FI108645B - Tumor necrosis factor alpha and beta receptors - Google Patents

Description

108645

Tumor Necrosis Factor-α and Tumor Necrosis Factor-B Receptors This patent application is distinct from patent application FI 920946.

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 catectin) and Tumor Necrosis Factor β (also known as lymphotoxin) are homologous mammalian endogenous secreted proteins capable of inducing a wide variety of cellular effects. Because of their great similarities in structure and function, these two cytokines are collectively referred to as "TNF". Complementary cDNA clones encoding TNF-α (Pennica et al., Nature 312 (1984) 724) and TNF-B (Gray 20 et al., Nature 312 (1984) 721) have been isolated. allows further characterization of the structural and biological properties of TNF.

TNF proteins initiate their biological activity in cells by binding to specific TNF-receptor (TNF-R) proteins expressed on the plasma membrane of a TNF-responsive cell. TNF-α and TNF-B were first shown to bind to a common receptor in human | »In the cervical cancer cell line ME-180 (Aggarwal et al., ***: Nature 318 (1985) 665). Estimates of the size of TNF-R as determined by affinity labeling studies ranged from 54 to 175 kDa (Creasey et al., Proc. Natl. Acad. Sci.

USA M (1987) 3293, Stauber et al., J. Biol. Chem. 263-1998 (19098); Hohmann et al., J. Biol. Chem. 264 (1989). Although the relationship between the two TNF-Rs with different molecular weights is not clear, Hohmann et al., 108645 2 al. (J. Biol. Chem. 264: 14927 (1989)) report that at least two different cell surface receptors for TNF are expressed in different cell types. These receptors have an apparent molecular weight of about 80 kDa and about 55-5 5 kDa, respectively. However, purification of cell surface TNF receptors to homogeneity has not been reported in any of the preceding publications.

In addition to cell surface receptors for TNF, soluble proteins have also been identified in human urine that are capable of binding to TNF (Peetre et al., Eur. J. Haematol. 41 (1988) 414; Seckinger et al., J.Exp.Med.

167: 1511 (1988); Seckinger et al., J. Biol. Chem. 264: 11966 (1989); British Patent Application 2,218,101 to 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 GB 2 218 101 A is Asp-Ser-Val-Cys-Pro-, which corresponds to the partial sequence disclosed later by Engelmann et al. (1989). The relationship between the above soluble urinary binding proteins was further clarified following the filing of the priority application of this patent when Engelmann et al. # V reporting. brought their identification and purification of another separate: · /. soluble urinary TNF - binding protein,. 25 having the N-terminal amino acid sequence of Val-Ala-Phe-Thr-Pro-1-1 (J. Biol. Chem. 265: 1531 (1990)). British Patent Specification 2 218 101 A and Engelmann et al. the two urinary proteins disclosed in the publication were shown to be immuno-related to two apparently distinct 30 cell surface proteins, the anti-binding protein F *: balance serum's ability to inhibit TNF binding to certain: cells.

'. Subsequently, two separate groups reported on the human. its 55 kDarn TNF-R molecular cloning and expression (Loetscher et al., Cell. 61: 351 (1990); 108645).

Schall et al. , Cell 61 (1990) 361). TNF-R of each group has the N-major amino acid sequence corresponding to Engelmann et al. In GB 2 218 101 A. (1989) and Engelmann et al. (1990) 5 partial amino acid sequences of the urinary binding protein.

To investigate the relationship between multiple forms of TNF-R and soluble urinary TNF-binding proteins, or to study the structural and biological properties of TNF-R and the role that TNF-10 R plays in responding to different cell populations, TNF or other cytokines, Purified TNF-R compositions are required for effective use, diagnosis or assay of TNF-Rs. 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 constitutively and continuously express high levels of TNF-R, which excluded the purification of the receptor. or sequencing of gene libraries ... for cloning 25 cDNA.

!.! SUMMARY OF THE INVENTION The present invention relates to a purified, biologically active mammalian tumor necrosis factor receptor (TNF-R) protein, particularly human TNF-R. The TNF-R protein of the present invention comprises an amino acid sequence selected from amino acids 1-x of Figure 2, wherein x is selected from amino acids 163-235. In particular, the invention relates to 1. soluble TNF receptor proteins.

. Compositions for use in diagnostic, assay, or anti-TNF-R formulations are also described. : for the production of antibodies comprising 4,106,645 effective amounts of soluble native receptor proteins or recombinant receptor proteins prepared by the methods described in the parent application.

Because TNF is capable of specifically binding TNF-5 receptors (TNF-Rs), purified TNF-R compositions are useful in diagnostic assays for TNF and in the production of anti-TNF receptor antibodies for use in diagnostics and therapy.

Other objects of the present invention will be apparent from the appended claims.

Brief description of the drawings

Figure 1 is a schematic representation of the coding region of various cDNA sequences encoding human and mouse TNF-R. The leader sequence is lined and the transmembrane region 15 is closed.

Figures 2-3 show a partial cDNA sequence of human TNF-R-clone-1 and the deduced amino acid sequence thereof. Nucleotides are numbered starting from the 5 'unread region. The amino acids are numbered from the beginning of the signal peptide sequence-20. Amino acids -2-1 -1 represent the putative signal peptide sequence. N-major leucine of mature TNF-R protein is underlined at position 1. Predicted transmembrane region. ': Amino acids 236-265 are likewise underlined. The C-terminals of various soluble TNF-Rs are indicated by an arrow (vertical-arrow 25).

• * ·, ···. Figures 4-6 show the cDNA sequence and its deduced amino acid sequence in murine TNF-R clone-11.

The amino acids '··· -22 to -1 representing the putative signal peptide sequence. The N-terminal valine of the mature TNF-R protein is underlined at position 1. The predicted transmembrane region amino acids:' 234-265 are also underlined.

DETAILED DESCRIPTION OF THE INVENTION., ..: Definitions * * (-ti; As used herein, the terms "TNF receptor" and 3581086455 "TNF-R" refer to proteins having amino acid sequences substantially similar to those of amino acid sequences of the native mammalian TNF receptor, and which are biologically active as defined below in that they are capable of binding to TNF molecules or transducing a biological signal triggered by TNF molecule binding to a cell, or of cross-reacting with anti- With 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-daltons. (kDa) 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 125 I-TNF-α with an apparent K i of about 5 x 10 9 M '. α, and that TNF-R bound I-TNF? with an apparent Ka of about 2 x 10 9 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 that exhibit at least some biological activity with TNF-R, e.g. ·. ·. 25 soluble TNF-R structures lacking transmem, ···. branial region (and secreted from the cell) but having> »! *. * ._ retention ability to bind TNF. The various bioequivalent protein and amino acid analogs are described in detail below.

30 The naming convention of TNF-R analogs used here follows the "*" * way in which a protein (eg TNF-R) is named after either hu (human) or mu (mouse), ____: and follows the delta (to indicate the deletion) and the C-major amino acid number. For example, hu-TNF-R-delta-235

* O -3 C

. 35 human TNF-R having Asp C-terminal amino acid 1086456 (i.e., a polypeptide having an amino acid sequence of 1 to 235 in Figure 2). In the absence of labeling in human or mouse species, TNF-R generically refers to mammalian TNF-R. Similarly, in the absence of any specific designation, deletion mutants include TNF-R, including all forms of TNF-R, including mutants and analogs having TNF-R biological activity.

As used in the context of the present invention, "soluble TNF-R" or "sTNF-R" refers to pro-10 proteins, or substantially equivalent analogs, whose amino acid sequence corresponds in whole or in part to the extracellular region of native TNF-R, e.g. TNF-R-delta-235, hu-TNF-R-delta-185, and hu-TNF-R-delta-163, or amino acid sequences substantially similar to those shown in Figure 2, amino acids 1-163, amino acids 1- 185, or amino acids 1 to 235, and which are biologically active in that they bind to a TNF ligand. Equivalent soluble TNF-R includes polypeptides that differ from these sequences by one or more substitutions, deletions, or additions, and which retain the ability to bind to TNF or to inhibit TNF signal transduction activity: intercellular binding of TNF receptors such as hu-TNF-R-delta-x, where x is selected from y. Of the 25 groups formed by any of the amino acids • · 163-235 in Figure 2. Analog deletions can be made to * TNF-R. Inhibition of TNF signal transduction activity can be determined by transfecting cells with recombinant TNF-R DNA sequences to obtain recombinant receptor expression. The cells are then contacted with TNF '' and the resulting metabolic effects are studied. If an effect is obtained that can be attributed to ligand function, the recombinant receptor will have a signal transduction. ; duktioaktiivisuutta. Exemplary procedures for determining it. 35 whether the polypeptide has signal transduction activity is disclosed by Idzerda et al., J.Exp.Med. 171: 861 (1990);

Curtis et al., Proc.Natl.Acad.Sei. USA 86: 3045 (1989);

Prywes et al. , EMBO J. 5: 2179 (1986); and Chou et al., J. Biol. Chem. 262: 1842 (1987). Alternatively, primary cells or cell lines expressing an endogenous TNF receptor and having 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 of production methods. residual protein impurities. However, such compositions may contain other proteins added as stabilizers, carriers or excipients. 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. 25 additions, whereby the additive effect here is to maintain the biological activity of TNF-R protein, ···, which can be * i !!! for example, determines one of the TNF-R binding assays shown in Example 1 below. Alternatively, • '' ··· 'nucleic acid subunits and analogs are "substantially 30" similar to the specific DNA sequences disclosed herein, if: (a) the DNA sequence is derived from a native mammalian / TNF-? The coding region of the 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 they encode a biocl. ·. 35 logically active TNF-R molecules; or DNA sequences which are degenerate as a result of the genetic code relative to the 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 (e.g., 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 and substantially free of native endogenous substances. In most bacterial cultures, e.g.

In E. coli, the expressed protein does not contain glycan.

The glycosylation pattern of the protein expressed in yeast may be different from that expressed in mammalian cells.

By "biologically active" as used throughout the specification, the property of TNF receptors means 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, e.g. as part of a hybrid receptor I '*, or to cross-react with anti-TNF-R antibodies produced against natural (i.e. non-recombinant) sources of TNF-R. Preferably, the biologically active TNF receptors encompassed by the present invention are capable of binding more than 0.1 nano-mole (nmol) of TNF per nanomolar receptor, and preferably more than 0.5 nanomolar of TNF per nano-mole. receptor binding assays (see below).

By "isolated DNA sequence" is meant a DNA polymer in the form of a single fragment, or as part of a larger 35 DNA construct derived from DNA that is distinct from the DNA sequence. at least once in substantially pure form, i.e., free from contaminating endogenous materials, and in an amount or concentration that permits the sequence and its partial nucleotide sequences to be identified, processed and recovered by conventional biochemical techniques, 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 unread DNA may be present in the 5 'or 3' open reading region when they do not interfere with the processing or expression of the codons.

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

Isolation of cDNA sequences encoding TNF-R: The coding sequence for TNF-R is obtained by isolation of the complementary DNA sequence (cDNA) encoding TNF-γ. 25 R from a recombinant cDNA or genomic DNA library. cDNA- I ·, ·> ·. The 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 30 mRNA as a template for double-stranded cDNA. • 'sex. The double-stranded cDNA is then packaged in a recombinant DNA vector which is introduced into an appropriate E coli strain for propagation. Mouse or other mammalian cell lines expressing 35 TNF-R can also be used. The TNF-R sequences contained in the cDNA library can be readily identified by screening the library with an appropriate nucleic acid probe capable of hybridizing to the TNF-R cDNA. Alternatively, DNA sequences encoding TNF-R proteins may be assembled by Liga-5 by performing synthetic oligonucleotide subunits corresponding, in whole or in part, to the sequence of Figures 2-3 or Figures 4-6 to obtain a complete coding sequence.

The human 10n TNF receptor cDNAs described in the context of the present invention were isolated by a direct expression cloning method. The cDNA library was constructed by first isolating mucus mRNA 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 which uses regulatory sequences derived 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. pCAV / NOT containing WI26-VA4 cDNA fragments vectors were transformed into E. coli strain * ·: V: DH5-a. Plating the transformants yielded approximately 800 colonies. 25 kegs per dish. The resulting colonies were harvested and • ·, ···. plasmid DNA was prepared from each pool, which was transfected into COS-7 cells essentially as shown in Figs. neet Cosman et al. (Nature 312 (1984) 7 68) and Luthman et al. (Nucl. Acids Res. 11, 1295 (1983)). Transfor- * ”pines expressing biologically active cell surface TNF receptors were identified by screening for their ability to bind * it / 125 I-TNF. In this screening approach, transfected and COS-7 cells were incubated in medium containing. With 125 I-TNF, cells were washed to remove unbound labeled 35 TNF, and the cell monolayers were contacted with 11,108,645 X-ray films to determine TNF binding levels as disclosed by Sims et al., Science 241: 585 (1988). the detected transfectants appear as dark spots against a relatively light background.

Using this approach, approximately 240,000 cDNAs were screened as pools of about 800 cDNA until a single transfectant pool assay showed positive sites for TNF binding. Frozen bacterial stock from this positive pool was cultured and mal-10 was left to obtain single colonies which were screened until a single clone (clone-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 in the in vitro diagnosis of specific conditions.

• ♦ ·, ·. As with most mammalian genes, mammalian TNF receptor receptors are thought to be encoded by multi-exogenous genes. Alternative mRNA constructs that can be considered to be the result of * · various post-transcriptional mRNA splicing events and which share common identity or identity regions with the cDNA sequences described 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 specific mammalian cDNA library 10864512 by cross-species hybridization.

Proteins and analogs

The present invention provides isolated recombinant mammalian TNF-R polypeptides. The 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 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-R according to the present invention includes, for example, TNF-R from 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-20 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 * I '·' of the primary protein. forms that 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 formation of 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.

35 Covalent derivatives are prepared by linking 108645 13 feminine functional groups to the TNF-R amino acid side chains or to the N or C terminus. Other TNF-R derivatives include covalent or aggressive conjugates of TNF-R or fragments thereof with other proteins or polypeptides (which are formed), for example, by synthesis in recombinant culture as N-major or C-major fusions. For example, the conjugated peptide may be a signal (or leader) polypeptide sequence in the N-terminal region of a protein that, during or after translation, directs the transfer of protein 10 from its site of synthesis to its site inside or outside the cell membrane or wall (e.g., yeast? ). TNF-R protein fusions may comprise peptides added to facilitate purification or recognition of TNF-R (e.g., poly-His). The TNF-15 receptor amino acid sequence can also be linked to the Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) peptide (Hopp et al., Bio / Technolqy 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 bovine mucosal enterokinase at the residue immediately following the Äsp-Lys pair. Fusion proteins blocked by this peptide can also resist intracellular degradation in L coli.

K '. TNF-R derivatives may also be used as a reagent ;;; wall in receptor-based immunoassays or as binding agents for affinity purification of TNF or other binding ligands. TNF-R derivatives may also be obtained using cross-linking agents such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide at cysteine and lysine residues.

»I

ti. Similarly, TNF-R proteins can be covalently bound via various active side groups to various non-soluble substrates, such as cyanobromide-activated, bisoxirane-activated, carbonyldiimidazole-activated or carboxyl-imidazole-activated or (with or without glial-5 rialdehyde cross-linking). Once bound to a substrate, TNF-R can be used to selectively bind anti-TNF-R antibodies or TNF (for assay or purification purposes).

The present invention also encompasses TNF-R, which comprises or does not comprise glycosylation of the associated native pattern. TNF-R expressed in yeast or mammalian expression systems, eg, 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 Ehc-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 20 site-directed mutation techniques. These analogue proteins can be produced in a homogeneous reduced carbohydrate form with good yield of yeast expression systems. N-j '·' of eukaryotic proteins. glycosylation centers are characterized by amino acid ·. *. A triplet Asn-Ai-Z, where Ai is any amino acid except Pro, and Z is Ser or Thr. In this sequence, aspartic acid provides a side chain amino group for carbohydrate co-attachment. Such a center can be eliminated by substituting another amino acid for Asn or 30 residues Z, deleting Asn or Z or inserting a bat between a non-Z amino acid Ai and Z, or an amino acid other than Asn Asn and Ai.

TNF-R derivatives may also be obtained by mutation. racket TNF-R or its subunits. In this sense, the TNF-R mutant is a polypeptide homologous to TNF-R, but differs in amino acid sequence from native TNF-R due to deletion, insertion or substitution.

Bioequivalent analogs of TNF-R proteins can be constructed, for example, by making various substitutions of residues or sequences, or by deleting end-to-end or internal residues or sequences that are not essential for biological activity. For example, cysteine residues may be deleted (e.g.

10 Cys178) or substituted 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 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 the deletion or insertion on biological activity should be considered. Substantially similar polypeptide sequences as defined above comprise

* I

I '. *. usually the same number of amino acid sequences, although · · *. ·, 25 C-terminal points for the construction of soluble TNF-Rs contain fewer amino acid sequences.

ΙΓ. Preferably, deletions and substitutions result in homologous or conservatively substituted sequences to maintain the biological activity of TNF-Rs, which means that a particular residue is replaced by a biologically similar residue. Examples of conservative substitutions include the placement of one aliphatic residue in place of another, such as Ile, Val, Leu or s.

Substitute for each other, or substitutions where one polar residue is replaced by another, such as with each other>

S i i I

! 108645 16

Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example 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 residues or sequences located at 10 ends or internal. 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 15 is called a soluble TNF-R molecule that retains its 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 20 transmembrane regions. Additional amino acids can be deleted from the transmembrane region while maintaining TNF binding activity. For example, hu-TNF-R-delta-183 comprising the sequence of amino acids 1-183 in Figure 2 and TNF-R-delta-163 comprising the sequence of amino acids 1-163 of Figure 2 , has the ability to bind TNF ligand, '· *. * as 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 suggests that either or both Cys157 and Cys163 residues are essential for the formation of an intramolecular disulfide bridge for the proper folding of TNF-R. Cys, which was deleted. ···, without any apparent negative 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 35 at the C-terminus relative to Cys163 could be expected to result in biologically active soluble 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 of 5 amino acids after Cys163. Other C major 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 oligo-10 nucleotide linkers. The resulting soluble TNF-R constructs 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 constructs are also contemplated herein. within the scope of the invention.

Naturally, the mutation nucleotide sequences constructed to express analog TNF-R must retain the reading sequence step of the code sequences, and preferably do not form complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hair eyes. . effect on receptor mRNA reading. Although the mutation site; can be predetermined, it is not necessary that the nature of the mutation itself is predetermined. For example, the optimization of?. *. * Mutants at a particular site? Aa can be subjected to random mutation of the target codon and followed by the expression of the TNF-R mutants for the desired activity.

Not all mutations in the nucleotide sequence encoding TNF-R are expressed in the final product, for example, ···, nucleotide substitutions may be made to enhance expression, primarily to avoid secondary structural loops in the copied mRNA (see EP-A- * "" 35 75 444A, which is incorporated herein by reference), or codon 18 108645 to obtain those which are easier to read by the selected host, e.g.

Mutations can be made at specific sites 5 by synthesizing oligonucleotides containing a 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 with the desired amino acid * 10 series, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutation procedures may 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 modifications are disclosed by Walder et al. (Gene 42: 133 (1986)); Bauer et al. (Gene 37: 73 (1985)); Craik (BioTechnologies 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 disclose suitable techniques and are incorporated herein by reference.

,. Both monovalent forms and polyvalent forms of TNF-R are useful in the compositions and methods of this invention. Multivalent forms of · · have multiple TNF-R binding sites for TNF ligand.

For example: bivalent soluble TNF-R may: consist of two consecutive repeats of amino acids 1 through 235 in Figure 2 separated by a fusion sequence region. Alternative polyvalent forms of step-30 can also be constructed, for example, by chemical attachment of TNF-R to any clinically acceptable carrier molecule, a polymer selected from the group consisting of Ficoll, polyethylene glycol or dextran, using conventional 'F Alternatively, TNF-R may be chemically coupled to biotin, after which the biotin-TNF-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 5 (DNP) or trinitrophenol (TNP) and the resulting conjugate purchased with anti-DNP or anti-TNP-IgM to form decameric conjugates having a TNF-R there are 10 in the case of healing centers.

Similarly, a recombinant chimeric antibody-10 antibody molecule 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 15 genes: TNF-R / human-kappa-light chain-chimera (TNF-R / C kappa) and TNF-R / human-gamma-heavy chain ju-chimeric (TNF). -R / Cgamma-i) · After duplication and reading of two chimeric genes, the gene products assemble into a single chimeric antibody molecule in which TNF-R is present at a valence of two to 20. Such multivalent forms of TNF-R may have enhanced binding affinity for TNF-ligand. For more details regarding such ki-. . construction of meric antibody molecules disclosed; WO 89/09622 and EP 315 062.

Recombinant TNF-R Expression Recombinant expression vectors can be used to amplify or express the DNA encoding TNF-R. Recombinant DNA expression vectors are replicable DNA constructs containing synthetic or cDNA-30-derived DNA fragments encoding mammalian TNF-R or bioequivalent analogues that are operatively linked. linked to appropriate transcriptional or translational regulatory units derived from mammalian, microbial, '* "· viral or insect genes. The transcriptional unit typically comprises a combination of (1) one or more genetic» · · »I t »108645 20 unit having a regulatory role in gene expression, for example, copy promoters or enhancers, (2) a structural or coding sequence that is copied to mRNA and read as a protein, and (3) appropriate copy-initiation and termination sequences as described in more detail below, such control units may comprise an operator sequence for controlling duplication which encodes suitable sequences mRNA ribosome binding centers The ability to replicate in a host, usually provided with the origin of replication, and a 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 (secretion leader sequence) is operably linked to the polypeptide 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 so that it is readable. Usually operatively linked means continuous, and for secretory lead sequences. . continuous and within the reading range. The structural units for use in yeast expression systems' '' preferably include a leader sequence that allows the host cell to secrete a read protein. Alternatively, when expressed, the recombinant protein without the leader or transport sequence may comprise: n-päämetioniinitähteen.

This residue may subsequently be cleaved from the lost * * 30 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,

I · I »I

108645 21 premature termination of transcription may also be desirable, for example, when it would lead to mutants with advantageous C-terminal inserts, such as deletion of the trans membrane region to obtain a soluble receptor that is not bound to the cell membrane. Due to the degenerate character of the code, considerable 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 culture of a suitable host microorganism, for example, from a bacterium such as H1 -Col or a yeast such as S-Cerevisiae which has been stably integrated ( as a result of transformation or transfection) into the chromosomal DNA of the recombinant copy unit or comprising the plasmid as an integral part of the plasmid present. Usually sys-. . the cells forming the theme are one strain transformant; *; offspring. The recombinant DNA expression themes 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, • S ·· ', but host cells transformed for TNF-R DNA cloning or amplification purposes do not need to express' ·' · TNF-R. The expressed TNF-R is either stored in the cell membrane or secreted into the culture supernatant, depending on the medium.

1 8 8 6 4 S

TNF-R DNA. Suitable host cells for expression of mammalian TNF-R are prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative 5 or gram-positive organisms, for example EL_coli 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 10 RNA sequences derived from the DNA constructs described above. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are described by Pouwels et al. ("Cloning Vectors: A Laboratory Manual", Elsevier, New York, 1985), 15 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 a host identifier. . the confirmation of this replication origin in the host • · • · ·; tamiseksi. Suitable prokaryotic hosts for transformation include: E. coli, Bacillus subtilis. Salmonella tvphimu- • * ·. *. * Rium and various species of the genus Pseudomonas, Streptomyces and • <· ** ,,, · Staphylococcus, although optional may be used. other.

• t I

Useful expression vectors for bacterial use 30 may comprise a selection marker and a bacterial origin of replication obtained from commercially available ··· plasmids comprising gene units from the well known cloning vector pBR322 (ATCC 37017).

Such commercial vectors include, for example, pKK223-3 'F *: 35 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Pro- *).

»I

108645 23 mega Biotec, Madison, WI, USA). These pBR322 "backbone" portions are linked to the appropriate promoter and the expressed structural sequence. JL-coli is typically transformed using pBR322 derivatives obtained from plasmid 5 from EL-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 β-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275 (1978) 615; and Goeddel et al., 1979, Nature 281: 544). , the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8: 4057 (1980); 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 are plasmid pHUB2 present in EL_coli. . strain JMB9 (ATCC 37092), and pPLc28 present in f EL coli RR1 (ATCC 53082).

The recombinant TNF-R proteins can also be expressed in yeast hosts, preferably Saccharomyces, such as S. cerevisiae. Other yeasts 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 a replication origin and a selection mark that allow transformation into both yeast and EL_coli, e.g., amp% t * l t ft I)

I t I

a t *

I I

24 of the E86 colin of the silicin resistance gene and the cerevisiae TRP1 or URA3 gene, which provides a selectable marker for a downstream structural sequence copy of a yeast mutant strain lacking the ability to grow with tryptophan and 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.Biol.Chem. 255 (1980) 2073) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 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 j1 * DNA sequences from pUC18 for selection and replication: · * 25 in E1 coli (Ampr gene and origin of replication) and yeast DNA sequences including the glucose-repressing ADH2 promoter and α-factor secretion. ADH2 promoter; ; the market has been described by Russell et al. (J. Biol. Chem. 258: 2674 (1982)) and Beier et al. (Nature 300 1982 724). The yeast 30 α-factor leader sequence that directs secretion of heterologous proteins can be inserted between the promoter and the structural gene to be expressed. See, e.g., Kurjan et al., Cell 30 (1982) 933; and Bitter et al., Proc.Natl. -

Acad. USA 81: 5330 (1984)). The leader sequence may be mo-*: * ': 35 diffused to include, near its 3'-end, one or more useful restriction sites to facilitate fusion of the leader sequence 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.Sei. USA 75, 1929 (1978), according to which 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 growth medium 10 containing 0.67% YNB using amino acids and bases as described by Sherman et al., "Cold Spring Harbor Laboratory, Manual of Methods in Yeast Genetics," Cold Spring Harbor, New York , 1986.

Host strains transformed with vectors comprising the ADH2-15 promoter 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 substrate glucose. The crude 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 COS-7 cell lines of &gt; 30 monkey kidney cells described by Gluz, ,,,,, man (Cell 23 (1981) 175) and other cell lines capable of expressing the appropriate vector, including, for example, L cells, C127, 3T3, Chinese Hams terrier ovary (CHO), HeLa and BHK cell lines. Mammalian-35 expression vectors may comprise untranslated units I »» I i) I I kuten kuten kuten8686868686 kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten86 kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten kuten8686868686 kuten 3'-sequences, and unread 5'- or 3'-sequences such as essential ribosome binding-5 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 which 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 · 250) sequence is included which extends • ·

From Hind3 to Bqll, located at the replication origin of the virus '··'. Further, the promoter, control and / or signal sequences of the mammalian genomic TNF-R- ... 'may be used, provided that such control sequences are compatible with the selected host cell. For more details on using a mammalian high expression vector. '··. the production of recombinant mammalian TNF receptor is shown in Examples 2 and 7 below. Exemplary vectors can be constructed as described by Okayama and Berg 108645 27.

(Mol.Cell.Biol. 3 (280) (1983)).

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

Preferably, recombinant DNA expression vectors comprising the TNF-R cDNA sequences are stably integrated into the host cell DNA. Increased amounts of expression product 10 are obtained by selection of cell lines containing amplified vector DNA. Cell lines containing amplified 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 sequence encoding the desired protein. Transformed or co-transformed host cells are then cultured at increasing concentrations of the known drug, which is selected for drug resistant cells. Such drug - resistant cells survive as concentrations of the toxic drug increase by producing run - *. saved the enzyme the drug inhibited, often as a result of the amplification of the gene encoding the enzyme. When ! drug resistance has been caused by growth in the copy number of the vector DNA encoding the enzyme which is inhibiting, while co-amplification of the vector DNA encoding the desired protein (TNF-R) in the DNA of the host cell.

In a preferred system, dihydrofolate reductase (DHFR) ·; ··· is used for such co-amplification. a gene that can be inhibited by the drug methotrexate (MTX). To achieve co-amplification, a host cell which lacks an active DHFR-encoding gene is either transformed with a vector comprising the DHFR and the desired DNA sequence encoding the desired protein 108645 28 or co-transformed with the vector. comprising a DNA sequence encoding DHFR and a vector comprising a DNA sequence encoding a desired protein. Transformed or co-transformed host cells are cultured in medium containing increasing levels of MTX and surviving cell lines are selected.

A particularly preferred co-amplification system utilizes the glutamine synthetase (GS) gene responsible for the synthesis of glutamate and ammonia using hydrolysis of ATP to ADP and phosphate to maintain the reaction. GS 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 a co-transformed vector comprising a DNA sequence encoding GS and a vector comprising a DNA sequence encoding the desired protein, followed by culturing the host cells in a medium containing increasing levels of MSX and selecting for surviving cells. The GS co-amplification system, appropriate recombinant expression vectors, and cell lines are described. in the following PCT patent applications: WO 87/04462, 25 WO 89/0136, WO 89/10404 and WO 86/05807.

\! The recombinant proteins are preferably expressed; ; by co-amplifying DHFR or GS in a mammalian host cell, such as Chinese hamster ovary (CHO) cells, or ... ', alternatively in a mouse myeloma cell line such as * * · 30 SP2 / 0Agl4 or a NSO or a rat myeloma cell line such as YB2 / 3.0-Ag20, which are disclosed in PCT Patent Application Publication No. · · ·; WO 89/10404 and WO 86/05807.

A preferred eukaryotic vector for expression of TNF-R DNA is disclosed in Example 2 below. This vector, called pCAV / NOT, was obtained in mammalian high.

• I I

I · 108645 29 'from 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 appropriate host / vector systems for expression of recombinant DNA reading products from the DNA sequences described above and then purifying them from the culture medium or cell extract.

For example, supernatants from systems that secrete recombinant protein into culture media may first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore ultrafiltration unit. After the concentration step 15, the concentrate can be charged 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. An alternative-V. a cation exchange step may be used. Suitable cation exchangers include various non-soluble matrices, which contain sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.

Finally, one or more reverse phase liquid chromatography (RP-HPLC) steps can be used to purify the TNF-R composition to <1 · · ... 1 using hydrophobic RP-HPLC. substrate, e.g., silica gel with ·; ··· dependent methyl groups or other aliphatic groups.

. ··. Some or all of the preceding purification steps in various combinations may also be used to obtain a homogeneous recombinant protein.

(MM

· · • »» ψ 1 108645 30

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. The 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 15 recombinant DNA from fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J.Chromatoq. 296: 171 (1984)). This publication describes two sequential reverse 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 cellular components, including proteins, in amounts and types, ·. ·. which depend on the purification methods used to recover human TNF-R. These components! are usually derived from yeast prokaryotes or *; // higher non-human eukaryotes, and are preferably present in harmless contaminating amounts of less than about 1% by weight. Furthermore, recombinant cell culture makes it possible to produce TNF-R which does not contain proteins normally associated with TNF ·; ·· R as it occurs naturally in its native species, eg, in cells, in cellular effluents. or food, what in liquids.

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

EXAMPLES Example 1 Binding Assays 5 A. Radiolabeling of TNF-α and TNF-B. Recombinant human TNF-α was expressed in the form of a fusion protein containing the hydrophilic octapeptide at the N-terminus as a yeast secreted protein and purified by affinity chromatography (Hopp et al., Bio / Technoloqy 6 10 (1988) 1204). Purified recombinant human TNF-β was purchased from R&D Systems (Minneapolis, MN). Both 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 15 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 m ) Na125I. This solution was then transferred to another glass tube containing 5 µg of TNF-α (or TNF-5) in 45 PBS for 20 minutes at 4 ° C. The reaction mixture was fractionated by gel filtration through 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). , 2% (w / v) sodium azide b 25 and 20 mM Hepes, pH 7.4 (binding medium). The final pool of 125I-TNF was diluted in binding medium to a working stock solution of 1 x 10 7 M and stored for one month at 4 ° C without any detectable loss of receptor binding activity. The specific activity is usually 1 x 10 6 scintillation per minute. mmol of TNF.

•; ··; B. Commitment to intact cells. Binding assays on intact cells were performed by two methods. by. In the first method, cells were first grown either by slurry (e.g. U937) or by attachment to tissue culture plates (eg WI26-VA4, COS cells expressing 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., J. Immunol. 132: 751 (1984)), essentially as described by Park et al. (J. Biol. Chem. 261 (198-6) 4-177). 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 migration of I-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 125 I-TNF by the plate binding assay described by Sims et al. (Science 241: 585 (1988)).

C. Binding Assays in Solid Phase. The means for detecting TNF-R was obtained by the ability of TNF-R to adsorb stably to nitrocellulose from detergent-20 extracts of human cells, while retaining TNF-binding activity. Cell extracts were prepared by mixing the cell pellet with 2x volume PBS containing 1% Triton X-100 and mixing with protease. inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 25 μΜ pepstatin, 10 μΜ leupeptin, 2 mM o-phenanthroline and 1 2 mM EGTA), vigorously vortexing. The mixture was incubated on ice for 30 minutes then centrifuged at 12,000 x g for 15 minutes at 8 ° C to remove dark matter and other debris. Two microliters aliquots of cell extracts were placed on dry BA85 / 21 nitrocellulose membranes (Schleicher & Schuell, Keene, ·; ···; NH) and allowed to dry. Membranes were incubated in tissue culture plates for 30 minutes in Tris (0.05 M) buffered saline (0.15 M) pH 7.5 containing 3% 35 (w / v) BSA, non- latches for specific binding centers- 4 I »I» • I * • t »» · · »»

I »I

• »» t · 108645 33 women. The membrane was then covered with a mixture of 5 x 10 11 M 125 I TNF in PBS containing 3% BSA and incubated for 2 hours at 4 ° C. After this time, the membranes were washed 3 times with PBS, dried and placed on 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 COS-7 cells Different human cell lines were screened for TNF-R expression by their ability to bind I-labeled 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 I-TNF binding, comprising approximately 4,000 high affinity centers (Ka = 1 x 10 10 M -1) and 15,000 low affinity centers (Ka = 1 x 10 8 M 1) cells. for.

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

• t ·

Cells were harvested by lysis of the cells in guan * * * * nidine hydrochloride solution and total RNA was isolated as previously described (March et al., Nature 315: 30641 (1985)).

Poly A + RNA was isolated from oligo-dT-cellulose chroma *; : topographically and 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 a '35 RNA cDNA hybrid using the reverse transcriptase enzyme and t I · t · I * * 34 108645 oligo-dT as a 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 fusion sequences (which have internal Not1 centers) that have been phosphorylated at one end only (Invitrogen) are added to 5 flat-ended cDNAs. 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 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 the phage JL_ coli strain c600 (hfl ') on a bacterial mat.

The phage DNA was purified from the resulting lambda-gt10 cDNA library and the cDNA inserts were cleaved by digestion with res - *. Done cleavage! ! After electrophoresis on an agarose gel, cDNA fragments larger than 2,000 bp were isolated.

The resulting cDNA fragments were ligated into the eukaryotic '<·· * expression vector, pCAV / NOT, designed to express the cDNA sequence inserted into its multicloning center, transfected into mammalian cells. PCAV / NOT was assembled in pDC201: (pMLSV derivative of Cosman et al., 1984, Nature 312 (768) 768), SV40 and cytomegalovirus DNA, and comprises, in sequence, from the origin of replication: (1) 35 SV40 sequences with coordinates 5171-270 including rep- »

• I

I * 1086 ^ 5 35 lysation primers, enhancer sequences, and early and late night promoters; (2) cytomegalovirus sequences including promoter and enhancer regions (nucleotides 671 to +63 of the sequence disclosed by Boechart et al.

5 (Cell 41: 521 (1985)); (3) adenovirus-2 sequences containing a first exon and part of an intron between a first and second exon of a three-part leader, a second exon and a third of a three-part leader sequence and a multicloning invention 10 (MCS) containing XhoI, KpnI,

Smal: lie, Notl: lie and Bgll: lie; (4) SV40 sequences coordinates 4127-4100 and 2770-2533, including polyadenylation and termination signals for early copying; (5) pBR322 derived sequences and VAI and VAII sequences from pDC201, wherein adenovirus sequences 10532-11156 include VAI and VAII genes followed by pBR322 sequences from 4363-2486 and 1094-375 which contain the ampicillin resistance gene and the origin of replication.

The resulting WI-26 VA4 cDNA library in pCAV / NOT transformed the L coli strain DH5, and then plated the recombinant DNA products to about 800 colonies per plate, allowing sufficient use of the plates, in order to obtain a total of about 50,000 colonies »» »! !. 25 per litter. Colonies were scraped from each plate; were pooled and plasmid DNA was prepared from each pool. The pooled DNA was then transfected with an untranslated layer of monkey COS-7 cells using DEAE-dextran and then chloroquine treatment as described in Luthman et al. (Nucl. Acids Res. 11 (1295) (1983)) and McCutchan et al. (J.Natl.Cancer Inst. 41 (1986) •; .. j 351). Cells were then grown in culture for 3 days. obtaining a transient expression of the inserted sequences. After three days, the cell culture supernatants were discarded and the TNF binding of the cell layers was determined from each plate 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 once 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 that was contacted with X-ray gene film for 72 hours at -70 ° C using an amplification filter. TNF-binding activity was visible on exposed films as a dark spot against a relatively even background.

After screening about 240,000 recombinant DNA products from booklet 15 in this manner, one transfectant pool was found to provide TNF binding sites which were clearly visible against the background exposure.

Plates of about 150 colonies were then prepared using a frozen stock of positive pool bacteria. Copies of these plates were made on nitrocellulose filters, then the plates were scraped and plasmid DNA transfected as described above for positive plate recognition V. order. Bacteria from single colonies of this dish I »·!. 1 25 copies of nitrocellulose were grown in 0.2 ml cultures, *. ! which were used to obtain plasmid DNA which was transfected into COS-7 cells as described above. In this manner, one clone, clone-1, capable of inducing the expression of human TNF-R on COS cells, was isolated. Illustrations »·

The 30 nection vector pCAV / NOT containing TNF-R DNA clone-1 has been deposited with the American Type Culture, ··· Collction, 12301 Parklawn Drive, Rockville, MD 20852, USA

(ref. 68088) as pCAV / NOT-TNF-R.

* * ♦ 35> i »I I * I»> »t I I I · * • ·» 108645 37

Example 3

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

The cDNA 5 encoding soluble hu-TNF-R-delta-235 (having the sequence amino acids 1 to 235 in Figure 2) was constructed by cleaving 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 site and Pvu2 cuts 20 nucleotides 5 'of the transmembrane region of the TNF-R coding region. TNF-R sequences of the 3'-terminal was synthesized uudelleenrakentami into two oligonucleotides which are soldered in order to obtain the next oligonukleotidiliittosekvenssin: 15 Bam HI Pvu two Bgl2 CTGÄAGGGAGCACTGGCGACTAAGGATCCA GACTTCCCTCGTGACCGCTGATTCCTAGGTCTAG AlaGluGlySerThrGlyAspLoppu this oligonukleotidiliittosekvenssissä 20 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, for isolation of soluble TNF-R as a whole by II · 25 NotI / BamHI digestion), this oligonucleotide was then lysed ·, then 840 bp NotI / With pvu2-TNF-R insert into Bgl2 / ~ '' 'l * Notl-cut pCAV / NOT to give psolho-... * TNF-R-delta-235 / CAVNOT transfected with COS-7 cells as described above. This expression vector induced the expression of? 30 * soluble human TNF-R capable of binding TNF.

* t I «i # I» I I t t · t

Iitti »l I * * | | · »* • ·» »» I I $ »108645 38

Example 4

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

The cDNA 5 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 pCAV / NOT-TNF-R from the multicloning site and Bgl2 cuts the TNF-R coding region at nucleotide 637, which is 237 10 nucleotides 5 'from the transmembrane region. The following oligonucleotide fusion sequences were synthesized:

Bgl2 5'-GATCTGTAACGTGGTGGCCATCCCTGGGÄATGCAAGCATGGÄTGC-3 '

15 ACATTGCACCACCGGTAGGGACCCTTACGTTCG

IleCysAsnValValAlalleProGlyAsnAlaSerMetAspAla

Notl 5'- AGTCTGCACGTCCACGTCCCCCACCCGGTGAGC -3 '

2 0 TACCTACGTCAGACGTGCAGGTGCAGGGGGTGGGCCACTCGCCGG

ValCysThrSerThrSerProThrArg End · * The above oligonucleotide fusion sequences construct I '. · Re-construct the 3' end of the receptor molecule to nucleotide • 25708, followed by the decision codon (underlined). These oligonucleotides were then ligated with the 640 bp Notl-TNF-R insert into Notl-cut pCAV / NOT to obtain • · ·. · *. an expression vector psol-TNF-R-delta-185 / CAVNOT was transfected into C0S-7 cells as described above. Here- 30 expression vector induced the expression of soluble human TNF-R capable of binding TNF.

• * ·; · 'Example 5

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

35 cDNA encoding soluble hu-TNF-R-delta-163

; 108645 39 was constructed by cutting the 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: 5

Bgl2 Notl 5'GATCTGTTGAGC -3 '

ACAACTCGCCGG

IleCys End 10 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 15 bp Not1-TNF-R insert into Not1-cut pCAV / -NOT- to give the expression vector psol-TNF-R-del-163 / CAVNOT transfected with COS- 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.

Example 6

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

• cDNA encoding soluble hu-TNF-R-delta-142

.Y: 25 (having the sequence of amino acids 1-142 in Figure 2) was constructed by cleaving the 550 bp fragment of pCAV / NOT-TNF-R with the restriction enzymes NotI and AlwN1. AlwN1 cleaves, * ··, the TNF-R coding region at nucleotide 549. The following oligonucleotide fusion sequence was synthesized: 40 108645

Bgl2 Not1

5'-CTGAAACATCAGACGTGGTGTGCAAGCCCTGTTAAA-3 'CTTGACTTTGTAGTCTGCACCACACGTTCGGGACAATTTCTAGA

End 5 This previous oligonucleotide fusion sequence rebuilds the 3 'end of the receptor molecule to nucleotide 579 (amino acid 142), followed by a decision codon (underlined). This oligonucleotide was then ligated with the 10,550 bp Notl / AlwN1-TNF-R insert into Notl / Bgl2-cut pCAV / NOT to obtain 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, which would be capable of binding TNF. It is believed that this particular structure was unable to express biologically active TNF-R because one or more of the essential cysteine residues (e.g., Cys157 or Cys163) necessary for intracellular binding (to form the correct tertiary structure of TNF-R) were eliminated. .

Example 7

Expression of soluble TNF receptors in CHO cells • ·: ** Soluble TNF receptor was expressed in Chinese; V hamster ovary (CHO) cells using the glutamine synthetase (GS) gene amplification system, essentially as described in PCT Patent Application Nos. WO 87/04462 and WO 89/01036. Briefly, CHO cells are transfected with II. · *. gene vector containing genes for both TNF-R and «· GS. CHO cells are selected for GS gene expression. The ability of 30 transfected DNA to confer resistance to low levels of methionine sulfoximine (MSX). GS- »* ··· * sequence amplification events in such cells selectively: ·; using higher concentrations of MSX. In this way; ·· continuous TNF-R sequences 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 WO 87/04462 and WO 89/01036, and which is the receiver-5 tavana from Celltech, Ltd., Berkshire, UK) was cut with the restriction enzyme BamHI and dephosphorylated with calf intestinal alkaline phosphatase (CIAP) to prevent internal re-ligation of the vector. The BamHI-cleaved pSVLGS.1 fragment was then ligated to the 2.4 kb BamHI-Bgl2 fragment of pEE6hCMV (described in PCT Patent Application Publication No. WO 89/01036 and also available from Celltech), which was cleaved Bgl2, BamHI and Fsp1 to avoid the formation of two fragments of the same size, resulting in a 15 11.2 kb vector, designated p6 / PSVLGS.1.

pSVLGS.l contains the glutamine synthetase selection marker gene under the control of the SV40 late promoter. The BamHI-Bq12 fragment from pEE6hCMV contains the major human cytomegalovirus immediate early promoter (hCMV), a poly-linker, and the SV40 early polyadenylation signal.

Soluble TNF-R coding sequences were inserted into p6 / -PSVLGS.l by cleaving the NotI-BamHI fragment from expression vector: psol-TNF-R / CAVNOT (prepared according to the above · #; Example 3), aligning the ends. Klenow and ligation with SmaI-dephosphorylated p6 / PS-VLGS.1, whereby the sol-TNF-R code sequences were inserted »» *. * ·. hCMV promoter control. This resulted in a single, * ··. a plasmid vector in which the SV40 / GS and hCMB / sol-TNF-R copy units are copied in opposite directions. This vector was designated psol-TNF-R / P6 / PSVLGS.

CHO-K1-> ·· * cells (available from ATCC Depository, Rockville, MD, ·: ··· with accession number CCL 61) were transfected with psol-TNF-R / P6 / PSVLGS. The CHO-K1 cell monolayer was grown to almost coexist with minimum requirement, ·. In 35 media (MEM), 10X (Gibco: 330-1581AJ), which did not contain 108645 42 glutamine free and supplemented with 10% dialyzed bovine serum (Gibco: 220-6300AJ), 1 mM sodium pyruvate (Sigma) ), MEM non-essential amino acids (Gibco: 320-1140AG), 5,500 μΜ asparagine and glutamate (Sigma) and nucleosides (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 10 phosphate constant precipitation essentially as described by Graham and van der Eb, Virology 52 (45) (1983). Cells were subjected to glycerol shock (15% glycerol in serum-free medium for about 1.5 minutes) approximately 4 hours after transfection as described essentially by Frost and Williams, Virology 91, (1978) 39, followed by serum-free washing. growth platform. One day later, 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 together in a selective medium. Processed; medium containing monolayer cells. From 25 points, soluble TNF-R activity was then determined using the binding assay described in Example 1 above. Assays showed that the colonies expressed soluble TNF-R biologically active.

To select for GS gene amplification, several MSX-resistant cell lines are transfected with psol-TNF-R / P6 / PSVLGS and grown at various concentrations of MSX.

··· * Approximately 1 x 106 cells from each cell line are plated at high concentrations of 100 μΜ, 250 μΜ, 500 μΜ and 1 mM MSX and incubated for 10-14 days. After 12 days at-35, colonies resistant to higher levels of MSX 108645 43 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 generated by a number of independent replication events. One or more of the most resistant cell lines are isolated from these 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 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 20 DNA fragment encoding the TNF receptor and suitable for cloning into the yeast expression vector pIXY120 was first generated by a polymerase chain reaction (PCR) amplification of the .1.11 extracellular portion of the full length receptor pCAV / NOT-TNF-R (ATCC 680). In this PCR amplification. The following primers were used: • »·» · • 1 • · ♦ »M • ·> · I ii 141 ·· • · * I ·» »t · ·» »· 108645 44 5'-main primer: 5'-TTCCGGTACCTTTGGATAAAAGAGACTACAAGGAC Asp718-> ProLeuAspLysArgAspTyrLysAsp 5 GACGATGACAAGTTGCCCGCCCAGGTGGCATTTACA-3 '

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

Bam> End <----------- TNF-R ------>

The 5 'major oligonucleotide primer used for amplification contained 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 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 / Technoloqy 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-coding strand of DNA code sequences that terminate the open reading region of the 25 receptor after nucleotide 704 (after the Asp residue preceding the trans membrane region). by importing the TAA termination codon (underlined. ··, tu). The stop codon is then followed by the BamHI restriction center.

The DNA sequences encoding TNF-R are then amplified. 30 PCR, essentially as described by Innis et al., Ed., "PCR Protocols: A Guide to Methods and Applicants," Academic Press, 1990.

·: **: The DNA fragment ti obtained by PCR encoding soluble human TNF-R was subcloned into the yeast expression vector 35 pIXY120 by digestion with BamHI and 108645 by PCR.

Asp718 restriction enzymes, digestion of pIXY120 with Bam-HI 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 JL_ 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? -Factor leader sequence, the FlagR-soluble TNF receptor and the stop codon. This fragment was ligated in vitro with EcoRI and BamHI cut plasmid pYEP352 (Hill et al., Yeast 2 (19869 163)) to give expression plasmid pIXY432, which was transformed into Eh1 coli strain RR1.

Soluble human TNF receptor secretion in yeast. For determination, pIXY424 was purified and introduced into the diploid S. cerevisiae yeast strain (XV2181) by electroporation and selection for plasmid-containing yeast * * ·, * ·, TRP1 + gene in medium lacking tryptophan. pIXY432-driven receptor secretion. To determine 30, the plasmid was introduced into the yeast strain PB149-6b by electroporation, followed by selection for the ··· * URA3 + gene containing plasmid by growth medium lacking uracil. Overnight cultures were grown at 30 ° C in an appropriate selective medium. PB149-6b / pIXY434 Transformants 108645 46 L were diluted 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 η 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 further horseradish peroxidase-conjugated anti-mouse IgG antibody. dilutions 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 receptors were secreted in both expression systems. Comparisons showed that the pIXY432 system secreted about 8-16 times more V soluble human TNF receptor than the pIXY424 system.

The supernatants were assayed for solubility of soluble TNF-R as described in Example 1 by their ability to bind 125 I-TNF-α and to block TNF-α binding. pIXY432-

• I I

supernatants were found to contain significant levels • * ·. ···. active soluble TNF-R.

Example 9. 30 Isolation of murine TNF-R cDNA sequences fin · I ·

Mouse TNF-R cDNA sequences were isolated from a cDNA library constructed from the mouse 7B9 cell line, ·; ··: an antigen-dependent helper T cell line; 6 mice by cross-species hydration to a human TNF-R probe. The cDNA library was constructed with lambda * * 108645 47 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 showed that the coding sequence for hii-15 ri-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 from purified recombinant TNF-R, e.g. human TNF-R, or transfected; V COS cells expressing high levels of TNF-R visit • #. #! T! 25 to form monoclonal antibodies against TNF-R using conventional techniques, for example those described in U.S. Patent No. 4,411,993. Such antibodies are likely to be useful as part of diagnostic assays for TNF or soluble TNF receptor.

To immunize mice, TNF-R immunogen is emulsified in complete Freund's booster and injected subcutaneously in an amount of 10-100 pg into Balb / c mice. After 10 to 12 days, immunized animals are boosted with 35 additional immunogens emulsified in incomplete Freund's I <108645 48 followed by intermittent boost according to a 1-2 week immunization program. Occasionally, serum samples are taken by counting blood from behind the eye or by cutting the tail end for analysis by blot-5 spot (antibody-sandwich) or ELISA (enzyme-linked immunosorbent assay). Other assay procedures are appropriate. Once an appropriate antibody titer 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. The hybridoma cell lines generated by this procedure are 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 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 the '1f !, syngeneic Balb / c mice. intraperitoneal to produce an intraperitoneal fluid containing high concentrations (&gt; 1 mg / ml) of an anti-TNF-R monoclonal antibody. The resulting monoclonal antibody can then be purified with ammonium sulfate by precipitation, ···, racket and then subjected to gel exclusion chromatography 1 and / or affinity chromatography on the antibody. 30 binding to protein A: from Staphylococcus aureus.

UM i «I <1 t · *» 1 1 »·

Claims (7)

108645 A purified biologically active mammalian TNF receptor (TNF-R) protein comprising an amino acid sequence selected from amino acids 1 to x of Figure 2, wherein x is selected from amino acids 163 to 235. TNF-R protein according to claim 1, characterized in that it is a soluble human TNF-R protein comprising the amino acid sequence 1 to 10 235 of Figure 2. The TNF-R protein of 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. TNF-R protein according to claim 1, characterized in that it is a soluble human TNF-R protein comprising the amino acid sequence 1-185 of Figure 2. The TNF-R protein of claim 1, characterized in that it is a soluble human TNF-R protein comprising the amino acid sequence 1-163 of Figure 2. t An antibody which specifically binds a biologically active, V; 25 with its mammalian TNF-R protein. A method for detecting TNF or TNF-R molecules or their interactions, characterized in that a composition comprising a TNF-R protein according to any one of claims 1 to 5 is used. • i »» f »t I I I» • '* * • | I »t» I I »account» i> »t I>« * I I »» 108645