FI105099B - Method for Preparation of a Biologically Active Tumor Necrosis Factor Receptor Protein of Mammalian Origin and DNA Encoding This - Google Patents

Description

105099

Cross-Reference to Related Applications This patent application is a partial continuation of U.S. Patent Application Serial No. 421,417, filed October 13, 1989, which is hereby incorporated by reference. Continuation of U.S. Patent Application Serial No. 405,370, filed September 11, 1989, which has now been rejected, and which is a partial continuation of U.S. Patent Application Serial No. 403,241, filed September 5, 1989, and is now rejected.

BACKGROUND OF THE INVENTION The present invention relates generally to cytokine receptors and more specifically to tumor necrosis factor receptors.

Tumor necrosis factor-α (TNF-α, also known as Cakectin) and tumor necrosis factor-β (TNF-β, also known as lymphotoxin) are homologous mammalian endogenous secreted proteins capable of inducing a wide variety of cell types. Because of their great similarities, in the structural and functional properties of the two cytokines they are referred to collectively as "TNF". Complementary Rice cDNA clones encoding TNF-α (Pennica et al., Nature 312 (1984) 724) and TNF-S (Gray et al., Nature 312 (1984) 721) have been isolated, allowing further characterization of the structural and biological properties of TNF.

: TNF proteins initiate their biological action on cells by binding to specific TNF receptor (TNF-R) proteins expressed on the plasma membrane of a TNF1-responsive cell. TNF-α and TNF-S were first shown to bind to a common receptor in the human cervical cancer cell line ME-180 (Aggarwal et al.

• 2 105099

Nature 318: 665 (1985)). Estimates of the size of TNF-R as determined by affinity labeling studies ranged from 54 to 175 kDar (Creasey et al., Proc. Natl.Acad.Sei. USA 84: 3293 (1987), Stauber et al., J.Biol.Chem. 263 5: 19098 (1988); Hohmann et al., J. Biol. Chem. 264 (1989) 14927). Although the relationship between the two TNF-Rs with different molecular weights is unclear, Hohmann et al. (J ♦ Biol.Chem. 264 (1989) 14927) reported that at least two different cell surface receptors for TNF are present in 10 different cell types. These receptors have an apparent molecular weight of about 80 kDa and about 55 -60 kDa, respectively. However, purification of cell surface TNF receptors to homogeneity has not been reported in any of the preceding publications.

There are also cell surface receptor TNFs

soluble proteins identified in human urine capable of binding to TNF (Peetre et al., Eur.-J. Haematol. 41 (1988) 414; Seckinger et al., J.Exp.Med. 167 (1988) 1511; Seckinger et al. al., J. Biol. Chem. 264: 2011966 (1989); UK Patent Application Publication No. 2 218 101 A

Seckinger et al .; Engelmann et al., J. Biol. Chem. 264: 11974 (1989)). The partial N-terminal amino acid sequence of the soluble urinary TNF binding protein disclosed in UK 2 218 101 A is Asp-Ser-Val-Cys-. 25 Pro-, corresponding to a partial sequence, later disclosed by Engelmann et al. (1989). The relationship between the above soluble urinary binding proteins was further clarified following the filing of the original strain application (US Serial No. 403,241), as described by Engelmann et al. reported having identified and purified a second soluble urinary TNF-binding protein having the N-terminal amino acid sequence of Val-Ala-Phe-Thr-Pro- (J. Biol. Chem. 265 (1990) 1531). In UK Publication 2,218,101 A and Engelmann et al. the two urinary proteins disclosed in Publication 35 were shown to be immunologically related to two apparently distinct cell surface proteins by the ability of antisera against binding proteins to inhibit TNF binding to certain cells.

5 Two separate groups have subsequently reported the molecular level cloning and expression of its 55 kDa TNF-R (Loetscher et al., Cell. 61: 351 (1990);

Schall et al., Cell 61: 361 (1990). TNF-R of each group has an N-major amino acid sequence corresponding to that described in UK 2 218 101 A, Engelmann et al. (1989) and Engelmann et al. (1990) the partial amino acid sequence of the urinary binding protein disclosed.

To clarify the relationship between multiple forms of TNF-R and soluble urinary TNF-binding proteins, or the role of TNF-R in the structural and biological properties and role of TNF-R in different cell populations in response to TNF or other cytokine stimulation , research, or effective use of TNF-Rs in therapy, diagnosis, or assays requires purified TNF-R compositions. However, such compositions can only be obtained in practical yields by cloning and expressing the genes encoding the receptors using recombinant DNA technology. However, attempts to purify the TNF-R molecule for use in biochemical analysis or for the cloning and expression of mammalian genes encoding TNF-R have been hampered by the lack of a suitable source of receptor protein or mRNA. Prior to the present invention, no cell line was known to express high levels of TNF-R constitutively and continuously, which precluded purification of the receptor for sequencing or construction of gene libraries for the purpose of cloning cDNA.

4, 105099

Summary of the Invention

The present invention provides isolated TNF receptors and DNA sequences encoding mammalian tumor necrosis factor receptors (TNF-R), particularly human TNF-R. Such DNA sequences include (a) cDNA clones having the nucleotide sequence derived from the coding region of the TNF-R gene of interest; (b) DNA sequences capable of hybridizing to cDNA clones from (a) under medium stringency and encoding biologically active TNF-R molecules; or (c) DNA sequences that are degenerate as a result of the genetic code with respect to the DNA sequences defined in (a) and (b) and which encode biologically active TNF-R molecules. In particular, the present invention provides DNA sequences encoding soluble TNF receptors.

The present invention also provides recombinant DNA expression vectors comprising the DNA sequences defined above, recombinant TNF-R-Mole-20 yeasts produced using recombinant DNA expression vectors, and methods of recombinant expression. -DNA-TNF-R-Molecules using expression vectors.

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

The present invention also provides compositions for use in the therapy, diagnosis, assay, or production of antibodies to TNF-R comprising effective amounts of soluble native (receptor proteins) or recombinant receptor proteins. , prepared by the above methods.

Due to the ability of TNF to specifically bind TNF receptors (TNF-Rs), purified TNF-R compositions are useful in diagnostic assays for TNF, and

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producing antibodies to the TNF receptor for use in diagnosis and therapy. In addition, purified TNF receptor compositions can be used directly in therapy to bind or capture TNF, providing a means to regulate the immune activities of this cytokine.

These and other aspects of the present invention will become apparent upon reading the following detailed description.

10 Brief description of the drawings

Figure 1 is a schematic representation of the coding region of various cDNA sequences encoding human nerve and mouse TNF-R. The leader sequence is vivolined and the transmem braane region is closed.

Figures 2-3 show a partial cDNA sequence and deduced amino acid sequence of human TNF-R clone-1. Nucleotides are numbered starting from the 5 'unread region. The amino acids are numbered at the beginning of the signal peptide sequence. The putative signal peptide sequence is represented by 20 amino acids -2-1. The mature TNF-R protein N-major leucine is underlined at position 1. The predicted transmembrane region of amino acids 236-265 is also underlined. The C-terminals of various soluble TNF-Rs are indicated by an arrow (vertical arrow).

Figures 4-6 show the cDNA sequence and deduced amino acid sequence thereof in murine TNF-R clone-11. The putative signal peptide sequence is represented by amino acids -22 to -1. The mature N-terminal valine of the mature TNF-R protein is underlined at position 1. The predicted transmembrane region of amino acids 30,234-265 is also underlined.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, the terms "TNF receptor" and "TNF-R" refer to proteins having amino acid 35 sequences which are substantially similar to the amino acid sequences of the 6105099 native mammalian TNF receptor and are biologically active. as defined below, in that they are capable of binding to TNF molecules or transducing a biological signal triggered by the binding of TNF to a cell, or of cross-reacting with anti-TNF-R antibodies raised against TNF- R from natural (i.e., non-recombinant DNA) sources. Mature full-length human TNF-R is a glycoprotein with a molecular weight of about 80 kilo-10 daltons (kDa). As used throughout the specification, the term "mature" refers to a protein expressed in a form lacking a leader sequence that can occur in full-length copies of the native gene. Experiments using COS cells transfected with cDNA encoding full-length human TNF-R showed that TNF-R bound 125I-TNF-α with an apparent Kα of about 5 x 10 9 M. '1, and that TNF-R bound 125I-TNF-B with an apparent Ka of about 2 x 109 M'1. The term "TNF receptor" or "TNF-R" includes, but is not limited to, analogs or subunits of native proteins having at least 20 amino acids and exhibiting at least some biological activity common to TNF-R, e.g., soluble TNF. -R structures lacking the transmembrane region (and secreted by the cell) but retaining the ability to bind TNF.

Various bioequivalent protein and amino acid analogues are described in detail below.

The naming convention of TNF-R analogs used herein follows the manner in which a protein (e.g., TNF-R) is named after either hu (human) or mu (mouse), V, and followed by delta (to indicate deletion) as well as the number of the major C-acid. For example, hu-TNF-R delta-235 refers to human TNF-R having an Asp235 C-terminal amino acid (i.e., a polypeptide having an amino acid sequence of 1 to 235 in Figure 2). In the absence of an entry in human or mouse species, 7105099 TNF-R generically refers to mammalian TNF-R. Similarly, in the absence of any specific designation, the term "deletion mutant" means TNF-R including all forms of TNF-R, including mutants and analogs having the biological activity of TNF-R.

As used in the context of the present invention, "soluble TNF-R" or "sTNF-R" refers to proteins, or substantially equivalent analogues, that have all or part of the extracellular region of native TNF-R, e.g., hu-TNF. -R-delta-235, hu-TNF-R-delta-185 and hu-TNF-R-delta-163, or amino acid sequences substantially similar to those of amino acids 1-163, amino acids 1-185, or amino acids 1-235 2, which are biologically active in that they bind to TNF ligand. Equivalent soluble TNF-R includes polypeptides that differ from these sequences by one or more substitutions, deletions, or additions and that retain the ability to bind to TNF or inhibit TNF signal transduction activity by binding of cell surface TNF receptor proteins, e.g. TNF-R delta-x, where x is selected from the group consisting of any of amino acids 163 to 235 in Figure 2. Analog deletions can be made · 25 to TNF-R. Inhibition of TNF signal transduction activity can be determined by transfecting cells with recombinant TNF-R DNA sequences to obtain recombinant DNA receptor expression. The cells are then contacted with TNF and the resulting metabolic effects are studied. If 30 gives an effect that can be attributed to the ligand *.! function, the recombinant DNA receptor has signal transduction activity. Exemplary procedures for determining whether a polypeptide has signal transduction activity are disclosed by Idzerda et al., J.Exp.fried. 171 · 861 (1990); 35 Curtis et al., Proc.Natl.Acad.Sci. USA 86: 3045 (1989); 8 105099

Prywes et al., EMBO J. 5: 2179 (1986); and Chou et al., J ♦ Biol.Chem. 262: 1842 (1987). Alternatively, primary cells or cell lines that express the endogenous TNF receptor and have a detectable biological response to TNF could also be used.

The term "isolated" or "purified" as used in this specification to determine the purity of a TNF-R protein or protein composition means that the protein or protein composition is substantially free of other natural or endogenous proteins and contains less than about 1% by weight. protein production residues from the production processes of. However, such compositions may contain other proteins added as stabilizers, carriers, excipients, or co-therapies. TNF-R is isolated if it can be detected as a single protein band in a polyacrylamide gel by silver staining.

The term "substantially similar" as used to define either amino acid or nucleic acid sequences means that a particular target sequence, for example a mutant sequence, deviates from the reference sequence by one or more substitutions, deletions or additions, whereby the effect of this is to maintain the TNF-R activity. · 25 determines, for example, one of the TNF-R binding assays shown in Example 1 below. Alternatively, nucleic acid subunits and analogs are "substantially similar" to the specific DNA sequences disclosed herein if: (a) the DNA sequence is derived from the coding region of the native mammalian TNF-R gene; (b) the DNA sequence is capable of hybridizing to the DNA sequences from (a) under moderate Ankara conditions (50 ° C, 2x SSC) and encoding biologically active TNF-R molecules; or DNA sequences that are degenerate as a result of the genetic code 355 to DNA sequences defined in (a) or (b) and which encode biologically active TNF-R molecules.

The term "recombinant DNA" as used herein means that the protein is derived from recombinant DNA (e.g.

5 microbial or mammalian expression systems. By "microbial" is meant 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 that is substantially free of native endogenous agents. The protein expressed in most bacterial cultures, e.g., EL coli, does not contain glycan. The glycosylation pattern of the protein expressed in yeast may be different from that expressed in mammalian cells.

As used throughout the specification, as a "biologically active" property of TNF receptors, it is meant that the particular molecule has an amino acid sequence sufficiently similar to the disclosed embodiments of the present invention to bind detectable amounts of TNF, to mediate TNF stimulation, for example, as part of a hybrid receptor construct, or to cross-react with anti-TNF-R antibodies produced against natural (i.e., non-recombinant) DNA against TNF-R. Preferably, the biologically active TNF receptors of the present invention are capable of binding more than 0.1 nanomolar (nmol) TNF per nanomolar receptor, and preferably more than 0.5 nanomolar TNF per nanomolar receptor in standard assays for binding (see 30 below).

By "isolated DNA sequence" is meant a DNA polymer in the form of a single fragment or part of a larger DNA construct derived from DNA isolated at least once in substantially pure form, i.e., 35 free contaminating endogenous materials and 10 105099 in an amount or concentration that permits the sequence. and its partial nucleotide sequences by conventional biochemical methods, for example using a cloning vector. Such sequences are preferably in the form of an open reading region not interrupted by internal unread sequences, or introns typically found in eukaryotic genes. Genomic DNA containing the essential sequences could likewise be used as a source of coding sequences. Sequences of unreaded DNA can occur 5 'or 3' of the open reading region when they do not interfere with the processing or expression of the coding regions.

By "nucleotide sequence" is meant a heteropolymer of deoxyribonucleotides. The DNA sequences encoding the proteins provided by the present invention may be assembled from cDNA fragments and short oligonucleotide linkers, or a series of oligonucleotides, to obtain a synthetic gene that can be expressed in a recombinant DNA copy unit.

Isolation of cDNA Sequences Encoded by TNF-R The TNF-R coding sequence is obtained by isolating a complementary DNA (cDNA) sequence encoding TNF-R from a recombinant cDNA or genomic DNA library. The cDNA library is preferably constructed by obtaining polyadenylated mRNA from a specific cell line expressing mammalian TNF-R, for example from human connective tissue formation cell line WI-26 VA4 (ATCC CCL 95.1) and using mRNA as a template for double-stranded cDNA. sex. The double-stranded cDNA is then packaged in a recombinant DNA-30 vector which is introduced into the appropriate E. Coli !! strain where it is multiplied. Mouse or other mammalian cell lines expressing TNF-R may also be used. TNF-R sequences contained in a cDNA library can be readily identified by screening the library with a purpose-35 nucleic acid probe capable of hybridizing to TNF-R cDNA. Alternatively, DNA sequences encoding TNF-R proteins may be assembled by ligation with synthetic oligonucleotide subunits which correspond wholly or partially to the sequence in Figures 2-3 or Figures 4-6 to obtain a complete coding sequence.

The human TNF receptor cDNAs of the present invention were isolated by the direct expression cloning method. The cDNA library was constructed by first isolating 10 mucosal mRNAs from the human connective tissue formation cell line WI-26 VA4. Polyadenylated RNA was isolated and used to prepare double stranded cDNA. The purified cDNA fragments were then ligated into pCAV / NOT vector DNA using control sequences obtained from pDC201 (a pMLSV derivative previously described by Cosman et al., Nature 312 (1984) 768), SV40. and cytomegalovirus DNA described in detail below in Example 2. pCAV / NOT has been deposited with the American Type Culture Collection under accession number ATCC 68014. The pCAV / NOT vectors containing the W26-VA4 cDNA fragments were transformed into coli- strain DH5-a. Transformants were plated to give approximately 800 colonies per plate. The resulting colonies were harvested and plasmid DNA was prepared from each pool, which was transfected into C0S-7 cells essentially as described by Cosman et al. (Nature 312: 768 (1984)) and Luthman et al. (Nucl. Acids Res. 11, 1295 (1983)). Transformants expressing biologically active cell surface TNF receptors were identified by screening for their ability to bind 125 I-TNF. In this screening approach, transfected V. COS-7 cells were incubated in medium containing 125 I-TNF, the cells were washed to remove unbound labeled TNF, and the cell monolayers were contacted with X-ray film to detect TNF binding concentrations as disclosed by Science. 12, 105099, 241 (1988) 585. Transfectants detected in this manner appear as dark spots against a relatively light background.

Using this approach, approximately 240,000 cDNAs were screened as pools of about 800 cDNAs until the assay of one of the 5 transfectant pools showed positive sites for TNF binding. Frozen bacterial stock from this positive pool was grown in culture and plated to obtain single colonies which were screened until a single clone (clone-10 ni-11) was identified that was capable of directing surface protein synthesis with detectable TNF binding activity. The sequence of cDNA clone-11 isolated by the above method is shown in Figures 4-6.

Further cDNA clones can be isolated from cDNA libraries of other mammalian species by cross-species hybridization. For use in hybridization, DNA encoding TNF-R may be covalently labeled with a detectable agent such as a fluorescent moiety, a radioactive atom or a chemiluminescent moiety by methods known to those skilled in the art. Such probes could also be used for in vitro diagnosis of specific conditions.

Like most mammalian genes, mammalian TNF receptors are presumably encoded by multi-exogenous genes. Alternative mRNA constructs that can be considered from various post-transcriptional mRNA splicing events and share broad identity or similarity domains with the cDNA sequences claimed herein are considered to be within the scope of the present invention.

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

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Proteins and analogs

The present invention provides isolated recombinant mammalian TNF-R polypeptides. The isolated TNF-R polypeptides of the present invention are substantially free of other contaminating materials of natural or endogenous origin and contain less than about 1% by weight of protein impurities from production processes. Native human TNF-R molecules are recovered from cell lysates as glycoproteins having an apparent molecular weight of about 80 kilodaltons (kDa) by SDS-PAGE. The TNF-R polypeptides of this invention may lack the associated native pattern glycosylation.

Mammalian TNF-Rs of the present invention include, for example, TNF-Rs of primates, humans, mice, dogs, cats, bovine animals, sheep, horses, and pigs. Mammalian TNF-Rs can be obtained by cross-species hybridization using single-stranded cDNA obtained from the human TNF-R DNA sequence as a hybridization probe to isolate TNF-R cDNA sequences from mammalian cDNA libraries.

The TNF-R derivatives encompassed by the invention also include various structural forms of the primary protein which retain biological activity. For example, due to the presence of ionizable amino and carboxyl groups, the TNF-R- protein may be in the form of acidic or basic salts, or it may be in neutral form. The 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 groups such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants. Covalent derivatives are prepared by linking specific 35 functional groups to the TNF-R amino acid side chains or the 14 or 105099 N- or C-terminus. Other TNF-R derivatives within the scope of this invention include covalent or aggregate conjugates of TNF-R or fragments thereof with other proteins or polypeptides (formed), for example, by synthesizing N-terminal or C- in recombinant culture. pääfuusioina. For example, the conjugated peptide may be a signal (or leader) polypeptide sequence in the N-terminal region of a protein that directs, during or after translation, the transfer of the protein from its synthesis site to its site within or outside the cell membrane or wall (e.g., yeast α-factor leader). TNF-R protein fusions may comprise peptides added to facilitate purification or recognition of TNF-R (e.g., poly-His). The TNF receptor amino acid sequence may also be linked to the Asp-Tyr-Lys-Asp-Asp-Asp-Asp-peptide peptide.

Lys (DYKDDDDK) (Hopp et al., Bio / Technology 6: 1204 (1988)). The latter sequence is highly antigenic and comprises an epitope which is reversibly bound by a specific monoclonal antibody, allowing rapid determination and easy purification of the expressed recombinant protein. This sequence is also specifically cleaved by the bovine mucosal enterokinase residue which immediately follows the Asp-Lys pair. The fusion proteins blocked by this peptide can also resist intracellular degradation of ε-25 in coli.

TNF-R derivatives may also be used as immunogens, reagents in receptor-based immunoassays, or as binding agents in affinity purification procedures for TNF or other binding ligands. TNF-R-30 derivatives may also be obtained using cross-linking agents such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide at cysteine and lysine residues. TNF-R proteins may likewise be covalently bound by side-reactive side groups. 35 various insoluble substrates, such as cyanobromo-activated, bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorption on polyolefin surfaces (with or without use of glutaraldehyde), may be used. For selective binding of TNF-R antibodies or TNF (for assay or purification purposes).

The present invention also encompasses TNF-R, which comprises or does not comprise glycosylation 10 according to the native pattern. TNF-R expressed in yeast or mammalian expression systems, e.g., COS-7 cells, may have the same or slightly different molecular weight and Gly cosylation pattern than native molecules, depending on the expression system. Expression of TNF-R DNA in bacteria such as coli yields non-glycosylated molecules. Functional mutant analogs of mammalian TNF-R having inactivated N-glycosylation centers can be produced by oligonucleotide synthesis and ligation or using site-directed mutation techniques. These analogue pro-20 proteins can be produced in a homogeneous reduced carbohydrate form with good yield using yeast expression systems. The N-glycosylation centers in eukaryotic proteins are characterized by the amino acid triplet Asn-A1-Z, where Ax is any amino acid except? 25 Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for the covalent attachment of a carbohydrate. Such a center can be eliminated by substituting another amino acid for Asn or residue Z, deleting Asn or Z, or inserting a non-Z-30 amino acid between Ax and Z, or an amino acid other than Asn I! Between Asn and A ^.

Similarly, TNF-R derivatives can be obtained by mutating TNF-R or its subunits. In this sense, the TNF-R mutant is a 35 polypeptide homologous to TNF-R which, however, differs in amino acid sequence from native TNF-R due to deletion, insertion or substitution.

Bioequivalent analogs of TNF-R proteins may be constructed, for example, by making various substitutions of residues 5 or sequences, or by deleting end-located or internal residues or sequences that are not essential for biological activity. For example, cysteine residues can be deleted (eg, Cys178) or replaced with other amino acids to prevent the formation of unnecessary or defective intramolecular disulfide bridges during renaturation. Other approaches for performing the mutation include modifying adjacent double-stranded amino acid residues to enhance expression in yeast systems that exhibit KEX2-15 protease activity. In general, substitutions should be made conservatively; i.e., the most preferred replacement amino acids are those which have physicochemical properties similar to those of the residue to be replaced. Similarly, when using a deletion or insertion strategy, the potential effect of deletion 20 or insertion on biological activity should be considered. Substantially similar polypeptide sequences as defined above generally include the same number of amino acid sequences, although C-terminal dots for the construction of soluble TNF-Rs contain fewer amino acid sequences.

To maintain the biological activity of TNF-Rs, deletions and substitutions will preferably result in homologous or conservatively substituted sequences, meaning that a particular residue is replaced by a biologically similar residue. Examples of conservative substitutions are the substitution of one aliphatic residue for another, such as Ile, Val, Leu or Ala, or substitutions where one polar residue is substituted for another such as Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, such as substitutions of whole regions with similar hydrophobic properties, are well known. In addition, explicit amino acid differences between human, mouse, and other mammalian TNF-Rs suggest other conservative substitutions that can be made without altering the essential biological properties of TNF-R.

TNF-R subunits can be constructed by deleting end-to-end or internal residues or sequences. Particularly preferred sequences are those in which the transmembrane region and the intracellular region of TNF-R are deleted or substituted by hydrophilic residues to facilitate receptor secretion into the cell culture medium. The resulting protein is called a soluble TNF-R molecule that has a ability to bind TNF. A particularly preferred soluble TNF-R construct is TNF-R delta-235 (amino acid sequence 1-23 in Figure 2), which comprises the entire extracellular region of TNF-R terminating in Asp235 immediately adjacent to the transmembrane region. Additional amino acids can be deleted from the transmembrane region while maintaining TNF binding activity. For example, hu-TNF-R-delta-183, which comprises the amino acid sequence 1-183 of Figure 2, and TNF-R-delta-163, which comprises the sequence of amino acids 1-163 of Figure 2, retain the ability to bind TNF-? ligand, / 25, determined using the binding assays described in Example 1 below. However, TNF-R-delta-142 does not retain the ability to bind TNF ligand. This indicates that either or both Cys157 and Cys163 residues are necessary to form an intramolecular disulfide bridge to properly fold TNF-R. Cys178, which deletes I! without any apparent adverse effect on the ability of soluble TNF-R to bind TNF, does not appear to be necessary for the correct folding of TNF-R. Thus, any deletion on the C-terminus to Cys163 would be expected to result in biologically active soluble '18505099 TNF-R. The present invention relates to such soluble TNF-R constructs which correspond wholly or partially to the extracellular domain of TNF-R terminating at any amino acid after Cys163. Other C-terminal deletions, such as TNF-R-delta-157, can be conveniently made by digesting TNF-R cDNA with appropriate restriction enzymes and, if necessary, rebuilding specific sequences using synthetic oligonucleotide linkers. The resulting soluble TNF-R structures are then inserted and expressed in appropriate expression vectors and assayed for their ability to bind TNF as described in Example 1. Biologically active soluble TNF-Rs derived from such structures are also contemplated herein. within the scope of the invention. The nucleotide sequences of the mutations constructed to express analog TNF-R must naturally retain the reading region of the coding sequences, and preferably do not form complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpin, mRNA. Although the mutation site can be predetermined, it is not necessary that the nature of the mutation itself be predetermined. For example, to select the optimum features for the mutants at a particular site 25, a random mutation of the target codon can be performed and the expressed TNF-R mutants screened for the desired activity.

Not all mutations in the nucleotide sequence encoding TNF-R are expressed in the final product, e.g., 30 nucleotide substitutions can be made to enhance expression, primarily to avoid secondary structural loops in the copied mRNA (see EP-A-75 444A, incorporated herein by reference). in order to obtain those that are easier to read by the selected host, 19 105099 e.g. well-known coli preference codons for expression per inch.

Mutations can be made at specific sites by synthesizing oligonucleotides containing the mutant sequence flanked by restriction sites which allow the fragments to be ligated to the native sequence. The rebuilt sequence obtained after ligation encodes an analog having the desired amino acid addition, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutation procedures can be used to obtain a modified gene in which certain codons have been altered according to the required substitution, deletion or insertion. Exemplary methods for making the above changes 15 are disclosed by Walder et al. (Gene 42: 133 (1986)); Bauer et al. (Gene 37: 73 (1985)); Craik (BioTechnics 1985: January 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462 Jul-20 disclose suitable techniques and are incorporated herein by reference.

Both monovalent forms and multivalent forms of TNF-R are useful in the compositions and methods of this invention. The multivalent forms contain multiple TNF-R binding sites for TNF ligand.

For example, a divalent soluble TNF-R may consist of two consecutive repeats of amino acids 1 to 235 in Figure 2, separated by a fusion sequence region. Alternative polyvalent forms can 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 can be chemically coupled to biotin, after which the biotin-TNF-105099 R conjugate is allowed to bind to avidin to give quaternary avidin / biotin / TNF-R molecules. TNF-R may also be covalently linked to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugate 5 precipitated with anti-DNP or anti-TNP-IgM to form decameric conjugates having a TNF-R binding center of 10 .

Similarly, a recombinant chimeric antibody antibody having variable regions substituted by TNF-R sequences in either the heavy or light chain of the immunoglobulin molecule, or both, and having non-modified constant regions may be formed. For example, chimeric TNF-R / IgGi can be produced from two chimeric genes: TNF-R / human kappa-light chain-chimera (TNF-15 R / Ckappa) and TNF-R / human-gammaj-heavy chain-chimera (TNF-R /CgaiBBa.1). After duplication and reading of two chimeric genes, the gene products assemble into a single chimeric antibody molecule in which TNF-R is bivalent. Such polyvalent forms of TNF-R form 20 may have enhanced binding affinity for TNF ligand. Further details regarding the construction of such chimeric antibody molecules are disclosed in WO 89/09622 and EP 315 062.

Recombinant TNF-R Expression The present invention provides recombinant DNA expression vectors for amplification or expression of DNA encoding TNF-R. Recombinant DNA expression vectors are replicable DNA constructs containing synthetic or cDNA-derived DNA fragments encoding mammalian TNF-R or bioequivalent analogues operatively linked to appropriate copy or reading regulatory units. , derived from mammalian, microbial, viral or insect genes. The copy unit generally comprises a combination of oh (1) one 35 or more genetic units having a regulatory moiety 21 in 105099 gene expression, for example copy promoters or enhancers, (2) a structural or coding sequence that is copied into mRNA and read and (3) appropriate copy and read initiation and termination sequences as described in detail below. Such regulatory units may comprise an operator sequence for controlling copy which encodes suitable mRNA ribosome binding centers. The ability to replicate in a host, usually brought with the origin of replication, and the selection gene to facilitate the identification of transformants may also be included. DNA regions are operatively linked when functionally linked to one another. For example, the signal peptide DNA (secreted leader sequence) is operably linked to the poly-15 peptide DNA if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the copy of the sequence; or the ribosome binding center is operably linked to a code sequence if it is positioned 20 so that reading is possible. Generally, operably linked means continuous, and for secretory leader sequences, continuous and within the reading range. Preferably, the structural units for use in yeast expression systems include a leader sequence that allows the host cell to secrete a protein that is read off.

Alternatively, when expressed without a leader or delivery sequence, the recombinant protein may comprise an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to yield the final product.

DNA sequences encoding mammalian TNF receptors to be expressed in a microorganism preferably do not contain introns that could prematurely terminate DNA copy to mRNA; however, premature termination of transcription may be desirable, for example, where it would result in mutants with advantageous C-terminus, for example, deletion of the trans membrane region to obtain a soluble receptor that is not bound to the cell membrane. Due to the nature of the code degenerate-5, significant variation in nucleotide sequences encoding the same amino acid sequence may occur. Other embodiments include sequences that are capable of hybridizing to sequences of a given cDNA under moderately stringent conditions (50 ° C, 2x SSC), and other sequences that hybridize to or are degenerate with biologically active TNF receptor polypeptides.

The recombinant TNF-R DNA is expressed or amplified in a recombinant DNA expression system comprising a substantially homogeneous monoculture of suitable host microorganisms, for example bacteria such as co-list or yeast such as cerevisiae, which have been stable integrated (by transformation or transfection). as a result), or comprising the recombinant DNA copy unit as a structural component of the present plasmid. Generally, the cells that make up the system are descendants of one parent transformant. The recombinant DNA expression systems defined herein express a heterologous protein when inducing regulatory units linked to the expressed DNA sequence or synthetic gene.

Transformed host cells are cells that have been transformed or transfected with TNF-R vectors constructed using recombinant DNA techniques. Transformed host cells normally express TNF-R, but host cells transformed for the purpose of cloning or amplifying TNF-R do not need to express TNF-R. The expressed TNF-R is deposited in the cell membrane or is secreted into the culture supernatant depending on the TNF-R DNA selected. Suitable host cells for the expression of mammalian TNF-23 105099 R are prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive organisms, for example 5 inches or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free reading systems could also be used to produce mammalian TNF-R using RNA sequences obtained from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast and mammalian

Pouwels et al. ("Cloning Vectors: A Laboratory Manual", Elsevier, New York, 1985), the relevant disclosure of which is incorporated herein by reference.

Prokaryotic expression hosts can be used to express TNF-R, which does not require extensive proteolysis and disulfide processing. Prokaryotic expression vectors generally comprise one or more phenotype selection markers, for example, a gene encoding proteins that confer antibiotic resistance or fulfill an autotrophic requirement, as well as confirmation of the host-recognized replication origin. Suitable prokaryotic hosts for transformants include E. Coli, Bacillus subtilis, Salmonella typhimurium, and various species of the genus Pseudomonas, Streptomyces, and Staphylococcus, although other options may be used.

Expression vectors useful for bacterial use may comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising gene units from the well-known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-353 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 24105099 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" portions are linked to the appropriate promoter and the expressed structural sequence. coli is typically transformed using pBR322 derivatives derived from E. coli (Bolivar et al., Gene 2 (1977) 95). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides a simple means for identifying transformed cells.

Conventional promoters used in recombinant microbial expression vectors include the S-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275 (1978) 615; and Goeddel et al., Nature 281 (1979) 544). , the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8 (1980) 4057; and EP-A-15 36 776), and the tac promoter (Maniatis, "Molecular Cloning: A Laboraroty Manual"). , Cold Spring Harbor Laboratory, 1982, p. 412). A particularly useful bacterial expression system utilizes the lambda-phage PL promoter and the heat lab cl857ts repressor. Plasmid vectors available from the 20 American Type Culture Collection containing lambda-PL promoter derivatives include plasmid pHUB2 present in E. Coli strain JMB9 (ATCC 37092) and pPLc28 present in E. coli RR1: (ATCC 53082).

Recombinant TNF-R proteins can also be expressed in yeast hosts, preferably from Saccharomyces, such as Cerevisiae. Yeasts of other genera such as Pichia or Kluyveromyces may also be used. Yeast vectors generally contain an origin of replication from the 2β yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding TNF-R, sequences for polyadenylation and termination of transcription, and a select gene. Preferably, the yeast vectors have an origin of replication and a selectable marker that permits transformation into both yeast and E. Coli, e.g., the E. Coli of the amphiphilic resistance gene and the S. Cerevisiae TRP1 or URA3 gene, which gives a selectable yeast mutant strain. ability to grow with tryptophan, as well as to induce downstream copy of a promoter derived from a highly expressed yeast gene. The presence of TRP1 or URA3 deficiency in the yeast host cell genome then provides an effective environment for detecting transformation from growth in the absence of tryptophan or uracil.

Suitable promoter sequences for yeast vectors include promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Blol.Chem. 255 (1980) 2073) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7 ( 1968) 149; and Holland et al., 15 Biochem., 17, 4900 (1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphate , pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al. EP-A-73,657.

Preferred yeast vectors can be assembled using DNA sequences from pUC18 for selection and replication in 25 E. Coli (Ampr gene and origin of replication) and yeast DNA sequences including the glucose-repressible ADH2 promoter and the α-factor secretion leader. The ADH2 promoter is described by Russell et al. (J. Biol. Chem. 258: 2674 (1982)) and Beier et al. (Nature 300 1982 724). The yeast 30-factor leader sequence that directs the secretion of heterologous proteins can be inserted between the promoter and the structural gene to be expressed. See, e.g., Kurjan et al., Cell 30: 933 (1982); and Bitter et al. , Proc.Natl. -Acad.Sci. USA 81: 5330 (1984)). The leader sequence may be modified to include, near its 3 'end, one or more useful restriction center leader sequences to facilitate fusion to the foreign genes.

Suitable yeast transformation protocols are known to those skilled in the art; exemplary techniques are described in Price 5 et al., Proc.Natl.Aca.Sci. USA 75, 1929 (1978), according to which the Trp * transformants are selected in selective medium containing 0.67% yeast nitrogen base, 0.5% casino acids, 2% glucose, 10 pg / ml adenine and 20 pg / ml uracil, or URA + - transformants in culture medium containing 0.67% YNB using 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 with vectors comprising the ADH2 promoter-15 can be grown for expression in rich medium containing 1% yeast extract, 2% peptone, and 1% or 4% glucose supplemented with 80 pg / ml adenine and 80 pg / ml uracil. Derepression of the ADH2 promoter occurs at the end of the growth substrate glucose. The impure yeast supernatants are recovered by filtration and stored at 4 ° C before further purification.

Various mammalian or insect cell culture systems are also preferably used to express recombinant protein. Expression of recombinant proteins in mammalian cells is particularly advantageous because such proteins are generally properly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines are the C0S-7 cell lines of monkey kidney cells described by Gluzman (Cell 23: 1981, 175) and other cell lines capable of expressing an appropriate vector, including, for example, L cells, C127, 3T3, Chinese Hamster Ovary (CH0), HeLa and BHK cell lines. Mammalian-35 expression vectors may comprise non-transcribed units 27-105099 such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other flanking non-transcribed 5 'or 3' sequences, and unreaded 5 'or 3' sequences such as essential ribosome binding centers, polyadenylation center, loop donor and recipient centers, and copy decision sequences. A review of baculovirus systems for production of heterologous proteins in insect cells is provided by Luckow and Summers, Bio / Technology 6 (1988) 47.

Control and copy control sequences in expression vectors to be used to transform vertebrate cells can be obtained from viral sources. For example, commonly used promoters and enhancers are obtained from polyoma, adenovirus-2, monkey-1540 (SV40) and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, the SV40 start site, early and late promoter, enhancer, splice and polyadenylation centers, can be used to obtain other genetic units required for expression of 20 heterologous DNA sequences. Early and late promoters are particularly useful because both are readily obtained from the virus as a fragment that also contains the SV40 viral replication origin (Fiers et al., Nature 273, 113 (1978)). Smaller or larger SV40-25 fragments may also be used provided that a sequence of about 250 bp extending from the Hind3 site to the Bg11 site located at the viral origin of replication is included. Further, the mammalian genomic TNF-R promoter, control, and / or signal sequences may be used provided such control sequences are compatible with the selected host cell. Further details regarding the use of a mammalian high expression vector for the production of recombinant mammalian TNF receptor are provided in Examples 2 and 7 below. Exemplary vectors can be constructed as described by Okayama and Berg (Mol.Cell.Biol. 3 (1983) 280).

A useful system for stable expression of high levels of mammalian receptor cDNA sequences in C127 mouse mammary epithelial cells can be constructed essentially as described by Cosman et al. (Mol. Immunol. 23, 935 (1986)).

In preferred aspects of the present invention, recombinant expression vectors comprising TNF-R cDNA sequences are stably integrated into the host cell DNA.

High levels of expression product are achieved by selection of cell lines containing amplified numbers of vector DNA. Cell lines having amplified numbers of vector DNA are selected, for example, by transforming the host cell with a vector comprising a DNA sequence encoding an enzyme which is inhibited by a known drug. The vector may also comprise a DNA sequence encoding the desired protein. Alternatively, the host cell may be co-transformed with another vector comprising a DNA-20 sequence encoding the desired protein. The transformed or co-transformed host cells are then cultured at increasing concentrations of the known drug, thereby selecting for drug-resistant cells. Such drug-resistant cells survive at increasing concentrations of the toxic drug by producing high levels of the enzyme that the drug inhibits, often as a result of the enhancement of the gene encoding the enzyme. Where drug resistance is caused by growth in the copy number of the vector DNA encoding the enzyme to be inhibited, co-amplification of the vector DNA encoding the desired protein (TNF-R) in the host cell DNA has occurred.

A preferred system for such co-amplification uses the dihydrofolate reductase (DHFR) gene, which can be inhibited by the drug methotrexate 35 (MTX). To achieve co-amplification, a host cell lacking 105099 active DHFR-encoding genes is either transformed with a vector comprising the DNA sequence encoding DHFR and the desired protein, or co-transformed with a vector comprising the desired DHFR-encoding DNA sequence and a vector comprising the desired protein more coding DNA sequence. Transformed or co-transformed host cells are cultured in medium containing increasing levels of MTX and surviving cell lines are selected.

10 In a particularly preferred co-gain system

uses the glutamine synthetase (GS) gene responsible for the synthesis of glutamate and ammonia using ATP hydrolysis to ADP and phosphate to maintain the reaction. GSi is inhibited by many different inhibitors, for example methionine sulfoximine (MSX). Thus, TNF-R

can be expressed at high concentrations by co-amplifying cells transformed with a vector comprising a DNA sequence for GS and the desired protein, or co-transformed with a vector comprising a DNA sequence encoding GSi and a vector comprising a DNA sequence encoding the desired protein , followed by culturing the host cells in medium containing increasing levels of MSX and selecting for surviving cells. The GS co-amplification system, appropriate recombinant DNA expression vectors, and cell lines are described in the following PCT patent applications WO 87/04462, WO 89/01016, WO 89/10404 and WO 86/05807.

The recombinant proteins are preferably expressed by co-amplification of DHFR or GS in a mammalian host cell, such as Chinese hamster ovary (CHO) cells, or alternatively in a murine myeloma cell line such as SP2 / 0Agl4 or NSO or a rat myeloma cell line such as YB2 / 3.0-Ag. disclosed in PCT patent applications WO 89/10404 and WO 86/05807.

30 105099

A preferred eukaryotic vector for expression of TNF-R DNA is disclosed below in Example 2. This vector, called pCAV / NOT, was obtained from the mammalian high expression vector pDC201 and contains control sequences from 5 of SV40, adenovirus-2, and human cytomegalovirus. ta.

Purification of recombinant TNF-R

Purified mammalian TNF receptors or analogs are prepared by culturing appropriate host / vector cyst templates to express recombinant DNA reading products from the DNA sequences of the present invention and then purifying them from culture medium or cell extracts.

For example, supernatants from systems that secrete recombinant protein into media may first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore ultrafiltration unit. After the concentration step, the concentrate can be loaded into a suitable purification matrix. For example, a suitable affinity matrix may comprise a TNF or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin such as a matrix or substrate having dependent diethylaminoethyl (DEAE) groups may be used. The matrices may be acrylamide, agarose, dextran, cellulose or other types conventionally used in protein purification. Alternatively, a cation exchange step may be used. Suitable cation exchangers include various non-soluble matrices comprising sulfopropyl or carboxymethyl groups.

Sulfopropyl groups are preferred.

Finally, one or more reverse phase high pressure liquid chromatography (RP-HPLC) steps using a hydro-35 phobic RP-HPLC medium, e.g., silica gel with 31-105099 dependent methyl groups or other aliphatic groups, can be used to further purify the TNF-R composition. Some or all of the preceding purification steps in various combinations may also be used to obtain a homogeneous recombinant protein.

The recombinant protein produced in the bacterial culture is usually first isolated by extraction of the cell pellets, followed by one or more steps of concentration, desalting, water ion exchange or size exclusion chromatography. Finally, high pressure liquid chromatography (HPLC) can be used in the final purification steps. Microbial cells used to express recombinant mammalian TNF-R can be disrupted by any convenient method, including freeze / thaw recycling, sonication, mechanical disruption, or the use of cell lysis agents.

Fermentation of yeast expressing mammalian TNF-R as a secreted protein greatly simplifies purification. Large-scale secreted recombinant DNA from fermentation can be purified by methods analogous to those reported by Urdal et al. (J. Chromatog. 296: 171 (1984)). This document describes two sequential reverse phase HPLC steps for purifying recombinant human GM-CSF on a preparative HPLC column.

Human TNF-R synthesized in recombinant culture is characterized by the presence of non-human cellular components, including proteins, in amounts and types that depend on the purification methods used to recover human TNF-R from the culture. These constituents 30 are usually of yeast prokaryotes or "higher non-human eukaryotes, and are preferably present in harmless contaminating amounts of less than about 1% by weight. Further, recombinant cell culture allows the production of TNF-R which does not contain proteins normally associated with TNF-32 105099 R as it occurs naturally in the native species, e.g., cells, cellular effluents or body fluids.

Administration of Soluble Recombinant DNA TNF-R The present invention provides methods of using therapeutic compositions comprising an effective amount of soluble TNF-R proteins and a suitable diluent or carrier, and methods for suppressing human TNF-dependent inflammatory responses, wherein the methods provide an effective amount of soluble TNF-R protein.

Purified soluble TNF-R protein for therapeutic use is administered to a patient, preferably a human, in a manner appropriate to the indication for treatment. Thus, for example, soluble TNF-R protein compositions may be administered by a single injection, by continuous fluid transfer, may be slowly released from the grafts, or may be administered by any other appropriate technique. Typically, the soluble TNF-R therapeutic agent is administered in the form of a composition comprising purified protein in association with physiologically acceptable carriers, excipients, or diluents. Such carriers are non-toxic to the recipients at dosages and concentrations employed. Usually in the preparation of such compositions: · 25 TNF-R is combined with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates such as glucose, sucrose or dextrins, and V. excipients. Examples of suitable diluents are neutral buffered saline or saline mixed with serum albumin of the same species. Preferably, the lyophilized product is formulated using appropriate filler solutions (e.g., sucrose) as diluents. Appropriate dosages may be determined in the assays. The amount and frequency of drug administration will, of course, depend on factors such as the nature and severity of the indication being treated, the response desired, the condition of the patient, etc.

Soluble TNF-R proteins are administered to inhibit TNF-dependent responses. TNF is believed to cause many different diseases or conditions, such as desertification and blood poisoning shock. In addition, other key cytokines 10 (IL-1, IL-2 and other colony stimulating factors) may also induce significant TNF production in the host. Soluble TNF-R compositions may therefore be used, for example, for the treatment of side effects associated with thinning or hemorrhagic shock, or cytokine therapy. Because of the primary portion that IL-1 and IL-2 play in TNF production, combination therapy using both IL-1 receptors and IL-2 receptors may be beneficial for the treatment of TNF-associated clinical indications.

The following examples are provided for illustrative purposes only and not for limitation.

examples

Example 1

Binding Assays A. Radiolabeling of TNF-α and TNF-B. Recombinant human TNF-α was expressed in the form of a fusion protein containing a hydrophilic octapeptide at the N-terminus as a yeast secreted protein and purified by affinity chromatography (Hopp et al., Bio / Technology 6: 1204 (1988)). Purified recombinant human TNF-β was purchased from R&D Systems (Minneapolis, MN). Both pro-1 'proteins were radiolabeled using a commercially available solid phase material, Iodo-Gen (Pierce). In this procedure, 5 µg of Iodo-Gen product was plated on 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 solution, 34 105099 pH 7.4, and 20 µl: IIa (2 mCi) Na125I. This solution was then transferred to another glass tube containing 5 µg TNF-α (or TNF-B) in 45 µl PBS for 20 minutes at 4 ° C. The reaction mixture was fractionated by gel filtration over 5 ml of 2 ml bed volume 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-10 vial). The final 125 I-TNF pool was diluted in binding medium to a stock stock solution of 1 x 10 7 M and stored for up to one month at 4 ° C with no detectable loss of receptor binding activity. The specific activity is usually 1 x 10 6 scintillation per minute / mmol TNF.

B. Commitment to intact cells. Binding assays on intact cells were performed by two methods. In the first method, cells were first grown either in slurry (e.g., U937) or by attachment to tissue-20 culture plates (e.g., WI26-VA4, COS cells expressing the recombinant TNF receptor). The adherent cells were then removed by treatment with 5 mM EDTA for 10 minutes at 37 ° C. Binding assays were then performed by the phthalate oil separation method (Dower et al., 25 J. Immunol. 132: 751 (1984)), essentially as described.

Park et al. (J. Biol. Chem. 261: 4177 (1986)). Non-specific binding of 125 I-TNF was measured in the presence of 200-fold or greater molar excess of unlabeled TNF. Sodium azide (0.2%) was included in the binding assay to prevent intracellular uptake of 125I-TNF. The second method tested the ability of COS cells transfected with a TNF-R-containing plasmid that expressed TNF receptors on its surface to bind 125I-TNF by the plate binding assay described by 35 Sims et al. (Science 241: 585 (1988)).

35 105099 C. Solid Binding Assays. The means for detecting TNF-R was obtained by the ability of TNF-R to adsorb stably to nitrocellulose from detergent extracts of human cells, while maintaining TNF-binding activity. Cell extracts were prepared by mixing the cell pellet with 2x volume of PBS containing 1% Triton X-100 and cocktail protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 μΜ pepstatin, 10 μΜ leupeptin, 2 mM o-phenanthroline). 2 mM EGTA), 10 vigorously vortexed. The mixture was incubated on ice for 30 minutes, then centrifuged at 12,000 x g for 15 minutes at 8 ° C to remove nuclei and other debris. Two microliters aliquots of cell extracts were placed on dry BA85 / 21 nitrocellulose amem-15 (Schleicher & Schuell, Keene, NH) dry and allowed to dry. Membranes were 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 non-specific binding centers. The membrane was then sealed with a mixture of 5 x 10-11 M 12SI TNF in PBS containing 3% BSA and incubated for 2 hours at 4 ° C with shaking. After this time, the membranes were washed 3 times with PBS, dried and placed on a Kodak X-Omat AR film for 18 hours at -70 ° C.

Example 2

Isolation of human TNF-R cDNA by direct expression of active protein in C0S-7 cells

Various human cell lines were screened for TNF-R expression by their ability to bind 125 I-labeled 30 TNF. The human connective tissue-forming cell line WI-26 VA4 was found to express a reasonable number of receptors per cell. Equilibrium binding studies showed that the cell line showed biphasic 125I-TNF binding, comprising approximately 4,000 high affinity centers (Ka = 1 x 36 105099 ΙΟ10 M11) and 15,000 low affinity centers (Ka = 1 x 108 M'1) per cell.

The size-indistinguishable cDNA library was constructed by reverse transcription of polyadenylated 5 mRNA isolated from total RNA extracted from human connective tissue WI-26 VA4 cells grown in Phytolacca Americana (pokeweed) mitogen. using standard techniques (Gubler et al., Gene 25 (1983) 263; Ausubel et al., Ed. 10, Current Protocols in Molecular Biology, Vol. 1, 1987).

Cells were harvested by lysing cells in guanidine hydrochloride solution and total RNA was isolated as previously described (March et al., Nature 315: 641 (1985)).

Poly A1 RNA was isolated by oligo-dT cellulose chromatography and the double-stranded cDNA was prepared by a method similar to that of Gubler and Hoffman (Gene 25: 263 (1983)). Briefly, poly A 'RNA was converted to RNA cDNA hybrid using reverse transcriptase and 20 oligo-dT as primer. The RNA-cDNA hybrid was then converted to double-stranded cDNA using RNase-H together with DNA polymerase-I. The ends of the resulting double-stranded cDNA were made flat using T4 DNA polymerase. EcoRI-25 fusion sequences (which have internal Not1 centers) that were phosphorylated at one end only (Invitrogen) are added to the cDNA with flat ends. The cDNA with the fusion sequences was treated with T4 polynucleotide kinase to phosphorylate the 5 'excess region of the fusion sequence, and the non-ligated fusion sequences were removed by running the cDNA on a Sepharose CL4B column.

!! The cDNA with the fusion sequences was ligated to an equimolar concentration of EcoRI cut and dephosphorylated stems of bacteriophage lambda-gt10 (Huynh et al., "DNA Cloning: A Practical Approach"; Glover, ed., IRL Press, pp. 49-78). ). The ligated DNA was packaged into phage particles 37-105099 using a commercially available kit to obtain a recombinant DNA product library (Stratagene Cloning Systems, San Diego, CA, USA). The recombinant DNA products were further amplified by plating a phage coli strain 5 c600 (hfl ') on a bacterial mat.

The phage DNA was purified from the resulting lambda-gt10 cDNA library and the cDNA inserts were excised by digestion with the restriction enzyme NotI. The digestion products were electrophoresed on an agarose gel, followed by isolation of cDNA fragments larger than 10,000 bp.

The resulting cDNA fragments were ligated into the eukaryotic expression vector pCAV / NOT, which was designed to express the cDNA sequences inserted into its multicloning center when transfected into mammalian cells. pCAV / NOT was constructed from 15 pDC201 (a pMLSV derivative previously described by Cosman et al., Nature 312: 768 (1984)), SV40, and cytomegalovirus DNA, and comprises sequentially copying from the origin of replication: (1) ) SV40 sequences coordinates 5171-270 including the rep-20 origin of priming, enhancer sequences, and early and late harboring promoters; (2) cytomegalovirus sequences including promoter and enhancer regions (nucleotides 671 to +63 of sequences disclosed by Boechart et al. (Cell 41: 521 (1985)); (3) adenovirus-2 sequences containing a first exon and a part of an intron between the first and second exons of a three-part leader sequence, a second exon and a third part of a three-part leader sequence and a multicloning invention (MCS) containing XhoI, KpnI, 30 Smal, NotI and BglII; (4) SV40 sequences coadminates 4127-4100 and 2770-2533 including polyadenylation and termination signals for early replication; (5) pBR322 derived sequences and VAI and VAII from pDC201, wherein -35 vectors 10532-11156 contain the VAI and VAII genes, followed by 1 38 1 0 5 0 9 9 pBR322 sequences from 4363-2486 and 1094-375 containing the ampicillin resistance gene and repli-k aatioalkukohdan.

The resulting WI-26 VA4 cDNA library in pCAV / NOT 5 transformed the E-coli strain DH5, then plating the recombinant DNA products yielded about 800 colonies per plate, giving sufficient plates to obtain about 50,000 colonies per screen. Colonies were scraped from each plate, pooled into pools, and plasmid DNA was prepared from each pool. The pooled DNA was then transfected with an as yet non-confluent layer of monkey C0S-7 cells using DEAE-dextran, followed by chloroquine treatment as described by Luthman et al. (Nucl. Acids Res. 11 (1983) 1595) and McCutchan et al. (J.Natl.Cancer Inst., 41, 351 (1986)). The cells were then grown in culture for 3 days to obtain a transient expression of the inserted sequences. After three days, cell culture supernatants were discarded and the TNF binding of the so-20 bone layers from each plate was determined as follows. Three ml of binding medium containing 1.2 x 10 * 11 M 125 I-labeled FlagR-TNF was added to each plate and the plates were incubated at 4 ° C for 120 minutes. This medium was then discarded and each plate was washed 25 times with cold binding medium (which did not contain labeled TNF) and twice with cold PBS. The edges of each dish were then broken to obtain a flat plate which was contacted with the X-ray film for 72 hours at -70 ° C using a reinforcement filter. TNF-binding activity was evident in the exposed films I as a dark spot against a relatively even background.

After approximately 240,000 recombinant DNA products from the library had been screened in this manner, one transfectant pool was found to provide TNF binding sites, which were clearly against background exposure.

39 105099 Plates of about 150 colonies were then prepared using a frozen pool of positive pool bacteria. Copies of these plates were made on nitrocellulose filters, followed by scraping and plasmid DNA transfected as described above to identify the positive plate. Bacteria from single colonies from a nitrocellulose copy of this dish were grown in 0.2 ml cultures used to obtain plasmid DNA which was transfected into COS-7 cells as described above. In this way, one clone, clone-1, capable of inducing the expression of human TNF-R on COS cells was isolated. The expression vector pCAV / NOT containing TNF-R DNA clone-1 has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA (accession number 68088) under the name pCAV / NOT- TNF-R.

Example 3

Construction of cDNA sequences encoding soluble hu-TNF-R-delta-235

20 cDNA encoding soluble hu-TNF-R-delta-235

(having the sequence amino acids 1 to 235 in Figure 2) was constructed by cutting the 840 bp fragment of pCAV / NOT-TNF-R with the restriction enzymes Not1 and Pvu2. Not1 cuts pCAV / NOT-TNF-R from the multicloning center and Pvu2 cuts: 25 nucleotides from the 25 TNF-R coding region 5 'of the transmembrane region. TNF-R sequences to the 3'-end of the reconstruction of two oligonucleotides was synthesized, which are soldered in order to obtain the next oligonukleotidiliittosekvenssin: 30 * Bam HI Pvu two Bgl2 CTGAAGGGAGCACTGGCGACTAAGGATCCA GACTTCCCTCGTGACCGCTGATTCCTAGGTCTAG AlaGluGlySerThrGlyAspLoppu 40 105 099 In this oligonukleotidiliittosekvenssissä is located away from the Pvu2- and Bgl2 restriction sites, it re-creates the 20 nucleotides of the TNF R followed by the decision codon (underlined) and the BamHI restriction site (for convenience in isolation of soluble TNF-R as a whole by NotI / BamHI digestion. This oligonucleotide was then ligated with 840 bp NotI / Pvu2-TNF-R- insert with Bgl2 / ~ Not1 cut pCAV / NOT to give psolhu-TNF-R-delta-235 / CAVN0T transfected with C0S-7 cells as described above This expression vector induces soluble human TNF Expression of -R capable of binding TNF.

Example 4

Construction of cDNA-15 sequences encoding soluble hu-TNF-R-delta-185

The cDNA encoding soluble hu-TNF-R-delta-185 (having the sequence amino acids 1 to 185 in Figure 2) was constructed by cleaving a 640 bp fragment of pCAV / NOT-TNF-R with the restriction enzymes NotI and Bgl2. Not1 cuts 20 pCAV / NOT-TNF-R from the multicloning site and Bgl2 cuts the TNF-R coding region at nucleotide 637, which is 237 nucleotides 5 'from the transmembrane region. Synthesized fol-lowing oligonukleotidiliittosekvenssit: - 25 Bgl2-GATCTGTAACGTGGTGGCCATCCCTGGGAATGCAAGCATGGATGC 5'-3 'NotI ACATTGCACCACCGGTAGGGACCCTTACGTTCG IleCysAsnValValAlalleProGlyAsnAlaSerMetAspAla 30' · 'AGTCTGCACGTCCACGTCCCCCACCCGGTGAGC 5' to 3 '♦ _

TACCTACGTCAGACGTGCAGGTGCAGGGGGTGGGCCACTCGCCGG

ValCysThrSerThrSerProThrArgEnd "105099 41

The above oligonucleotide fusion sequences rebuild the 3 'end of the receptor molecule to nucleotide 708, followed by the decision codon (underlined). These oligonucleotides were then ligated with 640 bp Notl-TNF-R-inser-5 into NotI-cut pCAV / NOT to obtain the expression vector psol-TNF-R-delta-185 / CAVNOT, which was transfected with C0S- 7 cells as described above. This expression vector induced the expression of soluble human TNF-R, which was capable of binding TNF.

Example 5

Construction of cDNA sequences encoding soluble hu-TNF-R-delta-163

The cDNA encoding soluble hu-TNF-R-delta-163 (having the sequence amino acids 1-163 in Figure 2) was constructed by cutting a 640 bp fragment of pCAV / NOT-TNF-R with the restriction enzymes Not1 and Bgl2 as described. in Example 4. The following oligonucleotide fusion sequence was synthesized: 20 Bgll2 Notl 5'GATCTGTTGAGC -3 '

ACAACTCGCCGG

IleCys End / 2525 This preceding oligonucleotide fusion sequence rebuilds the 3 'end of the receptor molecule to nucleotide 642 (amino acid 163), followed by a decision codon (underlined). This oligonucleotide was then ligated with the 640 bp NotI-TNF-R insert into NotI-cut pCAV / -30 NOT- to give the expression vector psol-TNF-R-del-I ta-163 / CAVNOT, which was transfected with C0S. -7 cells as described above. This expression vector induced the expression of soluble human TNF-R capable of binding TNF in the binding assay described in Example 1.

42 105099

Example 6

Construction of cDNA sequences encoding soluble hu-TNF-R-delta-142

The cDNA 5 encoding soluble hu-TNF-R-delta-142 (having the sequence of amino acids 1-142 in Figure 2) was constructed by digesting a 550 bp fragment of pCAV / NOT-TNF-R with the restriction enzymes NotI and AlwN1. AlwN1 cuts the TNF-R coding region at nucleotide 549. The following oligonucleotide fusion sequence was synthesized: 10 Bgl2 Notl

5'-CTGAAACATCAGACGTGGTGTGCAAGCCCTGTTAAA-3 'CTTGACTTTGTAGTCTGCACCACACGTTCGGGACAATTTCTAGA

End 15 This preceding oligonucleotide fusion sequence constructs at the 3 'end of the receptor molecule at nucleotide 579 (amino acid 142), followed by a decision codon (underlined). This oligonucleotide was then ligated with a 550 bp Notl / AlwN1-TNF-R insert into NotI / Bgl2-cut pCAV / NOT to give the expression vector psol-TNF-R-delta-142 / CAVNOT, which was transfected with COS- 7 cells as described above. This expression vector did not induce the expression of soluble human TNF-R capable of binding TNF. It is believed that this particular: structure was not able to express biologically active TNF-R because one or more essential cysteine residue (e.g., Cys157 or Cys163) necessary for intracellular binding (to form the correct tertiary structure of TNF-R) was present. eliminated.

Example 7 * Expression of 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, essentially as described in PCT Patent Application Nos. WO 87/0446243105099 and WO 89/013106. cells are transfected with an expression vector containing genes for both TNF-R and GS. CHO cells are selected for GS gene expression by the ability of the transfected DNA to confer resistance to low levels of methionine sulfoximine (MSX). GS sequence amplification events in such cells are selected using higher concentrations of MSX. TNF-R sequences that continue in this manner are also amplified and enhanced TNF-R expression is achieved.

The vector used in the GS expression system was psol-TNF-R / P6 / PSVLGS, constructed as follows. First pSVLGS.l vector (described in PCT Patent Application Publication Nos W0 87/04462 and W0 89/01036 and available from Celltech, Ltd., Berkshire, UK) lei-15 were modified with BamHI restriction enzyme and dephosphorylated with calf intestinal alkaline phosphatase (CIAP) to prevent internal re-ligation of the vector. The BamHI cut pSVLGS.1 fragment was then ligated to the 2.4 kb BamHI-Bgl2 fragment from pEE6hCMV (described in PCT-20 patent application WO 89/01036 and also available from Celltech), which was cut into Bgl2 , BamHI and Fsp1 to avoid the formation of two fragments of the same size, resulting in an 11.2 kb vector, designated p6 / PSVLGS.1.

** ·. PSVLGS.1 contains the glutamine synthetase selection marker gene in the control of the SV40 late promoter. The BamHI-Bgl2 fragment from pEE6hCMV contains the major human cytomegalovirus immediate early promoter (hCMV), a poly-linker, and the SV40 early polyadenylation signal. 30 Soluble TNF-R coding sequences were added to p6 / - · PSVLGS.1 by cutting the Not1-BamHI fragment from the expression vector psol-TNF-R / CAVNOT (prepared according to Example 3 above) by aligning the ends with Klenow and ligating. Smal-cut dephosphorylated p6 / PS-35 VLGS.1, whereby sol-TNF-R coding sequences were placed under control of the? 105099 44 hCMV promoter. This resulted in a single plasmid vector in which the SV40 / GS and hCMB / sol-TNF-R copy units are copied in opposite directions. This vector was named psol-TNF-R / P6 / PSVLGS.

5 psol-TNF-R / P6 / PSVLGS were transfected with CHO-K1 cells (available from the ATCC Depository, Rockville, MD under accession number CCL 61) as follows. The CHO-K1 cell monolayer was grown to almost overlap in minimal requirement medium (MEM), 10X (Gibco: 330-1581AJ), glutamine free and supplemented with 10% dialyzed bovine fetal serum (Gibco: 220-6300AJ). 1 mM sodium pyruvate (Sigma), with MEM-non-essential amino acids (Gibco: 320-1140AG), 500 μ40 asparagine and glutamate (Sigma) and nucleosideil-15α1 (30 μΜ adenosine, guanosine, cytidine and uridine and 10 μΜ thymidine) (Sigma).

Approximately 1 x 10 6 cells in a 10 cm petri dish were transfected with 10 µg psol-TNF-R / P6 / PSVLGS using calcium phosphate constant precipitation essentially as described by Graham and van der Eb, Ku-20, 456 (1983). Cells were exposed to glycerol shock (15% glycerol in serum-free medium for about 1.5 minutes) approximately 4 hours after transfection, essentially as described by Frost and Williams, Virology 91. 25 (1978) 39, after which they were washed with serum-free medium. One day later, the transfected cells were given fresh selective medium containing MSX at a final concentration of 25 μΜ. After 3-4 weeks, colonies of MSX-resistant surviving cells could be seen. The surviving colonies were transferred to 24-well plates and allowed to grow in selective medium. Wells from the treated medium from wells containing mononuclear cells were then assayed for soluble TNF-R · 35 using the binding assay described in Example 1, 10509945. Assays indicated that the colonies expressed biologically active soluble TNF-R.

To select for GS gene amplification, several MSX-resistant cell lines are transfected with psol-TNF-5 R / P6 / PSVLGS and grown at various concentrations of MSX. Approximately 1 x 106 cells from each cell line are plated at incremental concentrations of 100 μΜ, 250 μΜ, 500 μΜ and 1 mM MSX and incubated for 10-14 days. After 12 days, colonies resistant to higher levels of MSX appear. Surviving colonies are assayed for TNF-R activity using the binding assay described in Example 1 above. Each of these highly resistant cell lines contains cells that are generated by many independent validation events. Of these, cell line-15 is isolated from one or more of the most resistant cell lines. Fortified cells with high production efficiencies are then cloned by limiting dilution dilution. Cell cultures of transfectants secrete active soluble TNF-R.

Example 8

Expression of soluble human TNF-R in yeast Soluble human TNF-R was expressed in yeast using the expression vector pIXY432 obtained from the yeast expression vector pIXY120 and plasmid pYEP352. pIXY120: 25 is identical to pYaHuGM (ATCC 53157) except that it does not contain a cDNA insert and contains a poly-linker / multi-cloning center with an Nco I restriction site.

The DNA fragment encoding the TNF receptor and suitable for cloning into the yeast expression vector pIXY120 was first constructed by polymerase chain reaction (PCR) by amplifying the extracellular portion of the full length receptor pCAV / NOT-TNF-R (ATCC 680). The following primers were used for this PCR amplification: • · 46 1 0 5 0 9 9 5 'Primary: 5'-TTCCGGTACCTTTGGATAAAAGAGACTACAAGGAC Asp718-> ProLeuAspLysArgAspTyrLysAsp 5 GACGATGACAAGTTGCCCGCCCAGG

AspAspAspLys <--------TNF-R---------> 3 'Master Primer (Non-Data Sequence): 10 5'-CCCGGGATCCTTAGTCGCCAGTGCTCCCTTCAGCTGGG-3'

BamHl> End <-----------TNF-R------>

The 5 'major oligonucleotide primer used for amplification contained the 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 nucleotides encoding 8 amino acids from the FlagR peptide (AspTyrLysAspAspAspAspLys) fused to the sequence encoding the 5 'end of the mature receptor. The FlagR peptide (Hopp 20 et al., Bio / Technology 6: 1204 (1988)) is a highly antigenic sequence that reversibly binds monoclonal antibody M1 (ATCC HB 9259). The oligonucleotide used to generate the 3 'end of the PCR-derived fragment is a non-strand of DNA coding sequences that terminate the open reading region of the 25 receptor after nucleotide 704 (after the Asp residue preceding the trans membrane region) by introducing the TAA termination codon ( underlined). The stop codon is then followed by a BamHI restriction center. The DNA sequences encoding TNF-R are then amplified by PCR, essentially as described by Innis et al., Ed., "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 by digestion with BamHI and 47105099.

Asp718 restriction enzymes, digestion of pIXY120 with BamHI and Asp718 and ligation of the PCR fragment into the cut vector in vitro using T4 DNA ligase. The resulting construct (pIXY424) fused the open reading region of the FlagR-soluble TNF receptor 5 to the complete α-factor leader sequence and inserted the expression into the yeast regulated yeast alcohol dehydrogenase (ADH2) promoter. The nucleotide sequence of the soluble TNF receptor contained in pIXY424 with respect to the content of cDNA clone-1 was confirmed by DNA sequencing using the dideoxynucleotide chain method. pIXY424 was then transformed into E1 coli strain RR1.

Soluble human TNF receptor was also expressed and secreted in yeast in another vector. This second vector was constructed by recovering plasmid pIXY424 from E. coli and digestion with EcoRI and BamHI to isolate a fragment comprising the ADH2 promoter coding region, the α-factor leader, the Flag * soluble TNF receptor and the stop codon. This fragment was ligated in vitro with Eco RI and BamHI cut plasmid pYEP352 (Hill et al., Yeast 2 (19869,163)) to give expression plasmid pIXY432, which was transformed into E. Coli strain RR1.

To determine soluble human TNF receptor secretion from yeast, pIXY424 was purified and introduced into a diploid cerevisiae yeast strain (XV2181) by electroporation and selection of plasmid-containing yeast TRP1 * gene in tryptophan-lacking medium. To determine pIXY432-driven receptor secretion, the plasmid was introduced into the yeast strain PB149-6b by electroporation, followed by selection for the URA3 * gene contained in the plasmid by growth medium lacking uracil. Overnight cultures were grown at 30 ° C in appropriate selective media. PB149-6b / pIXY434 transformants were diluted to 1048105099 in YEP-1% glucose medium and grown at 30 ° C for 38-40 hours. Supernatants were prepared by removing the cells by centrifugation and filtering the supernatants through 0.45 μ filters.

The level of secreted receptor in the supernatants was determined by immuno-dot blot. Briefly, 1 μΐ of supernatants and dilutions of supernatants were dropped on nitrocellulose filters and allowed to dry. Non-specific protein binding was blocked with 3% BSA, followed by incubation of the filters with diluted M1 anti-FlagR antibody and removal of excess antibody by washing followed by horseradish peroxidase-conjugated anti-mouse IgG antibody. dilutions of the cultures were incubated with filters. Excess secondary antibodies were removed, after which the peroxidase substrates were added and the color development allowed for approximately 10 minutes before the substrate solution was removed.

The anti-FlagR reactive material found in the supernatants showed that significant levels of the receptor were secreted in both expression systems. Comparisons showed that the pIXY432 system secreted approximately 8-16 times more soluble human TNF receptor than the pIXY424 system. Supernatants were assayed for soluble TNF-R activity as described in Example 1 by their ability to bind 125 I-TNF-α and to block TNF-α binding. pIXY432 supernatants were found to contain significant levels of active soluble TNF-R.

Example 9 Isolation of murine TNF-R cDNA sequences Mouse TNF-R cDNA sequences were isolated from a cDNA library prepared from the mouse 7B9 cell line, an antigen-dependent helper T cell line obtained from From C57BL / 6 mice by cross-species hydration to the yeast-35 ni TNF-R probe. The cDNA library was constructed in lambda • 49505099 ZAP (Stratagene, San Diego), essentially as described above in Example 2, isolating polyadenylated RNA from 7B9 cells.

The double-stranded human TNF-R cDNA probe was prepared by cutting a NotI fragment of about 3.5 kb from human TNF-R clone-1 and 32 P-labeled cDNA using random primers (Boehringer-Mannheim). .

The mouse cDNA library was amplified once and a total of 900,000 plaques were screened, essentially as described in Example 2, with a human TNF-R cDNA probe.

About 21 positive plaques were purified and Sini copy containing plasmids containing Eco RI inserts were cut (Stratagene, San Diego). Nucleic acid sequencing of a portion of murine TNF-R clone II indicated that the coding sequence of murine TNF-R was approximately 88% homologous to the corresponding human TNF-R nucleotide sequence. The partial nucleotide sequence of murine TNF-R cDNA clone-11 is shown in Figures 4-5.

Example 10 Preparation of Monoclonal Antibodies Against TNF-R

Preparations of purified recombinant TNF-R, such as human TNF-R, or transfected COS cells expressing high levels of TNF-R are used to generate monoclonal antibodies to TNF-R. using, for example, those described in U.S. Patent No. 4,411,993. Such antibodies are likely to be useful in disrupting TNF binding to TNF receptors, for example, to alleviate toxic or other undesirable effects of TNF, or as part of diagnostic or research assays for TNF or soluble TNF.

To immunize the mice, TNF-R immunogen is emulsified in complete Freund's booster and injected subcutaneously in an amount of 10 to 35 to 100 pg into Balb / c mice. After 10 to 1299 days, immunized animals are boosted with an additional immunogen emulsified in incomplete Freund's, followed by a periodic boost according to the 1-2 week immunization program. Serum samples are then taken by counting blood from the back of the eye or by cutting the tail end for testing by blot-spot (antibody-sandwich) or ELISA (enzyme-linked immunosorbent assay). Other assay procedures are suitable. Once an appropriate antibody titer of 10 has been determined, positive animals are given an intravenous injection of antigen in saline. After 3-4 days, the animals are sacrificed, the spleen cells are harvested and fused to the murine myeloma cell line NS1. Hybridoma cell line 15 created by this procedure and plated on multiple microtiter plates in selective HAT medium (hypoxanthine, aminopterin and thymidine) to prevent proliferation of non-fused cells, myeloma hybrids and spleen cell hybrids.

The hybridoma clones thus formed can be screened by 20 ELISA for reactivity to TNF, for example, using modifications of the techniques disclosed by Engvall et al. , Immunochem. 8, 871 (1971); and disclosed in U.S. Patent No. 4,703,004. Positive clones are then injected into syngeneic Balb / c mice: ·. 25 intraperitoneal fluid to produce high concentrations (> 1 mg / ml) of anti-TNF-R monoclonal antibody. The resulting monoclonal antibody can then be purified by ammonium sulfate mixing and then subjected to gel exclusion chromatography and / or 30 or affinity chromatography based on the binding of the antibody to protein A from Staphylococcus aureus.

• · • ·

Claims (12)

105099 An isolated DNA sequence encoding a biologically active mammalian TNF receptor (TNF-R) protein selected from a) the DNA sequence encoding amino acids 1 to x of Figure 2, wherein x is selected from amino acids 163 to 235, b ) the DNA sequences of the coding region of the native mammalian TNF-R gene of Figure 2-3, c) DNA sequences capable of hybridizing to the sequences of position b) under moderately stringent conditions, i.e., 50 ° C in 2xSSC, or d) ) DNA sequences degenerate with respect to the sequences of b) or c) as a result of the genetic code. An isolated DNA sequence according to claim 1, characterized in that it encodes a soluble human TNF-R protein comprising the amino acid sequence 1 to 235 of Figure 2. An isolated DNA sequence according to claim 2, characterized in that amino acid residue 46 is replaced by amino acid Ile or Thr and amino acid residue 118 is replaced by amino acid residue V1 or Ile. The isolated DNA J * 25 sequence according to claim 1, characterized in that it encodes a soluble human TNF-R protein comprising the amino acid sequence 1 to 185 of Figure 2. The isolated DNA sequence of claim 1, characterized in that it encodes a soluble human TNF-R protein comprising the amino acid sequence 1-163 of Figure 2. Recombinant expression vector, characterized in that it contains the DNA sequence according to any one of claims 1 to 5. A process for the production of a biologically active mammalian TNF-R protein, characterized by culturing a suitable host cell containing the vector of claim 105099 under conditions promoting expression, and isolating and purifying the desired product. 8. The method of claim 7, wherein the purified biologically active mammalian TNF-R protein comprises an amino acid sequence selected from amino acid residues 1 to x of Figure 2, wherein x is selected from amino acids 163 to 235. 9. The method of claim 7, wherein the purified biologically active mammalian TNF-R protein comprises the amino acid sequence 1 to 235 of Figure 2. Method according to claim 7, characterized in that the purified biologically active mammalian TNF-R protein comprises the amino acid sequence 1 to 185 of Figure 2. The method of claim 7, wherein the purified biologically active mammalian TNF-R protein comprises the amino acid sequence 1-163 of Figure 2. A method for producing therapeutically or non-therapeutically useful antibodies that specifically bind to a purified biologically active mammalian TNF-R protein prepared by a method according to any one of claims 7 to 11, characterized by either (a) culturing a hybridoma cell. or (b) recovering antibodies that specifically bind to said TNF-R protein, suitably from 30 immunized animals. • · • <• · 53 105099