AU1757500A - TNF-alpha converting enzyme - Google Patents

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AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority

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Sc Related Art: Name of Applicant: Immunex Corporation Actual Inventor(s): ROY A BLACK, CHARLES RAUCH, CARL J MARCH, DOUGLAS P CERRETTI Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: TNF-alpha CONVERTING ENZYME Our Ref 609459 POF Code: 44735/44735 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 6006q Most, but not all, proteases recognise a specific amino acid sequence.

Some proteases primarily recognise residues located N-terminal of the cleaved bond, some recognise residues located C-terminal of the cleaved bond, and some proteases recognise residues on both sides of the cleaved bond.

Metalloprotease enzymes utilise a bound metal ion, generally Zn 2 to catalyse the hydrolysis of the peptide bond. Metalloproteases are implicated in joint destruction (the matrix metalloproteases), blood pressure regulation (angiotensin converting enzyme), and regulation of peptide-hormone levels (neutral endopeptidase-24.11).

SUMMARY OF THE INVENTION In one aspect the present invention provides an isolated and purified TACE polypeptide.

In particular the present invention provides an isolated and purified polypeptide comprising the sequence of amino acids 215 to 477 of SEQ ID 15 NO:2 and an isolated and purified polypeptide selected from the group consisting of a polypeptide comprising amino acids 18-Xaa of SEQ ID NO:2 wherein Xaa is an amino acid selected from the group consisting of amino acids 477 through 824.

The present invention also provides an isolated and purified antibody that 20 binds to the above polypeptides.

In a further aspect the present invention provides a method for detecting the TACE inhibiting activities of a molecule comprising mixing said molecule with a substrate, incubating a polypeptide as described above with the mixture and chromatographically determining the extent of substrate cleavage.

In yet a further aspect the invention provides a method of using a polypeptide as described above in a structure-based design of an inhibitor of said polypeptide, comprising the steps of determining the three-dimensional structure of such polypeptide, analysing the three-dimensional structure for the likely binding sites of substrates, synthesising a molecule that incorporates a predictive reactive site, and determining the polypeptide-inhibiting activity of the molecule.

k1 C %WINVWORfl',L. ODPYEN ET53781 DOC In yet a further aspect still the present invention provides a method for detecting the TNF-cleaving ability of a molecule, comprising incubating said molecule with a substrate that comprises the amino acid sequence Leu-Ala-GIn- Ala-Val-Arg-Ser-Ser, and determining the extent of substrate cleavage.

In an even further aspect the invention provides an isolated nucleic acid selected from the group consisting of: the coding region of a native mammalian TACE gene; cDNA comprising neucleotides 52-2472 of SEQ ID NO:1; nucleic acid that is at least 80% identical to the nucleic acid of (a) or and that encodes a polypeptide that converts TNF-a from the 26 kD form to the 17 kD form; and nucleic acid which is degenerate as a result of the genetic code to a nucleic acid defined in or and which encodes biologically active TACE.

15 In a further aspect the present invention provides a method of inhibiting the cleavage of TNF-ca from cell membranes in a mammal comprising administering to such mammal an effective amount of a compound that inhibits the TNF-a proteolytic activity of an enzyme comprising the sequence of amino acids 18-671 of SEQ ID NO:2.

20 In an even further aspect the present invention provides a method of inhibiting TNF-a cleavage from cell membranes comprising blocking the binding of TNF-x with an enzyme having the sequence of amino acids 18-671 of SEQ ID NO:2.

In a still further aspect the present invention provides a method for treating a mammal having a disease characterised by an overproduction or an upregulated production of TNF-a, comprising administering to the mammal a composition comprising an amount of a compound that effectively inhibits the TNF-a proteolytic activity of an enzyme comprising the sequence of amino acids 18-671 of SEQ ID NO:2.

Another aspect of the present invention includes a use of a non-antibody compound that inhibits the TNF-a proteolytic activity of an enzyme comprising the sequence of amino acids 18-671 of SEQ ID NO:2, for the manufacture of a ~UIUnommap NnnN~ tFTFIF.17RI DOC 1D medicament for inhibiting the cleavage of TNF-a from cell membranes in a mammal.

A further aspect is a use of a composition comprising an amount of a non-antibody compound that effectively inhibits the TNF-a proteolytic activity of an enzyme comprising the sequence of amino acids 18-671 of SEQ ID NO:2, for the manufacture of a medicament for treating a mammal having a disease characterised by an overproduction of an unregulated production of TNF-a.

*9 e M. r.'VNW m' faOY NDELETEl 378 DOC Most, but not all, proteases recognize a specific amino acid sequence. Some proteases primarily recognize residues located N-terminal of the cleaved bond, some recognize residues located C-terminal of the cleaved bond, and some proteases recognize residues on both sides of the cleaved bond. Metalloprotease enzymes utilize a bound metal ion, generally Zn2+, to catalyze the hydrolysis of the peptide bond. Metalloproteases are implicated in joint destruction (the matrix metalloproteases), blood pressure regulation (angiotensin converting enzyme), and regulation of peptide-hormone levels (neutral endopeptidase-24.11).

The invention pertains to biologically active TNF-a converting enzyme ("TACE") as an isolated and purified polypeptide. In addition, the invention is directed to isolated nucleic acids encoding TACE and to expression vectors comprising a cDNA encoding TACE. Within the scope of this invention are host cells that have been transfected or transformed with expression vectors that comprise a cDNA encoding TACE, and processes for producing TACE by culturing such host cells under conditions conducive to expression of TACE. By virtue of the purification of TACE, antibodies, and in particular, monoclonal antibodies against TACE are an aspect of the invention. In addition, assays utilizing TACE S to screen for potential inhibitors thereof, and methods of using TACE as a therapeutic agent for the treatment of diseases mediated by cell-bound TNF-a or other molecules are encompassed by the invention. Further, methods of using TACE in the design of inhibitors thereof are also an aspect of the invention.

The isolated and purified metalloprotease of the invention is capable of convering .TNF-a from the 26 kD membrane-bound form to the 17 kD form, and which has a molecular weight of between approximately 66 kD and approximately 97 kD. The cDNA sequence of TACE is shown in SEQ ID NO:1. The isolated and purified TNF-a converting enzyme ("TACE") comprises amino acids 18-824 of SEQ ID NO:2.

Inhibition of the TACE inhibits release of TNF-a into the serum and other extracellular spaces. TACE inhibitors would therefore have clinical utility in treating conditions characterized by over-production or upregulated production of TNF-a. A particularly useful TACE inhibitor for cenain pathological conditions would selectively inhibit TACE while not affecting TNF-B (also known as lymphotoxin) serum levels. The over-production or unregulated production of TNF-a has been implicated in certain conditions and diseases, for example, Systemic Inflammatory Response Syndrome, reperfusion injury, cardiovascular disease, infectious disease such as HIV infection and HIV neuropathy, obstetrical or gynecological disorders,- inflammatory disease/autoimmunity, allergic/atopic diseases, malignancy, transplants including organ transplant rejection or graft-versus-host disease, cachexia, congenital, dermatologic, neurologic, renal, toxicity, and metabolic/idiopathic diseases.

Inhibitors of TACE would prevent the cleavage of cell-bound TNF-a thereby reducing the level of TNF-a in serum and tissues. The present invention encompasses such an embodiment and comprises a method of inhibiting the cleavage of TNF-a from cell membranes in a mammal comprising administering to such mammal an effective amount of a compound that inhibits the TNF-a proteolytic activity of an enzyme comprising the sequence of amino acids from 18 to 671 through 824 of SEQ ID NO:2. In addition, the invention comprises a method for treating a mammal having a disease characterized by an overproduction or an upregulated production of TNF-a, comprising administering to the mammal a composition comprising an effective amount of a compound that inhibits the TNF-a proteolytic activity of an enzyme comprising the sequence of amino acids 18-824 of SEQ ID NO:2. Such inhibitors would be of significant clinical utility and could be potential therapeutics for treating the above-listed TNF-a-related disorders. Isolation and 15 purification of TACE would provide a significant advancement in the effort to develop inhibitors of such enzyme, and the treatment of TNF-associated diseases, and indeed, could lead to use of TACE itself as a therapeutic agent for certain physiological disorders. For example, in addition to TNF-a, other cytokines as well as cytokine receptors and several Sadhesion proteins may be released from the cell surface by TACE or related proteases.

TACE may be administered to modulate or remove cell surface cytokines, cytokine receptors and adhesion proteins involved in tumor cell growth, inflammation, or fertilization.

DETAILED DESCRIPTION OF THE INVENTION 25 A cDNA encoding human TNF-a converting enzyme ('TACE") has been isolated and is disclosed in SEQ ID NO:1. This discovery of the cDNA encoding human TACE enables construction of expression vectors comprising nucleic acid sequences encoding S TACE; host cells transfected or transformed with the expression vectors; biologically active human TACE as isolated and purified proteins; and antibodies immunoreactive with TACE.

Isolated and purified TACE polypeptides according to the invention are useful for detecting the TACE-inhibiting activity of a molecule. In such a method involving routine and conventional techniques, a molecule of unknown TACE-inhibiting activity is mixed with a substrate and incubated with a TACE polypeptide. The extent of substrate cleavage then can be determined chromatographically.

In addition, TACE polypeptides according to the invention are useful for the structure-based design of a TACE inhibitor. Such a design would comprise the steps of determining the three-dimensional structure of such TACE polypeptide, analyzing the threedimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predictive reactive site, and determining the TACE-inhibiting activity of the molecule.

Antibodies immunoreactive with TACE, and in particular, monoclonal antibodies against TACE, are now made available through the invention. Such antibodies may be useful for inhibiting TACE activity in vivo and for detecting the presence of TACE in a sample.

As used herein, the term "TACE" refers to a genus of polypeptides that are capable of converting the 26 kD cell membrane-bound form of TNF-a (that includes an intracellular region, a membrane region, and an extracellular region), into the soluble 17 kD form that comprises the C-terminal 156 residues of the TNF-a protein. TACE encompasses proteins having the amino acid sequence 18 to 824 of SEQ ID NO:2, as well as those proteins having a high degree of similarity (at least 80%, and more preferably 90% homology) with the amino acid sequence 18 to 824 of SEQ ID NO:2 and which proteins are biologically active. In addition, TACE refers to the biologically active gene products of the nucleotides 15 52-2472 of SEQ ID NO: 1. Further encompassed by the term "TACE" are the membranebound proteins (which include an intracellular region, a membrane region, and an extracellular region), and soluble or truncated proteins which comprise primarily the extracellular portion of the protein, retain biological activity and are capable of being secreted. Specific examples of such soluble proteins are those comprising the sequence of amino acids 18-671 of SEQ ID NO:2. Truncated versions are those having less than the extracellular portion of the protein and comprise, for example, amino acids 18-477 of SEQ ID NO:2, or that comprise substantially all of the catalytic domain, amino acids 215 to 477 of SEQ ID NO:2.

The isolated and purified TACE according to the invention has a molecular weight between about 66 kD and about 97 kD as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). More specifically, TACE was found to have a molecular weight of approximately 80 kD as determined by SDS-PAGE.

ooThe term "isolated and purified" as used herein, means that TACE is essentially free of association with other proteins or polypeptides, for example, as a purification product of recombinant host cell culture or as a purified product from a non-recombinant source. The term "substantially purified" as used herein, refers to a mixture that contains TACE and is essentially free of association with other proteins or polypeptides, but for the presence of known proteins that can be removed using a specific antibody, and which substantially purified TACE retains biological activity. The term. "purified TACE" refers to either the "isolated and purified" form of TACE or the "substantially purified" form of TACE, as both are described herein.

The term "biologically active" as it refers to TACE, means that the TACE is capable of converting the 26 kD cell form of TNF-a into the 17 kD form.

A "nucleotide sequence" refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct, that has been derived from DNA or RNA isolated at least once in substantially pure form free of contaminating endogenous materials) and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratiry, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA may be present or 3' from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.

A 'TACE variant" as referred to herein, means a polypeptide substantially homologous to native TACE, but which has an amino acid sequence different from that of 15 native TACE (human, murine or other mammalian species) because of one or more deletions, insertions or substitutions. The variant amino acid sequence preferably is at least 80% identical to a native TACE amino acid sequence, most preferably at least identical. The percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al.

20 (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). The preferred default parameters for the GAP program include: a unary comparison matrix (containing a value of 1 for identities and 0 for nonidentities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwanz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and no penalty for end gaps.

Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.

Conservative substitutions are well known in the art and include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Conventional procedures and methods can be used for making and using such variants. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are.well known and routinely performed.

Naturally occurring TACE variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the TACE protein, wherein the TACE proteolytic property is retained. Alternate splicing of mRNA may yield a truncated but biologically active TACE protein, such as a naturally occurring soluble form of the protein, for example, as shown in SEQ ID NO:4. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the TACE protein (generally from terminal amino acids).

As stated above, the invention provides isolated and purified, or homogeneous, TACE polypeptides, both recombinant and non-recombinant. Variants and derivatives of native TACE proteins that retain the desired biological activity may be obtained by mutations of nucleotide sequences coding for native TACE polypeptides. Alterations of the native amino acid sequence may be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides 15 containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of S the native sequence. Following ligation, the resulting reconstructed sequence encodes an S analog having the desired amino acid insertion, substitution, or deletion.

SAlternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); S Kunkel et al. (Methods in Enzymol. 154:367, 1987); and U.S. Patent Nos. 4,518,584 and 25 4,737,462 all of which are incorporated by reference.

TACE may be modified to create TACE derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of TACE may be prepared by linking the chemical moieties to functional groups on TACE amino acid side chains or at the N-terminus or C-terminus of a TACE polypeptide or the extracellular domain thereof. Other derivatives of TACE within the scope of this invention include covalent or aggregative conjugates of TACE or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugate may comprise a signal or leader polypeptide sequence the a-factor leader of Saccharomyces) at the N-terminus of a TACE polypeptide. The signal or leader peptide co-translationally or post-translationally directs transfer of the conjugate from its site of synthesis to a site inside or outside of the cell membrane or cell wall.

TACE polypeptide conjugates can comprise peptides added to facilitate purification and identification of TACE. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., BiolTechnology 6:1204, 1988.

The invention further includes TACE polypeptides with or without associated native-pattern glycosylation. TACE expressed in yeast or mammalian expression systems COS-7 cells) may be similar to or significantly different from a native TACE polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of TACE polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Glycosyl groups may be removed through conventional methods, in particular those utilizing glycopeptidase. In general, glycosylated TACE may be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).

Equivalent DNA constructs that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences not needed for biological activity are encompassed by the invention. For example, Nglycosylation sites in the TACE extracellular domain can be modified to preclude S glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are 20 characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate substitutions, additions or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an Nglycosylation site. Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Patent 5,071,972 and EP 276,846, hereby incorporated by reference.

In another example, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon renaturation. Other equivalents are prepared by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites.

Nucleic acid sequences within the scope of the invention include isolated DNA and RNA sequences that hybridize to the native TACE nucleotide sequences disclosed herein under conditions of moderate or high stringency, and which encode biologically active TACE. Conditions of moderate stringency, as known to those having ordinary skill in the art, and as defined by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed.

Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), include use of a prewashing solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 50C 60'C, 5 X SSC, overnight, preferably 55'C. Conditions of high stringency include higher temperatures of hybridization and washing. The skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as the length of the probe.

Due to the known degeneracy of the genetic code wherein more than one codon can encode the same amino acid, a DNA sequence may vary from that shown in SEQ ID NO:1 S and still encode a TACE protein having the amino acid sequence of SEQ ID NO:2. Such variant DNA sequences may result from silent mutations occurring during PCR amplification), or may be the product of deliberate mutagenesis of a native sequence.

The invention thus provides equivalent isolated DNA sequences encoding S biologically active TACE, selected from: the coding region of a native mammalian TACE gene; cDNA comprising the nucleotide sequence presented in SEQ ID NO:1; (c) 20 DNA capable of hybridization to a DNA of under moderately stringent conditions and which encodes biologically active TACE; and DNA which is degenerate as a result of the genetic code to a DNA defined in or and which encodes biologically active TACE. TACE proteins encoded by such DNA equivalent sequences are encompassed by the invention.

DNA that are equivalents to the DNA sequence of SEQ ID NO:1 will hybridize under moderately stringent or highly stringent conditions to the double-stranded native DNA sequence that encode polypeptides comprising amino acid sequences of 18 Xaa of SEQ ID NO:2, wherein Xaa is an amino acid from 671 to 824. Examples of TACE proteins encoded by such DNA, include, but are not limited to, TACE fragments (soluble or membrane-bound) and TACE proteins comprising inactivated N-glycosylation site(s), inactivated KEX2 protease processing site(s), or conservative amino acid substitution(s), as described above. TACE proteins encoded by DNA derived from other mammalian species, wherein the DNA will hybridize under conditions of moderate or high stringency to the complement of the cDNA of SEQ ID NO:1 are also encompassed.

Alternatively, TACE-binding proteins, such as the anti-TACE antibodies of the invention, can be bound to a solid phase such as a column chromatography matrix or a similar substrate suitable for identifying, separating or purifying cells that express the TACE on their surface. Adherence of TACE-binding proteins to a solid phase contacting surface can be accomplished by any means, for example, magnetic microspheres can be coated with TACE-binding proteins and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures are contacted with the solid phase that has TACEbinding proteins thereon. Cells having TACE on their surface bind to the fixed TACEbinding protein and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening or separating such TACE-expressing cells from solution.

Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-injurious to the cells and are preferably directed to cleaving the cell-surface binding partner.

Alternatively, mixtures of cells suspected of containing TACE-expressing cells first can be incubated with a biotinylated TACE-binding protein. Incubation periods are typically at least one hour in duration to ensure sufficient binding to TACE. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the **15 high affinity of biotin for avidin provides the binding of the TACE-binding cells to the beads. Use of avidin-coated beads is known in the art. See Berenson, et al. J. Cell.

i Biochem., 10D:239 (1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.

In the methods described above, suitable TACE-binding proteins are anti-TACE 20 antibodies, and other proteins that are capable of high-affinity binding of TACE. A preferred TACE-binding protein is an anti-TACE monoclonal antibody obtained, for example, as described in Example 4.

TACE polypeptides may exist as oligomers, such as covalently-linked or noncovalently-linked dimers or trimers. Oligomers may be linked by disulfide bonds formed between cysteine residues on different TACE polypeptides. In one embodiment of the invention, a TACE dimer is created by fusing TACE to the Fc region of an antibody IgG1) in a manner that does not interfere with biological activity of TACE. The Fc polypeptide preferably is fused to the C-terminus of a soluble TACE (comprising only the extracellular domain). General preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, by Ashkenazi et al. (PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), hereby incorporated by reference. A gene fusion encoding the TACE:Fc fusion protein is inserted into an appropriate expression vector.

TACE:Fc fusion proteins are allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between Fc polypeptides, yielding divalent TACE. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a TACE oligomer with as many as four TACE extracellular regions.

Alternatively, one can link two soluble TACE domains with a peptide linker.

Recombinant expression vectors containing a nucleic acid sequence encoding TACE can be prepared using well known methods. The expression vectors include a TACE DNA sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.

Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the TACE DNA sequence.

Thus, a promoter nucleotide sequence is operably linked to a TACE DNA sequence if the promoter nucleotide sequence controls the transcription of the TACE DNA sequence. The ability to replicate in the desired host cells, usually conferred by an origin of replication, and a selection gene by which transformants are identified, may additionally be incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are not naturally 15 associated with TACE can be incorporated into expression vectors. For example, a DNA sequence for a signal peptide (secretory leader) may be fused in-frame to the TACE sequence so that TACE is initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells enhances extracellular secretion of the TACE polypeptide. The signal peptide may be cleaved from the TACE 20 polypeptide upon secretion of TACE from the cell.

Suitable host cells for expression of TACE polypeptides include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985).

Cell-free translation systems could also be employed to produce TACE polypeptides using RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, for example, E. coli S or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella ryphimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E.

coli, a TACE polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant TACE polypeptide.

Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. To construct en expression vector using pBR322, an appropriate promoter and a TACE DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMI (Promega Biotec, Madison, WI, USA).

Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include P-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage X PL promoter and a c1857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the X PL promoter include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).

TACE polypeptides alternatively may be expressed in yeast host cells, preferably from the Saccharomyces genus S. cerevisiae). Other genera of yeast, such as Pichia, K. lactis or Kluyveromyces, may also be employed. Yeast vectors will often contain an 20 origin of replication sequence from a 24 yeast plasmid, an autonomously replicating S sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van den Berg et. al., BiolTechnology, 8:135-139 (1990). Another alternative is the glucoserepressible ADH2 promoter described by Russell et al. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E.

coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp' gene and origin of replication) into the above-described yeast vectors.

The yeast a-factor leader sequence may be employed to direct secretion of a TACE polypeptide. The a-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Nail. Acad. Sci. USA 81:5330, 1984; U. S. Patent 4,546,082; and EP 324,274.

Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 15:1929, 1978. The Hinnen et al. protocol selects for Trp transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 I.g/ml adenine and 20 gig/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promoter sequence may be grown for inducing expression in a "rich" medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ig/ml adenine and 80 gjg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.

Mammalian or insect host cell culture systems could also be employed to express recombinant TACE polypeptides. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 *2..20 (1988). Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL cell lines, and the CV-1/EBNA-1 cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).

Transcriptional and translational control sequences for mammalian host cell S expression vectors may be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978).

Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.

Exemplary expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986).

A useful high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in U.S. Patent Application Serial No. 07/701,415, filed May 16, 1991, incorporated by reference herein. The vectors may be derived from retroviruses. In place of the native signal sequence, a heterologous signal sequence may be added, such as the signal sequence for IL-7 described in United States Patent 4,965,195; the signal sequence for IL-2 receptor described in Cosman et al., Nature 312:768 (1984); the IL-4 signal peptide described in EP 367,566; the type I IL-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type I IL-1 receptor signal peptide described in EP 460,846.

15 An isolated and purified TACE protein according to the invention may be produced by recombinant expression systems as described above or purified from naturally occurring cells. TACE can be substantially purified, as indicated by a single protein band upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). One process for producing TACE comprises culturing a host cell transformed with an expression vector 20 comprising a DNA sequence that encodes TACE under conditions sufficient to promote expression of TACE. TACE is then recovered from culture medium or cell extracts, depending upon the expression system employed. As is known to the skilled artisan, procedures for purifying a recombinant protein will vary according to such factors as the type of host cells employed and whether or not the recombinant protein is secreted into the culture medium. For example, when expression systems that secrete the recombinant protein are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, silica gel having pendant methyl or other aliphatic groups) can be employed to further purify TACE. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide an isolated and purified recombinant protein.

In addition to recombinantly producing TACE, TACE may be isolated and purified from an activated monocytic cell line, THP-1. THP-1 cells typically produce more TNF-a than do HL-60 cells, and are a preferred source for TACE. Other sources for TACE may be used, and TACE may also be found in other types of cells that produce TNF-a. Once a source for TACE is identified, TACE may be isolated and purified by first optionally stimulating the source cells to produce TNF-a. Stimulation may not be necessary, however, it can be done using techniques that are well-known in the art. The cells are then harvested, washed, and plasma membranes isolated according to conventional procedures.

A particularly preferred method of isolating the plasma membranes is method number three as described in Maeda et. al., Biochim. et. Biophys. Acta, 211:115 (1983); except that dithiothreitol should not be included in this method since it was determined that dithiothreitol blocks TACE activity. Proteins from the cell membrane then can be J15 solubilized by suspending the membrane preparation in a dilute solution of non-ionic detergent, followed by brief homogenization. Phospholipids then can be extracted using conventional methods.

It is possible to utilize an affinity column comprising a TACE-binding protein to affinity-purify expressed TACE polypeptides. TACE polypeptides can be removed from an affinity column using conventional techniques, in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized. Example 4 describes a procedure for employing TACE of the invention to generate monoclonal antibodies directed against TACE.

Recombinant protein produced in bacterial culture is usually isolated by initial S 25 disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble o polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or o more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps.

Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Transformed yeast host cells are preferably employed to express TACE as a secreted polypeptide in order to simplify purification. Secreted recombinant polypeptide from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. Chromarog. 296:171, 1984). Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.

Antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to a target TACE mRNA sequence (forming a duplex) or to the TACE sequence in the double-stranded DNA helix (forming a triple helix) can be made according to the invention. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of TACE cDNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to create an antisense or a sense oligonucleotide, based upon a cDNA sequence for a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of complexes that block translation (RNA) or transcription (DNA) by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of TACE proteins. Antisense or sense oligonucleotides further comprise oligo-nucleotides having modified sugarphosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases.

Such oligonucleotides with resistant sugar linkages are stable in vivo capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those 20 oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oliginucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO4mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or or the double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656).

Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.

Alternatively, a sense or an antisense oligonucleotde may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.

Isolated and purified TACE or a fragment thereof, and in particular, the extracellular domain of TACE, may also be useful itself as a therapeutic agent in regulating the levels of certain cell surface proteins. In addition to TNF-a, other cytokines as well as cytokine receptors and several adhesion proteins may be released from the cell surface by TACE or related proteases. TACE or a fragment thereof, in particular, the extracellular domain of 15 TACE, may be administered to modulate or remove cell surface cytokines, cytokine receptors and adhesion proteins involved in tumor cell growth, inflammation, or fertilization. When used as a therapeutic agent, TACE can be formulated into pharmaceutical compositions according to known methods. TACE can be combined in admixture, either as the sole active material or with other known active materials, with 20 pharmaceutically suitable diluents Tris-HC1, acetate, phosphate), preservatives ."Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers.

Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition, such compositions can contain TACE complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or S incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of TACE.

TACE may be assayed using any of a variety of metalloprotease assays known in the art. In general, TACE can be assayed through the use of a peptide substrate that represents the natural cleavage site of TNF-a. For example, in order to detect the cleavage of a substrate by TACE, the substrate can be tagged with a fluorescent group on one side of the cleavage site and with a fluorescence-quenching group on the opposite side of the cleavage site. Upon cleavage by TACE, quenching is eliminated thus providing a detectable signal. Alternatively, the substrate may-be tagged with a colorimetric leaving group that more strongly absorbs upon cleavage. Alternatively, the substrate may have a thioester group synthesized into the cleavage site of the substrate so that upon cleavage by TACE, the thiol group remains and can be easily detected using conventional methods. A particularly preferred method of detecting TACE activity in a sample is described in Example 1, infra. Other methods of detecting TACE activity may be utilized without resorting to undue experimentation.

As further described in Example 1, infra, a quantitative assay for TACE also may be used which assay involves incubating the peptide substrate, at about 1 mM, with TACE at 37 'C for a fixed period of time; stopping the reaction by the addition of an acid or a metal chelator, and determining the extent of cleavage by HPLC analysis.

Within an aspect of the invention, TACE, and peptides based on the amino acid sequence of TACE, may be utilized to prepare antibodies that specifically bind to TACE. A specific example of such antibody preparation is described in Example 4 herein. The term "antibodies" is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab')2, and Fab fragments, as well as any recombinantly produced binding partners. Antibodies are defined to be specifically binding if they bind TACE with a Ka of greater than or equal to about 107 M1. Affinities of binding partners or antibodies 15 can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).

Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well-known in the art. In general, purified TACE, or a peptide based 20 on the amino acid sequence of TACE that is appropriately conjugated, is administered to the host animal typically through parenteral injection. The immunogenicity of TACE may be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to TACE or the TACE peptides. Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Patent Nos. 4,376,110 and 4,486,530.

Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol 1980. Briefly, the host animals, such as mice are injected intraperitoneally at least once, and preferably at least twice at about 3 week intervals with isolated and purified TACE or conjugated TACE peptide, optionally in the presence of adjuvant. Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given an intravenous boost of TACE or conjugated TACE peptide. Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as, 125I-TACE is added to each well followed by incubation. Positive wells can be subsequently detected by autoradiography. Positive clones can be grown in bulk culture and supematants are subsequently purified over a Protein A column (Pharmacia).

The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody 15 Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3:1-9 (1990) which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 2:394 (1989).' 20 Other types of "antibodies" may be produced using the information provided herein in conjunction with the state of knowledge in the art. For example, humanized antibodies that are capable of specifically binding TACE are also encompassed by the invention.

Once isolated and purified, the antibodies against TACE may be used to detect the presence of TACE in a sample using established assay protocols. Further, the antibodies of the invention may be used therapeutically to bind to TACE and inhibit its activity in vivo.

*.*The purified TACE according to the invention will facilitate the discovery of S inhibitors of TACE, and thus, inhibitors of excessive TNF-a release. The use of a purified TACE polypeptide in the screening of potential inhibitors thereof is important and can virtually eliminate the possibility of interfering reactions with contaminants. Such a screening assay for detecting the TACE-inhibiting activity of a molecule would typically involve mixing the potential inhibitor molecule with an appropriate substrate, incubating TACE that is at least substantially purified with the mixture, and determining the extent of substrate cleavage as, for example, described above. While various appropriate substrates may be designed for use in the assay, preferably, a peptidyl substrate is used, and which substrate comprises the amino acid sequence Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser (SEQ ID In addition, TACE polypeptides can also be used for structure-based design of TACE-inhibitors. Such structure-based design is also known as "rational drug design." The TACE polypeptides can be three-dimensionally analyzed by, tor example, X-ray crystallography, nuclear magnetic resonance or homology modeling, all of which are wellknown methods. The use of TACE structural information in molecular modeling software systems to assist in inhibitor design and inhibitor-TACE interaction is also encompassed by the invention. Such computer-assisted modeling and drug design may utilize information such as chemical conformational analysis, electrostatic potential of the molecules, protein folding, etc. For example, most of the design of class-specific inhibitors of metalloproteases has focused on attempts to chelate or bind the catalytic zinc atom.

Synthetic inhibitors are usually designed to contain a negatively-charged moiety to which is attached a series of other groups designed to fit the specificity pockets of the particular protease. A particular method of the invention comprises analyzing the three dimensional structure of TACE for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described above.

The following Examples provide an illustration of embodiments of the invention 15 and should not be construed to limit the scope of the invention which is set forth in the appended claims. In the following Examples, all methods described are conventional unless otherwise specified.

EXAMPLE 1 Purification of the TNF-a Converting Enzyme This Example describes a method for purifying TACE. The TACE was isolated and purified from the membranes of the human monocytic cell line, THP-1, (ATCC no. TIB 202) that had been stimulated to produce TNF-a. THP-1 cells were chosen because they produce more TNF-a than HL-60 cells, a more commonly used human monocytic cell line.

SApproximately 120 billion cells were stimulated using the procedure previously described 25 by Kronheim et al., Arch. Biochem. Biophys. 269:698 (1992), incorporated herein by reference. Two hours after stimulation, the cells were harvested by centrifugation. The *harvested cells were washed at least twice with Hanks balanced salt solution, and plasma membranes were isolated according to method number three as described by Maeda et. al., Biochim. et. Biophys. Acta, 731:115 (1983), except that dithiothreitol was not used, utilizing 1.25 ml of homogenization buffer per ml of cell pellet. It was determined that the standard procedure of Maeda et al., Id., utilizing dithiothreitol, failed to yield compounds having TACE activity (an assay for TACE activity is described below). Proteins were then solubilized by resuspending the membrane preparation in a solution of 1% octylglucoside, mM Tris-HCl (pH 1 mM MgCl2 and 30 mM NaCl and briefly homogenizing with a Brinkman Homogenizer (twice, five seconds each time). Phospholipids were then extracted by adding four volumes of ice-cold (0 acetone; after a thirty-minute incubation at 4 the acetone-extracted material was centrifuged at 1500 rpm for 10 minutes in a H1000B rotor.

Chromatography The pelleted material was dissolved in 450 ml of Buffer A (Buffer A comprises mM Tris-HCl (pH 7.5) and 1% octylglucoside (weight to volume percent)) and applied to a 120 ml column of DEAE-Sepharose fast-flow (Pharmacia) at 4 ml per minute. The column then was washed with 360 ml of Buffer A at 6 ml per minute, and protein was then eluted with an increasing gradient of NaCI (0-0.3 M) in Buffer A applied at 6 ml per minute over a period of 40 minutes. TACE was eluted with a NaCI concentration of about 50 to about 150 mM.

TACE was originally detected at this point by its ability to cleave recombinant 26 kD TNF-a fused to the "flag" Hopp, et al., Bio Technology, 6:1204 (1988) sequence of 8 amino acids at the amino-terminus. The gene encoding human TNF-a was spliced to DNA encoding the flag sequence, and this construct was placed in the pPL3 vector (C.

Maliszewski et al., Molec. Immunol., 25:429 (1987). The protein was then expressed in a 15 protease-deficient strain of E. coli Libby et al., DNA, 6:221 (1987) which was found necessary to prevent degradation of the precursor by the bacteria. After removal of growth medium, the bacteria were resuspended in 30 mM Tris-HCI (pH 5 mM EDTA, and the suspension was sonicated for about 30 seconds. The material was then centrifuged at 20,000 rpm in an SS34 rotor for 30 minutes, the supernatant fraction was discarded, and 20 the pellet was resuspended with 8 M urea in 10 mM Tris-HCl (pH The material was homogenized with 25 strokes in a dounce homogenizer and then centrifuged at 20,000 rpm in an SS34 rotor for 30 minutes. The supernatant fraction, which contained the precursor TNF-a, was then dialyzed four times against 10 mM Tris-HCI (pH 8).

This material was incubated at 37 'C for at least 4 hours with the TACE eluted from the DEAE-Sepharose, that had been treated with 1 mM N-methoxysuccinyl-Ala-Ala-Valchloro-methylketone, 10 Ig/ml leupeptin, and 1 mg/ml al-protease inhibitor, all of which are commercially available. The N-terminus of the resulting 17 kD product was found to be that of authentic TNF-a. After the initial identification of TACE in this way, it was found that the enzyme also cleaves an 8-residue peptide representing the segment Leu 73 -Ala 74 Gln 75 -Ala 76 1 -Val 77 -Arg 7 8 -Ser 79 -Ser 80 (SEQ ID NO:5) of TNF-a. Wherein the (1) illustrates the cleavage site. Based on this observation, a quantitative assay was established: the peptide, at 1 mM, was incubated with the enzyme at 37 'C for a fixed period of time, in the presence of 0.1 mM dichloroisocoumarin, 1 mM methoxysuccinyl- Ala-Ala-Pro-Val-chloromethyl-ketone, 10 gpg/ml leupeptin, 10 pM bestatin, and 1 mg/ml al-protease inhibitor (Sigma), all of which are commercially available. The reaction was then stopped by the addition of acid or a metal chelator. The extent of cleavage of this peptide, reflecting the amount of TACE present, was determined by applying the mixture to a Vydac C18 column and luting with a gradient of 0 to 30% acetonitrile over a period of minutes.

Material that eluted from the DEAE column with 0.05-0.25 M NaCl had about a 4fold higher specific activity than the starring material. The eluted material was sonicated and then shaken with wheat germ agglutinin-agarose (Vector Laboratories) for two hours at 4 Prior to use, the wheat germ agglutinin-agarose was washed with 5 column volumes of Buffer B (Buffer B comprises 10 mM Tris-HCl (pH 0.15 M NaCI, 0.1 mM MnC12, 0.1 mM CaC12, 1% octylglucoside and 10% glycerol); 1 ml of this resin was used for every 2 mg of protein in the sample, as determined by the BCA protein assay (Pierce).

After two hours, the resin was washed with 7 volumes of Buffer B, and material was then eluted with 5 column volumes of Buffer B plus 0.3 M acetylglucosamine (Sigma), with minute intervals between the application of each column volume.

Eluted fractions containing TACE activity had about a ten-fold higher specific activity than the starting material. These fractions were concentrated to about 5 ml with Centriprep-30 concentrators (Amicon) and then diluted three-fold with Buffer C (Buffer C comprises 10 mM Tris-HCl (pH 1 octylglucoside and 10% glycerol). The diluted material was sonicated (three 10-second bursts) and then loaded onto a MonoQ HR column (Pharmacia) at 0.5ml per minute. The column was then washed with 10 ml of Buffer C at 0.5 ml per minute, and material was eluted with a 0 to 0.25 M NaCI gradient in .:20 Buffer C at 0.5 ml per minute over a period of 30 minutes. TACE activity (detected at this stage and subsequently by incubation with the previously described peptide substrate in the absence of protease inhibitors) eluted with about 0.15M NaC1.

The NaCl concentration in the MonoQ fractions containing activity was reduced by at least ten-fold by diluting the material into Buffer C, and the material was then applied to a column of hydroxyapatite (American International Chemical, ceramic hydroxyapatite at the rate of 0.5 ml per minute. After washing with three column volumes of Buffer C, protein was eluted with a 0 to 50 mM gradient of sodium phosphate at 1 ml per minute over a period of 30 minutes. TACE eluted with about 15 mM sodium phosphate.

The TACE eluted from the hydroxyapatite column was then concentrated to about 100 pl with Centricon-50 concentrators (Amicon) and applied to a Bio-Rad SEC-400 sizing column (30cm). Protein was eluted with Buffer C run through the column at 0.5 ml per minute; TACE eluted at about 28 minutes.

The TACE eluted from the sizing column was diluted three-fold into Buffer D (Buffer D comprises 20 mM MES (pH 1% octyglucoside and 10% glycerol) and applied to a 1 ml column of Red 120-agrose (Signri) at 0.25 ml per minute. After the column was washed with 10 ml Buffer D, protein was eluted with a 0 to 1 M NaCl gradient in Buffer D at 0.25 ml per minute over a period of 60 minutes. TACE eluted with 0.2 to 0.3 M NaCI. Five percent of each eluted fraction was run on a SDS-polyacrylamide gel and silver staining showed that the predominant protein in the fractions with activity ran approximately midway between the 66 and 97 kD markers (Novex) on the gel, at approximately 80 kD.

Trifluoroacetic acid (TFA) was added to 0.2% (volume-to-volumne percentage) to a pool of the fractions containing the approximately 80 kD protein, and the mixture was then pumped onto a 2.1 x 5 cm C4 column, at approximately 100 pl per minute using a Shimadzu LC-10AD. Protein was eluted with a 0 to 100% gradient of acetonitrile in 0.1% TFA at 100 gil per minute over a period of 100 minutes. One minute fractions were collected and 5 to 10% of each fraction was run on a Novex SDS-polyacrylamide gel Fractions that eluted with about 70% acetonitrile and that contained a protein of approximately 80 kD were pooled and evaporated to dryness.

Generation of peptides and sequencing This pool of fractions then was dissolved in 200 !l of 50 mM Tris-HC1 (pH 1 15 mM EDTA, and an amount of endo-LYS-C (Promega) equal to about 1/50 of the amount of protein in the sample was added. The material was incubated at 37 'C overnight, and then a fresh aliquot of the same amount of endo-LYS-C was added for an additional 3 hours at 37

C.

The resulting peptides were separated by applying the material to a capillary C18 20 column at 20 pl per minute and eluting with an ascending gradient of acetonitrile per minute) in 0.1% TFA over a period of 200 minutes. Peptides were sequenced with an ABI 476 or an ABI 494 automated sequencer.

:4 EXAMPLE 2 25 Preparation of Isolated and Purified TACE This Example describes a method for further purifying the purified TACE as was 00 obtained using the procedures described above. Purified TACE obtained from the THP-1 cells may contain small amounts of human lysosomal 85 kD sialoglycoprotein (Biochem.

Biophys. Res. Commun. 184:604-611 (1992) and human lysosomal alpha-mannosidase (Biochem. Biophys. Res. Comm. 200:239-245 (1994) that can be removed using standard immunoadsorbant procedures, as described in, for example, Robert K. Scopes, Protein Purification--Principles and Practice (Springer-Verlag, 2nd edit), pp. 167-172. Using the procedures described in this Example 2, isolated and purified TACE can be obtained.

EXAMPLE 3 Cloning of Human TACE This example describes a procedure for isolating a DNA sequence encoding human TACE. A random primed cDNA library was generated from the commercially available cell line THP-1 (Amersham) using conventional methods. Polymerase chain reaction (PCR) (MuUis and Faloona, Meth. Enzymol. 155:335-350, 1987) amplifications were performed using the following primers: Primer 5'-AARTAYGTNATGTAYCC-3' SEQ ID NO:6 Primer 5'-CCRCARTCRCAYTCYTC-3' SEQ ID NO:7 Primer is based on the first five amino acids of Peptide with the addition of a triplet coding for lysine at the 5' end. Primer is antisense to a conserved amino acid sequence Glu-Glu-Cys-Asp-Cys-Gly (EECDCG) SEQ ID NO:8, which is found in a 15 homologous metalloprotease, bovine reprolysin 1 (GenBank Accession #Z21961).

Single stranded cDNA was amplified using the mixed oligonucleotides described above under standard PCR conditions. The PCR reaction products were fractionated by gel electrophoresis and DNA bands of approximately 180 bp were isolated and subcloned into commercially available pBLUESCRIPT. Sequencing revealed a clone that contained a 20 nucleotide sequence that codes for the amino acids Ile-Ala-Val-Ser-Gly-Asp-His-Glu-Asn- Asn-Lys (SEQ ID NO:9) and a nucleotide sequence that codes for amino acids Glu-Glu- Cys-Asp-Cys-Gly (EECDCG) (SEQ ID NO:8). This clone was termed the "30CD clone." The 30CD clone was sequenced and primers were generated based on this sequence. The primers then were used to detect TACE cDNA in phage library made from human KB cells.

This library was screened under conventional conditions using a probe based on the sequence. Positive hybridizing plaques were isolated and DNA fragments of these clones were sequenced. Sequencing provided a full length cDNA of human TACE which is shown in SEQ ID NO:1. Human TACE was found to be a type I transmembrane protein of 824 amino acids, including a N-terminal 17 amino acid signal peptide. The signal peptide is followed by an extracellular domain of 654 amino acids, a 23 amino acid transmembrane domain and a 130 amino acid cytoplasmic domain. An alternate spliced variant was cloned and sequenced and found to contain the same amino acid sequence as TACE, except that a bp fragment is deleted at the 5' end of the cytoplasmic domain, thus shifting the reading frame to encode a six amino acid cytoplasmic domain. The amino acid sequence of this variant is shown in SEQ ID NO:4, with the cDNA shown in SEQ ID NO:3.

EXAMPLE 4 Preparation of Antibodies Against TACE This Example describes a method for generating monoclonal antibodies against TACE. Balb/c mice are injected intraperitoneally on two occasions at 3 week intervals with 10 ug of isolated and purified TACE of Example 1 or peptides based on the amino acid sequence of TACE in the presence of RIBI adjuvant (RIBI Corp., Hamilton, Montana).

Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Three weeks later, mice are given an intrevenous boost of 3 ug of human TACE, or TACE peptide, suspended in sterile PBS. Three days later, mice are sacrificed and spleen cells fused with Ag8.653 myeloma cells (ATCC) following established protocols. Briefly, Ag8.653 cells are washed several times in serumfree media and fused to mouse spleen cells at a ratio of three spleen cells to one myeloma cell. The fusing agent is 50% PEG: 10% DMSO (Sigma). Fusion is plated out into twenty 96-well flat bottom plates (Coming) containing HAT supplemented DMEM media and allowed to grow for eight days. Supernatants from resultant hybridomas are collected and added to a 96-well plate for 60 minutes that is first coated with goat anti-mouse Ig.

Following washes, 125 1-TACE is added to each well, incubated for 60 minutes at room temperature, and washed four times. Positive wells can be subsequently detected by autoradiography at -70 'C using Kodak X-Omat S film. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).

Throughout the description and claims of this S specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

o SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Immunex Corporation (ii) TITLE OF INVENTION: TNF-a CONVERTING ENZYME (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Immunex Corporation STREET: 51 University Street CITY: Seattle STATE: WA COUNTRY: USA ZIP: 98101 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk oo: COMPUTER: Apple Macintosh OPERATING SYSTEM: Apple Operating System 7.5.2 SOFTWARE: Microsoft Word for Apple, Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: not yet assigned FILING DATE: 03-JUN-1996

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER:--to be assigned-- FILING DATE: 23-MAY-1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER:08/504,614 FILING DATE: 20-JUL-1995 vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/428,458 FILING DATE: 8-JUN-1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: Malaska, Stephen L.

REGISTRATION NUMBER: 32,655 REFERENCE/DOCKET NUMBER: 2507-WO (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (206) 587-0430 TELEFAX: (206) 233-0644 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2475 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 52..2472 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGAGGCAGT CTCTCCTATT CCTGACCAGC GTGGTTCCTT TCGTGCTGGC G CCG CGA Pro Arg 1 000 0 0 0t 0 *0 0

CCT

Pro

GAT

Asp

CAG

Gin 35

ACA

Thr

ACA

Thr

GAT

Asp

ACT

Thr

AGA

Arg 115

AAC

Asn

ATG

Met

CCG

Pro

TCT

Ser

CAT

His

CTA

Leu

TCA

Ser

GGT

Gly

GGA

Gly 100

GAT

Asp

ATA

Ile

TTA

Leu

GAT

Asp

TTG

Leu

TCG

Ser

CTA

Leu

AGT

Ser

AAA

Lys

CAC

His

GAT

Asp

GAG

Glu

GTT

Val

GAC

Asp

CTC

Leu

GTA

Vai

ACT

Thr

ACT

Thr 70

AAC

Asn

GTG

Val

GAT

Asp

CCA

Pro

TAT

Tyr 150

CCG

Pro

TCA

Ser

AGA

Arg

TTT

Phe

GAA

Glu

GAA

Glu

GTT

Va1

GTT

Vai

CTT

Leu 135

AAA

Lys

GGC

Gly

GAC

Asp

AAA

Lys 40

TCA

Ser

CGT

Arg

AGC

Ser

GGT

Gly

ATA

Ile 120

TGG

Trp

TCT

Ser

TTC

Phe

TAC

Tyr 25

AGA

Arg

GCT

Ala

TTT

Phe

GAG

Glu

GAG

Glu 105

ATC

Ile

AGA

Arg

GAA

Glu

GGC

Gly 10

GAT

Asp

GAT

Asp

TTG

Leu

TCA

Ser

TAC

Tyr 90

CCT

Pro

AGA

Arg

TTT

Phe

GAT

Asp

CCC

Pro

ATT

Ile

CTA

Leo

AAA

Lys

CAA

Gin 75

ACT

Thr

GAC

Asp A2'C Ile

GTT

Val

ATC

Ile 155

CAC

His

CTC

Leo

CAG

Gin

AGG

Arg 60

AAT

Asn

GTA

Val

TCT

Ser

AAC

Asn

AAT

Asn 140

AAG

Lvs CAG AGA CTC Gin Arg Leu TCT TTA TCT Ser Leu Ser ACT TCA ACA Thr Ser Thr 45 CAT TTT AAA His Phe Lys TTC AAG GTC Phe Lys Val AAA TGG CAG Lys Trp Gin AGG GTT CTA Arg Vai Leu 110 ACA GAT GGG Thr Asp Gly 125 GAT ACC AAA Asp Thr Lys AAT GTT TCA Asn Val Ser GAT AAT GAA

AAT

Asn

CAT

His

TTA

Leu

GTG

Val

GAC

Asp

GCC

Ala

GCC

Ala

GAC

Asp

CGT

Arg 160

ATC

Ile

GTA

Val

TAC

Tyr

GTG

Val

TTC

Phe

CAC

His

GAA

Glu

AAA

Lys 145

TTG

Leu

CAG

Gin

GAA

Glu

CTG

Leu

GTG

Val

TTC

Phe

ATA

Ile

TAT

Tyr 130

AGA

Arg

CAG

Gin GAG AAG CTT Glu Lys Leu 57 105 153 201 249 297 345 393 441 489 537 585 TCT CCA AAA GTG TGT GGT TAT TTA AAA GTG GAG TTG CTC Ser Pro Lys 165 Val Cys Gly Tyr Leu Lys Val Asp Asn Qiu Giu Leu Leo CCA AAA GGG TTA GTA GAC AGA GAA CCA CCT GAA GAG CTT GTT CAT CGA Pro Lys Gly 180 Leu Val Asp Arq Giu Pro Pro Giu Giu Leu Val His Arg 185 190

C.

C.

C

C

C. C

.C

CC

C

GTG Val I 195 TTG C Leu

GAG

Glu

GAC

Asp

GGA

Gly

AAA

Lys 275

GAA

Glu

GAT

Asp

TAC

Tyr

CCC

Pro

CCA

Pro 355

AAG

Lys

ACT

Thr

AA

.ys 3TG lal

%GT

Ser

ATC

Ile

ATA

Ile 260

CCT

Pro

AAG

Lys

ATA

Ile

CAA

Gin

AGA

Arg 340

GTT

Val

AAT

Asn

CAT

His

AGA

Arg

GTA

Va1

ACA

Thr

TAT

Tyr 245

CAG

Gin

GGT

Gly

GAT

Asp

GCT

Ala

GAT

Asp 325

GCA

Ala

GGG

Gly

TAT

Tyr

GAP

Glu AGA G Arg GCA C Ala 1 2 ACT I Thr 1 230 CGG 2 Arg 2

ATA

Ile

GAA

Glu

GCT

Ala

GAG

Glu 310

TTT

Phe

AAC

Asn

AAG

Lys

GGT

Gly

TTG

Leu 390

,CT

la

;AT

~sp !15

~CA

Lhr

AC

ksn 3AG lu

A.AG

Lys

TGG

Trp 295

GAA

Glu

GAT

Asp

AGC

Ser

AAA

Lys

AA.

Lyv 375

GGI

Gil GAC C Asp 1 200 CAT C His 2

AAT

Asn

ACT

Thr

CAG

Gin

CAC

His 280

GAT

Asp

GCA

Ala

ATG

Met

CAT

His

AAT

Asn 360

ACC

Thr

CAT

His

CA

ro

:GC

krg rAC Tyr

TCA

Ser

ATT

Ile 265

TAC

Tyr

GTG

Val

TCT

Ser

GGA

Gly

GGA

Gly 345

ATC

Ile

ATC

Ile

AAT

GAT C Asp I

TTC

Phe

TTA

Leu

TGG

Trp 250

CGC

Arg

AAC

Asn

AAG

Lys

AAA

Lys

ACT

Thr 330

GGT

Gly

TAT

Tyr

CTT

:CC

?ro

TAC

Tyr

ATA

Ile 235

GAT

Asp

ATT

Ile

ATG

Met

ATG

Met

GTT

Val 315

CTT

Leu

GTT

Val

TTG

Leu

ACP

ATG

Met

AGA

Arg 220

GAG

Glu

AAT

Asn

CTC

Leu

GCA

Ala

TTG

Leu 300

TGC

Cys

GGA

Glv

TGT

Cys

AAI

Asn

A.AC

AAG I Lys 2 205 TAC 2 Tyr

CTA

Leu

GCA

Ala 4

AAG

Lys

AAA

Lys 285

CTA

Leu

TTG

Leu

TTA

Leu

CCA

Pro

AGT

Ser 365

GAA

!LAC

ksn kTG let

ATT

Ile

GGT

Gly

TCT

Ser 270

AGT

Ser

GAG

Glu

GCA

Ala

GCT

Ala

AAG

Lys 350

GGT

Gly

GCT

ACG

Thr

GGC

Gly

GAC

Asp

TTT

Phe 255

CCA

Pro

TAC

Tyr

CAA

Gin

CAC

His

TAT

Tyr 335

GCT

Ala

TTG

Leu

GAC

TGT

:ys

AGA

Arg

AGA

Arg 240

AAA

Lys

CAA

Gin

CCA

Pro

TTT

Phe

CTT

Leu 320

GTT

Val

TAT

Tyr

ACG

Thr

CTC

AAA T Lys L 2 GGG C Gly C 225 GTT G Val I GGC I2 Gly

GAG

Glu

AAT

Asi

AGC

Ser 305

TTC

Phe

GGC

Gly

TAT

Tyr

AGC

Ser

GTT

Val 385

GAT

TA

,eu

;AA

;lu

;AT

~sp

[AT

ryr 3TA Ial

GAA

3iu 290

TTT

Phe

ACA

Thr

TCT

Ser

AGC

Ser

ACA

Thr 370

ACA

Thr

GGT

68.

729 777 825 873 921 969 1017 1065 1113 1161 1209 1257 1305 Leu Thr Lys Giu Ala Asp Leu 380 TTT GGA GCA GAA CAT GAT CCC Asn Phe Gly Ala Giu His Asp Pro Asp Gly 395 400 CTA GCA GAA Leu Ala Glu 405 TGT GCC CCG AAT Cys Ala Pro Asn GAC CAG GGA GGG Asp Gin Gly Gly TAT GTC ATG Tyr Val Met TAT CCC ATA GCT GTG AGT GGC GAT CAC GAG AAC AAT A.AG ATG TTT TCA Tyr Pro Ile Ala Val Set Gly Asp His Giu Asn Asn Lys Met Phe Ser 420 425 430 .00 000 0 0 0 0

AAC

Asn 435

GAG

Giu

GAT

Asp

ACC

Thr

GAG

Asp

AAG

Lys 515

TGC

Cys

GAG

Asp

CCT

Pro

ACT

Thr

GTG

Val1 595

AAG

Lys

CGA

Arg

TGC

Gys

TGT

Gys

GAA

Giu

TGC

Cys

AGG

Arg 500

AAG

Lys

ACA

Thr

ACT

Th r

TTC

Phe

GAG

Asp 580

CCC

Pro

CCC

Pro

GTA

Val1

AGT

Set

TTT

P he

GGA

Gly

TGC

Gys 485

AAC

Asn

TGC

Cys

GGT

Giy

GTT

Val1

TGC

Cys 565

AAG

Asn

TAT

Tyr

TGT

Cys

GAG

Gin

AAA

Lys

CAA

Gin

GAA

Glu 470

AAC

As n

AGT

Set

CAG

Gin

A.AT

As n

TGG

Cys 550

GAG

Giu

TCC

Set

GTC

Val1

ACA

Thr

GAT

Asp 630

CAA

Gin

GAA

Glu 455

GAG

Glu

AGC

Set

GCT

Pro

GAG

Giu

AGC

Set 535

TTG

Le u

AGG

Atg

TGC

Cys

GAT

Asp

GTA

Val 615

GTA

Val

TCA

Ser 440

GGC

Arg

TGT

Cvs

GAG

Asp

TGG

Gys

GCG

Al a 520

AGT

Set

GAT

Asp

GAA

Giu

KAG

Ly s

GOT

Al a 600

GGA

Giy AT T Ile

ATC

Ile

AGG

Set

GAT

Asp

TGC

Gys

TGT

Cys 505

ATT

Ile

GAG

Giu

GTT

Leu

GAG

Gin

GTG

Val1 585

GAA

Giu

TTT

Phe

GAA

Glu

TAT

Tyr

AAT

Asn

GGT

Pro

AG

Thr 490

AAA

Ly s

A.AT

Asn

TGG

Gys

GGG

Gly

GAG

Gin 570

TOG

Cys

CAA

Gin

TOT

Cys

CGA

Arg

AAG

Lys

AAA

Lys

GG

Gly 475

TTG

Leu

AAG

Asn

GGT

Ala

CCG

Pro

AAG

Lys 555

GTG

Leu

TGC

Cys

AAG

Lys

GAG

Asp

TTT

Phe 635

ACC

Thr

OTT

Val 460

ATG

Ile

AAG

Lys

TOT

Cys

ACT

Thr

GGT

Pro 540

TGT

Cys

GAG

Giu

AGO

Arg

AAC

As n

ATG

Met 620

TGG

Tro

ATT

Ile 445

TGT

Cys

ATG

Met

GAA

Giu

GAG

Gin

TGG

Cys 525

CCA

Pro

AAG

Lys

TGG

Set

GAG

Asp

TTA

Le u 605

AAT

As n

GAT

Asp

GAA

Giu

GGG

Gly

TAT

Tyr

GGT

Gly

TTT

P he 510

AAA

Lys

GGA

Gly

GAT

Asp

TGT

Gys

GTT

Leu 590

TTT

P he

GGC

Gly

TTC

Phe AGT AAG Set Lys AAG TG Asn Set GTG AAC Leu Asn 480 GTG GAG Val Gin 495 GAG ACT Giu Thr GGC GTG Gly Vai AAT OCT Asn Ala GGO AAA Giy Lys 560 GA TOT Ala Gys 57 TGC GGC Set Oly TTG AGG Leu Arg AAA TGT Lys Cys ATT GAG Ile Asp 640 GCC GAG Ala Gin 450 AGG GTG Arg Val 465 AAC GAG Asn Asp TGC AGT Gys Set 0CC GAG Ala Gin TOG TAG Ser Tyr 530 GAA OAT Giu Asp 545 TGC ATC Cys Ile AAT GAA Asn Giu GGC TOT Arg Cys A-AA GGA Lys Gly 610 GAG AAA Giu Lys 625 GAG CTG Gin Leu 1353 1401 1449 1497 1545 1593 1641 1689 1737 1785 1833 1881 1929 1977 2025 AGC ATC AAT ACT TTT OGA AAG TTT TTA GCA GAG AAG ATC OTT GGG TCT Ser Ile Asn Thr Phe Gly Lys Phe Leu Ala Asp Asn Ile Val Gly Set GTC CTG GTT TTC TCC TTG ATA TTT TGG ATT CCT TTC AGC ATT CTT GTC Val Leu Val Phe Ser Leu Ile Phe Trp Ile Pro Phe Ser Ile Leu Val 2073 660 665 67 0

S.

CAT TGT His Cys 675 TTT CAC Phe His GTT CGC Val Arg CAG CCT Gin Pro CAC CAG His Gin 740 ATG GAG Met Asp 755 GCT GCC Ala Ala GAA AAG Giu Lys AAA GAA Lys Giu GTG GAT Val Asp CCC AGT Pro Ser ATT ATC Ile Ile 710 GCC CCT Ala Pro 725 AGA ATG Arg Met GAG GAT Glu Asp AAG TCA Lys Ser GCT GCC Ala Ala 790 ACA GAG Thr Glu 805 AAG AAA Lys Lys 680 AAC GTC Asn Val 695 AAA CCC Lys Pro GTG ATC Val Ile GAC ACC Asp Thr GGG TTT Gly Phe 760 TTT GAG Phe Glu 775 TCC TTT Ser Phe TGC TAA Cys

TTG

Leu

GAA

Glu

TTT

Phe

CCT

Pro

ATC

Ile 745

GAG

Glu

GAT

Asp

GAT

Asp

ATG

Met

CCT

Pro

TCG

Ser 730

CAG

Gin

AAG

Lys

CTC

Leu

AAA

Lys

CTG

Leu

GCG

Ala 715

GCG

Ala

GA.A

Giu

GAC

Asp

ACG

Th r

CAG

Gin

AGC

Se r 700

CCC

Pro

CCA

Pro

GAC

Asp

CCC:

Pro

GAG

Asp 780

TAT

Tyr 685

AGC

Ser

GAG

Gin

GCA

Ala Ccc Pro

TTC

Phe 765

CAT

His

GAA

Giu

ATG

Met

ACT

Thr

GCT

Ala

AGC

Ser 750

GCA

Pro

CCG

Pro

TCT

Ser

GAT

Asp

CCA

Pro

CCA

Pro 735

ACA

Thr

AAT

Asn

GTC

Val

CTG

Leu

TCT

Se r

GGC

Gly 720

AAA

Lys

GAG

Asp

AGC

Se r

ACC

Thr TCT CTG Ser Leu 690 GCA TCG Ala Ser 705 CGC CTG Arg Leu CTG GAG Leu Asp TCA CAT Ser His AGC ACA Ser Thr 770 AGA AGT Arg Ser 785 2121 2169 2217 2265 2313 2361 2409 2457 2475 AAA CTG GAG CGT GAG A.AT GGT GTT GAG AGC Lys Leu Gin Arg Gin Asn Arg Val Asp Ser 800 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 807 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO;2: Arg Pro Pro Asp Asp Pro Gly Phe Gly Pro His Gin Arg Leu Giu 10 Leu Asp Ser Leu Leu Ser Asp Tyr Asp Ile Leu Ser Leu Ser Asn 25 Gin Gin His Ser Val Arg Lys Arg Asp Leu Gin Thr Ser Thr His 40 Vai Giu Thr Leu Leu Thr Phe Ser Ala Leu Lys Arg His Phe Lys Leu 55 Tyr Leu Thr Ser Ser Thr Glu Arg Phe Ser Gin Asn Phe Lys Val Vai 70 75 Val Val Asp Gly Lys Asn Glu Ser Glu Tyr Thr Val Lys Trp Gin Asp 90 Phe Phe Thr Gly His Val Val Gly Giu Pro Asp Ser Arg Val Leu Ala 100 105 110 His Ile Arg Asp Asp Asp Val Ile Ile Arg Ile Asn Thr Asp Giy Ala 115 120 125 Giu Tyr Asn Ile Giu Pro Leu Trp Arg Phe Val Asn Asp Thr Lys Asp 130 135 140 Lys Arg Met Leu Val Tyr Lys Ser Giu Asp Ile Lys Asn Vai Ser Arg 145 150 155 160 Leu Gin Ser Pro Lys Vai Cys Giy Tyr Leu Lys Vai Asp Asn Giu Giu 165 170 175 Leu Leu Pro Lys Gly Leu Val Asp Arg Glu Pro Pro Giu Giu Leu Vai 180 185 190 His Arg Vai Lys Arg Arg Ala Asp Pro Asp Pro Met Lys Asn Thr Cys *195 200 205 Lys Leu Leu Val Val Aia Asp His Arg Phe Tyr Arg Tyr Met Gly Arg 210 215 220 Giy Giu Giu Ser Thr Thr Thr Asn Tyr Leu Ile Giu Leu Ile Asp Arg 225 230 235 240 *Val Asp Asp Ile Tyr Arg Asn Thr Ser Trp Asp Asn Ala Giy Phe Lys 245 250 255 Gly Tyr Giy Ile Gin Ile Giu Gin Ile Arg Ile Leu Lys Ser Pro Gin **.260 265 270 Giu Vai Lys Pro Giy Giu Lys His Tyr Asn Met Ala Lys Ser Tyr Pro *275 280 285 Asn Giu Giu Lys Asp Ala Trp Asp Val Lys Met Leu Leu Giu Gin Phe 290 295 300 Ser Phe Asp Ile Aia Giu Giu Ala Ser Lys Val Cys Leu Ala His Leu 305 310 315 320 Phe Thr Tyr Gin Asp Phe Asp Met Gly Thr Leu Gly Leu Ala Tyr Val 325 330 335 Gly Ser Pro Arg Ala Asn Ser His Gly Gly Val Cys Pro Lys Ala Tyr 340 345 350 Tyr Ser Pro Val Gly Lys Lys Asn Ile Tyr Leu Asn Ser Gly Leu Thr 355 360 365 Se r Thr Lys Asn Tyr Gly 370 Thr Ile Leu Thr Lys Giu Ala Asp Leu 380 Val 385 Asp Val Phe Ala Arg 465 As n Cys Al a Ser Glu 545 Cys Asn Arg Lys Glu 625 Gin Gly Me t Ser Gin 450 Val1 Asp Se r Gin Tyr 530 Asp Ile Glu Cys Gly 610 Lys Leu Ser Thr Leu Tyr Asn 435 Giu Asp Thr Asp Lys 515 Cys Asp Pro Thr Val 595 Lys Arg Ser Val iis kia ?ro 420 :ys Cys Glu Cys Arg 500 Lys Thr Thr Phe Asp 580 Pro Pro Val1 I le Le u Glu Glu 405 Ile Ser Phe Gly Cys 485 As n Cys Gly Val Cys 565 As n Tyr Cys Gin Asn 645 Val .eu 390 -ys Ala Ly s iln Giu 470 As n Se r Gln Asn Cys 550 Giu Ser Val1 Th r AsD 630 Thr Gly Ala Val Gin Giu 455 Glu Ser Pro Giu Ser 535 Leu Arg Cys Asp Val1 615 Val Phe Hiis P ro Ser Se r 440 Arg Cys Asp Cys Al a 520 Ser Asp Giu Lys5 Al a 600 Gly Ile Glj As n Asn Gly 425 Ile Ser Asp Cys Cys 505 Ile Glu Leu Gin Val 585 Giu Phe Glu Lys I lE P he Glu 410 Asp Tyr Asn Pro Th r 490 Lys As n Cys Gly Gln 570 Cvs Gin Cvs Arc PhE 65( PhE Gly P 395 Asp C His C Lys I Lys Gly 475 Leu Asn Ala Pro Lys 555 Leu Cys Lys Asp Phe 635 Leu STrp ~la ;ln lu Lhr la 1 460 Ile Lys Thr Pro 540 Cys Glu Arg As r Met 620 T rj Al~ 114 Glu Gly Asn Ile 445 Cys Met Giu Gin Cys 525 Pro Lys Ser Asp Leu 605 Asn Asp Asp Pro His Gly As n 430 Glu Gly Tyr Gly Phe 510 Lys Gly Asp Cys Le u 590 Phe Glv Phe Asr Phe 67 0 Asp P 4 Lys TI 415 Lys I- Ser I AsnI Leu Val 495 Glu Gly Asn Gly Aia 575 Se r Le u Lys Ile Ile 655 Ser ro ~00 'yr ~et .ys 3er ks n 480 31n rhr Val1 Al a Lys 560 Cys Gly Arg Cys Asp 640 Val1 Ile Phe Ser Let 660 Leu Val His 675 Cys Val Asp Lys Leu Asp Lys Gin Giu Ser Leu Ser Leu Phe His Pro Ser Asn Val Glu Met Leu Ser Ser Met Asp Ser 690 695 700 Ala Ser Val Arg Ile Ile Lys Pro Phe Pro Ala Pro Gin Thr Pro Gly 705 710 715 720 Arg Leu Gin Pro Ala Pro Val Ile Pro Ser Ala Pro Ala Ala Pro Lys 725 730 735 Leu Asp His Gin Arg Met Asp Thr Ile Gin Glu Asp Pro Ser Thr Asp 740 745 750 Ser His Met Asp Glu Asp Gly Phe Glu Lys Asp Pro Phe Pro Asn Ser 755 760 765 Ser Thr Ala Ala Lys Ser Phe Glu Asp Leu Thr Asp His Pro Val Thr 770 775 780 Arg Ser Glu Lys Ala Ala Ser Phe Lys Leu Gin Arg Gin Asn Arg Val 785 790 795 800 Asp Ser Lys Glu Thr Glu Cys 805 805 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 2097 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO

FEATURE:

NAME/KEY: CDS LOCATION: 52..2094 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGAGGCAGT CTCTCCTATT CCTGACCAGC GTGGTTCCTT TCGTGCTGGC G CCG CGA 57 Pro Arg CCT CCG GAT GAC CCG GGC TTC GGC CCC CAC CAG AGA CTC GAG AAG CTT 105 Pro Pro Asp Asp Pro Gly Phe Gly Pro His Gin Arg Leu Glu Lys Leu 810 815 820 825 GAT TCT TTG CTC TCA GAC TAC GAT ATT CTC TCT TTA TCT AAT ATC CAG 153 Asp Ser Leu Leu Ser Asp Tyr Asp Ile Leu Ser Leu Ser Asn Ile Gin 830 835 840 CAG CAT TCG GTA AGA AAA AGA GAT CTA CAG ACT TCA ACA CAT GTA GAA 201 Gin His Ser Val Arg Lys Arg Asp Leu Gin Thr Ser Thr His Val Glu 845 850 855 ACA CTA CTA ACT TTT TCA GCT TTG AAA AGG CAT TTT AAA TTA TAC CTG Thr Leu Leu Thr Phe Ser Ala Leu Lys Arg His Phe 860 865 Lys Leu Tyr Leu 870 a ACA TCA AGT ACT GAA Thr Ser Ser Thr Glu 875 GAT GGT AAA AAC GAA Asp Gly Lys Asn Glu 890 ACT GGA CAC GTG GTT Thr Gly His Val Val 910 AGA GAT GAT GAT GTT Arg Asp Asp Asp Val 925 AAC ATA GAG CCA CTT Asn Ile Glu Pro Leu 940 ATG TTA GTT TAT AAA Met Leu Val Tyr Lys 955 TCT CCA AAA GTG TGT Ser Pro Lys Val Cys 970 CCA AAA GGG TTA GTA Pro Lys Gly Leu Val 990 GTG AAA AGA AGA GCT Val Lys Arg Arg Ala 1005 TTG GTG GTA GCA GAT Leu Val Val Ala Asp 1020 GAG AGT ACA ACT ACA Glu Ser Thr Thr Thr 1035 GAC ATC TAT CGG AAC Asp Ile Tyr Arg Asn 1050 GGA ATA CAG ATA GAG Gly Ile Gin Ile Glu CGT TTT TCA CAA AAT Arg Phe Ser Gin Asn 880 AGC GAG TAC ACT GTA Ser Glu Tyr Thr Val 895 GGT GAG CCT GAC TCT Gly Glu Pro Asp Ser 915 ATA ATC AGA ATC AAC Ile Ile Arg Ile Asn 930 TGG AGA TTT GTT AAT Trp Arg Phe Val Asn 945 TCT GAA GAT ATC AAG Ser Glu Asp Ile Lys 960 GGT TAT TTA AAA GTG Gly Tyr Leu Lys Val 975 GAC AGA GAA CCA CCT Asp Arg Glu Pro Pro 995 GAC CCA GAT CCC ATG Asp Pro Asp Pro Met 1010 CAT CGC TTC TAC AGA His Arg Phe Tyr Arg 1025 AAT TAC TTA ATA GAG Asn Tyr Leu Ile Glu 1040 ACT TCA TGG GAT AAT Thr Ser Trp Asp Asn 1055 CAG ATT CGC ATT CTC Gin Ile Arg Ile Leu 0 107

TTC

Phe

AAA

Lys 900

AGG

Arg

ACA

Thr

GAT

Asp

AAT

Asn

GAT

Asp 980

GAA

Glu

AAG

Lys

TAC

Tyr

CTA

Leu

GCA

Ala AAG GTC GTG GTG GTG Lys Val Val Val Val 885 TGG CAG GAC TTC TTC Trp Gin Asp Phe Phe 905 GTT CTA GCC CAC ATA Val Leu Ala His Ile 920 GAT GGG GCC GAA TAT Asp Gly Ala Glu Tyr 935 ACC AAA GAC AAA AGA Thr Lys Asp Lys Arg 950 GTT TCA CGT TTG CAG Val Ser Arg Leu Gin 965 AAT GAA GAG TTG CTC Asn Glu Glu Leu Leu 985 GAG CTT GTT CAT CGA Glu Leu Val His Arg 1000 AAC ACG TGT AAA TTA Asn Thr Cys Lys Leu 1015 ATG GGC AGA GGG GAA Met Gly Arg Gly Glu 1030 ATT GAC AGA GTT GAT Ile Asp Arg Val Asp 1045 GGT TTT AAA GGC TAT Gly Phe Lys Gly Tyr 297 345 393 441 489 537 585 633 681 729 777 825 1060 AAG TCT Lys Ser 5 1065 CCA CAA GAG GTA Pro Gin Glu Val 1080 TAC CCA AAT GAA Tyr Pro Asn Glu 1095 AAA CCT GGT Lys Pro Gly GAA AAG CAC TAC AAC Glu Lys His Tyr Asn 1085 ATG GCA AAA AGT Met Ala Lys Ser 1090 GAA AAG GAT GCT TGG GAT GTG AAG ATG TTG CTA GAG CAA TTT AGC TTT 969 Glu Lys Asp Ala Trp Asp Val Lys Met Leu Leu Glu Gin Phe Ser Phe 1100 1105 1110 GAT ATA GCT GAG GAA GCA TCT AAA GTT TGC TTG GCA CAC CTT TTC ACA 1017 Asp Ile Ala Glu Glu Ala Ser Lys Val Cys Leu Ala His Leu Phe Thr 1115 1120 1125 TAC CAA GAT TTT GAT ATG GGA ACT CTT GGA TTA GCT TAT GTT GGC TCT 1065 Tyr Gin Asp Phe Asp Met Gly Thr Leu Gly Leu Ala Tyr Val Gly Ser 1130 1135 1140 1145 CCC AGA GCA AAC AGC CAT GGA GGT GTT TGT CCA AAG GCT TAT TAT AGC 1113 Pro Arg Ala Asn Ser His Gly Gly Val Cys Pro Lys Ala Tyr Tyr Ser 1150 1155 1160 CCA GTT GGG AAG AAA AAT ATC TAT TTG AAT AGT GGT TTG ACG AGC ACA 1161 Pro Val Gly Lys Lys Asn Ile Tyr Leu Asn Ser Gly Leu Thr Ser Thr 1165 1170 1175 S AAG AAT TAT GGT AAA ACC ATC CTT ACA AAG GAA GCT GAC CTG GTT ACA 1209 Lys Asn Tyr Gly Lys Thr Ile Leu Thr Lys Glu Ala Asp Leu Val Thr 1180 1185 1190 ACT CAT GAA TTG GGA CAT AAT TTT GGA GCA GAA CAT GAT CCG GAT GGT 1257 Thr His Glu Leu Gly His Asn Phe Gly Ala Glu His Asp Pro Asp Gly 1195 1200 1205 CTA GCA GAA TGT GCC CCG AAT GAG GAC CAG GGA GGG AAA TAT GTC'ATG 1305 Leu Ala Glu Cys Ala Pro Asn Glu Asp Gin Gly Gly Lys Tyr Val Met S 1210 1215 1220 1225 TAT CCC ATA GCT GTG AGT GGC GAT CAC GAG AAC AAT AAG ATG TTT TCA 1353 Tyr Pro Ile Ala Val Ser Gly Asp His Glu Asn Asn Lys Met Phe Ser 1230 1235 1240 SAAC TGC AGT AAA CAA TCA ATC TAT AAG ACC ATT GAA AGT AAG GCC CAG 1401 Asn Cys Ser Lys Gin Ser Ile Tyr Lys Thr Ile Glu Ser Lys Ala Gin 1245 1250 1255 GAG TGT TTT CAA GAA CGC AGC AAT AAA GTT TGT GGG AAC TCG AGG GTG 1449 Glu Cys Phe Gin Glu Arg Ser Asn Lys Val Cys Gly Asn Ser Arg Val 1260 1265 1270 GAT GAA GGA GAA GAG TGT GAT CCT GGC ATC ATG TAT CTG AAC AAC GAC 1497 Asp Glu Gly Glu Glu Cys Asp Pro Gly Ile Met Tyr Leu Asn Asn Asp 1275 1280 1285 ACC TGC TGC AAC AGC GAC TGC ACG TTG AAG GAA GGT GTC CAG TGC AGT 1545 Thr Cys Cys Asn Ser Asp Cys Thr Leu Lys Glu Gly Val Gin Cys Ser 1290 1295 1300 1305 GAC AGG AAC AGT CCT TGC TGT AAA AAC TGT CAG TTT GAG ACT GCC CAG 1593 Asp Arg Asn Ser Pro Cys Cys Lys Asn Cys Gin Phe Glu Thr Ala Gin 1310 1315 1320 AAG AAG TGC CAG GAG GCG ATT AAT GCT ACT TGC AAA GGC GTG TCC TAC 1641 Lys Lys Cys Gin Glu Ala Ile Asn Ala Thr Cys Lys Gly Val Ser Tyr 1325 1330 1335 TGC ACA GGT AAT AGC AGT GAG TGC CCG CCT CCA GGA AAT GCT GXA GAT 1689 Cys Thr Gly Asn Ser Ser Glu Cys Pro Pro Pro Gly Asn Ala Glu Asp 1340 1345 1350 GAC ACT GTT TGC TTG GAT CTT GGC AAG TGT AAG GAT GGG AAA TGC ATC 1737 Asp Thr Val Cys Leu Asp Leu Gly Lys Cys Lys Asp Gly Lys Cys Ile 1355 1360 1365 CCT TTC TGC GAG AGG GAA CAG CAG CTG GAG TCC TGT GCA TGT AAT GAA 1785 Pro Phe Cys Glu Arg Glu Gin Gin Leu Glu Ser Cys Ala Cys Asn Glu 1370 1375 1380 1385 ACT GAC AAC TCC TGC AAG GTG TGC TGC AGG GAC CTT TCC GGC CGC TGT 1833 Thr Asp Asn Ser Cys Lys Val Cys Cys Arg Asp Leu Ser Gly Arg Cys 1390 1395 1400 GTG CCC TAT GTC GAT GCT GAA CAA AAG AAC TTA TTT TTG AGG AAA GGA 1881 Val Pro Tyr Val Asp Ala Glu Gin Lys Asn Leu Phe Leu Arg Lys Gly 1405 1410 1415 AAG CCC TGT ACA GTA GGA TTT TGT GAC ATG AAT GGC AAA TGT GAG AAA 1929 Lys Pro Cys Thr Val Gly Phe Cys Asp Met Asn Gly Lys Cys Glu Lys 1420 1425 1430 **ee CGA GTA CAG GAT GTA ATT GAA CGA TTT TGG GAT TTC ATT GAC CAG CTG 1977 Arg Val Gin Asp Val Ile Glu Arg Phe Trp Asp Phe Ile Asp Gin Leu S**1435 1440 1445 AGC ATC AAT ACT TTT GGA AAG TTT TTA GCA GAC AAC ATC GTT GGG TCT 2025 Ser Ile Asn Thr Phe Gly Lys Phe Leu Ala Asp Asn Ile Val Gly Ser 1450 1455 1460 1465 GTC CTG GTT TTC TCC TTG ATA TTT TGG ATT CCT TTC AGC ATT CTT GTC 2073 Val Leu Val Phe Ser Leu Ile Phe Trp Ile Pro Phe Ser Ile Leu Val 1470 1475 1480 CAT TGT GTA ACG TCG AAA TGC TGA 2097 His Cys Val Thr Ser Lys Cys ~1485 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 681 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Pro Arg Pro Pro Asp Asp Pro Gly Phe Gly Pro His Gin Arg Leu Glu 1 5 10 Lys Leu Asp Ser Leu Leu Ser Asp Tyr Asp I.le. Leu Ser Leu Ser Asn 25 Ile Gin Gin His Ser Val Arg Lys Arg Asp Leu Gin Thr Ser Thr His 40 Val Glu Thr Leu Leu Thr Phe Ser Ala Leu Lys 55 Arg His Phe Lys Leu a Tyr Val Phe His Glu Lys 145 Leu Leu His Lys Gly 225 Val Gly Glu Asn Ser 305 Phe Gly Tyr Leu Val Phe Ile Tyr 130 Arg Gin Leu Arg Leu 210 Glu Asp Tyr Val Glu 290 Phe Thr Ser Ser Thr Asp Thr Arg 115 Asn Met Ser Pro Val 195 Leu Glu Asp Gly Lys 275 Glu Asp Tyr Pro Pro 355 Ser Gly Gly 100 Asp lle Leu Pro Lys 180 Lys Val Ser Ile Ile 260 Pro Lys Ile Gin Arg 340 Val Ser Lys His Asp Glu Val Lys 165 Gly Arg Val Thr Tyr 245 Gin Gly Asp Ala Asp 325 Ala Gly Thr 70 Asn Val Asp Pro Tyr 150 Val Leu Arg Ala Thr 230 Arg Ile Glu Ala Glu 310 Phe Asn Glu Glu Val val Leu 135 Lys Cys Val Ala Asp 215 Thr Asn Glu Lys Trp 295 Glu Asp Ser Arg Ser Gly Ile 120 Trp Ser Gly Asp Asp 200 His Asn Thr Gin His 280 Asp Ala Met His Phe Glu Glu 105 Ile Arg Glu Tyr Arg 185 Pro Arg Tyr Ser Ile 265 Tyr Val Ser Gly Gly 345 Ile Ser Tyr 90 Pro Arg Phe Asp Leu 170 Glu Asp Phe Leu Trp 250 Arg Asn Lys Lys Thr 330 Gly Tyr Gln 75 Thr Asp Ile Val Ile 155 Lys Pro Pro Tyr Ile 235 Asp Ile Met Met Val 315 Leu Val Leu Asn Val Ser Asn Asn 140 Lys Val Pro Met Arg 220 Glu Asn Leu Ala Leu 300 Cys Gly Cys Asn Phe Lys Arg Thr 125 Asp Asn Asp Glu Lys 205 Tyr Leu Ala Lys Lys 285 Leu Leu Leu Pro SSer 365 Lys Trp Val 110 Asp Thr Val Asn Glu 190 Asn Met Ile Gly Ser 270 Ser Glu Ala Ala Lys 350 Gly Val Gin Leu Gly Lys Ser Glu 175 Leu Thr Gly Asp Phe 255 Pro Tyr Gin His Tyr 335 Ala Leu Val Asp Ala Ala Asp Arg 160 Glu Val Cys Arg Arg 240 Lys Gin Pro Phe Leu 320 Val Tyr Thr Lys Lys Asn 360 Ser Val 385 Asp Val Phe Ala Arg 465 Asn Cys Ala Ser Glu 545 Cys Asn Arg Lys Glu 625 Gin Gly rhr 370 rhr ;ly Met Ser Gln 450 Va1 Asp Ser Gin Tyr 530 Asp Ile Glu Cys Gly 610 Lys Leu Ser Lys Thr Leu Tyr Asn 435 Glu Asp Thr Asp Lys 515 Cys Asp Pro Thr Val 595 Lys Arg Ser Val Asn His Ala Pro 420 Cys Cys Glu Cys Arg 500 Lys Thr Thr Phe Asp 580 Pro Pro Va1 Ile Leu Tyr Glu Glu 405 Ile Ser Phe Gly Cys 485 Asn Cys Gly Val Cys 565 Asn Tyr Cys Gin Asn 645 Val Gly I Leu 390 Cys Ala Lys Gin Glu 470 Asn Ser Gln Asn Cys 550 Glu Ser Val Thr Asp 630 Thr Phe ,y;s 375 3ly Ala Val Gln Glu 455 Glu Ser Pro Glu Ser 535 Leu Arg Cys Asp Val 615 Val Phe Se Thr His Pro Ser Ser 440 Arg Cys Asp Cys Ala 520 Ser Asp Glu Lys Ala 600 Gly Ile Glj *Let Ile Leu Asn Phe Asn Glu 410 Gly Asp I 425 Ile Tyr Ser Asn Asp Pro Cys Thr 490 Cys Lys 505 Ile Asn Glu Cys Leu Gly Gin Gin 570 Val Cys 585 Glu Gin Phe Cys Glu Arg Lys Phe 650 Ile Phe rhr 3ly 395 Asp His Lys Lvs Gly 475 Leu Asn Ala Pro Lys 555 Leu Cys Lys Asp Phe 635 Lys 380 Ala Gin Glu Thr Va1 460 Ile Lys Cys Thr Pro 540 Cys Glu Arg Asn Met 620 Try Glu Glu I Gly Asn Ile 445 Cys Met Glu Gin Cys 525 Pro Lys Ser Asp Leu 605 Asn Asp Ala H-is Gly Asn 430 Glu Gly Tyr Gly Phe 510 Lys Gly Asp Cys Leu 590 Phe Gly Phe Asp I Asp I Lys 415 Lys 1 Ser Asn Leu Val 495 Glu Gly Asn Gly Ala 575 Ser Leu Lys Ile Leu ?ro 100 Tyr let Lys Ser Asn 480 Gin Thr Val Ala Lys 560 Cys Gly Arg Cys Asp 640 Leu Ala Asp Asn Ile Val 655 Trd Ile Pro Phe Ser Ile Leu Val His Cys Val Thr Ser 675 Lys Cys 680 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Ala Gin Ala Val Arg Ser Ser 1 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid e: STRANDEDNESS: single TOPOLOGY: linear (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: I 9 AARTAYGTNA TGTAYCC 17 INFORMATION FOR SEQ ID NO:7: o SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCRCARTCRC AYTCYTC 17 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Glu Cys Asp Cys Gly 1 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Ile Ala Val Ser Gly Asp His Glu Asn Asn Lys 1 5

S..

0So as*

Claims (9)

1. An isolated and purified TACE polypeptide.

2. An isolated and purified polypeptide according to claim 1, that has a molecular weight of about 80 kD.

3. An isolated and purified polypeptide according to claim 1, in non-glycosylated form.

4. An isolated and purified polypeptide according to claim 1 selected from the group consisting of a polypeptide comprising amino acids 18-Xaa of SEQ ID NO:2 wherein Xaa ia an amino acid selected from the group consisting of amino acids 671 through 824. Isolated and purified antibodies that bind to the polypeptide according to claim 1. Isolated and purified antibodies according to claim 5, wherein the antibodies are monoclonal antibodies.

7. A method for detecting the TACE-inhibiting activity of a molecule, comprising mixing said molecule with a substrate, incubating a polypeptide according to claim 1 with the mixture, and chromatographically determining the extent of substrate cleavage.

8. A method for detecting the TACE-inhibiting activity of a molecule according to claim 7, wherein the substrate comprises the amino acid sequence Lcu-Ala-Gln-Ala-Val- Arg-Ser-Ser.

9. A method of using a polypeptide according to claim 1 in a structure-based design of an inhibitor of said polypeptide, comprising the steps of determining the three-dimensional structure of such polypeptide. analyzing the three-dimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predictive reactive site, and determining the polypcptidc-inhibiting activity of the molecule. A method for detecting the TNF-cleaving ability of a molecule, comprising incubating said molecule with a substrate that comprises the amino acid sequence Lcu.Ala- Gln-Ala-Val.Arg-Scr-Ser, and determining the extent of substrate cleavage.

11. An isolated nucleic acid selected from the group consisting of: the coding region of a native mammalian TACE gene; cDNA comprising nucleoddes

52-2472 of SEQ ID NO:1; nucleic acid that is at least 80% identical to the nucleic acid of or and that encodes a polypcptide that converts TNF-a from the 26 kD form to the 17 kD form: and nucleic acid which is degenerate as a result of the genetic code to a nucleic acid defined in or and which encodes biologically active TACE. 12. An isolated nucleic acid according to claim 11. wherein the TACE is human TACE. 13. An isolated nucleic acid according to claim 11. which encodes a polypeptide comprising amino acids 18-671 of SEQ ID NO:2. 14. An expression vector that directs the expression of a nucleic acid sequence according to claim 11. 15. A host cell transfected or transformed with the expression vector according to claim 11. 16. A process for producing a TACE polypeptide, comprising culturing a host cell according to claim 15 under conditions promoting expression, and recovering the polypeptide from the culture medium. 17. A method of inhibiting the cleavage of TNF-a from cell membranes in a mammal comprising administering to such mammal an effective amount of a compound that inhibits the TNF-a proteolytic activity of an enzyme comprising the sequence of amino acids 18-671 of SEQ ID NO:2. 18. A method of inhibiting TNF-a cleavage from cell membranxes comprising blocking the binding of TNF-a with an enzyme having the sequence of amino acids 18-671 of SEQ ID NO:2. 19. A method for treating a mammal having a disease characterized by an overproduction or an upregulated production of TNF-a. comprising administering to the mammal a composition comprising an amount of a compound that effectively inhibits the TNF-a proteolytic activity of an enzyme comprising the sequence of amino acids 18-671 of SEQ ID NO:2. DATED: 18 February, 2000 PHILLIPS ORMONDE FITZPATRICK Attorneys for: P ORTO IMMUNEX CORPORATION