in combination with IL-1R and/or IL-2R may be preferred in the treatment of TNF-associated clinical indications. In the treatment of humans, soluble human TNFR is preferred. Either Type IIL-1R or Type IIIL-1R, or a combination thereof, may be used in accordance with the present invention to treat TNF-dependent inflammatory 20 diseases, such as arthritis. Other types of TNF binding proteins may be similarly used.

For treatment of arthritis, TNFR is administered in systemic amounts ranging from about 0.1 mg/kg/week to about 100 mg/kg/week. In preferred embodiments of the present invention, TNFR is administered in amounts ranging from about 0.5 mg/kg/week to about 50 mg/kg/week. For local intra-articular administration, dosages 25 preferably range from about 0.01 mg/kg to about 1.0 mg/kg per injection.

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

EXAMPLES Example 1 Expression and Purification of Soluble Human TNFRI The cloning of the cDNA for the 80 kD form of the human TNF receptor has been described in detail (Smith et al., Science 248:1019,1990). The expression vector pCAV/NOT-TNFR (ATCC 68088) containing the TNFR cDNA clone 1 was used to prepare and express a soluble human TNFRI as follows.

WO 94/06476 PCT/US93/08666 18 2 5 6 2 5 3 A cDNA encoding a soluble human TNFRIA235 (the primary translation product of which had the sequence of amino acids -22-235 of SEQ ID NO:l) was constructed by excising an 840 bp fragment from pCAV/NOT-TNFR with the restriction enzymes Notl and Pvu2. Notl cuts at the multiple cloning site of 5 pCAV/NOT-TNFR and Pvu2 cuts within the TNFR coding region 20 nucleotides 5' of the transmembrane region. In order to reconstruct the 3' end of the TNFR sequences, two oligonucleotides were synthesized and annealed to create the following oligonucleotide linker encoding amino acids corresponding to amino acids 229-235 of SEQ ID NO:l: Pvu2 BamHl Bgl2 ctgaagggagcactggcgacimggatcca GACTTCCCTCGTGACCGCTGATTCCTAGGTCTAG AlaGluGlySerThrGlyAspEnd This oligonucleotide linker has terminal Pvu2 and Bgl2 restriction sites, regenerates 20 nucleotides of the TNFR, followed by a termination codon (underlined) and a BamHl restriction site (for convenience in isolating the entire soluble TNFR by Notl/BamHl digestion). This oligonucleotide was then ligated with the 840 bp Notl/Pvu2 TNFR insert into Bgl2/Notl cut pCAV/NOT to yield psolhuTNFRA235/CAVNOT, which was transfected into COS-7 cells as described above. The host cells expressed a mature a soluble human TNFRI protein having the sequence of amino acids 1-235 which was capable of binding TNF.

Example 2 Construction and Expression of Soluble Human TNFR/Fc Fusion Protein A schematic diagram showing the construction of a recombinant soluble human TNFR:Fc expression vector is shown in Figure 1. The rhu TNFR:Fc fusion gene was created by ligating the following fragments into Bluescript®, a commercially available cloning vector (Stratagene): 1) An 867 bp Asp718-Pvu2 fragment from pCAV/NOT-TNFR (ATCC 68088) containing the cDNA encoding the truncated TNFR. 2) A 700 bp Styl-Spel fragment from plasmid pIXY498 coding for 232 amino acids of the Fc portion of human IgGl. Plasmid pIXY498 is a yeast expression vector containing the Fc fragment of human IgGl (see Figure 2). 3) An oligonucleotide linker, to fuse the truncated 19 human IgGl Fc fragment This linker was created by PCR (polymerase chain reaction) amplification using two primers, one having the sequence CCCCAGCTGAAGGGAGCACTGGCG ACX3AGCCCAAATCITGTGACAAAACTC (nucleotides 833-883 of SEQ ID NO: 3) which encodes the 3* end of the truncated TNF receptor and the 5' end of human IgGl, and the other having the sequence CGGTACGTGCTGTTGTTACTGC (SEQ ID NO:5), an antisense sequence encoding nucleotides 257-237 of human IgG 1. The template for this reaction was pIXY498. The reaction product was digested with Pvu2 and Styl, and a 115 bp fragment was isolated.

This construct was then digested with Notl and the resulting 1.4 kilobase fragment containing the rhu TNFR:Fc fusion DNA sequence was ligated into the Notl site of plasmid CAV/NOT/DHFR. Plasmid pCAV/NOT/DHFR was derived from plasmid pCAV/NOT by inserting the hamster dihydrofolate reductase DNA sequence (DHFR) into the Hpal site of pCAV/NOT (Figure 2). This construct was designated plasmid pCAVDHFRhuTNFRFc. The entire coding region sequence was confirmed by DNA sequencing and is depicted in Figure 2.

To prepare the host strain, DXB-11 CHO cells deficient in the expression of dihydrofolate reductase (DHFR) were obtained from Dr. Lawren Chasin at Columbia University. A bank of 100 vials of these cells was established, and representative vials were sent to Microbiological Associates for examination via the following procedures: Test Result All transfections and amplification steps were performed in a separate laboratory set aside for this purpose. Only mycoplasma-free cell lines were allowed into this facility.

Transfections were performed by mixing pCAVDHFRhuTNFRFc plasmid 35 DNA with LipofectinTM reagent from Gibco BRL. Approximately 10 >g of DNA was added to 10 cm petri dishes containing CHO DXB-11 cells. After the initial transfection, cells were selected for the expression of DHFR by subculturing in selective medium lacking glycine, hypoxanthine and thymidine. The resulting colonies 1. Transmission Electron Microscopy (TEM) 2. Sterility - Bacterial and Fungal 3. Mycoplasma 4. Mouse Antibody Production (MAP) Type A only, negative negative negative so 2 5 6 2 5 3 were then transferred to 24 well plates and analyzed for rhu TNFR:Fc expression. The highest expressing cultures were subjected to amplification by exposure to increasing concentrations of methotrexate (MTX). Cells able to grow at 25 nM MTX were cloned by limiting dilution in 96 well plates. The highest expressing clones were transferred to 5 suspension culture and the final selection of clone 4-4FC102A5-3 was made based on its high level of rhu TNFR:Fc expression under these conditions.

Example 3 Expression of Monomelic Soluble TNF Receptors in CHO Cells Soluble TNF receptor was expressed in Chinese Hamster Ovary (CHO) cells using the glutamine-synthetase (GS) gene amplification system, substantially as described in PCT patent application Nos. W087/04462 and W089/01036. Briefly, CHO cells are transfected with an expression vector containing genes for both TNFR 15 and GS. CHO cells are selected for GS gene expression based on the ability of the transfected DNA to confer resistance to low levels of methionine sulphoximine (MSX). GS sequence amplification events in such cells are selected using elevated MSX concentrations. In this way, contiguous TNFR sequences are also amplified and enhanced TNFR expression is achieved.

The vector used in the GS expression system was psolTNFR/P6/PSVLGS, which was constructed as follows. First, the vector pSVLGS.l (described in PCT Application Nos. W087/04462 and W089/01036, and available from Celltech, Ltd., Berkshire, UK) was cut with the BamHl restriction enzyme and-dephosphorylated with calf intestinal alkaline phosphatase (CLAP) to prevent the vector from religating to 25 itself. The BamHl cut pSVLGS.l fragment was then ligated to a 2.4 kb BamHl to Bgl2 fragment of pEE6hCMV (described in PCT Application No. W089/01036, also available from Celltech) which was cut with Bgl2, BamHl and Fspl to avoid two fragments of similar size, to yield an 11.2 kb vector designated p6/PSVLGS.l. pSVLGS.l contains the glutamine synthetase selectable marker gene under control of 30 the SV40 later promoter. The BamHl to Bgl2 fragment of pEE6hCMV contains the human cytomegalovirus major immediate early promoter (hCMV), a polylinker, and the SV40 early polyadenylation signal. The coding sequences for soluble TNFR were added to p6/PSVLGS.l by excising a Notl to BamHl fragment from the expression vector psolTNFR/CAVNOT (made according to Example 3 above), blunt ending with 35 Klenow and ligating with Smal cut dephosphorylated p6/PSVLGS.l, thereby placing the solTNFR coding sequences under the control of the hCM V promoter. This resulted in a single plasmid vector in which the SV40/GS and hCMB/solTNFR transcription WO 94/06476 PCT/US93/08666 256 25 3 units are transcribed in opposite directions. This vector was designated psolTNFR/P6/PSVLGS. psolTNFR/P6/PSVLGS was used to transfect CHO-K1 cells (available from ATCC, Rochville, MD, under accession number CCL 61) as follows. A monolayer of 5 CHO-K1 cells were grown to subconfluency in Minimum Essential Medium (MEM) 10X (Gibco: 330-1581AJ) without glutamine and supplemented with 10% dialysed fetal bovine serum (Gibco: 220-6300AJ), 1 mM sodium pyruvate (Sigma), MEM nonessential amino acids (Gibco: 320-1140AG), 500 nM asparagine and glutamate (Sigma) and nucleosides (30 uM adenosine, guanosine, cytidine and uridine and 10 jiM 10 thymidine)(Sigma).

Approximately 1 x 106 cells per 10 cm petri dish were transfected with 10 ug of psolTNFR/P6/PSVLGS by standard calcium phosphate precipitation, substantially as described by Graham & van der Eb, Virology 52:456 (1983). Cells were subjected to glycerol shock (15% glycerol in serum-free culture medium for approximately 1.5 15 minutes) approximately 4 hours after transfection, substantially as described by Frost & Williams, Virology 97:39 (1978), and then washed with serum-free medium. One day later, transfected cells were fed with fresh selective medium containing MSX at a final concentration of 25 uM. Colonies of MSX-resistant surviving cells were visible within 3-4 weeks. Surviving colonies were transferred to 24-well plates and allowed to grow 20 to confluency in selective medium. Conditioned medium from confluent wells were then assayed for soluble TNFR activity using standard binding assays. These assays indicated that the colonies expressed biologically active soluble TNFR.

In order to select for GS gene amplification, several MSX-resistant cell lines are transfected with psolTNFR/P6/PSVLGS and grown in various concentrations of MSX. 25 For each cell line, approximately lxlO6 cells are plated in gradually increasing concentrations of 100 uM, 250 uM, 500 uM and 1 mM MSX and incubated for 10-14 days. After 12 days, colonies resistant to the higher levels of MSX appear. The surviving colonies are assayed for TNFR activity. Each of these highly resistant cell lines contains cells which arise from multiple independent amplification events. From 30 these cells lines, one or more of the most highly resistant cells lines are isolated. The amplified cells with high production rates are then cloned by limiting dilution cloning. Mass cell cultures of the transfectants secrete active soluble TNFR.

Example 4 Effect of Soluble TNFR on Antigen-Induced Arthritis in Rats Lewis rats previously immunized with methylated bovine serum albumin (mBSA) in complete Freund's adjuvant develop antigen-induced arthritis (AIA) when IM 2 5 3 22 challenged with mBSA in knee joints. Administration of rhu TNFR?! monomer, recombinant murine soluble IL-1 receptor (rm IL-1R) or a combination of TNFR monomer plus rm IL-1R was shown to be effective in suppressing the effects of antigen-induced arthritis in rats.

Lewis rats were immunized in the hind flank with 0.5 mg mBSA in complete Freund's adjuvant. Twenty-one days later (day 0), the animals were injected in both hind knee joints with 50 |Jg mBSA in pyrogen-free saline. Groups of six rats were injected intra-articularly in both knee joints on that day and on the following 2 days (days 0,1 and 2) as indicated below in Table A: Table A Treatment and Dosage Schedule Group Treatment Dose 1 rhu TNFR/Fc 10 p.g 2 rhu TNFR/Fc 5 jig 3 rmu IL-1 Receptor 1 ng 20 4 TNFR Monomer 5 ng TNFR Monomer/rmu IL-1 R 10 p.g/1 p.g 6 Diluent (saline) Knee joint width was measured daily on days 0-6 relative to treatment. TNFR monomer was produced in CHO cells according to Example 2. The rhu TNFR:Fc used in this experiment was produced in BHK (hamster kidney) cells. This material is similar to the CHO cell-derived TNFR.

Figures 3 and 4 demonstrate that treatment with BHK-derived rhu TNFR:Fc at 30 the time of mBSA challenge and for two days following challenge resulted in a reduction of knee-joint swelling in comparison to diluent-treated control rats. A reduction in joint swelling and inflammation was observed in rats treated with 5 or 10 |ig BHK-derived rhu TNFR:Fc or 5 |lg TNFR monomer or 1 fxg of rmuIL-lR. Reduction in joint swelling was even more pronounced when rmuIL-lR and TNFR 35 monomer treatment was combined.

Histopathological examination of the joints harvested on day 6 was performed to confirm the degree of swelling. Histopathology scores were derived by evaluating knee joints and scoring their condition as follows: Grade 1, minimal, <10% of area WO 94/06476 PCT/US93/08666 6 2 5 3 affected; Grade 2, moderate, 10-50% of area affected; Grade 3, marked, at least 50%, b:: less than all, of area affected; Grade 4, maximal, total area severely affected. A variety of lesions/alterations involving five knee joint structures were evaluated: joint capsule, joint space, synovial membrane, articular cartilage, and subchondral bone.

Each structural alteration was scored from 1 to 4, and the scores were added and means were calculated. Histopathology results are expressed as the mean score in each treatment group.

The following Table B shows histopathology results, which also indicate that rhu TNFR:Fc, TNFR monomer and rmu IL-1R were effective in reducing the severity 10 of antigen-induced arthritis, and that a combination of rm IL-1 R and TNFR monomer was more effective than either receptor alone.

Table B Effect of rhu TNFR:Fc on Antigen Induced Arthritis in Rats Treatment Histopathology Score Number (Mean ± SD (SE)) Of Animals Saline 18.4 ± 4.9 (1.5) 1.0 *xg rmu IL-1R 13.1 ± 4.7 (1.7) .0 ^g TNFR monomer 12.8 ± 3.1 (1.1) 1.0 ng rmu IL-1R/10.0 p.g TNFR monomer 7.9 ± 5.2 (2.0) .0 ng TNFR monomer 13.4 ± 2.8 (1.0) .0 ^ig rhu TNFR:Fc (BHK) 13.4 ± 3.6 (1.3) 8 8 5 9 8 In summary, treatment with rhu TNFRFc, TNFR monomer, or rmu IL-1R at 30 the time of mBSA challenge and for two days following challenge resulted in a reduction of knee-joint swelling in comparison to diluent-treated control rats. A combination of both rmu IL- 1R and TNFR monomer resulted in greater reduction of swelling than either receptor molecule alone. Histopathology results also indicated that rhu TNFR/Fc, TNFR and rmu IL-1R were effective in reducing the severity of antigen-35 induced arthritis, and that a combination of rmu IL-1R and TNFR monomer was more effective than either receptor alone.

WO 94/06476 PCT/US93/08666 256 2 5 3 Example 5 Effect of Soluble TNFR on Collagen-Induced Arthritis in B10.RITI Mice BIO.RDI mice previously immunized with porcine type II collagen (CII) in 5 complete Freund's adjuvant consistently develop collagen-induced arthritis (CIA). Administration of rhu TNFR:Fc was shown to be effective in suppressing the symptoms of CIA in mice.

B10.RIII mice were immunized intradermally with 100 jig porcine type II collagen (CII) in complete Freund's adjuvant to induced arthritic symptoms.

Approximately 14-17 days post-immunization, symptoms of clinical arthritis began to appear in the mice, with 90-100% of the mice displaying severe arthritis by day 28.

Mice were injected irtraperitoneally with TNFR/Fc or PBS to determine the effect of soluble TNFR/Fc on CIA. Mice were assessed for symptoms of arthritis at 12 weeks post-immunization.

In a first experiment, TNFR/Fc was administered over the entire period of CIA development. Twelve mice were injected with 10 jxg TNFR/Fc, 3 days per week, from days 0 to 35. Twelve control mice were injected with PBS. Figure 5 shows that TNFR/Fc significantly reduced the incidence of arthritis when compared to controls. Upon cessation of treatment with TNFR/Fc, the mice developed arthritis.

In a second experiment, TNFR/Fc was administered during only the developmental stages of CIA on days -1-17 relative to immunization, as set forth in the following Table C.

Table C Effect of rhu TNFR:Fc Administered During Inductive Stage of CIA Treatment Incidence Onset Severity (Positive/Total) (Mean Day ± SE) (Mean ± SE) ng TNFR/Fc 10/10 Days-1,3 ng TNFR/Fc 8/10 35 Days -1 to 17 (alternate days) 100 m-1 PBS 10/10 Days -1 to 17 40 (alternate days) 24 ±2 10.5 ±0.5 21± 2 8.6 ± 0.6 18 ±1 10.6 ±0.4 WO 94/06476 PCT/US93/08666 2 5 6 2 5 3 These data show that TNFR/Fc delayed the onset of arthritis, but that CIA was unaltered in mice receiving 30 fig TNFR/Fc the day before and 3 days after immunization with type II collagen. Mice given 10 |ig TNFR/Fc, every other day, from day -1 to day 17 displayed a slight decrease in CIA incidence and severity versus 5 controls injected with PBS.

In a third experiment, TNFR/Fc was administered during only the progressive stages of CIA every other day on days 14-28 post-immunization as set forth in the following Table D.

Table D Effect of rhu TNFRrFc Administered During Progressive Stage of CIA Treatment Incidence Onset Severity (Positive/Total) (Mean Day ± SE) (Mean ± SE) ng TNFR/Fc 8/9 27 ±6 8.6 ±1.3 Days 14-28 (alternate days) 100 nl PBS 9/9 21 ±1 8.7 ±0.6 Days 14-28 (alternate days) These data show that mice given 10 ng TNFR/Fc, every other day, from days 14-28 showed a slight delay in CIA onset when compared to control animals. However, the incidence and severity of arthritis appears to be unaltered.

In summary, these experiments indicate that TNFR/Fc was effective in delaying 30 the onset of CIA when administered over the entire course of CIA development Example 6 Effect of Soluble TNFR on Collagen-Induced Arthritis in DBA/1 Mice The effect of soluble TNFR/Fc on CIA in DBA/1 mice previously immunized with porcine type II collagen (CII) in complete Freund's adjuvant was also tested. Administration of rhu TNFR:Fc was shown to be effective in suppressing the symptoms of CIA.

In this experiment, DBA/1 mice were immunized with 100 ng of CII and then 40 injected intraperitoneally with 50 |ig recombinant soluble human TNFR/Fc in sterile saline from day 21 to day 28. Control mice received sterile saline (vehicle) injections.

WO 94/06476 PCT/US93/08666 256 253 This treatment period was prior to the development of the clinical signs of CIA, but during the development of DTH responses to type II collagen and rapid IgG anti-CII production.

Both groups of mice were assessed for the development of CIA for 70 days, 5 and onset of CIA for 44-55 days post-immunization. Figures 6 and 7 show that TNFR/Fc significantly reduced the incidence of CIA compared with controls (28% vs. 86%; p<0.03), and reduced both arthritis index (a subjective measure of severity) and the number of involved joints. The antibody response to CII was significantly lower immediately post treatment with TNFR/Fc (day 28), but antibody levels were 10 equivalent at the conclusion of the experiment (day 70).

These results indicate that TNFR/Fc is effective in reducing the incidence of CIA in mice and may therefore be useful in the treatment arthritis.

ZD 6^93 27 SEQUENCE LISTING (I) GENERAL INFORMATION: (i) APPLICANT: Jacobs, Cindy A. (ii) TITLE OF INVENTION: Method of Treating TNF-Dependent Inflammation Using Tumor Necrosis Factor Antagonists (iii) NUMBER OF SEQUENCES: 5 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Immunex Corporation (5) STREET: 51 University Street (C) CITY: Seattle (D) STATE: Washington (E) COUNTRY: U.S.A.

(F) ZIP: 98101 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1641 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (G) CELL TYPE: Fibroblast (H) CELL LINE: WI-26 VA4 PATENT OFFICE -7 AUG 1996 received ! 40 45 50 55 60 PCT/ US93/08666 2 5 6 2 5 3 (vii) IMMEDIATE SOURCE: (A) LIBRARY: WI-26 VA4 (B) CLONE: Clone 1 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 88..1473 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 154..1470 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 88..153 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCGAGGCAGG CAGCCTGGAG AGAAGGCGCT GGGCTGCGAG GGCGCGAGGG CGCGAGGGCA 60 GGGGGCAACC GGACCCCGCC CGCATCC ATG GCG CCC GTC GCC GTC TGG GCC 111 Met Ala Pro Val Ala Val Trp Ala -22 -20 -15 GCG CTG GCC GTC GGA CTG GAG CTC TGG GCT GCG GCG CAC GCC TTG CCC 159 Ala Leu Ala Val Gly Leu Glu Leu Trp Ala Ala Ala His Ala Leu Pro -10 -5 1 GCC CAG GTG GCA TTT ACA CCC TAC GCC CCG GAG CCC GGG AGC ACA TGC 2 07 Ala Gin Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys 5 10 15 CGG CTC AGA GAA TAC TAT GAC CAG ACA GCT CAG ATG TGC TGC AGC AAA 2 5 Arg Leu Arg Glu Tyr Tyr Asp Gin Thr Ala Gin Met Cys Cys Ser Lys 20 25 30 TGC TCG CCG GGC CAA CAT GCA AAA GTC TTC TGT ACC AAG ACC TCG GAC 30 Cys Ser Pro Gly Gin His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp , 35 40 45 50 ACC GTG TGT GAC TCC TGT GAG GAC AGC ACA TAC ACC CAG CTC TGG AAC 35 Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gin Leu Trp Asn 55 60 65 TGG GTT CCC GAG TGC TTG AGC TGT GGC TCC CGC TGT AGC TCT GAC CAG 39 Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gin 70 75 80 GTG GAA ACT CAA GCC TGC ACT CGG GAA CAG AAC CGC ATC TGC ACC TGC 44 Val Glu Thr Gin Ala Cys Thr Arg Glu Gin Asn Arg lie Cys Thr Cys 85 90 95 AGG CCC GGC TGG TAC TGC GCG CTG AGC AAG CAG GAG GGG TGC CGG CTG 49 Arg Pro Gly Trp Tyr Cys Alt; Leu Ser Lys Gin Glu Gly Cys Arg Leu 100 105 110 TGC GCG CCG CTG CGC AAG TGC CGC CCG GGC TTC GGC GTG GCC AGA CCA 54 Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro 115 120 125 130 WO 94/06476 PCT/US93/08666 29 256253 GGA ACT GAA ACA TCA GAC GTG GTG TGC AAG CCC TGT GCC CCG GGG ACG 591 Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr 135 140 145 TTC TCC AAC ACG ACT TCA TCC ACG GAT ATT TGC AGG CCC CAC CAG ATC 639 Phe Ser Asn Thr Thr Ser Ser Thr Asp lie Cys Arg Pro His Gin lie 150 155 160 TGT AAC GTG GTG GCC ATC CCT GGG AAT GCA AGC ATG GAT GCA GTC TGC 687 Cys Asn Val Val Ala lie Pro Gly Asn Ala Ser Met Asp Ala Val Cys 165 170 175 ACG TCC ACG TCC CCC ACC CGG AGT ATG GCC CCA GGG GCA GTA CAC TTA 735 Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu 15 180 185 190 CCC CAG CCA GTG TCC ACA CGA TCC CAA CAC ACG CAG CCA ACT CCA GAA 783 Pro Gin Pro Val Ser Thr Arg Ser Gin His Thr Gin Pro Thr Pro Glu 195 200 205 210 CCC AGC ACT GCT CCA AGC ACC TCC TTC CTG CTC CCA ATG GGC CCC AGC 831 Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser 215 220 225 CCC CCA GCT GAA GGG AGC ACT GGC GAC TTC GCT CTT CCA GTT GGA CTG 879 Pro Pro Ala Glu Gly Ser Thr Gly Asp Phe Ala Leu Pro Val Gly Leu 230 235 240 ATT GTG GGT GTG ACA GCC TTG GGT CTA CTA ATA ATA GGA GTG GTG AAC 927 lie Val Gly Val Thr Ala Leu Gly leu Leu lie lie Gly Val Val Asn 245 250 255 TGT GTC ATC ATG ACC CAG GTG AAA AAG AAG CCC TTG TGC CTG CAG AGA 975 Cys Val lie Met Thr Gin Val Lys lys lys Pro Leu Cys Leu Gin Arg 35 260 265 270 GAA GCC AAG GTG CCT CAC TTG CCT GCC GAT AAG GCC CGG GGT ACA CAG 1023 Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gin 275 280 285 290 40 GGC CCC GAG CAG CAG CAC CTG CTG ATC ACA GCG CCG AGC TCC AGC AGC 1071 Gly Pro Glu Gin Gin His Leu Leu lie Thr Ala Pro Ser Ser Ser Ser 295 300 305 45 AGC TCC CTG GAG AGC TCG GCC AGT GCG TTG GAC AGA AGG GCG CCC ACT 1119 Ser Ser Leu Glu Ser Ser Ala Ser Ala leu Asp Arg Arg Ala Pro Thr 310 315 320 CGG AAC CAG CCA CAG GCA CCA GGC GTG GAG GCC AGT GGG GCC GGG GAG 1167 50 Arg Asn Gin Pro Gin Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu 325 330 335 GCC CGG GCC AGC ACC GGG AGC TCA GAT TCT TCC CCT GGT GGC CAT GGG 1215 Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly 55 340 345 350 ACC CAG GTC AAT GTC ACC TGC ATC GTG AAC GTC TGT AGC AGC TCT GAC 1263 Thr Gin Val Asn Val Thr Cys lie Val Asn Val Cys Ser Ser Ser Asp 355 360 365 370 60 WO 94/06476 PCI7US93/08666 50 256253 CAC AGC TCA CAG TGC TCC TCC CAA GCC AGC TCC ACA ATG GGA GAC ACA 1311 His Ser Ser Gin Cys Ser Ser Gin Ala Ser Ser Thr Met Gly Asp Thr 375 380 385 GAT TCC AGC CCC TCG GAG TCC CCG AAG GAC GAG CAG GTC CCC TTC TCC 1359 Asp Ser Ser Pro Ser Glu Ser Pro Ly3 Asp Glu Gin Val Pro Phe Ser 390 395 400 AAG GAG GAA TGT GCC TTT CGG TCA CAG CTG GAG ACG CCA GAG ACC CTG 1407 10 Lys Glu Glu Cys Ala Phe Arg Ser Gin Leu Glu Thr Pro Glu Thr Leu 405 410 415 CTG GGG AGC ACC GAA GAG AAG CCC CTG CCC CTT GGA GTG CCT GAT GCT 1455 Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala 15 420 425 430 GGG ATG AAG CCC AGT TAACCAGGCC GGTGTGGGCT GTGTCGTAGC CAAGGTGGGC 1510 Gly Met Lys Pro Ser 435 440 TGAGCCCTGG CAGGATGACC CTGCGAAGGG GCCCTGGTCC TTCCAGGCCC CCACCACTAG 1570 GACTCTGAGG CTCTTTCTGG GCCAAGTTCC TCTAGTGCCC TCCACAGCCG CAGCCTCCCT 1630 25 CTGACCTGCA G 1641 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 61 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu 40 -22 -20 -15 -10 Trp Ala Ala Ala His Ala Leu Pro Ala Gin Val Ala Phe Thr Pro Tyr -5 15 10 45 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gin 20 25 Thr Ala Gin Met Cys Cys Ser Lys Cys Ser Pro Gly Gin His Ala Lys 35 40 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 45 50 55 Ser Thr Tyr Thr Gin Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 55 60 65 70 Gly Ser Arg Cys Ser Ser Asp Gin Val Glu Thr Gin Ala Cys Thr Arg 75 80 85 90 60 Glu Gin Asn Arg lie Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 95 100 105 31 256 2 5 3 Ser Lys Gin Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg 110 115 120 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val 125 130 135 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 140 145 150 Asp lie Cys Arg Pro His Gin lie Cys Asn Val Val Ala lie Pro Gly 155 160 165 170 Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 15 175 180 185 Met Ala Pro Gly Ala Val His Leu Pro Gin Pro Val Ser Thr Arg Ser 190 195 200 Gin His Thr Gin Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser 205 210 215 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly 220 21-:> 230 Asp Phe Ala Leu Pro Val Gly Leu lie Val GJy Val Thr Ala Leu Gly 235 240 245 250 Leu Leu lie lie Gly Val Val Asn Cys Val He Met Thr Gin Val Lys 30 255 260 265 Lys Lys Pro Leu Cys Leu Gin Arg Glu Ala Lys Val Pro His Leu Pre 270 275 280 Ala Asp Lys Ala Arg Gly Thr Gin Gly Pro Glu Gin Gin His Leu Leu 285 290 295 lie Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser 300 305 310 40 Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gin Pro Gin Ala Pro Gly 315 320 325 330 Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser 45 335 340 345 Asp Ser Ser Pro Gly Gly His Gly Thr Gin Val Asn Val Thr Cys lie 350 355 360 50 Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gin Cys Ser Ser Gin 365 370 375 Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro 380 385 390 55 Lys Asp Glu Gin Val Pro Phe Ser Lys Glu Glu Cv: Ala Phe Arg Ser 395 400 405 410 Gin Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro 60 415 420 425 WO 94/06476 PCT/US93/08666 32 Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser 430 435 . (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1557 base pairs (B) TYPE: nucleic acid 10 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: 20 (B) CLONE: TNFR/Fc Fusion Protein (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1557 40 256253 (ix) FEATURE: (A) NAME/KEY: matjpeptide (B) LOCATION: 1..1554 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCG AGG CAG GCA GCC TGG AGA GAA GGC GCT GGG CTG CGA GGG CGC GAG 48 Ala Arg Gin Ala Ala Trp Arg Glu Gly Ala Gly Leu Arg Gly Arg Glu 35 1 5 10 15 GGC GCG AGG GCA GGG GGC AAC CGG ACC CCG CCC GCA TCC ATG GCG CCC 96 Gly Ala Arg Ala Gly Gly Asn Arg Thr Pro Pro Ala Ser Met Ala Pro 20 25 30 GTC GCC GTC TGG GCC GCG CTG GCC GTC GGA CTG GAG CTC TGG GCT GCG 144 Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu Trp Ala Ala 35 40 45 45 GCG CAC GCC TTG CCC GCC CAG GTG GCA TTT ACA CCC TAC GCC CCG GAG 192 Ala His Ala Leu Pro Ala Gin Val Ala Phe Thr Pro Tyr Ala Pro Glu 50 55 60 CCC GGG AGC ACA TGC CGG CTC AGA GAA TAC TAT GAC CAG ACA GCT CAG 240 50 Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gin Thr Ala Gin 65 70 75 80 ATG TGC TGC AGC AAA TGC TCG CCG GGC CAA CAT GCA AAA GTC TTC TGT 288 Met Cys Cys Ser Lys Cys Ser Pro Gly Gin His Ala Lys Val Phe Cys 55 85 90 95 ACC AAG ACC TCG GAC ACC GTG TGT GAC TCC TGT GAG GAC AGC ACA TAC 336 Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr 100 105 110 60 WO 94/06476 PCT/US93/08666 2 5 6 2 5 3 ACC CAG CTC TGG AAC TGG GTT CCC GAG TGC TTG AGC TGT GGC TCC CGC 384 Thr Gin Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg 115 120 125 TGT AGC TCT GAC CAG GTG GAA ACT CAA GCC TGC ACT CGG GAA CAG AAC 432 Cys Ser Ser Asp Gin Val Glu Thr Gin Ala Cys Thr Arg Glu Gin Asn 130 135 140 CGC ATC TGC ACC TGC AGG CCC GGC TGG TAC TGC GCG CTG AGC AAG CAG 480 Arg lie Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gin 145 150 155 160 GAG GGG TGC CGG CTG TGC GCG CCG CTG CGC AAG TGC CGC CCG GGC TTC 528 Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 15 165 170 175 40 45 50 55 GGC GTG GCC AGA CCA GGA ACT GAA ACA TCA GAC GTG GTG TGC AAG CCC 576 Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro 180 185 190 TGT GCC CCG GGG ACG TTC TCC AAC ACG ACT TCA TCC ACG GAT ATT TGC 624 Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp lie Cys 195 200 205 AGG CCC CAC CAG ATC TGT AAC GTG GTG GCC ATC CCT GGG AAT GCA AGC 672 Arg Pro His Gin lie Cys Asn Val Val Ala lie Pro Gly Asn Ala Ser 210 215 220 ATG GAT GCA GTC TGC ACG TCC ACG TCC CCC ACC CGG AGT ATG GCC CCA 720 Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro 225 230 235 240 GGG GCA GTA CAC TTA CCC CAG CCA GTG TCC ACA CGA TCC CAA CAC ACG 768 Gly Ala Val His Leu Pro Gin Pro Val Ser Thr Arg Ser Gin His Thr 245 250 255 CAG CCA ACT CCA GAA CCC AGC ACT GCT CCA AGC ACC TCC TTC CTG CTC 816 Gin Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu 260 265 270 CCA ATG GGC CCC AGC CCC CCA GCT GAA GGG AGC ACT GGC GAC GAG CCC 864 Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro 275 280 285 AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT GAA 912 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 290 295 300 CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC 960 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Fro Pro Lys Pro Lys Asp 305 310 315 320 a: CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC 1008 Tr r Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 325 330 335 GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC 1056 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 340 345 350 34 256253 GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 355 360 GAG CAG TAC AAC Glu Gin Tyr Asn 365 1104 AGC ACG TAC CGG GTG GTC AGC GTC CTC ACC GTC CTG Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 370 375 380 CAC CAG GAC TGG His Gin Asp Trp 1152 CTG 10 Leu 385 AAT GGC AAG GAC TAC AAG TGC AAG GTC TCC AAC Asn Gly Lys Asp Tyr Lys Cys Lys Val Ser Asn 390 395 AAA GCC CTC CCA Lys Ala Leu Pro 400 1200 GCC CCC ATG CAG AAA ACC ATC TCC AAA GCC AAA GGG Ala Pro Met Gin Lys Thr lie Ser Lys Ala Lys Gly 405 410 CAG CCC CGA GAA Gin Pro Arg Glu 415 1248 CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAT GAG Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 420 425 CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 435 440 GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 450 455 460 CTG ACC AAG AAC 1296 Leu Thr Lys Asn 430 CCC AGG CAC ATC 1344 Pro Arg His lie 445 AAC TAC AAG ACC 1392 Asn Tyr Lys Thr ACG 30 Thr 465 CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 470 475 CTC TAC AGC AAG Leu Tyr Ser Lys 480 1440 CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 485 490 GTC TTC TCA TGC Val Phe Ser Cys 495 1488 40 45 TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 500 505 TCC CTG TCT CCG GGT AAA TGA Ser Leu Ser Pro Gly Lys 515 (2) INFORMATION FOR SEQ ID NO:4: CAG AAG AGC CTC Gin Lys Ser Leu 510 1536 1557 50 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 518 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 55 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ala Arg Gin Ala Ala Trp Arg Glu Gly Ala Gly Leu 15 10 60 Gly Ala Arg Ala Gly Gly Asn Arg Thr Pro Pro Ala 25 Arg Gly Arg Glu • 15 Ser Met Ala Pro 30 40 55 ^ 6 2 5 3 Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu Trp Ala Ala 3 5 40 45 Ala His Ala Leu Pro Ala Gin Val Ala Phe Thr Pro Tyr Ala Pro Glu 50 55 60 Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gin Thr Ala Gin 65 70 75 80 Met Cys Cys Ser Lys Cys Ser Pro Gly Gin His Ala Lys Val Phe Cys 85 90 95 Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr 100 105 110 Thr Gin Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg 115 120 125 Cys Ser Ser Asp Gin Val Glu Thr Gin Ala Cys Thr Arg Glu Gin Asn 130 135 140 Arg lie Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gin 145 150 155 160 Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 165 170 175 Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro 180 185 190.

Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp lie Cys 195 200 205 Arg Pro His Gin lie Cys Asn Val Val Ala lie Pro Gly Asn Ala Ser 210 215 220 Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro 225 230 235 240 Gly Ala Val His Leu Pro Gin Pro Val Ser Thr Arg Ser Gin His Thr 245 250 255 Gin Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu 45 260 265 270 Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro 275 280 285 50 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 290 295 300 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 305 310 315 320 Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 325 330 335 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 60 340 345 350 36 56 2 5 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn 355 360 365 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp 5 370 375 380 Leu Asn Gly Lys Asp Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 385 390 395 400 Ala Pro Met Gin Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu 405 410 415 55 Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 420 425 430 Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Arg His lie 435 440 445 Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 450 455 460 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 465 470 475 480 Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys 485 490 495 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu 500 505 510 Ser Leu Ser Pro Gly Lys 515 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid 40 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 45 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES 50 (vii) IMMEDIATE SOURCE: (B) CLONE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CGGTACGTGC TGTTGTTACT GC 25629J 37

Claims (11)

WHAT WE CLAIM IS:

1. The use, in the manufacture of a medicament, of a TNF antagonist selected from the group consisting of a soluble Type 1 TNF receptor, and a chimeric antibody comprising a TNF receptor and the constant domain of an immunoglobulin molecule as an active ingredient in a composition for the treatment of TNF-medicated inflammatory diseases.

2. Use according to claim 1, wherein the TNF-mediated inflammatory disease is arthritis.

3. Use according to claim 2, wherein the mammal is a human.

4. Use according to claim 3, wherein the TNF antagonist is soluble human TNFR.

5. Use according to claim 4, wherein the soluble human TNFR is selected from the group consisting of soluble human Type I TNFR and soluble human Type II TNFR.

6. Use according to claim 2, wherein TNFR is administered in combination with IL-1R.

7. The use, in the manufacture of a medicament of a soluble human TNFR as an active ingredient in a composition for treating arthritis in a mammal, wherein the amount to be given ranges from substantially 0.1 mg/kg/week to substantially 100 mg/kg/week.

8. Use according to claim 7, wherein the amount of soluble human TNFR ranges from substantially 0.5 mg/kg/week to substantially 50 mg/kg/week .r, ~ 256293 38

9. Use of a compound for treating TNF-mediated inflammatory diseases substantially as herein described with reference to the accompanying examples.

10. A method for treating arthritis in a mammal substantially as herein described with reference to accompanying examples 4 and 5.

11. Use according to claim 1 or claim 7 substantially as herein described with reference to the accompanying examples.