AU752369B2 - TNF-alpha converting enzyme - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

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

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.555

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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- 60 0 6 q TNF-a CONVERTING ENZYME This application is a divisional from parent application 63781/96, the entire content of which is incorporated herein by reference.

FIELD OF THE INVENTION The invention is directed to purified and isolated TNF-a convening enzyme, the nucleic acids encoding such enzyme, processes for production of recombinant TNF-a convertases, pharmaceutical compositions containing such enzymes, and their use in various assays and therapies.

BACKGROUND OF THE INVENTION Tumor necrosis factor-a (TNF-a, also known as cachectin) is a mammalian protein capable of inducing a variety of effects on numerous cell types. TNF-a was initially characterized by its ability to cause lysis of tumor cells and is produced by activated cells such as mononuclear phagocytes, T-cells, B-cells, mast cells and NK cells. There are two forms of TNF-a, a type II membrane protein of relative molecular mass 26,000 (26 kD) and a soluble 17 kD form generated from the cell-bound protein by proteolytic cleavage.

TNF-a is a principal mediator of the host response to gram-negative bacteria.

Lipopolysaccharide (LPS, also called endotoxin), derived from the cell wall of gramnegative bacteria, is a potent stimulator of TNF-a synthesis. Because the deleterious effects which can result from an over-production or an unregulated-production of TNF-a are extremely serious, considerable efforts have been made to control or regulate the serum level of TNF-a. An important part in the effor to effectively control serum TNF-a levels is the understanding of the mechanism of TNF-a biosynthesis.

The mechanism by which TNF-a is secreted has not previously been elucidated.

S" Kriegler et al. Cell, 53:45 (1988) conjectured that TNF-a "secretion" is due to the converting of the 26 kD membrane-bound molecule by a then unknown proteolytic enzyme or protease. Scuderi et. al., J. Immunology, 143:168 (1989), suggested that the release of TNF-a from human leukocyte cells is dependent on one or more serine proteases, a S 30 leukocyte elastase or trypsin. A serine protease inhibitor, p-toluenesulfonyl-L-arginine methyl ester, was found to suppress human leukocyte TNF-a release in a concentrationdependent manner. Scuderi et. al. suggested that an arginine methyl ester competes for the arginine-binding site in the enzyme's reactive center and thereby blocks hydrolysis. The lysine and phenylalanine analogs of the inhibitor reportedly failed to mimic the arginine methyl ester. However, it was never shown that this compound acted by inhibiting a protease that cleaves the 26 kD TNF. More recently, it has been reported that metalloprotease inhibitors block the release of TNF from THP-1 cells. See Mohler et al., Nature 370:218 (1994); Gearing et al., Nature, 370:555 (1994); and McGeehan et al., Nature, 370:568 (1994).

1B 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 Zn2+, 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.

k'R C WINV40P.')9n!nDNOET LTE%178 DOC 1c 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-Gln-Ala-Val-Arg- Ser-Ser, and determining the extent of substrate cleavage.

In an even further aspect of the invention provides an isolated and purified TACE polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:9; a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:9; a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 477; .i a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 671; a fragment of SEQ ID NO:2 such that a polypeptide with an amino acid sequence consisting of said fragment is capable of converting TNF-a from the 26 20 kD form to the 17 kD form; a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:9; a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 477; 25 a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 671; a fragment of SEQ ID NO:4 such that a polypeptide with an amino acid sequence consisting of said fragment is capable of converting TNF-a from the 26 kD form to the 17 kD form; an amino acid sequence that is at least 80% identical to the amino acid Ssequence of any of and an amino acid sequence that is at least 90% identical to the amino acid sequence of any of S19q onaXSharon\SJJspeci\spo17575.doc 1d The present invention further provides an isolated nucleic acid selected from the group consisting of: nucleic acid encoding a TACE polypeptide comprising an amino acid sequence selected from the group consisting of: (al) SEQ ID NO:2; (a2) SEQ ID NO:4; (a3) SEQ ID NO:9; (a4) a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:9; a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 215 to amino acid 477; (a6) a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 477; 15 (a7) a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 671; (a8) a fragment of SEQ ID NO:2 such that a polypeptide with an amino acid sequence consisting of said fragment is capable of converting STNF-a from the 26 kD form to the 17 kD form; (a9) a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:9; (a10) a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 215 to amino acid 477; (all) a fragment of SEQ ID NO:4, the fragment comprising the amino S. 25 acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 477; (a12) a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 671; (a13) a fragment of SEQ ID NO:4 such that a polypeptide with an amino acid sequence consisting of said fragment is capable of converting TNF-a from the 26 kD form to the 17 kD form; (a14) an amino acid sequence that is at least 80% identical to the amino acid sequence of any of (al) (al3); and an amino acid sequence that is at least 90% identical to the amino acid sequence of any of (al) (a13); le nucleic acid that is at least 80% identical to the nucleic acid of and that encodes a polypeptide that converts TNF-a from the 26 kD form to the 17 kD form; 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; and nucleic acid that hybridizes under conditions of moderate stringency to the nucleic acid of any of and that encodes a polypeptide that converts TNF-a from the 26 kD form to the 17 kD form.

In a further aspect the present invention provides 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.

In an even further aspect of the present invention provides a method of inhibiting TNF-a cleavage from cell membranes comprising blocking the binding of TNF-a 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 S: comprising an amount of a compound that effectively inhibits the TNF-alp proteolytic activity of an enzyme comprising the sequence of amino acids 18-671 of SEQ ID NO:2.

S. 25 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 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 nonantibody 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.

IC W:\ilona\Sharon\SJJspeci\spol7575.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 Zn 2 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 inventon petains 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 i: 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 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 Sencompassed 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 converting 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 certain 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 adhesion 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 Senables 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 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 25 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.

The 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 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 non- 25 identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of SProtein 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 an 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 5 containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, 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 .: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 glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are 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 N- 25 glycosylation 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 50'C 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 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 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.

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 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.

S. Prokaryotes include gram negative or gram positive organisms, for example, E. coli o or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, 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 15 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 2. yeast plasmid, an autonomously replicating 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. 2:149, 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 (Ampr 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. Natl. 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 75:1929, 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 pg/ml adenine and 20 Ig/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 15 adenine and 80 Ig/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, BiolTechnology 6:47 (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. 21:935, 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 II 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, 231:115 (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 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 20 affinity column using conventional techniques, in a high salt elution buffer and then S 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 25 disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or S 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 W091/06629) 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 S..target nucleic acid sequence by any gene transfer method, including, for example, CaPO 4 mediated 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 oligonucleotide 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 incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such composition's 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, infr. 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 S a Ka of greater than or equal to about 107 M- 1 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, 125 I-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 supernatants 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 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 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.

EXAMLE 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 S 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 NaC1 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-HC1 (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 NaCl 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-HCl (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-HCl (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 4 -Val 77 -Arg 78 -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 ig/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 Cl8 column and eluting 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 NaCI had about a 4fold higher specific activity than the starting 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-HCI (pH 0.15 M NaCI, 0.1 mM MnCl2, 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 Buffer C at 0.5 ml per minute over a period of 30 minutes. TACE activity (detected at this S 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 ll 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 (Sigma) 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 gl 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 p. 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.

C

EXAMPLE 2 25 Preparation of Isolated and Purified TACE This Example describes a method for further purifying the purified TACE as was 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) (Mullis 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 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 (Coring) 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, 1 25 I-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 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.

S

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 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: L..2472 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: atg Met 1 agg cag tct Arg Gin Ser otc Leu 5 cta ttc otg acc Leu Phe Leu Thr gtg gtt Oct ttc Val Val Pro Phe gtg otg Val Len gcg ccg cga Ala Pro Arg gag aag ott Glu Lys Len oct Pro ccg gat gao cog Pro Asp Asp Pro ggc Gly 25 ttc ggc coo cac Phe Gly Pro His cag aga otc Gin Arg Leu tct tta tct Ser Leu Ser gat tot ttg Asp Ser Leu otc tca Leu Ser 40 gao tao gat att Asp Tyr Asp Ile otc Leu aat ato Asn Ile cag cag oat tcg Gin Gin His Ser gta Val 55 aga aaa aga gat Arg Lys Arg Asp cag act toa aca Gin Thr Ser Thr cat gta gaa aca cta His Vai Glu Thr Leu ota Leu 70 act ttt tca gct Thr Phe Ser Ala ttg Leu 75 aaa agg cat ttt Lys Arg His Phe aaa Lys 192 240 288 tta tao ctg aca Len Tyr Len Thr toa Ser agt act gaa cgt Ser Thr Glu Arg toa caa aat ttc Ser Gin Asn Phe aag gto Lys Val 95 a gtg gtg gtg Vai Val Vai gao ttc ttc Asp Phe Phe 115 gat Asp 100 ggt aaa aac gaa Gly Lys Asn Glu ago Ser 105 gag tao act gta Glu Tyr Thr Vai aaa tgg cag Lys Trp Gin 110 agg gtt ota Arg Val Len act gga cac gtg Thr Gly His Val gtt Vai 120 ggt gag cot gao Gly Glu Pro Asp tct Ser 125 goc cac Ala His 130 ata aga gat gat Ile Arg Asp Asp gat Asp 135 gtt ata ato aga Val Ile Ile Arg ato Ile 140 aac aca gat ggg Asn Thr Asp Gly 432 qcc gaa tat aac ata gag cca ott tgg aga ttt gtt aat gat acc aaa Ala Glu Tyr Asn Ile Glu Pro Leu Trp Arg Phe Val Asn Asp Thr Lys 160 150 155 gac aaa a Asp Lys A cgt ttg c Arg Leu G gag ttg c Glu Leu L.

1 gtt cat c Val His T~ 210 tgt aaa t Cys Lys I 225 aga ggg Arg Gly aga gtt Arg Val aaa ggc Lys Gly caa gag Gin Glu 290 cca aat Pro Asn 305 ttt agc 1008 Phe Ser ctt ttc 1056 Leu Phe gtt ggc 1104 Val Gly tat tat 1152 Tyr Tyr 370 acg agc 1200 ga rg ag in :tc ,eu .95 :ga ~rg :ta ~eu 3aa 3iu gat 3 .sp tat Tyr 275 gt a Val1 gaa Gli ttt Ph ac~ Th; tc Se ag Se atg t Met L 1 tct c Ser P 180 cca a Pro I gtg ValI ttg Leu gag Giu gac Asp 260 gga *Gly aaa *Lys gaa 1Giu gat SAsp a tac r Tyr 340 t ccc r Pro 5 cca Pro ta e u 65 :ca 'ro iaa *ys ~aa ,ys jtg ~a 1 agt Ser 245 atc Ile ata Ile cct Pro aag Lys atz IlE 32 ca~ G1i ag Ar gt Va gtt tat aaa tct g Val Tyr Lys Ser G .1 aaa gtg tgt ggt t Lys Val Cys Gly T 185 ggg tta gta gac a Gly Leu Val Asp A~ 200 aga aga gct gac c Arg Arg Ala Asp F 215 gta gca gat cat Val Ala Asp His 230 aca act aca aatI Thr.Thr Thr Asn tat cgg aac act Tyr Arg Asn Thr 265 cag ata gag cag Gin Ile Giu Gin 280 ggt gaa aag cac Gly Glu Lys His 295 gat gct tgg gat Asp Aia Trp Asp 310 gct gag gaa gca Aia Giu Glu Ala Asp Phe Asp Met 345 gca aac agc cat g Ala Asn Ser His 360 t ggg aag aaa aat 1 Gly Lys Lys Asn 375 aa iu 70 at yr gat atc aag aat Asp tta Leu Ile Lys Asn gtt tca Val Ser 175 ga gaa c rg Glu F ca gat c ro Asp *gc ttcI rg Phe 235 ac tta 'yr Leu .50 .ca tgg ,er Trp att cgc Ile Arg tac aac Tyr Asn gtg aag Vai Lys 315 tct aaa Ser Lys 330 gga act Gly Thr aa gtg g ys Val I ,ca cct 'ro Pro 205 .cc atg ?ro Met ac aga ryr Arg ata gag Ile Glu gat aat Asp Asn att ctc Ile Leu 285 atg gca Met Ala 300 atg ttg Met Leu gtt tgc Vai Cys ctt gga Leu Giy gtt tgt ~at ~sp ;aa 3iu aag Lys tac Tyr cta Leu gca Ala 2-70 a ag Lys aaa Lys cta Le.

tt Let tt' Le' 35 cc aat As n gag Glu aac Asn atg Met att Ile 255 ggt Gly t ct Ser agt Ser gac iGit .Al 33 a gc u Al 0 a aa gaa Glu ctt Leu acg Thr ggc Giy 240 gac Asp t t t Phe cca *Pro tac *Tyr caa .i Gin 320 a cac a His t tat a Tyr g gct 528 576 624 672 720 768 816 912 gga ggt Gly Gly Val Cys Pro Lys Ala 365 atc Ile atc ttg Le u 380 aca aat As n aag agt Ser gaa ggt Gly gct ttg Leu gac aca aag aat tat ggt aaa acc Lys Asn Tyr Gly L-ys Thr Ile Leu Thr Lys G,'u Ala Asp 385 ctg qtt 1248 Leu Val ccg gat 1296 Pro Asp tat gtc 1344 Tyr Val atg ttt 1392 Met Phe 450 aag gcc 1440 Lys Ala 465 tcg agg 1488 Ser Arg aac aac 1536 Asn Asn cag tqc 1584 Gin Cys 390 aca act cat Thr Thr His 405 ggt cta gca Giv Leu Ala gaa ttg gga cat aat Giu Leu Gly His Asn 410 gaa tgt gcc ccg aat Giu Cys Ala Pro Asn 395 ttt Phe gga gca Gly Ala 400 gaa cat gat Giu His Asp 415 420 atg tat Met Tyr 435 ccc ata gct gtg Pro Ile Ala Val 440 425 agt ggc Ser Gly tca atc Ser Ile gag gac cag gga ggg aaa Giu Asp Gin Gly Gly Lys 430 gat cac gag aac aat aag Asp His Giu Asn Asn Lys 445 tat aag acc att gaa agt Tyr Ly's Thr Ile Giu Ser tca aac tgc agt Ser Asn Cys Ser cag gag tgt ttt Gin Giu Gys Phe aaa Lys 455 caa Gin 460 gtg gat gaa Vai Asp Giu 485 gac acc tgc Asp Thr Cys 500 agt gac agg Ser Asp Arg 470 gga Gly caa gaa cgc agc aat aaa Gin Giu Arg Ser Asn Lys 475 gaa gag tgt gat cct ggc Giu Giu Cys Asp Pro Giy gtt tgt ggg aac Val Cys Giy Asn 480 atc atg tat ctg Ile Met Tyr Leu 490 tgc aac agc gac tgc acg Cys Asn Ser Asp Cys Thr 505 aac agt cct tgc tgt aaa Asn Ser Pro Cys Cys Lys ttg aag gaa Leu Lys Giu 510 495 ggt gtc Giy Val As n 515 act gcc cag 1632 Thr Aia Gin 530 gtg tcc tac 1680 Val Ser Tyr 545 qct gaa gat 1728 Ala Giu Asp aag aag tgc cag Lys Lys Cys Gin 535 tgc aca ggt aat Cys Thr Gly Asn 550 gac act gtt tgc Asp Thr Val Cys 520 gag Giu tgt cag ttt gag Cys Gin Phe Giu 525 act tgc aaa ggc Thr Cys Lys Gly gcg att aat gct Ala Ile Asn Aia 540 agc agt gag tgc Ser Ser Giu Cys 555 ttg gat ctt ggc Leu Asp Leu Giy 570 agg gaa cag cag Arai Giu Gin Gin ccg cct Pro Pro aaa tgc 1776 Lys Cys tgt aat 1824 cca gga aat Pro Gly Asn 560 565 atc cct ttc Ile Pro Phe tgc gag aag tgt aag gat ggg Lys Cys Lys Asp Gly 575 ctg gag tcc tgt gca Leu Giu Ser Cys Ala 580 act gac 585 aac tcc tgc aag Asn Ser Cys Lys gtg tgc tgc agg Val Cys Cys Arg 605 590 gac ctt tcc Asp Leu Ser Cys Asn Gliu Thr Asp 600 ggc cgc 1872 Gly Arg 610 agg aaa 1920 Arg Lys 625 tgt gag 1968 Gys Glu gac cag.

2016 Asp Gin gtt ggg 2064 Val Gly tgt gtg ccc tat gtc Cys Val Pro Tyr Val 615 gga aag ccc tgt aca Gly Lys Pro Cys Thr 630 aaa cga gta cag gat Lys Arg Val Gin Asp 645 ctg agc atc aat act Leu Ser Ile Asn Thr gat got gaa caa aag aactta Asp Ala Giu Gin Lys Asn Leu 620 gta gga ttt tgt gac atg aat Val Gly Phe Cys Asp Met Asn ttt ttg Phe Leu ggc aaa Gly Lys 640 635 gta att gaa cga Val Ile Giu Arg 650 ttt gga aag ttt Phe Gly Lys Phe ttt tgg gat ttc att Phe Trp Asp Phe Ile 655 tta gca gac aac atc Leu Ala Asp Asn Ile tot Ser 675 660 gtc Val ctg Leu 665 gtt ttc tc ttg Val Phe Ser Leu 680 gtg gat aag aaa Vai Asp Lys Lys 670 ata ttt tgg att cot ttc ago Ile Phe Trp Ile Pro Phe Ser 685 ttg gat aaa cag tat gaa tct Leu Asp Lys Gin Tyr Giu Ser att ott gtc cat tgt 2112 Ile Leu Val His Cys 690 otg tot ctg ttt cac 2160 Leu Ser Leu Phe His 705 tct goa tog gtt cgc 2208 Ser Aia Ser Val Arg 695 ccc agt Pro Ser 710 att ato Ile Ile aac gto gaa Asn Vai Glu aaa ccc ttt Lys Pro Phe atg Met 715 700 otg ago ago Leu Ser Ser atg gat Met Asp 720 725 ggc cgc 2256 Gly Arg aaa ctg 2304 Lys Leu gao toa 2352 Asp Ser 770 ago ago 2400 Ser Ser 785 aco aga 2448 Thr Arg otg cag Leu Gin 740 gao cac Asp His cct goc cot gtg ato Pro Ala Pro Val Ile 745 cag aga atg gao aco Gin Arg Met Asp Thr 730 cct Pro ato Tie cct gog ccc cag act oca Pro Aia Pro Gin Thr Pro 735 tcg gog oca goa got oca Ser Aia Pro Ala Ala Pro 750 cag gaa gao ccc ago aca Gin Giu Asp Pro Ser Thr 755 cat His atg gao gag gat Met Asp Glu Asp 775 760 ggg Gly ttt Phe 765 gag aag gao coo ttc cca aat Glu Lys Asp Pro Phe Pro Asn aca got goc aag Thr Ala Ala Lys 790 agt gaa aag got Ser Giu Lys Ala 805 tca Ser qcc ttt gag gat otc Phe Glu Asp Leu 795 too ttt aaa otg 780 acg Thr gac cat Asp His ccg gto Pro Val 800 aat cgt cag cgt cag Ala Ser Phe Lys Leu 810 Gin Arg Gin Asn Arg 815 28a gtt gac age aaa gaa aca gag tgc taa 2475 Val Asp Ser Lys Glu Thr Glu Cys 820 r r r INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 824 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION SEQ ID NO: 2 Met Arg 1 Ala Pro Glu Lys Asn Ile His Val Leu Tyr Val Val Asp Phe Ala His 130 Ala Glu 145 Asp Lys Arg Leu Glu Leu Val His 210 Cys Lys 225 Arg Gly Arg Val Lys Gly Gin Glu 290 Pro Asn 305 Phe Ser Gin Ser Arg Pro Leu Asp Gin Gin Glu Thr Leu Thr Val Asp 100 Phe Thr 115 Ile Arg Tyr Asn Arg Met Gin Ser 180 Leu Pro 195 Arg Val Leu Leu Glu Glu Asp Asp 260 Tyr Gly 275 Val Lys Glu Glu Phe Asp Leu Leu Phe Leu Thr Ser Val Pro Asp Ser Leu His Ser Leu Leu 70 Ser Ser Gly Lys Gly His Asp Asp Ile Glu 150 Leu Val 165 Pro Lys Lys Gly Lys Arg Val Val 230 Ser Thr 245 Ile Tyr Ile Gin Pro Gly Lys Asp 310 Ile Ala Asp Leu Val 55 Thr Thr Asn Val Asp 135 Pro Tyr Val Leu Arg 215 Ala Thr Arg Ile Glu 295 Ala Glu Pro Ser 40 Arg Phe Glu Glu Val 120 Val Leu Lys Cys Val 200 Ala Asp Thr Asn Glu 280 Lys Trp Glu Gly 25 Asp Lys Ser Arg Ser 105 Gly Ile Trp Ser Gly 185 Asp Asp His Asn Thr 265 Gin His Asp Ala Phe Tyr Arg Ala Phe 90 Glu Glu Ile Arg Glu 170 Tyr Arg Pro Arg Tyr 250 Ser Ile Tyr Val Ser Gly Asp Asp Leu 75 Ser Tyr Pro Arg Phe 155 Asp Leu Glu Asp Phe 235 Leu Trp Arg Asn Lys 315 Lys Pro Ile Leu Lys Gin Thr Asp Ile 140 Val Ile Lys Pro Pro 220 Tyr Ile Asp Ile Met 300 Met Val His Leu Gin Arg Asn Val Ser 125 Asn Asn Lys Val Pro 205 Met Arg Glu Asn Leu 285 Ala Leu Cys Gin Ser Thr His Phe Lys 110 Arg Thr Asp Asn Asp 190 Glu Lys Tyr Leu Ala 270 Lys Lys Leu Leu Arg Leu Ser Phe Lys Trp Val Asp Thr Val 175 Asn Glu Asn Met Ile 255 Gly Ser Ser Glu Ala Leu Ser Thr Lys Val Gin Leu Gly Lys 160 Ser Glu Leu Thr Gly 240 Asp Phe Pro Tyr Gin 320 His Val Pro Phe Val Leu 29a 325 330 335 Leu Phe Thr Tyr Gin Asp Phe Asp Met Gly Thr Leu Gly Leu Ala Tyr 340 345 350 Val Gly Ser Pro Arg Ala Asn Ser His Gly Gly Val Cys Pro Lys Ala 355 360 365 Tyr Tyr Ser Pro Val Gly Lys Lys Asn Ile Tyr Leu Asn Ser Gly Leu 370 375 380 Thr Ser Thr Lys Asn Tyr Gly Lys Thr Ile Leu Thr Lys Glu Ala Asp 385 390 395 400 Leu Val Thr Thr His Glu Leu Gly His Asn Phe Gly Ala Glu His Asp 405 410 415 Pro Asp Gly Leu Ala Glu Cys Ala Pro Asn Glu Asp Gin Gly Gly Lys 420 425 430 Tyr Val Met Tyr Pro Ile Ala Val Ser Gly Asp His Glu Asn Asn Lys 435 440 445 Met Phe Ser Asn Cys Ser Lys Gin Ser Ile Tyr Lys Thr Ile Glu Ser 450 455 460 Lys Ala Gln Glu Cys Phe Gln Glu Arg Ser Asn Lys Val Cys Gly Asn A...465 470 475 480 SSer Arg Val Asp Glu Gly Glu Glu Cys Asp Pro Gly Ile Met Tyr Leu 485 490 495 *Asn Asn Asp Thr Cys Cys Asn Ser Asp Cys Thr Leu Lys Glu Gly Val 500 505 510 Gin Cys Ser Asp Arg Asn Ser Pro Cys Cys Lys Asn Cys Gin Phe Glu 515 520 525 *Thr Ala Gin Lys Lys Cys Gin Glu Ala Ile Asn Ala Thr Cys Lys Gly S530 535 540 SV'al Ser Tyr Cys Thr Gly Asn Ser Ser Glu Cys Pro Pro Pro Gly Asn 545 550 555 560 Ala Glu Asp Asp Thr Val Cys Leu Asp Leu Gly Lys Cys Lys Asp Gly 565 570 575 Lys Cys Ile Pro Phe Cys Glu Arg Glu Gin Gin Leu Glu Ser Cys Ala 580 585 590 Cys Asn Glu Thr Asp Asn Ser Cys Lys Val Cys Cys Arg Asp Leu Ser 595 600 605 Gly Arg Cys Val Pro Tyr Val Asp Ala Glu Gin Lys Asn Leu Phe Leu 610 615 620 Arg Lys Gly Lys Pro Cys Thr Val Gly Phe Cys Asp Met Asn Gly Lys 625 630 635 640 Cys Glu Lys Arg Val Gin Asp Val Ile Glu Arg Phe Trp Asp Phe Ile 645 650 655 Asp Va 1.

Ile Le u -705 Ser Gly Lys :Asp

*T

GIn (I Y Leu 690 Se r Ala Arg Leu Ser 770 Ser Arg Len Ser 660 Ser Vai 675 Val His Leu Phe Ser Val Leu Gin 740 Asp His 755 His Met Thr Ala Ser Gin TII.e A s 1 Thu I he Giy Lys P1he Len Ala Asp Asn I I e 665 67-0 L en Cys His Arg 725 Pro Gin Asp Ala Lys 805 ValI Val Pro 71i0 Ile Ala Arg Glu Lys 790 Ala r iI Asp 695 Ser Ile Pro Met Asp 775 Ser Ala S ~e I.- 680 Lys Asn Lys Val1 Asp 760 Gly Phe Se r Lenu Lys Val Pro Ile 745 Thr Phe Glu Phe fle Len Gin Ph e 730 Pro Ile Gin Asp Lys 810 Phe Asp Met 715 Pro Ser Gin Lys Len 795 Len Tr ,fp Lys 700 Len Al a Ala Glu Asp 780 Thr Gin I Ie 685 Gin Se r Pro Pro Asp 765 Pro Asp Arg P r Tyr Ser Gin Al a 750 Pro Ph e His Gin Gin Met Thr 735 Ala Ser Pro Pro Asn S I.- Ser Asp 720 Pro Pro Thr As n Val1 800 Arg Asp Ser Lys Gin Thr Gin Cys 820 INFORMATION FORL SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 2097 base pairs TYPE: nucleic acid STRANDEDNESS: single (D).TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL:

NO

(ix) FEATURE: NAME/KEY:

CDS

LOCATION: L.2094 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: atg agg oag tct oto ota ttc ctg aco Met Arg Gin Ser Leu Leu Phe Leu Thr ago Se r gtg gtt oct ttc Vai Val Pro Phe otg Leu gcg ccg cga Ala Pro Arg cct Pro ocg gat gao ccg Pro Asp Asp Pro ggo Gly 25 ttc ggc 000 cac Phe Gly Pro His tag aga ctc Gin Arg Leu gag aag ott gat tot ttg Glu Lys Leu Asp Ser Leu oto toa Leu Ser gao tao gat att oto tot tta tot Asp Tyr Asp Ile Leu Ser Leu Ser 96:. 144 192 240 aat ato Asn Ile oag oag oat tog gta aga aaa aga gat Gin Gin His Ser Vai Arg Lys Arg Asp cag aot toa aoa Gin Thr Ser Thr oat gta gaa aoa ota ota act ttt toa got ttg aaa agg oat ttt aaa His Vai Giu Thr Leu Leu Thr Phe Ser Ala Leu Lys Arg His Phe Lys

S

S..

S S S

S

tta tac ctg aca Leu Tyr Leu Thr tca Ser agt act gaa ogt Ser Thr Giu Arg tca caa aat ttc Ser Gin Asn Phe aag gtc Lys Val gtg gtg gtg Val Val Val gao ttc ttc Asp Phe Phe 115 gat Asp 100 ggt aaa aac gaa Gly Lys Asn Glu agc Ser 105 gag tao act gta Glu Tyr Thr Val aaa tgg cag Lys Trp Gin 110 agg qtt cta Arg Val Leu act gga cac gtg Thr Gly His Val gtt Va1 120 ggt gag cot gao Gly Giu Pro Asp tot Ser 125 gcc cac Ala His 130 ata aga gat gat Ile Arg Asp Asp gtt ata ato aga Val Ile Ile Arg atc Ile 140 aac aca gat ggg Asn Thr Asp Gly goc Ala 145 gaa tat aao ata Glu Tyr Asn Ile gag Glu 150 oca ott tgg aga Pro Leu Trp Arg gtt aat gat aco Vai Asn Asp Thr aaa Lys 160 gao aaa aga atg Asp Lys Arg Met tta Leu 165 gtt tat aaa tct Val Tyr Lys Ser gaa Glu 170 gat ato aag aat Asp Ile Lys Asn gtt toa Val Ser 175 ogt ttg cag Arg Leu Gin gag ttg otc Glu Leu Leu 195 oca aaa gtg tgt Pro Lys Vai Cys ggt Gly 185 tat tta aaa gtg Tyr Leu Lys Val gat aat gaa Asp Asn Glu 190 gaa gag ott Glu Giu Leu 336 384 432 480 528 576 624 672 720 768 816 oca aaa ggg tta Pro Lys Giy Leu gta Val 200 gao aga gaa oca Asp Arg Giu Pro cct Pro 205 gtt oat Vai His 210 oga gtg aaa aga Arg Val Lys Arg aga Arg 215 got gac cca gat Ala Asp Pro Asp cCC Pro 220 atg aag aac acg Met Lys Asn Thr

S.

a S aaa tta ttq gtg Lys Leu Leu Val gta Val 230 gca gat cat cgc Ala Asp His Arg tao aga tao atg Tyr Arg Tyr Met ggc Gly 240 aga ggg gaa gag Arg Giy Glu Glu agt Ser 245 aca act aca aat Thr Thr Thr Asn tao Tyr 250 tta ata gag ota Leu Ile Giu Leu att gao Ile Asp 255 aga gtt gat Arg Val Asp gao Asp 260 ato tat ogg aac Ile Tyr Arg Asn act Thr 265 toa tgg gat aat Ser Trp Asp Asn gca ggt ttt Ala Gly Phe 270 aag tot oca Lys Ser Pro aaa Lys k ggo tat Gly Tyr 275 gga ata cag ata Gly Ile Gin Ile gag Glu 280 cag att cgo att Gin Ile Arg Ile otc Leu 285 oaa gag Gin Glu 290 gta aaa cot ggt Val Lys Pro Gly gaa Glu 295 aag cac tao aao Lys His Tyr Asn atg Met 300 gca aaa agt tao Ala Lys Ser Tyr 864 912 960 aat gaa gaa aag Asn Giu Giu Lys gat Asp 310 got tgg gat gtg Ala Trp Asp Vai aag Lys 315 atg ttg ota gag Met Leu Leu Glu caa Gin 320 ttt 100E ago ttt gat ata got gag gaa goa tot aaa gtt tgo ttg goa cac Phe Asp Ile 325 Ala Giu Giu Ala Ser 330 Lys Val Cys Leu Ala His 335 ctt ttc 1056 Leu Phe gtt ggc 1104 Val Gly aca tac Thr Tyr 340 tot ccc Ser Pro 355 caa gat ttt gat atq Gin Asp Phe Asp Met 345 gga act ctt gga tta got tat Gly Thr Leu Gly Leu Ala Tyr 350 gga ggt gtt tgt cca aag got Gly Gly Val Cys Pro Lys Ala aga gca aac ago Arg Ala Asn Ser 360 gtt ggg aag aaa Val Gly Lys Lys cat His tat tat ago 1152 Tyr Tyr Ser 370 acg ago aca 1200 Thr Ser Thr 385 oca Pro aag aat tat Lys Asn Tyr 390 375 ggt aaa Gly Lys aat atc tat ttg aat Asn Ile Tyr Leu Asn 380 aco ato ctt aca aag Thr Ile Leu Thr Lys 395 cat aat ttt gga goa His Asn Phe Gly Ala agt ggt ttg Ser Giy Leu gaa gct gao Giu Ala Asp 400 gaa oat gat Giu His Asp 415 ctg gtt 1248 Leu Val cog gat 1296 Pro Asp tat gto 1344 Tyr Vai atg ttt 1392 Met Phe 450 aag gc 1440 Lys Ala 465 tog agg 1488 Ser Arg aac aao 1536 Asn Asn aoa act cat gaa ttg gga Thr Thr His Giu Leu'Giy 405 ggt cta goa gaa tgt gco Giy Leu Ala Giu Cys Ala 410 cog aat Pro Asn 420 atg tat Met Tyr 435 000 ata got gtg Pro Ile Ala Val 440 425 agt Ser gag gac cag gga ggg aaa Giu Asp Gin Gly Gly Lys 430 gat oao gag aac aat aag Asp His Giu Asn Asn Lys ggo Gly 445 toa aao tgo agt Ser Asn Cys Ser cag gag tgt ttt Gin Giu Cys Phe aaa Lys 455 caa Gin tca atc tat aag aco Ser Ile Tyr Lys Thr 460 att gaa agt Ile Giu Ser tgt ggg aac Cys Gly Asn 480 gtg gat gaa Val Asp Giu 485 gao aco tgc Asp Thr Cys 470 gga Gly oaa gaa ogo agc Gin Giu Arg Ser gaa gag tgt gat Giu Giu Cys Asp aat Asn 475 cag tgc agt 1584 Gin Cys Ser 515 act gcc cag aaa gtt Lys Vai 500 gao Asp tgc aac ago gao Cys Asn Ser Asp 505 aac agt cot tgc Asn Ser Pro Cys 520 490 tgc Cys agg Arg oct ggc ato atg tat ctg Pro Giy Ilie Met Tyr Leu 495 acg ttg aag gaa ggt gto Thr Leu Lys Giu Gly Vai tgt aaa aac tgt Cys Lys Asn Cys 525 att aat got act 510 cag ttt gag Gin Phe Giu tgc aaa ggc Cys Lys Gly aag aag tgc cag gag gog 1632 Thr Ala 530 Gin Lys Lys Cys Gin Giu Ala Ile Asn Ala Thr 535 540 gtg tc 1680 Val Ser 545 got gaa 1728 Ala Glu aaa tgc 1776 Lys Cys tgt aat 1824 Cys Asn ggc cgo 1872 Gly Arg 610 agg aaa 1920 Arg Lys 625 tgt gag 1968 Cys Glu gac cag 2016 Asp Gin gtt ggg 2064 Val Gly att ctt 2097 Ile Leu 690 tac tgc aca ggt Tyr Cys Thr Gly 550 gat gao act gtt Asp Asp Thr Val 565 ato cct ttc tgo Ile Pro Phe Cvs aat ago agt gag tgc ccq cct Asn Ser Ser Giu Cys Pro Pro 555 tgo ttg gat ctt ggc aag tgt Cys Leu Asp Leu Gly Lys Cys oca gga aat Pro Gly Asn 560 aag gat ggg Lys Asp Gly 575 gag agg gaa Glu Arg Glu 585 570 cag Gin cag otg gag too tgt gca Gin Leu Giu Ser Cys Ala 590 gaa Glu 595 tgt Cys 580 act gac aac tcc tgc Thr Asp Asn Ser Cys 600 gtg ccc tat gtc gat Val Pro Tyr Val Asp aag gtg tgc tgc agg Lys Val Cys Cys Arg 605 got gaa caa aag aac Ala Giu Gin Lys Asn 620 gga ttt tgt gac atg Gly Phe Cys Asp Met gac ott too Asp Leu Ser tta ttt ttg Leu Phe Leu aat ggc aaa Asn Gly Lys 640 gga aag ccc Gly Lys Pro aaa oga gta Lys Arg Val 645 otg ago ato Leu Ser Ile tgt Cys 630 gta Val 635 cag gat gta att gaa Gin Asp Val Ile Glu 650 aat act ttt gga aag Asn Thr Phe Gly Lys 665 gtt ttc too ttg ata Val Phe Ser Leu Ile oga Arg ttt Phe ttt tgg gat ttc att Phe Trp Asp Phe Ile 655 tta gca gao aac ato Leu Ala Aso Asn Ile 660 tot gto Ser Val 675 gto cat Val His ctg Leu 670 ttt tgg att cct Phe Trp Ile Pro ttc ago Phe Ser a 00 a 680 tgt gta acg tcg Cys Val Thr Ser 685 aaa tgc tga Lys Cys INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 698 amino acids TYPE: amino acid TOPOLOGY linear (ii) MOLECULE TYPE protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4 Met Arg Gin Ser Leu Leu Phe Leu Thr Ser Val Val Pro Phe Val Leu 1 10 Ala Pro Arg Pro Pro Asp Asp Pro Giy Phe Gly Pro His Gin Arg Leu 25 Giu Lys Leu Asp Ser Leu Leu Ser Asp Tyr Asp Ile Leu Ser Leu Ser 40 Asn Ile Gin Gin His Ser Val Arg Lys Arq Asp Leu Gin Thr Ser Thr 55 His Val Giu Thr Leu Leu Thr Phe Ser Ala Leu Lys Arq His Phe Lys 70 75 Leu Tyr Leu Thr Ser Ser Thr Glu Arg Phe Ser Gin Asn Phe Lys Val 90 Val Val Val Asp Gly Lys Asn Glu Ser Glu Tyr Thr Val Lys Trp Gin 100 105 110 Asp Phe Phe Thr Gly His Val Val Gly Glu Pro Asp Ser Arg Val Leu 115 120 125 Ala His Ile Arq Asp Asp Asp Val Ile Ile Ar Ile Asn Thr Asp Gly 130 135 140 Ala Glu Tyr Asn Ile Glu Pro Leu Trp Arg Phe Val Asn Asp Thr Lys 145 150 155 160 Asp Lys Arq Met Leu Val Tyr Lys Ser Giu Asp Ile Lys Asn Val Ser 165 170 175 Arg Leu Gin Ser Pro Lys Val Cys Gly Tyr Leu Lys Val Asp Asn Glu 180 185 190 ,~Glu Leu Leu Pro Lys Gly Leu Val Asp Arg Glu Pro Pro Glu Glu Leu 195 200 205 lVal His Arg Val Lys Arg Arq Ala Asp Pro Asp Pro Met Lys Asn Thr 210 215 220 ys Lys Leu Leu Val Val Ala Asp His Arg Phe Tyr Arg Tyr Met Gly .225 230 235 240 Arg Gly Glu Giu Ser Thr Thr Thr Asn Tyr Leu Ile Glu Leu Ile Asp 245 250 255 e...Arg Val Asp Asp Ile Tyr Arg Asn Thr Ser Trp Asp Asn Ala Gly Phe 00..

260 265 270 Lys Gly Tyr Gly Ile Gin Ilie Glu Gin Ile Arg Ile Leu Lys Ser Pro 275 280 285 Giu Val Lys Pro Giy Glu Lys His Tyr Asn Met Ala Lys Ser Tyr 290 295 300 ':Pro Asn Glu Glu Lys Asp Ala Trp Asp Val Lys Met Leu Leu Glu Gin 305 310 315 320 Phe Ser Phe Asp Ile Ala Glu Giu Ala Ser Lys Val Cys Leu Ala His 325 330 335 Leu Phe Thr Tyr Gin Asp Phe Asp Met Gly Thr Leu Gly Leu Ala Tyr 340 345 350 Val Gly Ser Pro Arq Ala Asn Ser His Gly Gly Val Cys Pro Lys Ala 355 360 365 Tyr Tyr Ser Pro Val Gly Lys Lys Asn Ile Tyr Leu Asn Ser Gly Leu 370 375 380 Thr Ser Thr Lys Asn Tyr Gly Lys Thr Ile Leu Thr Lys Glii Ala Asp 385 390 395 400 Leu Val Thr Thr His Giu Leu Gly His Asn Phe Gly Ala Giu His Asp 405 410 Pro Asp Gly Leu Ala Glu Cys Ala Pro Asn Glu Asp Gin Gly Gly Lys 420 425 430 Tyr Val Met Tyr Pro Ile Ala Val Ser Gly Asp His Glu Asn Asn Lys 435 440 445 Met Phe Ser Asn Cys Ser Lys Gin Ser Ile Tyr Lys Thr Ile Glu Ser 450 455 460 Lys Ala Gin Glu Cys Phe Gin Glu Arg Ser Asn Lys Val Cys Gly Asn 465 470 475 480 Ser Arg Val Asp Glu Gly Glu Glu Cys Asp Pro Gly Ile Met Tyr Leu 485 490 495 Asn Asn Asp Thr Cys Cys Asn Ser Asp Cys Thr Leu Lys Glu Gly Val 500 505 510 Gin Cys Ser Asp Arg Asn Ser Pro Cys Cys Lys Asn Cys Gin Phe Glu 515 520 525 Thr Ala Gin Lys Lys Cys Gin Glu Ala Ile Asn Ala Thr Cys Lys Gly 530 535 540 S Val Ser Tyr Cys Thr Gly Asn Ser Ser Glu Cys Pro Pro Pro Gly Asn 545 550 555 560 *5$ Ala Glu Asp Asp Thr Val Cys Leu Asp Leu Gly Lys Cys Lys Asp Gly 565 570 575 Lys Cys Ile Pro Phe Cys Glu Arg Glu Gin Gin Leu Glu Ser Cys Ala 580 585 590 Cys Asn Glu Thr Asp Asn Ser Cys Lys Val Cys Cys Arg Asp Leu Ser 595 600 605 Gly Arg Cys Val Pro Tyr Val Asp Ala Glu Gin Lys Asn Leu Phe Leu 610 615 620 Arg Lys Gly Lys Pro Cys Thr Val Gly Phe Cys Asp Met Asn Gly Lys S 625 630 635 640 Cys Glu Lys Arg Val Gin Asp Val Ile Glu Arg Phe Trp Asp Phe Ile 645 650 655 Asp Gin Leu Ser Ile Asn Thr Phe Gly Lys Phe Leu Ala Asp Asn Ile 660 665 670 Val Gly Ser Val Leu Val Phe Ser Leu Ile Phe Trp Ile Pro Phe Ser 675 680 685 ile Leu Val His Cys Val Thr Ser Lys Cys 690 695 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 Gln Ala Val Arg Ser Ser 1 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iii) HYPOTHETICAL: NO 0* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AARTAYGTNA TGTAYCC 17 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: S* 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 NO:8r Glu Glu Cys Asp Cys Gly 1 4 P t INFORMATION FOR SEQ ID NO:9: Ci) 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 0* .04 0 s 6'0#0

Claims (18)

1. An isolated and purified TACE polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:9; a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:9; a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 477; a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 671; a fragment of SEQ ID NO:2 such that a polypeptide with an amino acid sequence consisting of said fragment is capable of converting TNF-a from the 26 kD form to the 17 kD form; a fragment of SEQ ID. NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:9; a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 477; a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 671; a fragment of SEQ ID NO:4 such that a polypeptide with an amino acid 25 sequence consisting of said fragment is capable of converting TNF-a from the 26 kD form to the 17 kD form; an amino acid sequence that is at least 80% identical to the amino acid sequence of any of and S* an amino acid sequence that is at least 90% identical to the amino acid 30 sequence of any of

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. Isolated and purified antibodies that bind to the polypeptide according to any 41 Isolated and purified antibodies according to claim 4, wherein the antibodies are monoclonal antibodies.

6. An isolated nucleic acid selected from the group consisting of: nucleic acid encoding a TACE polypeptide comprising an amino acid sequence selected from the group consisting of: (al) SEQ ID NO:2; (a2) SEQ ID NO:4; (a3) SEQ ID NO:9; (a4) a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:9; a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 215 to amino acid 477; (a6) a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 477; S.(a7) a fragment of SEQ ID NO:2, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 671; (a8) a fragment of SEQ ID NO:2 such that a polypeptide with an 20 amino acid sequence consisting of said fragment is capable of converting TNF-a from the 26 kD form to the 17 kD form; (a9) a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:9; (a10) a fragment of SEQ ID NO:4, the fragment comprising the amino 25 acid sequence of SEQ ID NO:2 from amino acid 215 to amino acid 477; (all) a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 477; (a12) a fragment of SEQ ID NO:4, the fragment comprising the amino acid sequence of SEQ ID NO:2 from amino acid 18 to amino acid 671; 30 (a13) a fragment of SEQ ID NO:4 such that a polypeptide with an amino acid sequence consisting of said fragment is capable of converting TNF-a from the 26 kD form to the 17 kD form; (a14) an amino acid sequence that is at least 80% identical to the amino acid sequence of any of (al) (al and (a15) an amino acid sequence that is at least 90% identical to the amino acid sequence of any of (al) (a13); IC W:\lona\Sharon\SJJspecispo17575.doc 42 nucleic acid that is at least 80% identical to the nucleic acid of and that encodes a polypeptide that converts TNF-a from the 26 kD form to the 17 kD form; 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; and nucleic acid that hybridizes under conditions of moderate stringency to the nucleic acid of any of and that encodes a polypeptide that converts TNF-a from the 26 kD form to the 17 kD form.

7. An isolated nucleic acid according to claim 6, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:9.

8. An expression vector that directs the expression of at least one nucleic acid sequence according to any of claims 6 to 7.

9. A recombinant host cell comprising the nucleic acid according to any of claims 6 to 7. 15 10. A process for producing a TACE polypeptide comprising culturing a host cell according to claim 9 under conditions promoting expression.

11. An isolated and purified polypeptide produced by the process of claim 20 12. A method for detecting the TNF-cleaving ability of a polypeptide of any of claims 1 to 3 and claim 11, comprising incubating said polypeptide with a substrate that comprises the amino acid sequence Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser (SEQ ID NO:5), and determining the extent of substrate cleavage. 25 13. A method of using a polypeptide according to any of claims 1 to 3 and claim 11 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 polypeptide-inhibiting activity of the molecule. W:Ulona\Sharon\SJJspeci\spo17575.doc 43

14. A method for detecting the TACE-inhibiting activity of a molecule, comprising mixing said molecule with a substrate and a polypeptide according to any of claims 1 to 3 and claim 11, and determining the extent of substrate cleavage. A method for identifying compounds that inhibit TNF-a cleavage comprising: mixing the polypeptide of any of claims 1 to 3 and claim 11 with a test compound and a substrate that is cleavable by said polypeptide; and determining whether the test compound inhibits the cleavage of said substrate by said polypeptide.

16. A method for identifying compounds that inhibit TNF-a cleavage comprising: mixing a test compound with a membrane-bound substrate and the polypeptide of any of claims 1 to 3 and claim 11; and determining whether the test compound inhibits the cleavage of said substrate by said polypeptide.

17. The method of any of claims 14 to 16 wherein the polypeptide is provided by recombinant cells containing at least one expression vector that directs the 20 expression of at least one nucleic acid encoding said polypeptide.

18. A method for inhibiting the cleavage of a substrate by the polypeptide of any of claims 1 to 3 and claim 11, comprising mixing said polypeptide, said substrate, and a compound, wherein the compound inhibits the cleavage of said substrate by said 25 polypeptide. .00 19. A method of inhibiting TNF-a cleavage from cell membranes comprising .blocking the binding of TNF-a with an enzyme comprising the sequence of SEQ ID *NO:9, wherein the binding is blocked by a compound selected by a process 0 30 comprising: mixing one or more compounds to be tested with a substrate and a polypeptide of any of claims 1 to 3 and claim 11; and identifying which compounds inhibit the cleavage of said substrate by said polypeptide. W:llona\Sharon\SJJspeci\spo7575.doc A method of inhibiting the cleavage of TNF-a from cell membranes in a mammalcomprising administering to such mammal an effective amount of a compound that inhibits TACE activity, wherein the compound is selected by a process comprising: mixing one or more compounds to be tested with a substrate and a polypeptide of any of claims 1 to 3 and claim 11; and identifying which compounds inhibit the cleavage of said substrate by said polypeptide.

21. 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 TACE activity, wherein the compound is selected by a process comprising: mixing one or more compounds to be tested with a substrate and a polypeptide of any of claims 1 to 3 and claim 11; and identifying which compounds inhibit the cleavage of said substrate by S* said polypeptide. 20 22. The method of any of claims 18 to 21 wherein the compound is an antibody that binds to the polypeptide of any of claims 1 to 3 and claim 11.

23. The method of any of claims 14 to 22 wherein the substrate is selected from the group consisting of cytokines, cytokine receptors, and adhesion molecules.

24. The method of any of claims 14 to 23, wherein the substrate comprises the amino acid sequence Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser (SEQ ID 6* S. 25. The method of any of claims 14 to 24 wherein the substrate is TNF-a.

26. A compound that inhibits TACE activity for use in medicine, wherein the compound is selected by a process comprising: mixing one or more compounds to be tested with a substrate and a polypeptide of any of claims 1 to 3 and claim 11; and identifying which compounds inhibit the cleavage of said substrate by said polypeptide. IC W:UIonakShamn\SJJspeci\spo17575.doc

27. The use of a compound that inhibits TACE activity in the manufacture of a medicament for treating tumours, inflammation, or fertility disorders, wherein the compound is selected by a process comprising: mixing one or more compounds to be tested with a substrate and a polypeptide of any of claims 1 to 3 and claim 11; and identifying which compounds inhibit the cleavage of said substrate by said polypeptide. DATED: 24 October, 2000 PHILLIPS ORMONDE FITZPATRICK Attorneys for: IMMUNEX CORPORATION 0@ 0 o *0*S S L:ilona\Sharon\SJJspeci\spo17575.doc