WO1991003553A1 - TUMOR NECROSIS FACTOR-α AND -β RECEPTORS - Google Patents

Abstract

Tumor necrosis factor receptor proteins, DNAs and expression vectors encoding TNF receptors, and processes for producing TNF receptors as products of recombinant cell culture, are disclosed.

Description

TITLE

Tumor Necrosis Factor-α and -β Receptors

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Serial No. 421,417, fil October 13, 1989, now pending, which is a continuation-in-part of U.S. application Seri No. 405,370, filed September 11, 1989, now abandoned, which is a continuation-in-part U.S. application Serial No. 403,241, filed September 5, 1989, now abandoned.

BACKGROUND OF THE TNVENTTON

The present invention relates generally to cytokine receptors and more specifically tumor necrosis factor receptors. Tumor necrosis factor-α (TNFα, also known as cachectin) and tumor necrosis facto β (TNFβ, also known as lymphotoxin) are homologous mammalian endogenous secreto proteins capable of inducing a wide variety of effects on a large number of cell types. T great similarities in the structural and functional characteristics of these two cytokines ha resulted in their collective description as "TNF." Complementary cDNA clones encodi TNFα (Pennica et al., Nature 312:724, 1984) and TNFβ (Gray et al., Nature 312:721, 198 have been isolated, permitting further structural and biological characterization of TNF.

TNF proteins initiate their biological effect on cells by binding to specific T receptor (TNF-R) proteins expressed on the plasma membrane of a TNF-responsive ce TNFα and TNFβ were first shown to bind to a common receptor on the human cervic carcinoma cell line ME-180 (Aggarwal et al., Nature 318:665, 1985). Estimates of the size the TNF-R determined by affinity labeling studies ranged from 54 to 175 kDa (Creasey et Proc. Natl. Acad. Sci. USA 84:3293, 1987; Stauber et al., J. Biol. Chem. 263:19098, 198 Hohmann et al., J. Biol. Chem. 264:14927, 1989). Although the relationship between the TNF-Rs of different molecular mass is unclear, Hohmann et al. (J. Biol. Chem. 264:1492 1989) reported that at least two different cell surface receptors for TNF exist on different c types. These receptors have an apparent molecular mass of about 80 kDa and about 55- kDa, respectively. None of the above publications, however, reported the purification homogeneity of cell surface TNF receptors.

In addition to cell surface receptors for TNF, soluble proteins from human uri capable of binding TNF have also been identified (Peetre et al., Eur. J. Haematol. 47:41 1988; Seckinger et al., J. Exp. Med. 167:1511, 1988; Seckinger et al., /. Biol. Che 264:11966, 1989; UK Patent Application, Publ. No. 2 218 101 A to Seckinger et a Engelmann et al., J. Biol. Chem. 264:11974, 1989). The soluble urinary TNF binding protein disclosed by UK 2218 101 A has a partial N-terminal amino acid sequence of Asp- Ser-Val-Cys-Pro-, which corresponds to the partial sequence disclosed later by Engelmann et al. (1989). The relationship of the above soluble urinary binding proteins was further elucidated after original parent application (U.S. Serial No. 403,241) of the present application was filed, when Engelmann et al. reported the identification and purification of a second distinct soluble urinary TNF binding protein having an N-terminal amino acid sequence of Val-Ala-Phe-Thr-Pro- (J. Biol. Chem. 265:1531, 1990). The two urinary proteins disclosed by the UK 2218 101 A and the Engelmann et al. publications were shown to be immunochemically related to two apparently distinct cell surface proteins by the ability of antiserum against the binding proteins to inhibit TNF binding to certain cells.

More recently, two separate groups reported the molecular cloning and expression of a human 55 kDa TNF-R (Loetscher et al., Cell 61:351, 1990; Schall et al., Cell 61:361, 1990). The TNF-R of both groups has an N-terminal amino acid sequence which corresponds to the partial amino acid sequence of the urinary binding protein disclosed by UK 2218 101 A, Engelmann et al. (1989) and Englelmann et al. (1990).

In order to elucidate the relationship of the multiple forms of TNF-R and soluble urinary TNF binding proteins, or to study the structural and biological characteristics of TNF- Rs and the role played by TNF-Rs in the responses of various cell populations to TNF or other cytokine stimulation, or to use TNF-Rs effectively in therapy, diagnosis, or assay, purified compositions of TNF-R are needed. Such compositions, however, are obtainable in practical yields only by cloning and expressing genes encoding the receptors using recombinant DNA technology. Efforst to purify the TNF-R molecule for use in biochemical analysis or to clone and express mammalian genes encoding TNF-R, however, have been impeded by lack of a suitable source of receptor protein or mRNA. Prior to the present invention, no cell lines were known to express high levels of TNF-R constitutively and continuously, which precluded purification of receptor for sequencing or construction of genetic libraries for cDNA cloning.

SUMMARY OF THE TNVENTTON

The present invention provides isolated TNF receptors and DNA sequences encoding mammalian tumor necrosis factor receptors (TNF-R), in particular, human TNF-Rs. Such DNA sequences include (a) cDNA clones having a nucleotide sequence derived from the coding region of a native TNF-R gene; (b) DNA sequences which are capable of hybridization to the cDNA clones of (a) under moderately stringent conditions and which encode biologically active TNF-R molecules; or (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 TNF-R molecules. In particular, the present invention provides DN sequences which encode soluble TNF receptors.

The present indention also provides recombinant expression vectors comprising th DNA sequences defined above, recombinant TNF-R molecules produced using th recombinant expression vectors, and processes for producing the recombinant TNF- molecules using the expression vectors.

The present invention also provides isolated or purified protein composition comprising TNF-R, and, in particular, soluble forms of TNF-R.

The present invention also provides compositions for use in therapy, diagnosis, assa of TNF-R, or in raising antibodies to TNF-R, comprising effective quantities of solubl native or recombinant receptor proteins prepared according to the foregoing processes.

Because of the ability of TNF to specifically bind TNF receptors (TNF-Rs), purifie

TNF-R compositions will be useful in diagnostic assays for TNF, as well as in raisin antibodies to TNF receptor for use in diagnosis and therapy. In addition, purified TN receptor compositions may be used directly in therapy to bind or scavenge TNF, thereb providing a means for regulating the immune activities of this cytokine.

These and other aspects of the present invention will become evident upon referenc to the following detailed description.

BRIEF DESCRIPTION OF THE PRAWINCrS

Figure 1 is a schematic representation of the coding region of various cDNAs encoding human and murine TNF-Rs. The leader sequence is hatched and the transmembrane region is solid.

Figures 2-3 depict the partial cDNA sequence and derived amino acid sequence of th human TNF-R clone 1. Nucleotides are numbered from the beginning of the 5' untranslate region. Amino acids are numbered from the beginning of the signal peptide sequence. Th putative signal peptide sequence is represented by the amino acids -22 to -1. The N-termin leucine of the mature TNF-R protein is underlined at position 1. The predicte transmembrane region from amino acids 236 to 265 is also underlined. The C-termini various soluble TNF-Rs are marked with an arrow (t).

Figures 4-6 depict the cDNA sequence and derived amino acid sequence of murin TNF-R clone 11. The putative signal peptide sequence is represented by amino acids -22 to 1. The N-t^-minal valine of the mature TNF-R protein is underlined at position 1. Th predicted transmembrane region from amino acids 234 to 265 is also underlined. DETAILED DESCRIPTION OF THE TNVENTTON

Definitions

As used herein, the terms "TNF receptor" and "TNF-R" refer to proteins having amino acid sequences which are substantially similar to the native mammalian TNF receptor amino acid sequences, and which are biologically active, as defined below, in that they are capable of binding TNF molecules or transducing a biological signal initiated by a TNF molecule binding to a cell, or cross-reacting with anti-TNF-R antibodies raised against TNF- R from natural (i.e., nonrecombinant) sources. The mature full-length human TNF-R is a glycoprotein having a molecular weight of about 80 kilodaltons (kDa). As used throughout the specification, the term "mature" means a protein expressed in a form lacking a leader sequence as may be present in full-length transcripts of a native gene. Experiments using COS cells transfected with a cDNA encoding full-length human TNF-R showed that TNF-R bound ¹²5l-TNFα with an apparent K_a of about 5 x 10⁹ M"¹, and that TNF-R bound ¹²5l- TNFβ with an apparent K_a of about 2 x 10⁹ M^*1. The terms "TNF receptor" or "TNF-R" include, but are not limited to, analogs or subunits of native proteins having at least 20 amino acids and which exhibit at least some biological activity in common with TNF-R, for example, soluble TNF-R constructs which are devoid of a transmembrane region (and are secreted from the cell) but retain the ability to bind TNF. Various bioequivalent protein and amino acid analogs are described in detail below. The nomenclature for TNF-R analogs as used herein follows the convention of naming the protein (e.g., TNF-R) preceded by either hu (for human) or mu (for murine) and followed by a Δ (to designate a deletion) and the number of the C-terminal amino acid. For example, huTNF-RΔ235 refers to human TNF-R having Asp²³⁵ as the C-terminal amino acid (i.e., a polypeptide having the sequence of amino acids 1-235 of Figure 2). In the absence of any human or murine species designation, TNF-R refers generically to mammalian TNF-R. Similarly, in the absence of any specific designation for deletion mutants, the term TNF-R means all forms of TNF-R, including mutants and analogs which possess TNF-R biological activity.

"Soluble TNF-R" or "sTNF-R" as used in the context of the present invention refer to proteins, or substantially equivalent analogs, having an amino acid sequence corresponding to all or part of the extracellular region of a native TNF-R, for example, huTNF-RΔ235, huTNF-RΔ185 and huTNF-RΔ163, or amino acid sequences substantially similar to the sequences of amino acids 1-163, amino acids 1-185, or amino acids 1-235 of Figure 2, and which are biologically active in that they bind to TNF ligand. Equivalent soluble TNF-Rs 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 huTNF-RΔx, wherein x is selected from the group consisting of any one of amino acids 163-235 of Figur 2. Analogous deletions may be made to muTNF-R. Inhibition of TNF signal transductio activity can be determined by transfecting cells with recombinant TNF-R DNAs to obtai recombinant receptor expression. The cells are then contacted with TNF and the resultin metabolic effects examined. If an effect results which is attributable to the action of th ligand, then the recombinant receptor has signal transduction activity. Exemplary procedure for determining whether a polypeptide has signal transduction activity are disclosed b Idzerda et al., J. Exp. Med. 777:861 (1990); Curtis et al., Proc. Natl. Acad. Sci. US 86:3045 (1989); Prywes et al., EMBO J.5:2179 (1986) and Chou et al., J. Biol. Che 262:1842 (1987). Alternatively, primary cells or cell lines which express an endogenou TNF receptor and have a detectable biological response to TNF could also be utilized.

The term "isolated" or "purified", as used in the context of this specification to defin the purity of TNF-R protein or protein compositions, means that the protein or protei composition is substantially free of other proteins of natural or endogenous origin an contains less than about 1% by mass of protein contaminants residual of productio processes. Such compositions, however, can contain other proteins added as stabilizer carriers, excipients or co-therapeutics. TNF-R is isolated if it is detectable as a single protei band in a polyacrylamide gel by silver staining.

The term "substantially similar," when used to define either amino acid or nucleic aci sequences, means that a particular subject sequence, for example, a mutant sequence, varie from a reference sequence by one or more substitutions, deletions, or additions, the net effe of which is to retain biological activity of the TNF-R protein as may be determined, f example, in one of the TNF-R binding assays set forth in Example 1 below. Alternativel nucleic acid subunits and analogs are "substantially similar" to the specific DNA sequence disclosed herein if: (a) the DNA sequence is derived from the coding region of a nativ mammalian TNF-R gene; (b) the DNA sequence is capable of hybridization to DN sequences of (a) under moderately stringent conditions (50"C, 2x SSC) and which encod biologically active TNF-R molecules; or DNA sequences which are degenerate as a result the genetic code to the DNA sequences defined in (a) or (b) and which encode biologicall active TNF-R molecules.

"Recombinant," as used herein, means that a protein is derived from recombina (e.g., microbial or mammalian) expression systems. "Microbial" refers to recombina proteins made in bacterial or fungal (e.g., yeast) expression systems. As a produc "recombinant microbial" defines a protein produced in a microbial expression system which essentially free of native endogenous substances. Protein expressed in most bacteri cultures, e.g., E. coli, will be free of glycan. Protein expressed in yeast may have 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 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-TNF-R antibodies raised against TNF-R from natural (i.e., nonrecombinant) sources. Preferably, biologically active TNF receptors within the scope of the present invention 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). "Isolated 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. Genomic DNA containing the relevant sequences could also be used as a source of coding sequences. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. DNA sequences encoding the proteins provided by this invention can be 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

Isolation of cDNAs Encoding TNF-R

The coding sequence of TNF-R is obtained by isolating a complementary DNA (cDNA) sequence encoding TNF-R from a recombinant cDNA or genomic DNA library. A cDNA library is preferably constructed by obtaining polyadenylated mRNA from a particular cell line which expresses a mammalian TNF-R, for example, the human fibroblast cell line WI-26 VA4 (ATCC CCL 95.1) and using the mRNA as a template for synthesizing double stranded cDNA. The double stranded cDNA is then packaged into a recombinant vector, which is introduced into an appropriate E. coli strain and propagated. Murine or other mammalian cell lines which express TNF-R may also be used. TNF-R sequences contained in the cDNA library can be readily identified by screening the library with an appropriate nucleic acid probe which is capable of hybridizing with TNF-R cDNA. Alternatively, DNAs encoding TNF-R proteins can be assembled by ligation of synthetic oligonucleotide subunits corresponding to all or part of the sequence of Figures 2-3 or Figures 4-6 to provide complete coding sequence.

The human TNF receptor cDNAs of the present invention were isolated by the metho of direct expression cloning. A cDNA library was constructed by first isolating cytoplasmi mRNA from the human fibroblast cell line WI-26 VA4. Polyadenylated RNA was isolate and used to prepare double-stranded cDNA. Purified cDNA fragments were then ligated int pCAV/NOT vector DNA which uses regulatory sequences derived from pDC201 (a derivativ of pMLSV, previously described by Cosman et al., Nature 572:768, 1984), SV40 an cytomegalovirus DNA, described in detail below in Example 2. pCAV/NOT has bee deposited with the American Type Culture Collection under accession No. ATCC 68014. Th pCAV/NOT vectors containing the WI26-VA4 cDNA fragments were transformed into E. col strain DH5α. Transformants were plated to provide approximately 800 colonies per plate The resulting colonies were harvested and each pool used to prepare plasmid DNA fo transfection into COS-7 cells essentially as described by Cosman et al. (Nature 372:768 1984) and Luthman et al. (Nucl. Acid Res. 77:1295, 1983). Transformants expressin biologically active cell surface TNF receptors were identified by screening for their ability t bind ¹²⁵I-TNF. In this screening approach, transfected COS-7 cells were incubated wit medium containing ¹²⁵I-TNF, the cells washed to remove unbound labeled TNF, and the cel monolayers contacted with X-ray film to detect concentrations of TNF binding, as disclose by Sims et al, Science 247:585 (1988). Transfectants detected in this manner appear as dar foci against a relatively light background.

Using this approach, approximately 240,000 cDNAs were screened in pools o approximately 800 cDNAs until assay of one transfectant pool indicated positive foci for TN binding. A frozen stock of bacteria from this positive pool was grown in culture and plated t provide individual colonies, which were screened until a single clone (clone 11) wa identified which was capable of directing synthesis of a surface protein with detectable TN binding activity. The equence of cDNA clone 11 isolated by the above method is depicted i Figures 4-6.

Additional cDNA clones can be isolated from cDNA libraries of other mammalia species by cross-species hybridization. For use in hybridization, DNA encoding TNF-R ma be covalently labeled with a detectable substance such as a fluorescent group, a radioactiv atom or a chemiluminescent group by methods well known to those skilled in the art. Suc probes could also be used for in vitro diagnosis of particular conditions.

Like most mammalian genes, mammalian TNF receptors are presumably encoded b multi-exon genes. Alternative mRNA constructs which can be attributed to different mRN splicing events following transcription, and which share large regions of identity or similarit with the cDNAs claimed herein, are considered to be within the scope of the present invention.

Other mammalian TNF-R cDNAs are isolated by using an appropriate human TNF-R DNA sequence as a probe for screening a particular mammalian cDNA library by cross- species hybridization.

Proteins and Analogs

The present invention provides isolated recombinant mammalian TNF-R polypeptides. Isolated TNF-R polypeptides of 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 residual of production processes. The native human TNF-R molecules are recovered from cell lysates as glycoproteins having an apparent molecular weight by SDS-PAGE of about 80 Mlodaltons (kDa). The TNF-R polypeptides of this invention are optionally without associated native-pattern glycosylation. Mammalian TNF-R of the present invention includes, by way of example, primate, human, murine, canine, feline, bovine, ovine, equine and porcine TNF-R. Mammalian TNF- Rs can be obtained by cross species hybridization, using a single stranded cDNA derived from the human TNF-R DNA sequence as a hybridization probe to isolate TNF-R cDNAs from mammalian cDNA libraries. Derivatives of TNF-R within the scope of the 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 TNF-R 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 TNF-R amino acid side chains or at the N- or C-termini. Other derivatives of TNF-R within the scope of this invention include covalent or aggregative conjugates of TNF-R 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- 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 α-factor leader). TNF-R protein fusions can comprise peptides added to facilitate purification or identification of TNF-R (e.g., poly-His). The amino acid sequence of TNF receptor 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 latt sequence is highly antigenic and provides an epitope reversibly bound by a specifi monoclonal antibody, enabling rapid assay and facile purification of expressed recombina protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at t residue immediately following the Asp-Lys pairing. Fusion proteins capped with this pepti may also be resistant to intracellular degradation in E. coli.

TNF-R derivatives may also be used as immunogens, reagents in receptor-base immunoassays, or as binding agents for affinity purification procedures of TNF or oth binding ligands. TNF-R derivatives may also be obtained by cross-linking agents, such M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at cysteine and lysi residues. TNF-R proteins may also be covalently bound through reactive side groups various insoluble substrates, such as cyanogen bromide-activated, bisoxirane-activate carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing polyolefin surfaces (with or without glutaraldehyde cross-linking). Once bound to substrate, TNF-R may be used to selectively bind (for purposes of assay or purification) ant TNF-R antibodies or TNF.

The present invention also includes TNF-R with or without associated native-patte glycosylation. TNF-R expressed in yeast or mammalian expression systems, e.g., COS cells, may be similar or slightly different in molecular weight and glycosylation pattern th the native molecules, depending upon the expression system. Expression of TNF-R DN in bacteria such as E. coli provides non-glycosylated molecules. Functional mutant analo of mammalian TNF-R having inactivated N-glycosylation sites can be produced oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques. The analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yie using yeast expression systems. N-glycosylation sites in eukaryotic proteins a characterized by the amino acid triplet Asn-Ai-Z, where Ai is any amino acid except Pro, a Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covale attachment of carbohydrate. Such a site can be eliminated by substituting another amino ac for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between A] a Z, or an amino acid other than Asn between Asn and Ai.

TNF-R derivatives may also be obtained by mutations of TNF-R or its subunits. TNF-R mutant, as referred to herein, is a polypeptide homologous to TNF-R but which h an amino acid sequence different from native TNF-R because of a deletion, insertion substitution. Bioequivalent analogs of TNF-R proteins may be constructed by, for exampl making various substitutions of residues or sequences or deleting terminal or internal residu or sequences not needed for biological activity. For example, cysteine residues can be delet (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 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, generally comprise a like number of amino acids sequences, although C-terminal truncations for the purpose of constructing soluble TNF-Rs will contain fewer amino acid sequences. In order to preserve the biological activity of TNF- Rs, deletions and substitutions will preferably 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 Gin 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 TNF-Rs is suggestive of additional conservative substitutions that may be made without altering the essential biological characteristics of TNF-R.

Subunits of TNF-R may be constructed by deleting terminal or internal residues or sequences. Particularly preferred sequences include those in which the transmembrane region and intracellular domain of TNF-R 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 TNF-R molecule which retains its ability to bind TNF. A particularly preferred soluble TNF-R construct is TNF-RΔ235 (the sequence of amino acids 1-235 of Figure 2), which comprises the entire extracellular region of TNF-R, 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, huTNF- RΔ183 which comprises the sequence of amino acids 1-183 of Figure 2, and TNF-RΔ163 which comprises the sequence of amino acids 1-163 of Figure 2, retain the ability to bind TNF ligand as determined using the binding assays described below in Example 1. TNF- RΔ142, 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 TNF-R. Cys¹⁷⁸, which was deleted without any apparent adverse effect on the ability of the soluble TNF-R to bind TNF, does not appear to be essential for proper folding of TNF-R. Thus, any deletion C-terminal to Cys¹⁶³ would be expected t result in a biologically active soluble TNF-R. The present invention contemplates suc soluble TNF-R constructs corresponding to all or part of the extracellular region of TNF- terminating with any amino acid after Cys¹⁶³. Other C-terminal deletions, such as TNF FΔ157, may be made as a matter of convenience by cutting TNF-R cDNA with appropriat restriction enzymes and, if necessary, reconstructing specific sequences with syntheti oligonucleotide linkers. The resulting soluble TNF-R constructs are then inserted an expressed in appropriate expression vectors and assayed for the ability to bind TNF, a described in Example 1. Biologically active soluble TNF-Rs resulting from suc constructions are also contemplated to be within the scope of the present invention.

Mutations in nucleotide sequences constructed for expression of analog TNF-R must of course, preserve the reading frame phase of the coding sequences and preferably will no create complementary regions that could hybridize to produce secondary mRNA structure 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 th mutation per se be predetermined. For example, in order to select for optimum characteristic of mutants at a given site, random mutagenesis may be conducted at the target codon and th expressed TNF-R mutants screened for the desired activity.

Not all mutations in the nucleotide sequence which encodes TNF-R will be expresse in the final product, for example, nucleotide substitutions may be made to enhanc expression, primarily to avoid secondary structure loops in the transcribed mRNA (see EP 75,444A, incorporated herein by reference), or to provide codons that are more readil translated by the selected host, e.g., the well-known E. coli preference codons for E. co expression. Mutations can be introduced at particular loci by synthesizing oligonucleotide containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of th native sequence. Following ligation, the resulting reconstructed sequence encodes an analo having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can b employed to provide an altered gene having particular codons altered according to th substitution, deletion, or insertion required. Exemplary methods of making the alterations s forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:7 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 an 4,737,462 disclose suitable techniques, and are incorporated by reference herein.

Both monovalent forms and polyvalent forms of TNF-R are useful in th compositions and methods of this invention. Polyvalent forms possess multiple TNF- binding sites for TNF ligand. For example, a bivalent soluble TNF-R may consist of two tandem repeats of amino acids 1-235 of Figure 2, separated by a linker region. Alternate polyvalent forms may also be constructed, for example, by chemically coupling TNF-R to any clinically acceptable carrier molecule, a polymer selected from the group consisting of Ficoll, polyethylene glycol or dextran using conventional coupling techniques. Alternatively, TNF-R may be chemically coupled to biotin, and the biotin-TNF-R conjugate then allowed to bind to avidin, resulting in tetravalent avidin/biotin/TNF-R molecules. TNF-R 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 TNF-R binding sites.

A recombinant chimeric antibody molecule may also be produced having TNF-R 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 TNF-R/IgGi may be produced from two chimeric genes — a TNF- R/human K light chain chimera (TNF-R/C_K) and a TNF-R/human γi heavy chain chimera (TNF-R/Cγ.ι). Following transcription and translation of the two chimeric genes, the gene products assemble into a single chimeric antibody molecule having TNF-R displayed bivalently. Such polyvalent forms of TNF-R may have enhanced binding affinity for TNF ligand. Additional details relating to the construction of such chimeric antibody molecules are disclosed in WO 89/09622 and EP 315062.

Expression of Recombinant TNF-R

The present invention provides recombinant expression vectors to amplify or express DNA encoding TNF-R. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding mammalian TNF-R 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, and a 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 operabl linked to a coding sequence if it controls the transcription of the sequence; or a ribosom binding site is operably linked to a coding sequence if it is positioned so as to permi translation. Generally, operably linked means contiguous and, in the case of secretor leaders, contiguous and in reading frame. Structural elements intended for use in yeas expression systems preferably include a leader sequence enabling extracellular secretion o translated protein by a host cell. Alternatively, where recombinant protein is expresse without a leader or transport sequence, it may include an N-terminal methionine residue. Thi residue may optionally be subsequently cleaved from the expressed recombinant protein t provide a final product.

DNA sequences encoding mammalian TNF receptors which are to be expressed in microorganism will preferably contain no introns that could prematurely terminat transcription of DNA into mRNA; however, premature termination of transcription may b desirable, for example, where it would result in mutants having advantageous C-termina truncations, for example, deletion of a transmembrane region to yield a soluble receptor no bound to the cell membrane. Due to code degeneracy, there can be considerable variation i nucleotide sequences encoding the same amino acid sequence. Other embodiments includ sequences capable of hybridizing to the sequences of the provided cDNA under moderatel stringent conditions (50°C, 2x SSC) and other sequences hybridizing or degenerate to thos which encode biologically active TNF receptor polypeptides.

Recombinant TNF-R DNA is expressed or amplified in a recombinant expressio system comprising a substantially homogeneous monoculture of suitable hos microorganisms, for example, bacteria such as E. coli or yeast such as S. cerevisiae, whic have stably integrated (by transfrrmation or transfection) a recombinant transcriptional un into chromosomal DNA or can, _ne recombinant transcriptional unit as a component of resident plasmid. Generally, cells constituting the system are the progeny of a singl ancestral transformant. Recombinant expression systems as defined herein will expres heterologous protein upon induction of the regulatory elements linked to the DNA sequenc or synthetic gene to be expressed. Transformed host cells are cells which have been transformed or transfected wit

TNF-R vectors constructed using recombinant DNA techniques. Transformed host cell ordinarily express TNF-R, but host cells transformed for purposes of cloning or amplifyin TNF-R DNA do not need to express TNF-R. Expressed TNF-R will be deposited in the ce membrane or secreted into the culture supernatant, depending on the TNF-R DNA selecte Suitable host cells for expression of mammalian TNF-R include prokaryotes, yeast or high eukaryotic cells under the control of appropriate promoters. Prokaryotes include gra negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cel include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed to produce mammalian TNF-R 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 TNF-R 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 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, 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 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 β- 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. 8:4057, 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 λ PL promoter and cI857ts thermolabile repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λ PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).

Recombinant TNF-R 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 Kluyveromyces may also be employed. Yeast vectors will generally contain an origin of replication from the 2μ yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA encoding TNF-R, sequences for polyadenylation and transcriptio termination and a selection gene. Preferably, yeast vectors will include an origin o replication and selectable marker permitting transformation of both yeast and E. coli, e.g., th ampicillin resistance gene of E. coli and S. cerevisiae TRP1 or URA3 gene, which provides selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, and promoter derived from a highly expressed yeast gene to induce transcription of a structur sequence downstream. The presence of the TRP1 or URA3 lesion in the yeast host ce genome then provides an effective environment for detecting transformation by growth in th absence of tryptophan or uracil. Suitable promoter sequences in yeast vectors include the promoters fo 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; an Holland et al., Biochem. 77:4900, 1978), such as enolase, glyceraldehyde-3-phosphat dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphat isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters f 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 f selection and replication in E. coli (Amp^r gene and origin of replication) and yeast DN sequences including a glucose-repressible ADH2 promoter and α-factor secretion leader. Th ADH2 promoter has been described by Russell et al. (J. Biol. Chem. 258:2674, 1982) an Beier et al. (Nature 300:724, 1982). The yeast α-factor leader, which directs secretion heterologous proteins, can be inserted between the promoter and the structural gene to b expressed. See, e.g., Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proc. Natl. Aca Sci. USA 57:5330, 1984. The leader sequence may be modified to contain, near its 3' en 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; a exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:192

1978, selecting for Trp⁺ transformants in a selective medium consisting of 0.67% yea nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil URA+ tranformants in medium consisting of 0.67% YNB, with amino acids and bases described by Sherman et al., Laboratory Course Manual for Methods in Yeast Genetics, Co Spring Harbor Laboratory, Cold Spring Harbor, New York, 1986.

Host strains transformed by vectors comprising the ADH2 promoter may be gro for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% or 4 glucose supplemented with 80 μg/ml adenine and 80 μg/ml uracil. Derepression of the AD 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 by Gluzman (Cell 23:175, 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 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and 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 SV40 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 S V40 viral origin of replication (Fiers et al., Nature 273:113, 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 TNF-R 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 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 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 TNF-R 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, b transforming a host cell with a vector comprising a DNA sequence which encodes an enzym which is inhibited by a known drug. The vector may also comprise a DNA sequence whic encodes a desired protein. Alternatively, the host cell may be co-transformed with a secon vector which comprises the DNA sequence which encodes the desired protein. Th transformed or co-transformed host cells are then cultured in increasing concentrations of th known drug, thereby selecting for drug-resistant cells. Such drug-resistant cells survive i increased concentrations of the toxic drug by over-production of the enzyme which i inhibited by the drug, frequently as a result of amplification of the gene encoding the enzym Where drug resistance is caused by an increase in the copy number of the vector DN encoding the inhibitable enzyme, there is a concomitant co-amplification of the vector DN encoding the desired protein (TNF-R) in the host cell's DNA.

A preferred system for such co-amplification uses the gene for dihydrofolate reductas (DHFR), which can be inhibited by the drug methotrexate (MTX). To achieve c amplification, a host cell which lacks an active gene encoding DHFR is either transforme with a vector which comprises DNA sequence encoding DHFR and a desired protein, or co-transformed with a vector comprising a DNA sequence encoding DHFR and a vect comprising a DNA sequence encoding the desired protein. The transformed or c transformed host cells are cultured in media containing increasing levels of MTX, and tho cells lines which survive are selected.

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

Recombinant proteins are preferably expressed by co-amplification of DHFR or GS a mammalian host cell, such as Chinese Hamster Ovary (CHO) cells, or alternatively in murine myeloma cell line, such as SP2/0-Agl4 or NS0 or a rat myeloma cell line, such YB2/3.0-Ag20, disclosed in PCT applications WO/89/10404 and WO 86/05807.

A preferred eukaryotic vector for expression of TNF-R DNA is disclosed below Example 2. This vector, referred to as pCAV/NOT, was derived from the mammalian hi expression vector pDC201 and contains regulatory sequences from SV40, adenovirus-2, and human cytomegalovirus.

Purification of Recombinant TNF-R Purified mammalian TNF receptors 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, supernatants from systems which secrete recombinant 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 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 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 TNF-R 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 TNF-R 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 TNF-R as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). This reference describes two sequential, reversed-phase HPLC steps for purification of recombinant human GM-CSF on a preparative HPLC column.

Human TNF-R 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 TNF-R from the culture. Thes components ordinarily will be of yeast, prokaryotic or non-human higher eukaryotic origi and preferably are present in innocuous contaminant quantities, on the order of less tha about 1 percent by weight. Further, recombinant cell culture enables the production of TN R free of proteins which may be normally associated with TNF-R as it is found in nature i its species of origin, e.g. in cells, cell exudates or body fluids.

Therapeutic Administration of Recombinant Soluble TNF-R

The present invention provides methods of using therapeutic compositions comprisin an effective amount of soluble TNF-R proteins and a suitable diluent and carrier, and metho for suppressing TNF-dependent inflammatory responses in humans comprising administerin an effective amount of soluble TNF-R protein.

For therapeutic use, purified soluble TNF-R protein is administered to a patien preferably a human, for treatment in a manner appropriate to the indication. Thus, f example, soluble TNF-R protein compositions can be administered by bolus injectio continuous infusion, sustained release from implants, or other suitable technique. Typicall a soluble TNF-R therapeutic agent will be administered in the form of a compositio comprising purified protein in conjunction with physiologically acceptable carriers, excipien or diluents. Such carriers will be nontoxic to recipients at the dosages and concentratio employed. Ordinarily, the preparation of such compositions entails combining the TNF- with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 1 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipient Neutral buffered saline or saline mixed with conspecific serum albumin are exempla appropriate diluents. Preferably, product is formulated as a lyophilizate using appropria excipient solutions (e.g., sucrose) as diluents. Appropriate dosages can be determined trials. The amount and frequency of administration will depend, of course, on such factors the nature and severity of the indication being treated, the desired response, the condition the patient, and so forth. Soluble TNF-R proteins are administered for the purpose of inhibiting TN dependent responses. A variety of diseases or conditions are believed to be caused by TN such as cachexia and septic shock. In addition, other key cytokines (IL-1, IL-2 and oth colony stimulating factors) can also induce significant host production of TNF. Solub TNF-R compositions may therefore be used, for example, to treat cachexia or septic shock to treat side effects associated with cytokine therapy. Because of the primary roles EL-l a IL-2 play in the production of TNF, combination therapy using both IL-1 receptors or IL receptors may be preferred in the treatment of TNF-associated clinical indications. The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

Example 1

Binding Assays

A. Radiolabeling ofTNFa and TNFβ. Recombinant human TNFα, in the form of a fusion protein containing a hydrophilic octapeptide at the N-terminus, was expressed in yeast as a secreted protein and purified by affinity chromatography (Hopp et al., Bio/Technology 6:1204, 1988). Purified recombinant human TNFβ was purchased from R&D Systems (Minneapolis, MN). Both proteins were radiolabeled using the commercially available solid phase agent, IODO-GEN (Pierce). In this procedure, 5 μg of IODO-GEN were plated at the bottom of a 10 x 75 mm glass tube and incubated for 20 minutes at 4°C with 75 μl of 0.1 M sodium phosphate, pH 7.4 and 20 μl (2 mCi) Na ¹²⁵I. This solution was then transferred to a second glass tube containing 5 μg TNFα (or TNFβ) in 45 μl PBS for 20 minutes at 4°C. The reaction mixture was fractionated by gel filtration on a 2 ml bed volume of Sephadex G- 25 (Sigma) equilibrated in Roswell Park Memorial Institute (RPMI) 1640 medium containing 2.5% (w/v) bovine serum albumin (BSA), 0.2% (w/v) sodium azide and 20 mM Hepes pH 7.4 (binding medium). The final pool of ¹²⁵I-TNF was diluted to a working stock solution of 1 x 10"⁷ M in binding medium and stored for up to one month at 4°C without detectable loss of receptor binding activity. The specific activity is routinely 1 x 10⁶ cpm/mmole TNF.

B. Binding to Intact Cells. Binding assays with intact cells were performed by two methods. In the first method, cells were first grown either in suspension (e.g., U 937) or by adherence on tissue culture plates (e.g., WI26-VA4, COS cells expressing the recombinant TNF receptor). Adherent cells were subsequently removed by treatment with 5mM EDTA treatment for ten minutes at 37 degrees centigrade. Binding assays were then performed by a pthalate oil separation method (Dower et al., /. Immunol. 732:751, 1984) essentially as described by Park et al. (/. Biol. Chem. 267:4177, 1986). Non-specific binding of ¹²⁵I- TNF was measured in the presence of a 200-fold or greater molar excess of unlabeled TNF. Sodium azide (0.2%) was included in a binding assay to inhibit internalization of ¹²⁵I-TNF by cells. In the second method, COS cells transfected with the TNF-R-containing plasmid, and expressing TNF receptors on the surface, were tested for the ability to bind ¹²⁵I-TNF by the plate binding assay described by Sims et al. (Science 247:585, 1988).

C. Solid Phase Binding Assays. The ability of TNF-R to be stably adsorbed to nitrocellulose from detergent extracts of human cells yet retain TNF-binding activity provided a means of detecting TNF-R. Cell extracts were prepared by mixing a cell pellet with a 2 x volume of PBS containing 1% Triton X-100 and a cocktail of protease inhibitors (2 m phenylmethyl sulfonyl fluoride, 10 μM pepstatin, 10 μM leupeptin, 2 mM o-phenanthrolin and 2 mM EGTA) by vigorous vortexing. The mixture was incubated on ice for 30 minute after which it was centrifuged at 12,000x g for 15 minutes at 8'C to remove nuclei and othe debris. Two microliter aliquots of cell extracts were placed on dry BA85/21 nitrocellulos membranes (Schleicher and Schuell, Keene, NH) and allowed to dry. The membranes wer incubated in tissue culture dishes for 30 minutes in Tris (0.05 M) buffered saline (0.15 M pH 7.5 containing 3% w/v BSA to block nonspecific binding sites. The membrane was the covered with 5 x 10'¹¹ M ¹²5l-TNF in PBS + 3% BSA and incubated for 2 hr at 4°C wit shaking. At the end of this time, the membranes were washed 3 times in PBS, dried an placed on Kodak X-Omat AR film for 18 hr at -70°C.

Example 2 Isolation of Human TNF-R cDNA by Direct Expression of Active Protein in COS-7 Cells

Various human cell lines were screened for expression of TNF-R based on thei ability to bind ¹²⁵I-labeled TNF. The human fibroblast cell line WI-26 VA4 was found t express a reasonable number of receptors per cell. Equilibrium binding studies showed tha the cell line exhibited biphasic binding of ¹²⁵I-TNF with approximately 4,000 high affinit sites (K_a = 1 x 10" M-^l) and 15,00 low affinity sites (K_a = 1 x 10⁸ M-l) per cell.

An unsized cDNA library was constructed by reverse transcription of polyadenylate mRNA isolated from total RNA extracted from human fibroblast WI-26 VA4 cells grown i the presence of pokeweed mitogen using standard techniques (Gubler, et al., Gene 25:263 1983; Ausubel et al., eds., Current Protocols in Molecular Biology, Vol. 1, 1987). The cell were harvested by lysing the cells in a guanidine hydrochloride solution and total RN isolated as previously described (March et al., Nature 375:641, 1985).

Poly A⁺ RNA was isolated by oligo dT cellulose chromatography and double stranded cDNA was prepared by a method similar to that of Gubler and Hoffman (Gen 25:263, 1983). Briefly, the poly A+ RNA was converted to an RNA-cDNA hybrid b reverse transcriptase using oligo dT as a primer. The RNA-cDNA hybrid was then converte into double-stranded cDNA using RNAase H in combination with DNA polymerase I. Th resulting double stranded cDNA was blunt-ended with T4 DNA polymerase. To the blunt ended cDNA is added EcoRI linker-adapters (having internal Notl sites) which wer phosphorylated on only one end (Invitrogen). The linker-adaptered cDNA was treated wit T4 polynucleotide kinase to phosphorylate the 5' overhanging region of the linker-adapter an unligated linkers were removed by running the cDNA over a Sepharose CL4B column. Th linker-adaptered cDNA was ligated to an equimolar concentration of EcoRI cut an dephosphorylated arms of bacteriophage λgtlO (Huynh et al, DNA Cloning: A Practical Approach, Glover, ed., IRL Press, pp. 49-78). The ligated DNA was packaged into phage particles using a commercially available kit to generate a library of recombinants (Stratagene Cloning Systems, San Diego, CA, USA). Recombinants were further amplified by plating phage on a bacterial lawn of E. coli strain c600(hfl^_).

Phage DNA was purified from the resulting λgtlO cDNA library and the cDNA inserts excised by digestion with the restriction enzyme Notl. Following electrophoresis of the digest through an agarose gel, cDΝAs greater than 2,000 bp were isolated.

The resulting cDΝAs were ligated into the eukaryotic expression vector pCAV/ΝOT, which was designed to express cDΝA sequences inserted at its multiple cloning site when transfected into mammalian cells. pCAV/ΝOT was assembled from pDC201 (a derivative of pMLSV, previously described by Cosman et al., Nature 312: 768, 1984), SV40 and cytomegalovirus DΝA and comprises, in sequence with the direction of transcription from the origin of replication: (1) SV40 sequences from coordinates 5171-270 including the origin of replication, enhancer sequences and early and late promoters; (2) cytomegalovirus sequences including the promoter and enhancer regions (nucleotides 671 to +63 from the sequence published by Boechart et al. (Cell 47:521, 1985); (3) adenovirus-2 sequences containing the first exon and part of the intron between the first and second exons of the tripartite leader, the second exon and part of the third exon of the tripartite leader and a multiple cloning site (MCS) containing sites for Xhol, Kpnl, Smal, Νotl and Bgll; (4) SV40 sequences from coordinates 4127-4100 and 2770-2533 that include the polyadenylation and termination signals for early transcription; (5) sequences derived from pBR322 and virus-associated sequences VAI and VAII of pDC201, with adenovirus sequences 10532-11156 containing the VAI and VAII genes, followed by pBR322 sequences from 4363-2486 and 1094-375 containing the ampicillin resistance gene and origin of replication.

The resulting WI-26 VA4 cDΝA library in pCAV/ΝOT was used to transform E. coli strain DH5α, and recombinants were plated to provide approximately 800 colonies per plate and sufficient plates to provide approximately 50,000 total colonies per screen. Colonies were scraped from each plate, pooled, and plasmid DΝA prepared from each pool. The pooled DΝA was then used to transfect a sub-confluent layer of monkey COS-7 cells using DEAE-dextran followed by chloroquine treatment, as described by Luthman et al. (Nucl. Acids Res. 77:1295, 1983) and McCutchan et al. (J. Natl. Cancer Inst. 41:351, 1986). The cells were then grown in culture for three days to permit transient expression of the inserted sequences. After three days, cell culture supernatants were discarded and the cell monolayers in each plate assayed for TΝF binding as follows. Three ml of binding medium containing 1.2 x 10-H _ I25ι_iabeled FLAG®-TΝF was added to each plate and the plates incubated at 4^βC for 120 minutes. This medium was then discarded, and each plate was washed once with cold binding medium (containing no labeled TNF) and twice with cold PBS. The edge of each plate were then broken off, leaving a flat disk which was contacted with X-ray fil for 72 hours at -70°C using an intensifying screen. TNF binding activity was visualized o the exposed films as a dark focus against a relatively unifoπn background. After approximately 240,000 recombinants from the library had been screened in thi manner, one transfectant pool was observed to provide TNF binding foci which were clearl apparent against the background expo. ure.

A frozen stock of bacteria from the positive pool was then used to obtain plates o approximately 150 colonies. Replicas of these plates were made on nitrocellulose filters, an the plates were then scraped and plasmid DNA prepared and transfected as described above t identify a positive plate. Bacteria from individual colonies from the nitrocellulose replica o this plate were grown in 0.2 ml cultures, which were used to obtain plasmid DNA, whic was transfected into COS-7 cells as described above. In this manner, a single clone, clone 1 was isolated which was capable of inducing expression of human TNF-R in COS cells. Th expression vector pCAV/NOT containing the TNF-R cDNA clone 1 has been deposited wit the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, US (Accession No. 68088) under the name pCAV NOT-TNF-R.

Example 3 Construction of cDNAs Encoding Soluble huTNF-RΔ235

A cDNA encoding a soluble huTNF-RΔ235 (having the sequence of amino acids 1 235 of Figure 2) was constructed by excising an 840 bp fragment from pCAV/NOT-TNF- with the restriction enzymes Notl and Pvu2. Notl cuts at the multiple cloning site pCAV/NOT-TNF-R and Pvu2 cuts within the TNF-R coding region 20 nucleotides 5' of th transmembrane region. In order to reconstruct the 3' end of the TNF-R sequences, tw oligonucleotides were synthesized and annealed to create the following oligonucleotide linke

Pvu2 BamHl Bgl2 CTGAAGGGAGCACTGGCGACTAAGGATCCA

GACTTCCCTCGTGACCGCTGATTCCTAGGTCTAG AlaGluGlySerT rGlyAspEnd

This oligonucleotide linker has terminal Pvu2 and Bgl2 restriction sites, regenerates 2 nucleotides of the TNF-R, followed by a termination codon (underlined) and a Bam restriction site (for convenience in isolating the entire soluble TNF-R by Notl/Bam digestion). This oligonucleotide was then ligated with the 840 bp Notl/Pvu2 TNF-R inse into Bgl2/Notl cut pCAV/NOT to yield psolhuTNF-RΔ235/CAVNOT, which w transfected into COS-7 cells as described above. This expression vector induced expression of soluble human TNF-R which was capable of binding TNF.

Example 4 Construction of cDNAs Encoding Soluble huTNF-RΔ185

A cDNA encoding a soluble huTNF-RΔ185 (having the sequence of amino acids 1- 185 of Figure 2) was constructed by excising a 640 bp fragment from pCAV NOT-TNF-R with the restriction enzymes Notl and Bgl2. Notl cuts at the multiple cloning site of pCAV/NOTNF-R and Bgl2 cuts within the TNF-R coding region at nucleotide 637, which is 237 nucleotides 5' of the transmembrane region. The following oligonucleotide linkers were synthesized:

Bgl2 5'-GATCTGTAACGTGGTGGCCATCCCTGGGAATGCAAGCATGGATGC-3'

ACATTGCACCACCGGTAGGGACCCTTACGTTCG IleCysAsnValValAlalleProGlyAsnAlaSerMetAspAla

Notl

5'- AGTCTGCACGTCCACGTCCCCCACCCGGTGAGC -3' TACCTACGTCAGACGTGCAGGTGCAGGGGGTGGGCCACTCGCCGG ValCysThrSerThrSerProThrArgEnd

The above oligonucleotide linkers reconstruct the 3' end of the receptor molecule up to nucleotide 708, followed by a termination codon (underlined). These oligonucleotides were then ligated with the 640 bp Notl TNF-R insert into Notl cut pCAV/NOT to yield the expression vector psolTNFRΔ185/CAVNOT, which was transfected into COS-7 cells as described above. This expression vector induced expression of soluble human TNF-R which was capable of binding TNF.

Example 5 Construction of cDNAs Encoding Soluble huTNF-RΔ163

A cDNA encoding a soluble huTNF-RΔ163 (having the sequence of amino acids 1-

163 of Figure 2) was constructed by excising a 640 bp fragment from from pCAV/NOT- TNF-R with the restriction enzymes Notl and Bgl2 as described in Example 4. The following oligonucleotide linkers were synthesized:

Bgl2 Notl

5'-GATCTGT-EGΔGC -3' ACAACTCGCCGG IleCysEnd This above oligonucleotide linker reconstructs the 3' end of .he receptor molecule up t nucleotide 642 (amino acid 163), followed by a termination codon (underlined). Thi oligonucleotide was then ligated with the 640 bp Notl TNF-R insert into Notl c pCAV/NOT to yield the expression vector psolTNFRΔ 163/CAVNOT, which was transfecte into COS-7 cells as described above. This expression vector induced expression of solubl human TNF-R which was capable of binding TNF in the binding assay described in Exampl 1.

Example 6

Construction of cDNAs Encoding Soluble huTNF-RΔ142

A cDNA encoding a soluble huTNF-RΔ142 (having the sequence of amino acids 1 142 of Figure 2) was constructed by excising a 550 bp fragment from from pCAV/NO TNF-R with the restriction enzymes Notl and AlwNl. AlwNl cuts within the TNF- coding region at nucleotide 549. The following oligonucleotide linker was synthesized:

Bgl2 Notl 5'-CTGAAACATCAGACGTGGTGTGCAAGCCCTGTTAAA-3'

CTTGACTTTGTAGTCTGCACCACACGTTCGGGACAATTTCTAGA

End

This above oligonucleotide linker reconstructs the 3' end of the receptor molecule up t nucleotide 579 (amino acid 142), followed by a termination codon (underlined). Th oligonucleotide was then ligated with the 550 bp Notl/AlwNl TNF-R insert into Notl/Bgl cut pCAV/NOT to yield the expression vector psolTNFRΔ 142/CAVNOT, which w transfected into COS-7 cells as described above. This expression vector did not induce expression of soluble human TNF-R which was capable of binding TNF. It is believed th this particular construct failed to express biologically active TNF-R because one or mo essential cysteine residue (e.g., Cys^l57 or Cys^l63) required for intramolecular bonding (f formation of the proper tertiary structure of the TNF-R molecule) was eliminated.

Example 7 Expression of Soluble TNF Receptors in CHO Cells

Soluble TNF receptor was expressed in Chinese Hamster Ovary (CHO) cells usi the glutamine-synthetase (GS) gene amplification system, substantially as described in PC patent application Nos. WO87/04462 and WO89/01036. Briefly, CHO cells are transfect with an expression vector containing genes for both TNF-R and GS. CHO cells are select 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 TNF-R sequences are also amplified and enhanced TNF-R 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. WO87/04462 and WO89/01036, 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 itself. The BamHl cut pSVLGS.1 fragment was then ligated to a 2.4 kb BamHl to Bgl2 fragment of pEE6hCMV (described in PCT Application No. WO89/01036, 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 the SV40 later promoter. The BamHl to Bgl2 fragment of pEEόhCMV contains the human cytomegalovirus major immediate early promoter CMV), a polylinker, and the SV40 early polyadenylation signal. The coding sequences for soluble TNF-R 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 Klenow and ligating with Smal cut dephosphorylated p6/PSVLGS.l, thereby placing the solTNF-R coding sequences under the control of the hCMV promoter. This resulted in a single plasmid vector in which the SV40/GS and hCMB/solTNF-R transcription 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 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 non-essential amino acids (Gibco: 320- 1140AG), 500 μM asparagine and glutamate (Sigma) and nucleosides (30 μM adenosine, guanosine, cytidine and uridine and 10 μM thymidine)(Sigma). Approximately 1 x 10⁶ cells per 10 cm petri dish were transfected with 10 ug of psolTNFR/PoVPSVLGS 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 minutes) approximately 4 hours after transfection, substantially as described by Frost & Williams, Virology 91:39 (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 to confluency in selective mediu Conditioned medium from confluent wells were then assayed for soluble TNF-R activit using the binding -.ssay described in Example 1 above. These assays indicated that th colonies expressed biologically active soluble TNF-R. In order to select for GS gene amplification, several MSX-resistant cell lines a transfected with psolTNFR/P6/PS VLGS and grown in various concentrations of MSX. F each cell line, approximately 1x10^ cells are plated in gradually increasing concentrations 100 uM, 250 uM, 500 uM and 1 mM MSX and incubated for 10-14 days. After 12 day colonies resistant to the higher levels of MSX appear. The surviving colonies are assayed f TNF-R activity using the binding assay described above in Example 1. Each of these highl resistant cell lines contains cells which arise from multiple independent amplification event From these cells lines, one or more of the most highly resistant cells lines are isolated. T amplified cells with high production rates are then cloned by limiting dilution cloning. Ma cell cultures of the transfectants secrete active soluble TNF-R.

Example 8 Expression of Soluble Human TNF-R in Yeast

Soluble human TNF-R was expressed in yeast with the expression vector pIXY43 which was derived from the yeast expression vector pIXY120 and pi mid ρYEP35 pIXY120 is identical to pYαHuGM (ATCC 53157), except that it contains no cDNA inse and includes a polylinker/multiple cloning site with a Ncol restriction site.

A DNA fragment encoding TNF receptor and suitable for cloning into the yea expression vector pIXY120 was first generated by polymerase chain reaction (PC amplification of the extracellular portion of the full length receptor from pCA V/NOT-TNF- (ATCC 68088). The following primers were used in this PCR amplification:

5' End Primer 5'-TTCCGGTACCTTTGGATAAAAGAGACTACAAGGAC

Asp718->Pro euAspLysArgAspTyrLysAsp

GACGATGACAAGTTGCCCGCCCAGGTGGCATTTACA-3' AspAspAspLys< TNF-R >

3' End Primer (antisense)

5 ' -CCCGGGATCCTTAGTCGCCAGTGCTCCCTTCAGCTGGG-3 ' BamHl>End< TNF-R- > The 5' end oligonucleotide primer used in the amplification included an Asp718 restriction site at its 5' end, followed by nucleotides encoding the 3' end of the yeast α-factor leader sequence (Pro-Leu-Asp-Lys-Arg) and those encoding the 8 amino acids of the FLAG^® peptide (AspTyrLysAspAspAspAspLys) fused to sequence encoding the 5' end of the mature receptor. The FLAG® peptide (Hopp et al., Bio /Technology 6:1204, 1988) is a highly antigenic sequence which reversibly binds the monoclonal antibody Ml (ATCC HB 9259). The oligonucleotide used to generate the 3' end of the PCR-derived fragment is the antisense strand of DNA encoding sequences which terminate the open reading frame of the receptor after nucleotide 704 of the mature coding region (following the Asp residue preceding the transmembrane domain) by introducing a TAA stop codon (underlined). The stop codon is then followed by a BamHl restriction site. The DNA sequences encoding TNF-R are then amplified by PCR, substantially as described by Innis et al., eds., PCR Protocols: A Guide to Methods and Applications (Academic Press, 1990).

The PCR-derived DNA fragment encoding soluble human TNF-R was subcloned into the yeast expression vector pIXY120 by digesting the PCR-derived DNA fragment with BamHl and Asp718 restriction enzymes, digesting pIXY120 with BamHl and Asp718, and ligating the PCR fragment into the cut vector in vitro with T4 DNA ligase. The resulting construction (pDCY424) fused the open reading frame of the FLAG®-soluble TNF receptor in-frame to the complete α-factor leader sequence and placed expression in yeast under the aegis of the regulated yeast alcohol dehydrogenase (ADH2) promoter. Identity of the nucleotide sequence of the soluble TNF receptor carried in pIXY424 with those in cDNA clone 1 were verified by DNA sequencing using the dideoxynucleotide chain termination method. pD Y424 was then transformed into E. coli strain RR1.

Soluble human TNF receptor was also expressed and secreted in yeast in a second vector. This second vector was generated by recovering the pIXY424 plasmid from E. coli and digesting with EcoRI and BamHl restriction enzymes to isolate the fragment spanning the region encoding the ADH2 promoter, the α-factor leader, the FLAG^®-soluble TNF receptor and the stop codon. This fragment was ligated in vitro into EcoRI and BamHl cut plasmid pYEP352 (Hill et al., Yeast 2:163 (1986)), to yield the expression plasmid pIXY432, which was transformed into E.coli strain RR1.

To assess secretion of the soluble human TNF receptor from yeast, pIXY424 was purified and introduced into a diploid yeast strain of S. cerevisiae (XV2181) by electroporation and selection for acquisition of the plasmid-borne yeast TRP1+ gene on media lacking tryptophan. To assess secretion of the receptor directed by pEXY432, the plasmid was introduced into the yeast strain PB149-6b by electroporation followed by selection for the plasmid-borne URA3⁺ gene with growth on media lacking uracil. Overnight cultures were grown at 30^* C in the appropriate selective media. The PB149-6b/pIXY434 transformants were diluted into YEP-1% glucose media and grown at 30^βC for 38-40 hours Supernatants were prepared by removal of cells by centrifugation, and filtration o supematants through 0.45μ filters.

The level of secreted receptor in the supernatants was determined by immuno-dotblot Briefly, 1 ul of supernatants, and dilutions of the supernatants, were spotted ont nitrocellulose filters and allowed to dry. After blocking non-specific protein binding with 3% BSA solution, the filters were incubated with diluted Ml anti-FLAG^® antibody, exces antibody was removed by washing and then dilutions of horseradish peroxidase conjugate anti-mouse IgG antibodies were incubated with the filters. After removal of excess secondar antibodies, peroxidase substrates were added and color development was allowed to procee for approximately 10 minutes prior to removal of the substrate solution.

The anti-FLAG® reactive material found in the supernatants demonstrated tha significant levels of receptor were secreted by both expression systems. Comparison demonstrated that the pIXY432 system secreted approximately 8-16 times more solubl human TNF receptor than the pIXY424 system. The supernatants were assayed for solubl TNF-R activity, as described in Example 1, by their ability to bind ^l25I-TNFα and bloc TNFα binding. The pIXY432 supernatants were found to contain significant levels of activ soluble TNF-R.

Example 9

Isolation of Murine TNF-R cDNAs

Murine TNF-R cDNAs were isolated from a cDNA library made from murine 7B cells, an antigen-dependent helper T cell line derived from C57BL/6 mice, by cross-specie hybridization with a human TNF-R probe. The cDNA library was constructed in λZA

(Stratagene, San Diego), substantially as described above in Example 2, by isolatin polyadenylated RNA from the 7B9 cells.

A double-stranded human TNF-R cDNA probe was produced by excising a approximately 3.5 kb Notl fragment of the human TNF-R clone 1 and ³²P-labeling th cDNA using random primers (Boehringer-Mannheim).

The murine cDNA library was amplified once and a total of 900,000 plaques wer screened, substantially as described in Example 2, with the human TNF-R cDNA prob Approximately 21 positive plaques were purified, and the Bluescript plasmids containin EcoRl-linkered inserts were excised (Stratagene, San Diego). Nucleic acid sequencing of portion of murine TNF-R clone 11 indicated that the coding sequence of the murine TNF- was approximately 88% homologous to the corresponding nucleotide sequence of huma TNF-R. A partial nucleotide sequence of murine TNF-R cDNA clone 11 is set forth in Figures 4-5.

Example 10 Preparation of Monoclonal Antibodies to TNF-R

Preparations of purified recombinant TNF-R, for example, human TNF-R, or transfected COS cells expressing high levels of TNF-R are employed to generate monoclonal antibodies against TNF-R using conventional techniques, for example, those disclosed in U.S. Patent 4,411,993. Such antibodies are likely to be useful in interfering with TNF binding to TNF receptors, for example, in ameliorating toxic or other undesired effects of TNF, or as components of diagnostic or research assays for TNF or soluble TNF receptor.

To immunize mice, TNF-R immunogen is emulsified in complete Freund's adjuvant and injected in amounts ranging from 10-100 μg subcutaneously into Balb/c mice. Ten to twelve days later, the immunized animals are boosted with additional immunogen emulsified in incomplete Freund's adjuvant and periodically boosted thereafter on a weekly to biweekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay (antibody sandwich) or ELISA (enzyme-linked immunosorbent assay). Other assay procedures are also suitable. Following detection of an appropriate antibody titer, positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and fused to the murine myeloma cell line NS1. Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a HAT selective medium (hypoxanthine, aminopterin, and thymidine) to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivity with TNF- R, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochem. δ:871 (1971) and in U.S. Patent 4,703,004. Positive clones are then injected into the peritoneal cavities of syngeneic Balb/c mice to produce ascites containing high concentrations (>1 mg/ml) of anti-TNF-R monoclonal antibody. The resulting monoclonal antibody can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography, and/or affinity chromatography based on binding of antibody to Protein A of Staphylococcus aureus.

Claims

1. An isolated DNA sequence encoding a biologically active mammalian TNF receptor (TNF-R) protein.

2. An isolated DNA sequence according to claim 1 , selected from the group consisting of:

(a) cDNA clones having a nucleotide sequence derived from the coding region of a native mammalian TNF-R gene;

(b) DNA sequences capable of hybridization to the clones of (a) under moderately stringent conditions (50°C, 2 x SSC) and which encode biologically active TNF-R protein; 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 TNF-R protein.

3. An isolated DNA sequence according to claim 1 which encodes a soluble human TNF-R protein.

4. An isolated DNA sequence according to claim 3, wherein the soluble human

TNF-R protein has an amino acid sequence comprises the sequence of amino acid residues 1- x of Figure 2, wherein x is selected from the group consisting of amino acids 163-235

5. An isolated DNA sequence according to claim 3, wherein the soluble human TNF-R protein comprises the sequence of amino acids 1-235 of Figure 2.

6. A DNA sequence according to claim 5, wherein amino acid residue 46 is selected from the group consisting of He and Thr and amino acid residue 118 is selected from the group consisting of Val and He.

7. An isolated DNA sequence according to claim 3, wherein the soluble human TNF-R protein comprises the sequence of amino acids 1-185 of Figure 2.

8. An isolated DNA sequence according to claim 3, wherein the soluble human TNF-R protein comprises the sequence of amino acids 1-163 of Figure 2.

9. A recombinant expression vector comprising a DNA sequence according to any one of claims 1-8.

10. A process for preparing a biologically active mammalian TNF receptor (TNF-R) protein, comprising culturing a suitable host cell comprising a vector according to claim 8 under conditions promoting expression.

11. A purified biologically active mammalian TNF receptor (TNF-R) protein composition.

12. A purified biologically active soluble human TNF-R protein composition.

13. A purified biologically active TNF-R protein composition according to claim 12, comprising the sequence of amino acid residues 1-235 of Figure 2.

14. A purified biologically active TNF-R protein composition according to claim 12, comprising the sequence of amino acid residues 1-185 of Figure 2.

15. A purified biologically active TNF-R protein composition according to claim 12, comprising the sequence of amino acid residues 1-163 of Figure 2.

16. A composition for regulating immune responses in a mammal, comprising an effective amount of a mammalian TNF-R protein composition according to claim 11 , and a suitable diluent or carrier.

17. A method for regulating immune responses in a mammal, comprising administering an effective amount of a composition according to claim 16.

18. The method of claim 17, wherein the TNF-R protein is human TNF-R and the mammal to be treated is a human.

19. The use of mammalian TNF-R protein in preparing a pharmaceutical composition suitable for parenteral administration to a human patient for regulating immune responses.

20. An assay method for detection of TNF or TNF-R molecules or the interaction thereof, comprising use of a protein composition according to claim 11.

21. Antibodies immunoreactive with mammalian TNF receptors.