Truncated aggrecanase molecules Number:7,150,983 from the United States Patent and Trademark Office (PTO) owispatent Truncated aggrecanase proteins and nucleotides sequences encoding them as well as processes for producing them are disclosed. Additionally, aggrecanases with amino acid mutations that lead to increased stability and expression levels in comparison with wild-type or native aggrecanases are also disclosed. Aggrecanases of the invention are especially useful for development of compositions for treatment of diseases such as osteoarthritis. Methods for developing inhibitors of the aggrecanase enzymes and antibodies to the enzymes for treatment of conditions characterized by the degradation of aggrecan are also disclosed.<H aggrecanase, molecules, Truncated, aggre, 7150983.html, Patent, pto, 7150983

Title: Truncated aggrecanase molecules

Abstract: Truncated aggrecanase proteins and nucleotides sequences encoding them as well as processes for producing them are disclosed. Additionally, aggrecanases with amino acid mutations that lead to increased stability and expression levels in comparison with wild-type or native aggrecanases are also disclosed. Aggrecanases of the invention are especially useful for development of compositions for treatment of diseases such as osteoarthritis. Methods for developing inhibitors of the aggrecanase enzymes and antibodies to the enzymes for treatment of conditions characterized by the degradation of aggrecan are also disclosed.

Patent Number: 7,150,983 Issued on 12/19/2006 to Georgiadis, et al.

Inventors: Georgiadis; Katy E. (Belmont, MA), Crawford; Tara K. (Cambridge, MA), Tomkinson; Kathleen N. (Cambridge, MA), Collins-Racie; Lisa A. (Acton, MA), Corcoran; Christopher J. (Arlington, MA), Freeman; Bethany A. (Arlington, MA), Lavallie; Edward R. (Harvard, MA)
Assignee: Wyeth (Madison, NJ)

Appl. No.: 10/358,283

Filed: February 5, 2003

Current U.S. Class: 435/226 ; 435/69.7; 536/23.2

Current International Class: C12N 9/64 (20060101); C07H 21/04 (20060101); C12P 21/04 (20060101)

Field of Search: 435/226,69.7 536/23.2

References Cited [Referenced By]

U.S. Patent Documents


4419446	December 1983	Howley et al.
4816567	March 1989	Cabilly et al.
6326162	December 2001	Miller et al.
6391610	May 2002	Apte et al.
6451575	September 2002	Arner et al.
6521436	February 2003	Arner et al.

Foreign Patent Documents


0 123 289	Oct., 1984	EP
0 155 476	Sep., 1985	EP
0 177 343	Sep., 1986	EP
WO 86/00639	Jan., 1986	WO
99/05291	Feb., 1999	WO
WO 00/53774	Sep., 2000	WO
03/062263	Jul., 2003	WO
2004/011637	Feb., 2004	WO

Other References

(Abbaszade et al. (1999) J.Biol. Chem., vol. 274 (33), pp. 23443-23450. cited by examiner .
Hurskainen et al. (J. Biol. Chem., 1999, vol. 274 (36), pp. 25555-2563. cited by examiner .
Altschul et al., "Basic . . . ," J.Mol. Biol., vol. 215, p. 403-410, (Oct. 5, 1990). cited by other .
Ausubel, Wiley & Sons, Current Protocols in Molecular Biology, N.Y., 6.3.1-6.3.6, (1989). cited by other .
Brandt and Mankin, "Pathogenesis . . .," Textbook of Rheumatology, WB Saunders, P.A., p. 1355-1373, (1993). cited by other .
Clackson et al., "Making . . .," Nature, vol. 352, p. 624-628, (Aug. 15, 1991). cited by other .
Flannery et al., "Identification . . .," JBC, vol. 267 (No. 2), p. 1008-1014, (Jan. 15, 1992). cited by other .
Fosang et al., "Neutrofil . . . ," Biochem. J., vol. 304, p. 347-351, (Dec. 1, 1994). cited by other .
Gething and Sambrook, "Cell-surface . . .," Nature, vol. 293, p. 620-625, (Oct. 22, 1981). cited by other .
Gossen and Bujard, "Tight . . . ," PNAS USA, vol. 89, p. 5547-5551, (Jun. 15, 1992). cited by other .
Gough et al., "Structure . . . ," EMBO J., vol. 4 (No. 3), p. 645-653, (Mar. 1985). cited by other .
Hughes et al., "Monocolonal . . . ," Biochem. J., vol. 305, p. 799-804, (Feb. 1, 1995). cited by other .
Jang et al., "Initiation . . .," J. of Virol., vol. 63 (No. 4), p. 1651-1660, (Apr. 1989). cited by other .
Kaufman and Sharp, "Amplification . . .," J. Mol. Biol., vol. 159, p. 601-621, (Aug. 25, 1982). cited by other .
Kaufman et al., "Coamplification . . .," Mol. and Cellular Bio., vol. 5 (No. 7), p. 1750-1759, (Jul. 1985). cited by other .
Kaufman and Sharp, "Construction . . .," Mol. and Cellular Bio., vol. 2(No. 11), p. 1304-1319, (Nov. 1982). cited by other .
Kaufman, "Identification . . .," PNAS USA, vol. 82, p. 689-693, (Feb. 1985). cited by other .
Kaufman et al., "Improved . . .," Nuc. Acids Res., vol. 19 (No. 16), p. 4485-4490, (Aug. 25, 1991). cited by other .
Kohler and Milstein, "Continuous . . .," Nature, vol. 256, p. 495-497, (Aug. 7, 1975). cited by other .
Laemmli, "Cleavage . . .," Nature, vol. 227, p. 680-685, (Aug. 15, 1970). cited by other .
Lohmander et al., "The Structure . . .," Arthritis & Rheumatism, vol. 36 (No. 9), p. 1214-1222, (Sep. 1993). cited by other .
MacLean et al., "Costs . . .," J. of Rheumatology, vol. 25 ( No. 11), p. 2213-2218, (Nov. 1998). cited by other .
Maniatis et al., Molecular Cloning: A Laboratory Manual, p. 387-389, (1982). cited by other .
Marks et al., "By-passing . . .," J. Mol. Biol., vol. 222, p. 581-597, (Dec. 5, 1991). cited by other .
Mercuri et al., "Recombinant . . .," JBC, vol. 274 ( No. 45), p. 32387-32395, (Nov. 5, 1999). cited by other .
Miller et al., "An Insect . . .," Genetic Engineering, vol. 8, Plenum Press, p. 277-298, (1986). cited by other .
Morinaga et al., "Improvement . . .," Bio/Technology, 84, p. 636-639, (Jul. 1984). cited by other .
Needleman and Wunsch, "A General . . .," J. Mol. Biol., vol. 48, p. 443-453, (Mar. 1970). cited by other .
Oakley et al., "A Simplified . . .," Analytical Biochemistry, vol. 105, p. 361-363, (Jul. 1, 1980). cited by other .
Okayama and Berg, "High-Efficiency . . .," Mol. and Cellular Bio., vol. 2 ( No. 2), p. 161-170, (Feb. 1982). cited by other .
Sandy et al., "Catabolism . . .," JBC, vol. 266 ( No. 14), p. 8683-8685, (May 15, 1991). cited by other .
Sandy et al., "The Structure . . .," J. Clin. Invest., vol. 89, p. 1512-1516, (May, 1992). cited by other .
Steele et al., "Expression . . .," Protein Engineering, vol. 13 (No. 6), p. 397-405, (Jun. 2000). cited by other .
Taniguchi et al., "Expression . . .," PNAS USA, vol. 77 ( No. 9), p. 5230-5233, (Sep. 1980). cited by other .
Tortorella et al., "Purification . . .," Science, vol. 284, p. 1664-1666, (Jun. 4, 1999). cited by other .
Towbin et al., "Electrophoretic . . .," PNAS USA, vol. 76 ( No. 9), p. 4350-4354, (Sep. 1979). cited by other .
Urlaub and Chasin, "Isolation . . .," PNAS USA, vol. 77 ( No. 7), p. 4216-4220, (Jul. 1980). cited by other .
Wong et al., "Human . . .," Science, vol. 228 ( No. 4701), p. 810-815, (May 17, 1985). cited by other .
Cal, Santiago, et al., "Cloning, Expression Analysis . . .," GENE, vol. 283, 2002, pp. 49-62. cited by other .
Cal, Santiago, et al., "Identification, Characterization, and . . .," The Journal of Biological Chemistry and Molecular Biology, Inc., vol. 276, No. 21, May 25, 2001, pp. 17932-17940. cited by other .
Caterson, Bruce et al., "Mechanisms Involved in Cartilage . . .," Matrix Biology, vol. 19, 2000, pp. 333-344. cited by other .
Clark, Melody et al., "Adamts9, A Novel Member . . .," GENOMICS, vol. 67, 2000, pp. 343-350. cited by other .
Flannery, Carl et al., "Autocatalytic Cleavage of AMAMTS-4 . . .," The Journal of Biological Chemistry, vol. 277, No. 45, Nov. 8, 2002, pp. 42775-42780. cited by other .
Gao, Gui et al., "Activation of the Proteolytic . . .," The Journal of Biological Chemistry, vol. 277, Mar. 29, 2002, No. 13, pp. 11034-11041. cited by other .
Hashimoto, Gakuji et al., "Inhibition of Adamts4 (Aggrecanase-1) . . .," Federation of European Biochemical Societies, vol. 494, 2001, pp. 192-195. cited by other .
Kuno,Kouji et al., "ADAMTS-1 Protein Anchors at the . . .," The Journal of Biological Chemistry, vol. 273, No. 22, May 29, 1998, pp. 13912-13917. cited by other .
Rodriguez-Manzaneque, Juan Carlos et al., "Characterization of Meth-1/ADAMTS1 . . .," The Journal of Biological Chemistry, vol. 275, No. 43, Oct. 27, 2000, pp. 33471-33479. cited by other .
Somerville, Robert et al., "Characterization of ADAMTS-9 . . .," The Journal of Biological Chemistry, vol. 278, No. 11, Mar. 14, 2003, pp. 9503-9513. cited by other .
Tortorella, Mickey et al., "The Thrombospondin Motif of Aggrecanase-1 . . .," The Journal of Biological Chemistry, vol. 275, No. 33, Aug. 18, 2000, pp. 25791-25797. cited by other .
Vazquez, Francisca et al., "METH-1, A Human Ortholog . . .," vol. 274, No. 33, Aug. 13, 1999, pp. 23349-23357. cited by other .
Kuno et al., "ADAMTS-1 Is an Active Metalloproteinase Associated with the Extracellular Matrix," J. Biol. Chem., 274(26):18821-18826, 1999. cited by other .
Myers, E.W. and Miller, W., Optical alignments in linear space, CABIOS, 4(1):11-17 (1988). cited by other .
Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). cited by other .
Kashiwagi et al., "Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing," J. Biol. Chem., 279(11):10109-10119 (2004). cited by other .
"Partial European Search Report" Nov. 16, 2005, European patent application 03710886.7. cited by other.

Primary Examiner: Slobodyansky; Elizabeth
Attorney, Agent or Firm: Kirkpatrick & Lockhart Nicholson Graham LLP

Parent Case Text

RELATED APPLICATIONS

This application relies on the benefit of priority of U.S. provisional patent application Ser. No. 60/354,592 filed Feb. 5, 2002, the entire disclosure of which is incorporated by reference herein.

Claims

We claim:

1. An isolated, recombinantly-produced or chemically-synthesized aggrecanase consisting of an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 8, or fragments thereof, wherein said fragments retain aggrecanase activity.

2. An isolated, recombinantly-produced or chemically-synthesized fusion protein consisting of: comprising the aggrecanase of claim 1 and at least one peptide tag.

Description

FIELD OF THE INVENTION

The present invention relates to the discovery of truncated aggrecanase molecules, processes for producing them, and methods employing these molecules to develop inhibitors to aggrecanase enzymes. The invention further relates to the development of inhibitors of, as well as antibodies to, aggrecanase enzymes. These inhibitors and antibodies may be useful for the treatment of various aggrecanase-associated conditions including osteoarthritis.

BACKGROUND OF THE INVENTION

Aggrecan is a major extracellular component of articular cartilage. It is a proteoglycan responsible for providing cartilage with its mechanical properties of compressibility and elasticity. The loss of aggrecan has been implicated in the degradation of articular cartilage in arthritic diseases such as osteoarthritis.

Osteoarthritis is a debilitating disease which affects at least 30 million Americans (MacLean et al., J Rheumatol 25:2213 2218 (1998)). Osteoarthritis can severely reduce quality of life due to degradation of articular cartilage and the resulting chronic pain. An early and important characteristic of the osteoarthritic process is loss of aggrecan from the extracellular matrix (Brandt and Mankin, "Pathogenesis of Osteoarthritis," Textbook of Rheumatology, W B Saunders Company, Philadelphia, Pa., pgs. 1355 1373 (1993)). The large, sugar-containing portion of aggrecan is thereby lost from the extra-cellular matrix, resulting in deficiencies in the biomechanical characteristics of the cartilage.

A proteolytic activity termed "aggrecanase" is believed to be responsible for the cleavage of aggrecan, thereby having a role in cartilage degradation associated with osteoarthritis and inflammatory joint disease. Research has been conducted to identify the enzymes responsible for the degradation of aggrecan in human osteoarthritic cartilage. Aggrecan contains two N-terminal globular domains, G1 and G2, separated by a proteolytically sensitive interglobular domain, followed by a glycosaminoglycan attachment region and a C-terminal globular domain, G3. At least two enzymatic cleavage sites have been identified within the interglobular domain of aggrecan. One enzymatic cleavage site within the interglobular domain of aggrecan (Asn.sup.341-Phe.sup.312) has been observed to be cleaved by several known metalloproteases. Flannery et al., J Biol Chem 267:1008 1014 (1992); Fosang et al., Biochemical J. 304:347 351 (1994). Cleavage at a second aggrecan cleavage site within aggrecan (Glu.sup.373-Ala.sup.374) due to IL-1 induced cartilage aggrecan cleavage results in the generation of an aggrecan fragment found in human synovial fluid (Sandy et al., J Clin Invest 89:1512 1516 (1992); Lohmander et al., Arthritis Rheum 36: 1214 1222 (1993); Sandy et al., J Biol Chem 266: 8683 8685 (1991)). Aggrecan cleavage at (Glu.sup.373-Ala.sup.374) has been attributed to aggrecanase activity (Sandy et al., J Clin Invest 89:1512 1516 (1992). This Glu.sup.373-Ala.sup.374 cleavage site will be referred to as the aggrecanase cleavage site.

Recently, identification of two enzymes, aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-11) within the "a Disintegrin and Metalloproteinase with Thrombospondin motifs" (ADAMTS) family, have been identified which are synthesized by IL-1 stimulated cartilage and cleave aggrecan at the Glu.sup.373-Ala.sup.374 site (Tortorella et al., Science 284:1664 1666 (1999); Abbaszade et al., J Biol Chem 274: 23443 23450 (1999)). Aggrecanase-1 is reported to include at least six domains: signal; propeptide; catalytic; disintegrin; TSP type-1 motif and C-terminal. Aggrecanase-2 is also a multidomain protein. It is reported to have a signal sequence; a prodomain; a metalloproteinase domain; a disintegrin domain and a spacer domain between a TSP motif and a TSP sub motif in the C-terminal of the protein. It was generally believed that the TSP domains and the spacer domain are critical for substrate recognition. Specifically, Tortorella et al. reported that "this region may serve to bind aggrecanase-1 to the glycosaminoglycans of the aggrecan substrate." See Tortorella et al., Science 284:1664 1666 (1999).

It is contemplated that there are other, related enzymes in the ADAMTS family which are capable of cleaving aggrecan at the Glu.sup.373-Ala.sup.374 bond and could contribute to aggrecan cleavage in osteoarthritis. It is possible that these enzymes could be synthesized by osteoarthritic human articular cartilage. However, it has been difficult to develop inhibitors and treatment therapies to treat diseases that involve aggrecan cleavage because aggrecanases have been difficult to isolate and purify in large amounts due to poor stability of these molecules and generally low expression levels. Therefore, there is a need to identify novel forms of aggrecanases and further develop ways to isolate and purify aggrecanase proteins in large amounts in order to investigate their role in disease states and also to develop therapies and compositions to treat diseases involving aggrecan cleavage.

SUMMARY OF THE INVENTION

The present invention is directed to truncated aggrecanase proteins and variants and fragments thereof; nucleotide sequences which encode truncated aggrecanase enzymes of the invention and fragments and variants thereof; and processes for the production of truncated aggrecanases. Truncated aggrecanases of the invention are biologically active and have greater stability and higher expression levels than their full-length counterparts. More specifically, the invention features truncated aggrecanase-1 and aggrecanase-2 enzymes that are more stable and show higher levels of expression than full-length aggrecanase-1 and aggrecanase-2 enzymes, respectively; nucleic acid sequences encoding truncated aggrecanases-1 and 2 of the invention and fragments and variants thereof; and methods of producing truncated aggrecanases 1 and 2, or fragments and variants thereof.

In one embodiment, truncated aggrecanases of the invention comprise aggrecanases that have at least one TSP domain deleted. In another embodiment, truncated aggrecanases of the invention comprise aggrecanases that have at least two TSP domains deleted. Although, TSP domains in aggrecanases have been thought to be important for substrate recognition, and therefore, for the ability of aggrecanase to recognize and subsequently cleave aggrecan, truncated aggrecanase proteins of the invention are biologically active despite deletion of one or both TSP domains in the proteins.

Truncated aggrecanases of the invention have greater stability and are expressed at higher levels compared with the full-length aggrecanase proteins, thereby facilitating isolation, purification, and use of aggrecanases of the invention in the development of inhibitors and therapies for treatment of diseases. Accordingly, in one embodiment, the invention comprises methods for producing large amounts of purified truncated aggrecanases that may be used for development of inhibitors and treatment therapies.

The invention further includes compositions comprising truncated aggrecanases of the invention and use of such compositions for the development of inhibitors of aggrecanases for treatment of diseases including osteoarthritis. In addition, the invention includes methods for identifying and developing inhibitors of aggrecanase which block the enzyme's activity. The invention also includes antibodies to these enzymes, in one embodiment, for example, antibodies that block aggrecanase activity. These inhibitors and antibodies may be used in various assays and therapies for treatment of conditions characterized by the degradation of articular cartilage. In one embodiment, inhibitors are peptide molecules that bind aggrecanases.

This invention provides amino acid sequences of biologically active truncated aggrecanase molecules that have greater stability compared with the full-length aggrecanase protein.

In one aspect, the invention features biologically active truncated aggrecanase-2 molecules that have at least one TSP domain deleted, such as a protein with an amino acid sequence from amino acid #1 (Met) through amino acid #753 (Glu) of SEQ ID NO: 4; from amino acid #1 (Met) through amino acid #752 (Pro) of SEQ ID NO: 6; and from amino acid #1 (Met) through amino acid #628 (Phe) of SEQ ID NO: 8, and variants and fragments thereof, including substitution mutants, that exhibit aggrecanase activity.

The invention also features truncated aggrecanase-1 molecules that have at least one TSP domain deleted. An example includes an aggrecanase-1 protein with an amino acid sequence from amino acid #1 (Met) through amino acid #520 (Ala) of SEQ ID NO: 13, fragments, and variants thereof including substitution mutants that exhibit aggrecanase activity.

In another embodiment, the invention features biologically active truncated aggrecanase-2 proteins with at least two TSP domains deleted comprising, for example, a protein with an amino acid sequence from amino acid #1 through amino acid #527 (His) of SEQ ID NO: 10 (FIG. 10), variants, and fragments thereof including substitution mutants that exhibit aggrecanase activity.

Truncated aggrecanases with one or both TSP domains deleted are biologically active and are more stable than the full-length aggrecanase enzymes.

The invention also features nucleic acid molecules that encode truncated aggrecanases of the invention. For example, nucleic acid molecules encoding truncated aggrecanase-2 molecules of the invention include: nucleotide #1 through nucleotide #2259 of SEQ ID NO: 3 (FIG. 3), which encodes a polypeptide set forth in SEQ ID NO: 4; nucleotide #1 through nucleotide #2256 of SEQ ID NO: 5 (FIG. 5), which encodes a polypeptide set forth in SEQ ID NO: 6; nucleotide #1 through nucleotide #1884 of SEQ ID NO: 7 (FIG. 7), which encodes the polypeptide set forth in SEQ ID NO: 8; and nucleotide #1 through nucleotide #1701 of SEQ ID NO: 9 (FIG. 9), which encodes the polypeptide set forth in SEQ ID NO: 10. Nucleic acid molecules of the invention further include fragments and variants of SEQ ID NOs: 3, 5, 7, and 9 which encode truncated aggrecanase-2 molecules of the invention, nucleotide sequences that hybridize under moderate to stringent conditions with nucleotide sequences of SEQ ID NOs: 3, 5, 7, or 9 and fragments or variants thereof, naturally occurring allelic sequences, and equivalent degenerative codon sequences of aggrecanase-2 nucleic acid sequences disclosed herein.

Nucleic acid molecules encoding truncated aggrecanase-1 molecules of the invention include, for example, nucleotides which encode the polypeptides of SEQ ID NOs: 12 and 13; set forth in FIGS. 23 and 24 respectively, fragments and variants of nucleic acids that encode truncated aggrecanase-1 molecules of the invention, nucleotide sequences that hybridize under moderate to stringent conditions to nucleic acid sequences that encode truncated aggrecanase-1 molecules of the invention, for example, nucleic acid sequences of FIGS. 23 and 24, or fragments and variants thereof, naturally occurring allelic sequences and equivalent degenerative codon sequences of aggrecanase-1 encoding nucleic acid sequences. A nucleic acid sequence for full-length aggrecanase-1 molecule is found in Genbank under Accession No.: NM.sub.--005099 (FIG. 22). Therefore, one skilled in the art can use this sequence to generate a full-length aggrecanase-1 molecule set forth in SEQ ID NO: 11, or use part of the nucleotide sequence to generate a truncated protein, for example, the aggrecanase protein set forth in FIG. 12 or 13. For example nucleotide #407 through nucleotide #2132 of the published NM.sub.--005099 sequence (FIG. 23) (SEQ ID NO: 32) may be used for generating the truncated aggrecanase-1 protein of SEQ ID NO: 12. Similarly, nucleotide #1 through nucleotide #1967 of the published NM.sub.--005099 sequence (FIG. 24) (SEQ ID NO: 33) may be used for the generation of truncated aggrecanase molecule of FIG. 13.

In another aspect, the invention includes aggrecanase molecules that comprise mutations that increase stability and expression levels of truncated aggrecanase molecules compared with their full-length counterparts. Aggrecanases with mutations in their active sites are particularly useful for the synthesis of inhibitors of aggrecanases. Accordingly, in one embodiment, the invention features an aggrecanase-2 molecule with a mutation at amino acid 411 (E411-Q411 mutation) in the active site within the catalytic domain. The amino acid sequence of an aggrecanase-2 molecule with the E411-Q411 mutation is shown in FIG. 21 (SEQ ID NO: 30). Mutations that lead to increased stability of the aggrecanase proteins in comparison with their wild-type counterparts can be made in both truncated as well as full-length aggrecanase proteins. It is contemplated that mutations that alter stability of aggrecanase molecules can be made within the catalytic domain of an aggrecanase protein or outside the catalytic domain. Aggrecanase proteins carrying such mutations may be found in nature or may be generated artificially. One skilled in the art can test the effect of a mutation on the stability of an aggrecanase molecule by one of many assays provided. Mutations of the invention include, for example, amino acid substitutions or modifications. Amino acid mutations in aggrecanases of the invention can be generated by mutagenesis, chemical alteration, or by alteration of DNA sequence used to produce the polypeptide.

Aggrecanase-1 molecules can also be generated to include mutations in the catalytic domain in order to increase their stability. For example, FIG. 17 features flag-tagged truncated aggrecanase-1 proteins that include an E-to-Q amino acid mutation in the catalytic domain of truncated aggrecanase-1 molecules, the wild-type sequences of which are set forth in SEQ ID NO: 12 and 13, thereby leading to increased stability of aggrecanase-1 proteins compared to full-length wild-type aggrecanase-1 protein, the sequence of which is set forth in SEQ ID NO: 11. These aggrecanases are particularly useful for the development as well as identification of novel inhibitors of aggrecanases.

The invention further features truncated and/or mutant aggrecanase family members and aggrecanase-like proteins with deletions and/or substitution mutations, where a deletion or an amino acid substitution mutation occurs in a region of the protein comparable to that of aggrecanases of the invention.

The invention further includes variants and equivalent degenerative codon sequences of nucleic acid sequences described herein that encode biologically active truncated aggrecanase polypeptides. Additionally, the invention includes nucleic acid molecules that hybridize under moderate to stringent conditions to the nucleic acids of the invention, allelic variants and substitution and deletion mutants of nucleic acids molecules described herein. In one embodiment, truncated aggrecanases and/or aggrecanases carrying at least one amino acid substitution encoded by nucleic acid molecules of the invention are more stable than the corresponding full-length aggrecanase protein and can be expressed at higher levels than the full-length aggrecanase protein. In one embodiment, mutations are introduced in nucleic acid molecules encoding aggrecanases of the invention that lead to mutations; for example, amino acid substitutions, in the protein encoded by the nucleic acid carrying the mutation. One example of such a mutation is a nucleic acid sequence encoding an aggrecanase-2 protein with an E-to-Q mutation in the active site of molecule. In another embodiment, the invention includes aggrecanase-1 nucleic acid molecules that encode aggrecanase-1 proteins comprising an E-to-Q mutation in the catalytic domain, thereby leading to generation of molecules with greater stability, longer half-lives and increased levels of expression as compared to the full-length aggrecanases 1 and 2.

It is expected that other species have DNA sequences that are similar or identical to human aggrecanase enzymes described herein. Accordingly, the invention further includes methods for obtaining other nucleic acid molecules encoding truncated aggrecanases or aggrecanase-like molecules or aggrecanases with amino acid substitutions that alter their biological activity, from humans as well as non-human species. In one embodiment, a method for isolating a nucleic acid sequence encoding an aggrecanase of the invention involves utilizing a nucleic acid sequence disclosed herein or variants or fragments thereof; for example, SEQ ID NO: 1 or a fragment or a variant thereof; SEQ ID NO: 3 or a fragment or a variant thereof; SEQ ID NO: 5 or a fragment or a variant thereof; SEQ ID NO: 7 or a fragment or a variant thereof; SEQ ID NO: 9 or a fragment or a variant thereof; SEQ ID NO: 31 or a fragment or a variant thereof; SEQ ID NO: 32 or a fragment or variant thereof; or SEQ ID NO: 33 or a fragment or a variant thereof, to design probes for screening libraries for the corresponding gene from other species or coding regions of genes that encode proteins/peptides with aggrecanase activity. Therefore, the invention includes DNA sequences from other species, which are homologous to human aggrecanase sequences, or fragments or variants thereof, and can be obtained using at least one of the DNA sequences provided herein. In addition, the present invention includes DNA sequences that encode fragments or variants of aggrecanases of the invention. The present invention may also include functional fragments of the aggrecanase protein, and DNA sequences encoding such functional fragments, as well as functional fragments of other related proteins. The ability of such a fragment to function is determinable by an assay of the protein in one of many biological assays described for the assay of the aggrecanase protein.

In another aspect, the invention provides methods for producing isolated truncated aggrecanases of the invention. In one embodiment, a human aggrecanase protein of the invention or a variant or fragment thereof may be produced, for example, by culturing a cell transformed with a DNA sequence: from nucleotide #1 through nucleotide #2259, set forth in SEQ ID NO: 3; or from nucleotide #1 through nucleotide #2256, set forth in FIG. 5; or from nucleotide #1 through nucleotide #1884, set forth in FIG. 7; or from nucleotide #1 through nucleotide #1701, set forth in FIG. 9, and recovering and purifying from the culture medium an aggrecanase-2 protein characterized by the amino acid sequence set forth in: FIG. 4 from amino acid #1 (Met) through amino acid #753 (Glu); FIG. 6 from amino acid #1 (Met) through amino acid #752 (Pro); FIG. 8 from amino acid #1 (Met) through amino acid #628 (Phe); or FIG. 10 from amino acid #1 (Met) through amino acid #567 (His). Similarly, nucleic acid sequences expressing truncated aggrecanase-1 molecules; for example, nucleic acid sequences set forth in FIGS. 23 and 24 can be used for the production of truncated aggrecanase-1 proteins set forth in FIGS. 12 and 13, where the aggrecanase-1 proteins are substantially free from other proteinaceous materials with which they are co-produced.

In another embodiment, truncated aggrecanase proteins of the invention may be produced by culturing a cell transformed with a full-length DNA sequence for aggrecanase, and recovering truncated aggrecanase proteins from the culture medium. Accordingly, in one embodiment, a truncated aggrecanase-2 protein including amino acid #1 (Met) to amino acid #753 (Glu), set forth in SEQ ID No: 4, is recovered from the culture medium of a cell transformed with a full-length nucleic acid molecule for aggrecanase-2; for example, nucleic acid molecule set forth in SEQ ID NO: 1. In another embodiment, a truncated aggrecanase-2 molecule including amino acid #1 (Met) to amino acid #752 (Pro), is recovered from culture medium of cells transformed with a nucleic acid molecule for full-length aggrecanase-2. Truncated aggrecanase protein of SEQ ID NO: 4 results from cleavage of the full-length aggrecanase protein, set forth in SEQ ID NO: 2, at E.sup.753-G.sup.754, yielding a 55 kDa protein. The nucleotide and amino acid sequences of the full-length aggrecanase-2 molecule are set forth in SEQ ID NO: 1 and SEQ ID NO: 2 respectively (Accession Nos: NM.sub.--007038 and NP.sub.--008969) (FIGS. 1A 1C and 2).

Truncated aggrecanases of the invention that are purified from a culture medium are substantially free from other proteinaceous materials. A recovered purified aggrecanase protein having at least one TSP domain deleted generally exhibits proteolytic aggrecanase activity by cleaving aggrecan, as disclosed. Therefore, truncated proteins of the invention may be further characterized by the ability to demonstrate aggrecan proteolytic activity in an assay which determines the presence of an aggrecan-degrading molecule. These assays or the development thereof is within the knowledge of one skilled in the art. Such assays may involve contacting an aggrecan substrate with a truncated aggrecanase molecule and monitoring the production of aggrecan fragments (see, for example, Hughes et al., Biochem J 305: 799 804 (1995); Mercuri et al., J. Bio Chem. 274:32387 32395 (1999)). For production in mammalian cells, a DNA sequence used for expression of a truncated aggrecanase of the invention further comprises a DNA sequence encoding a suitable propeptide 5' to and linked in frame to the nucleotide sequence encoding the aggrecanase enzyme.

The invention also provides antibodies that bind to isolated aggrecanase proteins of the invention. In one embodiment, such an antibody reduces, inhibits or antagonizes aggrecanase activity. The invention further provides methods for developing and identifying inhibitors of aggrecanase activity comprising the use of a truncated aggrecanase protein with amino acid sequence chosen from, for example, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 13 or a fragment or a variant thereof. In one embodiment, inhibitors of aggrecanase activity prevent cleavage of aggrecan.

Additionally, the invention provides pharmaceutical compositions for inhibiting the proteolytic activity of aggrecanases, wherein the compositions comprise at least one antibody according to the invention and at least one pharmaceutical carrier. The invention also provides methods for inhibiting aggrecanase activity in a mammal comprising administering to the mammal an effective amount of a pharmaceutical composition according to the invention to inhibit aggrecanase activity.

In another embodiment, the invention includes methods for identifying or developing inhibitors of aggrecanases and the inhibitors produced thereby. In one embodiment, inhibitors of the invention prevent binding of an aggrecanase to an aggrecan molecule. In another embodiment, inhibitors of the invention prevent cleavage of aggrecan by aggrecanase.

The method may entail the identification of inhibitors based on an assay comprising combining at least one aggrecanase protein of this invention with at least one test sample; and determining if the test sample inhibits activity of the aggrecanase protein. The test sample may comprise known or unknown samples, and these samples may be peptides, proteins, chemical compounds (often referred to as small molecules), or antibodies. They may be selected for testing individually, or in batches, such as from a library. The art provides aggrecanase activity assays that could be easily utilized in such a method. Assays for inhibitors may involve contacting a mixture of aggrecan and the inhibitor with an aggrecanase molecule followed by measurement of the aggrecanase inhibition; for instance, by detection and measurement of aggrecan fragments produced by cleavage at an aggrecanase-susceptible site.

The method may also entail the determination of binding sites based on at least one of the amino acid sequences of aggrecanase and the three-dimensional structure of aggrecanase, and optionally aggrecan. Based on this information, one could develop or identify a candidate molecule that may inhibit aggrecanase activity based on a structural analysis, such as predicted structural interaction with the binding site. In one embodiment, such a molecule may comprise a peptide, protein, chemical compound, or antibody. Candidate molecules may be later assayed for actual inhibitory activity of the aggrecanase enzyme, as discussed.

Another aspect of the invention therefore provides pharmaceutical compositions containing a therapeutically effective amount of aggrecanase inhibitors in a pharmaceutically acceptable vehicle.

Aggrecanase-mediated degradation of aggrecan in cartilage has been implicated in osteoarthritis and other inflammatory diseases. Therefore, these compositions of the invention may be used in the treatment of diseases characterized by the degradation of aggrecan and/or an up regulation of aggrecanase. The compositions may be used in the treatment of these conditions or in the prevention thereof.

The invention further includes methods for treating patients suffering from conditions characterized by a degradation of aggrecan or preventing such conditions. These methods, according to the invention, entail administering to a patient needing such treatment an effective amount of a composition comprising an aggrecanase inhibitor which inhibits the proteolytic activity of aggrecanase enzymes.

The DNA sequences of the present invention are useful, for example, as probes for the detection of mRNA encoding aggrecanase in a given cell population. Thus, the present invention includes methods of detecting or diagnosing genetic disorders involving aggrecanases, or disorders involving cellular, organ, or tissue disorders in which aggrecanase is irregularly transcribed or expressed. The DNA sequences may also be useful for preparing vectors for gene therapy applications as described below.

A further aspect of the invention includes vectors comprising a DNA sequence as described above in operative association with an expression control sequence therefor. These vectors may be employed in a novel process for producing an aggrecanase protein of the invention in which a cell line transformed with a DNA sequence encoding an aggrecanase protein in operative association with an expression control sequence therefor, is cultured in a suitable culture medium and an aggrecanase protein is recovered and purified therefrom. This process may employ a number of known cells, both prokaryotic and eukaryotic in origin, as host cells for expression of the polypeptide. The vectors may be used in gene therapy applications. In such use, the vectors may be transfected into the cells of a patient ex vivo, and the cells may be reintroduced into a patient. Alternatively, the vectors may be introduced into a patient in vivo through targeted transfection.

Additional aspects of the disclosure will be set forth in part in the description, and in part be obvious from the description, or may be learned from practicing the invention. The invention is set forth and particularly pointed out in the claims, and the disclosure should not be construed as limiting the scope of the claims. The following detailed description includes exemplary representations of various embodiments of the invention, which are not restrictive of the invention as claimed. The accompanying figures constitute a part of this specification and, together with the description, serve to illustrate embodiments and not limit the invention.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A 1C show the nucleotide sequence of a full-length aggrecanase-2/ADAMTS-5 molecule (SEQ ID NO: 1).

FIG. 2 shows the amino acid sequence of a full length aggrecanase-2/ADAMTS-5 molecule (SEQ ID NO: 2) encoded by nucleotide #1 through nucleotide #2915 of SEQ ID NO: 1.

FIG. 3 shows the nucleotide sequence of a truncated aggrecanase-2 protein of the invention from nucleotide #1 through nucleotide #2259 (SEQ ID NO: 3).

FIG. 4 shows the amino acid sequence of a truncated aggrecanase-2 protein of the invention from amino acid #1 through amino acid #753 (SEQ ID NO: 4), encoded by nucleotide sequence set forth in SEQ ID NO: 3.

FIG. 5 shows the nucleotide sequence of a truncated aggrecanase-2/ADAMTS-5 of the invention from nucleotide #1 through nucleotide #2256 (SEQ ID NO: 5).

FIG. 6 shows the amino acid sequence of a truncated aggrecanase-2 protein of the invention from amino acid #1 through amino acid #752 (SEQ ID NO: 6), encoded by nucleotide sequence set forth in SEQ ID NO: 5.

FIG. 7 shows the nucleotide sequence of a truncated aggrecanase-2 molecule of the invention from nucleotide #1 through nucleotide #1884 (SEQ ID NO: 7).

FIG. 8 shows the amino acid sequence of a truncated aggrecanase-2 protein of the invention from amino acid #1 through amino acid #628 (SEQ ID NO: 8), encoded by the nucleotide sequence set forth in SEQ ID NO: 7.

FIG. 9 shows the nucleotide sequence of a truncated aggrecanase-2 molecule of the invention from nucleotide #1 through nucleotide #1701 (SEQ ID NO: 9).

FIG. 10 shows the amino acid sequence of a truncated aggrecanase-2 protein of the invention from amino acid #1 through amino acid #567 (SEQ ID NO: 10), encoded by the nucleotide sequence set forth in SEQ ID NO: 9.

FIG. 11 shows the amino acid sequence of a full-length aggrecanase-1 molecule (SEQ ID NO: 11).

FIG. 12 shows the amino acid sequence of a truncated aggrecanase-1 molecule including amino acid #1 through amino acid #575 (Pro) (SEQ ID NO: 12).

FIG. 13 shows the amino acid sequence of a truncated aggrecanase-1 molecule including amino acid #1 through amino acid #520 (Ala) (SEQ ID NO: 13).

FIG. 14 shows the nucleotide sequence of a recombinant truncated aggrecanase-2 protein comprising a peptide linker and a streptavidin tag (SEQ ID NO: 14).

FIG. 15 shows the amino acid sequence of a recombinant truncated aggrecanase-2 protein (SEQ ID NO: 15), encoded by nucleotide sequence set forth in SEQ ID NO: 14.

FIG. 16 shows aggrecanase activity of conditioned medium from CHO cells expressing wild-type (ADAMTS-5 WT) or active-site mutant (ADAMTS-5 ASM) as detected by the aggrecanase ELISA assay.

FIG. 17 shows a schematic representation of flag-tagged truncated aggrecanase-1 molecules with an E-to-Q mutation in the catalytic domain.

FIG. 18 shows a schematic representation of streptavidin tagged truncated aggrecanase-2 molecules.

FIG. 19 shows a western blot with expression of truncated aggrecanase-2 proteins in comparison with a full-length aggrecanase-2 protein.

FIG. 20 shows percent aggrecan cleavage by truncated aggrecanase-2 molecules of the invention in a 3B3 ELISA assay.

FIG. 21 shows the amino acid sequence for a full-length ADAMTS-5 protein with an E-to-Q mutation at amino acid position 411 (SEQ IS NO: 30).

FIGS. 22A and 22B show a nucleic acid sequence encoding a full-length aggrecanase-1 protein (SEQ ID NO: 31).

FIG. 23 shows a nucleic acid sequence encoding a truncated aggrecanase-1 molecule; for example, the protein set forth in FIG. 12 (SEQ ID NO: 32).

FIG. 24 shows a nucleic acid sequence encoding a truncated aggrecanase-1 molecule; for example, the protein set forth in FIG. 13 (SEQ ID NO: 33).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "aggrecanase" refers to a family of polypeptides that are capable of cleaving the aggrecan protein. Generally, these are proteins that cleave aggrecan at the Glu.sup.373-Ala.sup.374 aggrecanase cleavage site. Aggrecanases of the present invention encompass but are not limited to the sequences of SEQ ID NO: 11 (aggrecanase-1) and SEQ ID NO: 2 (aggrecanase-2). The term "aggrecanase" includes naturally occurring variants SEQ ID NOs: 11 and 2, as well as fragments of the sequences encoded by SEQ ID NOs: 11 and 2 that are active in at least one of the assays provided. For example, included in this definition are amino acid sequences substantially similar or substantially identical to the amino acid of SEQ ID NOs: 11 or 2 or fragments thereof; or an amino acid sequence at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of SEQ ID NO: 11 or 2, or a fragment thereof.

The term aggrecanase further includes the proteins encoded by the nucleic acid sequence of SEQ ID NO: 31 and 1 (aggrecanase-1 and 2 respectively) disclosed, fragments and variants thereof. In one embodiment, the nucleic acids of the present invention will possess a sequence which is either derived from, or is a variant of a natural aggrecanase encoding gene, or a fragment thereof.

The term "antibody" refers to an immunoglobulin, or a fragment thereof, and encompasses any polypeptide comprising an antigen-binding site. The term includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. It also includes, unless otherwise stated, antibody fragments such as Fab, F(ab').sub.2, Fv, scFv, Fd, dAb, and other antibody fragments which retain the antigen binding function.

The term "biological activity" refers to at least one cellular process interrupted or initiated by an aggrecanase enzyme binding to aggrecan. Generally, biological activity refers to proteolytic cleavage of aggrecan by aggrecanase. Aggrecanase activities include, but are not limited to, binding of aggrecanase to aggrecan and cleavage of aggrecan by aggrecanase. Activity can also include a biological response resulting from the binding to or cleavage of aggrecan by aggrecanases of the invention.

The term "deletion" as used herein is the removal of at least one amino acid from the full-length amino acid sequence of an aggrecanase. The term deletion also refers to removal of nucleotides from a nucleic acid sequence encoding an aggrecanase, thereby resulting in a nucleic acid that encodes a truncated protein.

A deletion in a nucleic acid molecule encoding an aggrecanase or an aggrecanase protein can be made anywhere in the nucleic acid molecule or the protein as desirable. For example, a deletion may occur anywhere within the protein; for example, N-terminus, C-terminus or any other part of an aggrecanase protein, and can include removal of at least one amino acid; for example, from about 1 to about 5 amino acids, from about 5 to about 10 amino acids, from about 10 to about 20 amino acids, from about 20 to about 30 amino acids, from about 30 to about 50 amino acids, from about 50 to about 100 amino acids, from about 100 to about 150 amino acids, from about 150 to about 200 amino acids, from about 200 to about 250 amino acids, from about 250 to about 300 amino acids, from about 300 to about 350 amino acids, from about 350 to about 400 amino acids, from about 400 to about 450 amino acids, from about 450 to about 500 amino acids, or greater than 500 amino acids.

Deletions can also be made in nucleic acid molecules that encode aggrecanases of the invention; for example, deletions can be made in the region of a nucleic acid that encodes for a TSP domain of an aggrecanase. Such deletions typically encompass the 3' region of a nucleic acid molecule encoding an aggrecanase of the invention. However, it is contemplated that deletions can be made anywhere in a nucleic acid expressing an aggrecanase molecule. One skilled in the art can test truncated aggrecanases of the invention for activity in one of many assays disclosed.

An amino acid deletion according to the invention comprises the removal of at least one amino acid from the N-terminus of an aggrecanase protein. In another embodiment, a deletion comprises removal of at least one amino acid from the C-terminus of an aggrecanase protein. In yet another embodiment, a deletion comprises removal of amino acids from a region lying between N-terminal end and C-terminal end of an aggrecanase molecule. In one embodiment, such a deletion involves removal of an entire domain of an aggrecanase of the invention. For example, aggrecanases of the invention comprising a deletion include aggrecanase-1 molecules that have one TSP domain deleted or aggrecanase-1 molecules that have two TSP domains deleted, or aggrecanase-2 molecules that have one TSP domain deleted, or aggrecanase-2 molecules that have two TSP domains deleted. Aggrecanases according to the invention may comprise deletion of all TSP domains within an aggrecanase protein. It is contemplated that other domains within aggrecanase molecules may also be deleted to generate biologically active truncated aggrecanases that are more stable than the full-length counterpart.

The term "fragment" as used herein, refers to a portion of an aggrecanase protein of the invention, for example, a portion of amino acid sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, 11, 12, and 13. In one embodiment, a fragment of a protein refers to an amino acid sequence that has aggrecanase activity in one of many assays provided. The term "fragment" also includes nucleotide sequences that are long enough to encode peptides that exhibit aggrecanase activity. However, fragments of a nucleotide sequence may or may not encode protein fragments that retain aggrecanase biological activity. Protein and nucleic acid fragments of the invention include portions of other nucleic acid molecules or proteins that are substantially identical to at least one portion of the nucleotide sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 31, 32, or 33, or at least one portion of amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 11, 12, or 13, respectively. Fragments of nucleic acid sequences may range, for example, from at least about 20 nucleotides, from at least about 50 nucleotides, from at least about 100 nucleotides, from at least about 150 nucleotides, from at least about 200 nucleotides, from at least about 250 nucleotides, from at least about 300 nucleotides, from at least about 400 nucleotides, from at least about 500 nucleotides, from at least about 600 nucleotides, from at least about 700 nucleotides, from at least about 800 nucleotides, up to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 1. In one embodiment, nucleic acid fragments encode peptides that have aggrecanase activity. Protein fragments may range, for example, from at least about 5 amino acids, from at least about 10 amino acids, from at least about 20 amino acids, from about 30 amino acids, from about 40 amino acids, from about 50 amino acids, from at least about 100 amino acids, from at least about 150 amino acids, from at least about 200 amino acids, from at least about 250 amino acids, from at least about 300 amino acids, from at least about 350 amino acids, from at least about 400 amino acids, up to the entire length of the amino acid sequence set forth in SEQ ID NO: 2. Fragments of nucleic acids of the invention can arise from 3' portions, 5' portions or any other part of a nucleic acid sequence. Similarly, protein fragments can arise from N-terminus portion, C-terminus portion or any other part of a protein. In one embodiment, fragments of proteins retain aggrecanase activity. In another embodiment, protein fragments of the invention arise from a portion of a protein that has aggrecanase activity.

The term "effective amount" refers to a dosage or an amount of a composition of at least one aggrecanase inhibitor or antibody of the invention that is sufficient to treat a patient.

The term "inhibit" or "inhibition" of aggrecanase or aggrecanase activity refers to a reduction, inhibition of otherwise diminution of at least one activity of aggrecanase due to binding of an inhibitor to the aggrecanase or aggrecan. The reduction, inhibition, or diminution of binding can be measured by one of many assays provided. Inhibition of aggrecanase activity does not necessarily indicate a complete negation of aggrecanase activity. A reduction in activity can be, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In one embodiment, inhibition is measured by a reduction in the detection of cleavage products of aggrecan. Inhibitors of the present invention include, but are not limited to, antibodies, proteins, peptides, and chemical compounds (often referred to as small molecules).

The term "isolated" describes a nucleic acid molecule or polypeptide molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived. The term "isolated" also refers to an aggrecanase protein according to the invention which is free from association with other proteases and retains aggrecanase proteolytic activity. In addition, the term "isolated" refers to nucleic acid molecules that encode aggrecanases of the invention and are free from other cellular material and contaminants.

The term "neoepitope antibody" refers to an antibody that specifically recognizes a new N- or C-terminal amino acid sequence generated by proteolytic cleavage but which does not bind to such an epitope on the intact (uncleaved) substrate.

The term "operative association" with an expression control sequence generally refers to the presence of a specific nucleotide sequence or sequences that control or affect transcription rate or efficiency of a nucleotide molecule linked to the sequence. For example, a promoter sequence that is located proximally to the 5' end of an aggrecanase coding nucleotide sequence may be in operative association with the aggrecanase encoding nucleotide sequence. Expression control sequences include, but are not limited to, for example, promoters, enhancers, and other expression control sequences, or any combination of such elements, either 5' or 3' to an aggrecanase encoding nucleotide sequence in order to control its expression. Not all of these elements are required, however. A skilled artisan can select the appropriate expression control sequences, for example, depending on desired expression levels for the aggrecanases of the invention.

The term "specific binding" of an antibody means that the antibody binds to at least one aggrecanase molecule of the present invention and the antibody will not show any significant binding to molecules other than at least one novel aggrecanase molecule. The term is also applicable where, e.g., an antigen binding domain of an antibody is specific for a particular epitope, which is represented on a number of antigens, and the specific binding member (the antibody) carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope. Therefore, it is contemplated that an antibody of the invention will bind to an epitope on multiple novel aggrecanase proteins. Typically, the binding is considered specific when the affinity constant K.sub.a is higher than 10.sup.8 M.sup.-1. An antibody is said to "specifically bind" to an antigen if, under appropriately selected conditions, such binding is not substantially inhibited, while at the same time non-specific binding is inhibited. The conditions are usually defined in terms of concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of additional molecules associated with the binding reaction (e.g., serum albumin, milk casein), etc. Such conditions are well known in the art, and a skilled artisan using routine techniques can select appropriate conditions.

The term "stability" as used herein, generally refers to a decrease in the rate of degradation of a protein, thereby increasing its half-life, solubility, and/or expression levels. Several factors affect protein stability in vitro and in vivo, for example, pH, salt concentration, temperature, protein degradation, for example by proteases, metal ions, autocatalysis of proteins, hydrophobicity etc. In one embodiment, the invention includes truncated aggrecanases that are more stable than their full-length counterparts. In another embodiment, the invention includes aggrecanase active-site mutants that are more stable than their wild-type counterparts. Conditions that make a protein more stable generally include conditions that keep the protein in a folded conformation for longer than normal, thereby preserving its biological activity for a longer period of time. An increase in stability of a protein generally increases its half-life and expression levels, thereby making it possible to purify the protein in large amounts for therapeutic purposes and for development of inhibitors.

The term "highly stringent" or "high stringency" describes conditions for hybridization and washing used for determining nucleic acid-nucleic acid interactions. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. The stringency conditions are dependent on the length of the nucleic acid and the base composition of the nucleic acid and can be determined by techniques well known in the art. Generally, stringency can be altered or controlled by, for example, manipulating temperature and salt concentration during hybridization and washing. For example, a combination of high temperature and low salt concentration increases stringency. Such conditions are known to those skilled in the art and can be found in, for example, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1 6.3.6, (1989). Both aqueous and nonaqueous conditions as described in the art can be used. One example of highly stringent hybridization conditions is hybridization in 6.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C., followed by at least one wash in 0.2.times.SSC, 0.1% SDS at 50.degree. C. A second example of highly stringent hybridization conditions is hybridization in 6.times.SSC at about 45.degree. C., followed by at least one wash in 0.2.times.SSC, 0.1% SDS at 55.degree. C. Another example of highly stringent hybridization conditions is hybridization in 6.times.SSC at about 45.degree. C., followed by at least one wash in 0.2.times.SSC, 0.1% SDS at 60.degree. C. A further example of highly stringent hybridization conditions is hybridization in 6.times.SSC at about 45.degree. C., followed by at least one wash in 0.2.times.SSC, 0.1% SDS at 65.degree. C. Highly stringent conditions include hybridization in 0.5M sodium phosphate, 7% SDS at 65.degree. C., followed by at least one wash at 0.2.times.SSC, 1% SDS at 65.degree. C.

The phrase "moderately stringent" or "moderate stringency" hybridization refers to conditions that permit a nucleic acid to bind a complementary nucleic acid that has at least about 60%, at least about 75%, or at least about 85%, identity to the nucleic acid, with greater than about 90% identity to the nucleic acid especially preferred. Moderately stringent conditions comprise but are not limited to, for example, hybridization in 50% formamide, 5.times. Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree. C., followed by washing in 0.2.times.SSPE, 0.2% SDS, at 65.degree. C. (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).

The phrase "substantially identical" or "substantially similar" means that the relevant amino acid or nucleotide sequence will be identical to or have insubstantial differences (through conserved amino acid substitutions) in comparison to the sequences which are disclosed. Nucleotide and polypeptides of the invention include, for example, those that are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical in sequence to nucleic acid molecules and polypeptides disclosed.

For polypeptides, at least 20, 30, 50, 100, or more amino acids will be compared between the original polypeptide and the variant polypeptide that is substantially identical to the original. For nucleic acids, at least 50, 100, 150, 300, or more nucleotides will be compared between the original nucleic acid and the variant nucleic acid that is substantially identical to the original. Thus, a variant could be substantially identical in a region or regions, but divergent in others, while still meeting the definition of "substantially identical." Percent identity between two sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al., J. Mol. Biol., 215:403 410 (1990), the algorithm of Needleman and Wunsch, J. Mol. Biol., 48:444 453 (1970), or the algorithm of Meyers and Miller, Comput. Appl. Biosci., 4:11 17 (1988).

The term "treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventative measures). Treatment may regulate aggrecanase activity or the level of aggrecanase to prevent or ameliorate clinical symptoms of at least one disease. The inhibitors and/or antibodies may function by, for example, preventing the interaction or binding of aggrecanase to aggrecan, or by reducing or inhibiting aggrecanase activity.

The term "truncated" as used herein, refers to nucleo