WO2002042439A2

WO2002042439A2 - Aggrecanase adamts9

Info

Publication number: WO2002042439A2
Application number: PCT/US2001/049814
Authority: WO
Inventors: Lisa Anne Racie; Natalie Constance Twine; Michael John Agostino; Neil Michael Wolfman; Elisabeth Ann Morris
Original assignee: Genetics Institute, Llc
Priority date: 2000-10-27
Filing date: 2001-10-25
Publication date: 2002-05-30
Also published as: AU2002239680A1; WO2002042439A3

Abstract

Aggrecanase proteins and the nucleotides sequences encoding them as well as processes for producing them are disclosed. 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.

Description

TITLE OF THE INVENTION

AGGRECANASE MOLECULES

The present mvention relates to the discovery of nucleotide sequences encoding novel aggrecanase molecules, the aggrecanase proteins and processes for producing them. The mvention further relates to the development of inhibitors of, as well as antibodies to the 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. Osteoarthritis is a debilitating disease which affects at least 30 million Americans [MacLean et al. J Rheumatol 25:2213-8. (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, KD. and Man in HJ. Pathogenesis of Osteoarthritis. in Textbook of Rheumatology, WB 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 thought to be responsible for the cleavage of aggrecan thereby having a role in cartilage degradation associated with osteoarthritis and inflammatory joint disease. Work has been conducted to identify the enzyme responsible for the degradation of aggrecan in human osteoarthritic cartilage. Two enzymatic cleavage sites have been identified within the interglobular domain of aggrecan. One (Asn³ l-Phe³⁴²) is observed to be cleaved by several known metalloproteases [Flannery, CR et al. J Biol Chem 267:1008-14. 1992; Fosang, AJ et al. Biochemical J. 304:347-351. (1994 1. The aggrecan fragment found in human synovial fluid, and generated by IL-1 induced cartilage aggrecan cleavage is at the Glu³⁷³-Ala3⁷⁴ bond [Sandy, JD, et al. J Clin Invest 69:1512-1516. (1992); Lohmander LS, et al. Arthritis Rheum 36: 1214-1222. (1993); Sandy JD et al. J Biol Chem. 266: 8683-8685. (1991)], indicating that none of the known enzymes are responsible for aggrecan cleavage in vivo.

Recently, identification of two enzymes , aggrecanase- 1 (AD AMTS 4) and aggrecanase -2 (ADAMTS-11) within the "Disintegrin-like and Metalloprotease with Thrombospondin type 1 motif" (ADAM-TS) family have been identified which are synthesized by IL-1 stimulated cartilage and cleave aggrecan at the appropriate site [Tortorella MD, et al Science 284: 1664-6. (1999); Abbaszade, I, et al. J Biol Chem 274: 23443-23450. (1999)]. It is possible that these enzymes could be synthesized by osteoarthritic human articular cartilage. It is also contemplated that there are other, related enzymes in the ADAM-TS family which are capable of cleaving aggrecan at the Glu³⁷³-Ala3⁷⁴ bond and could contribute to aggrecan cleavage in osteoarthritis.

SUMMARY OF THE INVENTION

The present invention is directed to the identification of aggrecanase protein molecules capable of cleaving aggrecanase, the nucleotide sequences which encode the aggrecanase enzymes, and processes for the production of aggrecanases. These enzymes are contemplated to be characterized as having proteolytic aggrecanase activity. The invention further includes compositions comprising these enzymes as well as antibodies to these enzymes. In addition, the invention includes methods for developing inhibitors of aggrecanase which block the enzyme's proteolytic activity. These inhibitors and antibodies may be used in various assays and therapies for treatment of conditions characterized by the degradation of articular cartilage.

The nucleotide sequence of the aggrecanase molecule of the present invention is set forth in SEQ ID NO: 8. In a further embodiment, the nucleotide sequence of the aggrecanase molecule of the present invention is set forth SEQ ID NO: 6 from nucleotide # 1 to #5605. Other embodiments of the nucleotide sequence of the invention comprise the sequences of SEQ ID NO: 1, SEQ ID NO. 2, SEQ ID NO: 3, SEQ ID NO 4 and SEQ ID NO: 5. The invention further includes equivalent degenerative codon sequences of these nucleotide sequences, as well as fragments thereof which exhibit aggrecanase activity. The amino acid sequence of an isolated aggrecanase molecule of the present invention is set forth in SEQ ID NO:9. In a further embodiment, the amino acid sequence of an isolated aggrecanase molecule comprises the sequence set forth in SEQ ID. No. 7. The invention further includes fragments of the amino acid sequence which encode molecules exhibiting aggrecanase activity. In another embodiment the amino acid sequences of an isolated aggrecanase molecule of the present invention comprises the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 7 from amino acid #1 to #139. The human aggrecanase protein or a fragment thereof may be produced by culturing a cell transformed with a DNA sequence of SEQ ID NO: 8 or SEQ ID NO: 6 comprising nucleotide # 1 to #5605 and recovering and purifying from the culture medium a protein characterized by the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 7, respectively, substantially free from other proteinaceous materials with which it is co-produced. In another embodiment the human aggrecanase protein or a fragment thereof may be produced by culturing a cell transformed with a DNA sequence of SEQ ID NO: 8 or SEQ ID NO: 6 comprising nucleotide # 1 to #466 of SEQ ID NO:6 and recovering and purifying from the culture medium a protein characterized by the amino acid sequence set forth respectively in SEQ ID NO:9 or SEQ ID NO: 7 comprising amino acid #1-139 substantially free from other proteinaceous materials with which it is co-produced. For production in mammalian cells, the DNA sequence 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 includes methods for obtaining the full length aggrecanase molecules, the DNA sequence obtained by this method and the protein encoded thereby. The method for isolation of further sequence involves utilizing the aggrecanase sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 6 from nucleotide # 1 to #5605 to design probes for screening using standard procedures known to those skilled in the art.

It is expected that other species have DNA sequences homologous to human aggrecanase enzyme. The invention, therefore, includes methods for obtaining the DNA

sequences encoding other aggrecanase molecules , the DNA sequences obtained by those

methods, and the protein encoded by those DNA sequences. This method entails

utilizing the nucleotide sequence of the invention or portions thereof to design probes to

screen libraries for the corresponding gene from other species or coding sequences or

fragments thereof from using standard techniques. Thus, the present invention may

include DNA sequences from other species, which are homologous to the human

aggrecanase protein and can be obtained using the human sequence. 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 assay of the

protein in the biological assays described for the assay of the aggrecanase protein.

The aggrecanase proteins of the present invention may be produced by culturing

a cell transformed with the DNA sequence of SEQ ID NO: 8 or the sequence of SEQ JD

NO. 6 comprising nucleotide # 1 to # 5605 or comprising nucleotide # 1 to #466 of SEQ

ID NO: 6 and recovering and purifying aggrecanase protein from the culture medium. In the first embodiment the protein comprises the amino acid sequence of SEQ ID NO:9 . In

the latter embodiments the protein comprises respectively, amino acid #1 to #1610 of

SEQ ID NO:7 and amino acid #1 to #139 of SEQ ID No:7. In further embodiments the

nucleotide sequences set forth in SEQ ID NOS: 1, 2, 3, 4, and 5 are utilized in the

expression of the aggrecanase molecules. The purified expressed protein is substantially

free from other proteinaceous materials with which it is co-produced, as well as from

other contaminants. The recovered purified protein is contemplated to exhibit proteolytic

aggrecanase activity cleaving aggrecan. Thus, the 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 the 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)]

In another embodiment, the invention includes methods for developing inhibitors

of aggrecanase and the inhibitors produced thereby. These inhibitors prevent cleavage of

aggrecan. The method may entail the determination of binding sites based on the three

dimensional structure of aggrecanase and aggrecan and developing a molecule reactive

with the binding site. Candidate molecules are assayed for inhibitory activity. Additional

standard methods for developing inhibitors of the aggrecanase molecule are known to

those skilled in the art. Assays for the inhibitors 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.

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 upregulation of aggrecanase. The

compositions may be used in the treatment of these conditions or in the prevention

thereof.

The invention 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.

Still a further aspect of the invention are DNA sequences coding for expression of

an aggrecanase protein. Such sequences include the sequence of nucleotides in a 5' to 3'

direction illustrated in SEQ ID NO: 1 comprising nucleotide # 1 to # 1506 or comprising

nucleotide # 1 to #1028 of SEQ ID NO: 2 or comprising nucleotide # 1 to #1254 of SEQ ID. NO:3 or comprising nucleotide #1 to #687 of SEQ ID NO: 4 or comprising nucleotide

# 1 to #466 of SEQ ID NO: 5 or comprising nucleotide # 1 to #5605 of SEQ ID NO:6 , the nucleotide sequence of SEQ ID NO: 8 and DNA sequences which, but for the degeneracy of the genetic code, are identical to the DNA sequence set forth above, and

encode an aggrecanase protein. Further included in the present invention are DNA

sequences which hybridize under stringent conditions with the DNA sequence of SEQ ID

NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, or

SEQ ID NO: 8 and encode a protein having the ability to cleave aggrecan. Preferred

DNA sequences include those which hybridize under stringent conditions [see, T.

Maniatis et al, Molecular Cloning (A Laboratory Manual). Cold Spring Harbor

Laboratory (1982), pages 387 to 389]. It is generally preferred that such DNA sequences

encode a polypeptide which is at least about 80% homologous, and more preferably at

least about 90% homologous, to the sequence of set forth in SEQ ID NO: 9 or in SEQ ID

NO: 7 from amino acid #1 to #139 or amino acid #1 to #1610. Finally, allelic or other variations of the sequence of SEQ ID NO: 7 from nucleotide #1 to #466 or from #1 to #

5605 or the sequence of SEQ ID NO:9, whether such nucleotide changes result in

changes in the peptide sequence or not, but where the peptide sequence still has

aggrecanase activity, are also included in the present invention. The present invention also includes fragments of the DNA sequences shown in SEQ ID NO: 1, SEQ ID NO: 2,

SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:8 which

encode a polypeptide which retains the activity of aggrecanase.

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 the aggrecanase, 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 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.

Still a further aspect of the invention are aggrecanase proteins or polypeptides.

Such polypeptides are characterized by having an amino acid sequence including the

sequence illustrated in SEQ ID NO. 7 comprising amino acid #1 to #139 or amino acids

#1 to #1610, the sequence of SEQ ID NO:9 or variants of the amino acid sequences of

SEQ ID NO.7 or SEQ ID NO:9, including naturally occurring allelic variants, and other

variants in which the protein retains the ability to cleave aggrecan characteristic of

aggrecanase molecules. Preferred polypeptides include a polypeptide which is at least about 80% homologous, and more preferably at least about 90% homologous, to the amino acid sequence shown in SEQ ID NO. 7 comprising amino acid #1 to #139 or

comprising #1 to #1610 or the sequence of SEQ ID NO: 9. Finally, allelic or other

variations of these sequences of SEQ ID NO. 7or SEQ ID NO: 9 , whether such amino

acid changes are induced by mutagenesis, chemical alteration, or by alteration of DNA

sequence used to produce the polypeptide, where the peptide sequence still has

aggrecanase activity, are also included in the present invention. The present invention

also includes fragments of the amino acid sequence of SEQ ID NO. 7 or SEQ ID NO:9

which retain the activity of aggrecanase protein.

The purified proteins of the present inventions may be used to generate antibodies,

either monoclonal or polyclonal, to aggrecanase and/or other aggrecanase -related

proteins, using methods that are known in the art of antibody production. Thus, the

present invention also includes antibodies to aggrecanase or other related proteins. The

antibodies may be useful for detection and/or purification of aggrecanase or related

proteins, or for inhibiting or preventing the effects of aggrecanase. The aggrecanase of

the invention or portions thereof may be utilized to prepare antibodies that specifically

bind to aggrecanase.

DETAILED DESCRIPTION OF THE INVENTION

The nucleotide sequence of the human aggrecanase of the present invention comprises the sequence set forth in SEQ ID NO: 8. In a further embodiment, the nucleotide sequence of the human aggrecanase of the present invention comprises nucleotides # 1 to # 5605 of SEQ ID NO: 6. In another embodiment the nucleotide sequence comprises nucleotide #1-466 of SEQ ID NO:6. Other embodiments comprise

the nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ

ID NO:4, and SEQ ID NO: 5. The human aggrecanase protein sequence comprises the

sequence set forth in SEQ ID NO:9. In a further embodiment, a human aggrecanase of

the present invention comprises amino acids # 1 to # 1610 set forth in SEQ ID NO. 7. In

another embodiment the human aggrecanase sequence if the invention comprises amino acids #1 to #466 of SEQ ID NO: 7. Further sequences of the aggrecanase of the present

invention may be obtained using the sequences of SEQ ID NO. 6 comprising nucleotides

# 1 to # 466 or nucleotides #1 to # 5605 to design probes for screening for the full

sequence using standard techniques.

The aggrecanase proteins of the present invention, include polypeptides

comprising the amino acid sequence SEQ ID NO:9 or of SEQ ID NO.7 from amino acid

#1 to #139 or from #1 to #1610 and having the ability to cleave aggrecan.

The aggrecanase proteins recovered from the culture medium are purified by

isolating them from other proteinaceous materials from which they are co-produced and from other contaminants present. The isolated and purified proteins may be

characterized by the ability to cleave aggrecan substrate. The aggrecanase proteins

provided herein also include factors encoded by the sequences similar to those of SEQ ID

NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 or

SEQ ID NO: 8, but into which modifications or deletions are naturally provided (e.g.

allelic variations in the nucleotide sequence which may result in amino acid changes in the polypeptide) or deliberately engineered. For example, synthetic polypeptides may wholly or partially duplicate continuous sequences of the amino acid residues of SEQ ID

NO. 7 or SEQ ID NO: 9. These sequences, by virtue of sharing primary, secondary, or

tertiary structural and conformational characteristics with aggrecanase molecules may

possess biological properties in common therewith. It is know, for example that

numerous conservative amino acid substitutions are possible without significantly

modifying the structure and conformation of a protein, thus maintaining the biological

properties as well. For example, it is recognized that conservative amino acid

substitutions may be made among amino acids with basic side chains, such as lysine (Lys

or K), arginine (Arg or R) and histidine (His or H); amino acids with acidic side chains,

such as aspartic acid (Asp or D) and glutamic acid (Glu or E); amino acids with

uncharged polar side chains, such as asparagine (Asn or N), glutamine (Gin or Q), serine

(Ser or S), threonine (Thr or T), and tyrosine (Tyr or Y); and amino acids with nonpolar

side chains, such as alanine (Ala or A), glycine (Gly or G), valine (Nal or N), leucine (Leu

or L), isoleucine (He or I), proline (Pro or P), phenylalanine (Phe or F), methionine (Met

or M), tryptophan (Trp or W) and cysteine (Cys or C). Thus, these modifications and

deletions of the native aggrecanase may be employed as biologically active substitutes for

naturally-occurring aggrecanase and in the development of inhibitors other polypeptides in therapeutic processes. It can be readily determined whether a given variant of

aggrecanase maintains the biological activity of aggrecanase by subjecting both

aggrecanase and the variant of aggrecanase, as well as inhibitors thereof, to the assays described in the examples. Other specific mutations of the sequences of aggrecanase proteins described

herein involve modifications of glycosylation sites. These modifications may involve O-

linked or N-linked glycosylation sites. For instance, the absence of glycosylation or only

partial glycosylation results from amino acid substitution or deletion at asparagine-linked

glycosylation recognition sites. The asparagine-linked glycosylation recognition sites

comprise tripeptide sequences which are specifically recognized by appropriate cellular

glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or

asparagine-X-serine, where X is usually any amino acid. A variety of amino acid

substitutions or deletions at one or both of the first or third amino acid positions of a

glycosylation recognition site (and/or amino acid deletion at the second position) results

in non-glycosylation at the modified tripeptide sequence. Additionally, bacterial

expression of aggrecanase-related protein will also result in production of a non-

glycosylated protein, even if the glycosylation sites are left unmodified.

The present invention also encompasses the novel DNA sequences, free of

association with DNA sequences encoding other proteinaceous materials, and coding for

expression of aggrecanase proteins. These DNA sequences include those depicted in SEQ

ID NO: 8 or SEQ ID NO: 1 in a 5' to 3' direction and those sequences which hybridize

thereto under stringent hybridization washing conditions [for example, 0.1X SSC, 0.1%

SDS at 65°C; see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold

Spring Harbor Laboratory (1982), pages 387 to 389] and encode a protein having

aggrecanase proteolytic activity. These DNA sequences also include those which comprise the DNA sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 and those which hybridize thereto under stringent

hybridization conditions and encode a protein which maintain the other activities

disclosed for aggrecanase.

Similarly, DNA sequences which code for aggrecanase proteins coded for by the

sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:

5, or SEQ TD NO: 6 comprising nucleotide # 1 to # 466 or comprising nucleotide # 1 to

#5605 of SEQ ID NO 6 or SEQ ID NO: 8 or aggrecanase proteins which comprise the

amino acid sequence of SEQ ID NO: 9 or SEQ ID NO.7 from amino acid # 1-139 or #1 to

#1610, but which differ in codon sequence due to the degeneracies of the genetic code or

allelic variations (naturally-occurring base changes in the species population which may

or may not result in an amino acid change) also encode the novel actors described herein. Variations in the DNA sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID

NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8 comprising

nucleotide # 1 to # 466 or comprising nucleotide # 1 to #5605 of SEQ ID NO:7 which are

caused by point mutations or by induced modifications (including insertion, deletion, and

substitution) to enhance the activity, half-life or production of the polypeptides encoded are also encompassed in the invention.

Another aspect of the present invention provides a novel method for producing

aggrecanase proteins. The method of the present invention involves culturing a suitable

cell line, which has been transformed with a DNA sequence encoding a aggrecanase

protein of the invention, under the control of known regulatory sequences. The transformed host cells are cultured and the aggrecanase proteins recovered and purified from the culture medium. The purified proteins are substantially free from other proteins

with which they are co-produced as well as from other contaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinese hamster

ovary cells (CHO). The selection of suitable mammalian host cells and methods for

transformation, culture, amplification, screening, product production and purification are

known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), or

alternatively, Kaufman et al, Mol. Cell. Biol.. 5(7): 1750-1759 (1985) or Howley et al,

U.S. Patent 4,419,446. Another suitable mammalian cell line, which is described in the

accompanying examples, is the monkey COS-1 cell line. The mammalian cell CV-1 may

also be suitable.

Bacterial cells may also be suitable hosts. For example, the various strains of E. coli (e.g., HB101, MC1061) are well-known as host cells in the field of biotechnology.

Various strains of B . subtilis. Pseudomonas. other bacilli and the like may also be

employed in this method. For expression of the protein in bacterial cells, DNA encoding

the propeptide of Aggrecanase is generally not necessary.

Many strains of yeast cells known to those skilled in the art may also be available as host cells for expression of the polypeptides of the present invention. Additionally,

where desired, insect cells may be utilized as host cells in the method of the present

invention. See, e.g. Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein.

Another aspect of the present invention provides vectors for use in the method of expression of these novel aggrecanase polypeptides. Preferably the vectors contain the full novel DNA sequences described above which encode the novel factors of the

invention. Additionally, the vectors contain appropriate expression control sequences

permitting expression of the aggrecanase protein sequences. Alternatively, vectors

incorporating modified sequences as described above are also embodiments of the present

invention. Additionally, the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3,

SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:8 or other sequences

encoding aggrecanase proteins could be manipulated to express composite aggrecanase

molecules. Thus, the present invention includes chimeric DNA molecules encoding an

aggrecanase protein comprising a fragment from SEQ ID NO: 8 or SEQ ID NO: 6

comprising nucleotide # 1 to # 466 or comprising nucleotide # 1 to #5605 of SEQ ID

NO: 6 linked in correct reading frame to a DNA sequence encoding another aggrecanase polypeptide.

The vectors may be employed in the method of transforming cell lines and contain

selected regulatory sequences in operative association with the DNA coding sequences of

the invention which are capable of directing the replication and expression thereof in

selected host cells. Regulatory sequences for such vectors are known to those skilled in

the art and may be selected depending upon the host cells. Such selection is routine and

does not form part of the present invention.

Various conditions such as osteoarthritis are known to be characterized by

degradation of aggrecan. Therefore, an aggrecanase protein of the present invention

which cleaves aggrecan may be useful for the development of inhibitors of aggrecanase. The invention therefore provides compositions comprising an aggrecanase inhibitor. The inhibitors may be developed using the aggrecanase in screening assays involving a

mixture of aggrecan substrate with the inhibitor followed by exposure to aggrecan. The

compositions may be used in the treatment of osteoarthritis and other conditions

exhibiting degradation of aggrecan.

The invention further includes antibodies which can be used to detect aggrecanase

and also may be used to inhibit the proteolytic activity of aggrecanase.

The therapeutic methods of the invention includes administering the aggrecanase

inhibitor compositions topically, systemically, or locally as an implant or device. The

dosage regimen will be determined by the attending physician considering various factors

which modify the action of the aggrecanase protein, the site of pathology, the severity of

disease, the patient's age, sex, and diet, the severity of any inflammation, time of

administration and other clinical factors. Generally, systemic or injectable administration

will be initiated at a dose which is minimally effective, and the dose will be increased

over a preselected time course until a positive effect is observed. Subsequently,

incremental increases in dosage will be made limiting such incremental increases to such

levels that produce a corresponding increase in effect, while taking into account any

adverse affects that may appear. The addition of other known factors, to the final

composition, may also effect the dosage.

Progress can be monitored by periodic assessment of disease progression. The

progress can be monitored, for example, by x-rays, MRI or other imaging modalities,

synovial fluid analysis, and/or clinical examination. The following examples illustrate practice of the present invention in isolating

and characterizing human aggrecanase and other aggrecanase-related proteins, obtaining

the human proteins and expressing the proteins via recombinant techniques.

EXAMPLES

EXAMPLE 1 Isolation of DNA

Potential novel aggrecanase family members were identified using a database screening

approach. Aggrecanase-1 [Science284: 1664-1666 (1999)] has at least six domains: signal, propeptide, catalytic domain, disintegrin, tsp and c-terminal. The catalytic domain contains a zinc binding signature region, TAAHELGHVKF and a "MET turn"which are responsible for protease activity. Substitutions within the zinc binding region in the number of the positions still allow protease activity, but the histidine (H) and glutamic acid (E) residues must be present. The thrombospondin domain of Aggrecanase-1 is also a critical domain for substrate recognition and cleavage. It is these two domains that determine our classification of a novel aggrecanase family member. The coding region of the Aggrecanase-1 DNA sequence was used to query against the GeneBank ESTs focusing on human ESTs using TBLASTN. The resulting sequences were the starting point in the effort to identify full length sequence for potential family members. The nucleotide sequence of the aggrecanase of the present invention is comprised of one EST (A1479925) that contains homology over the catalytic domain and zinc binding motif of Aggrecanase-1(ADAMTS4). AI479925 was used to search the public database using the algorithm BLASTX, which searches a protein sequence database using all six conceptual translations of a nucleotide sequence query. AI479925 was shown to have 98% homology to KIAA1312 over 83 bps. The KIAA1312 sequence was used to query the public databases with the algorithm BLASTX and found to have 44% identity to ADAMTS-1. KIAA1312 was sequenced by the Kazusa DNA Research Institute. KIAA1312 appears to be 5' truncated missing the signal and propeptide. KIAA1312 contains the catalytic domain, disintegrin, tsp type I motif and c-terminal spacer(found in ADAMTS4. It is with these criteria that candidate #7 (KIAA1312) is considered a novel Aggrecanase family member. GenBank deposit (for ADAMTS9) showed identity to EST7 and KIAA1312. By

alignment with other family members, ADAMTS9 appears to have intact 5P signal and propetide sequences, but is 3P truncated in comparison to KIAA1312. A full-length

EST7 sequence has been constructed using the initiator met from ADAMTS9 and the

translational stop found in KIAA1312.

This human aggrecanase sequences were isolated from a dT-primed cDNA library constructed in the plasmid vector pED6-dpc2. cDNA was made from human small intestine RNA purchased from Clontech. The probe to isolate the aggrecanase of the present invention was generated from the sequence obtained from the database search. The sequence of the probe was as follows: EST7_1 ,_4,_8,_9

5'-GACAGCTTTTACGATCGCCCATGAGCTG-3' EST7 8B 5'-TTAATGCTGCTACGTCACCAGCCAGGTTA-3'

The DNA probe was radioactively labeled with ³²P and used to screen the human small intestine dT-primed cDNA library, under high stringency hybridization/washing conditions, to identify clones containing sequences of the human candidate #7.

Nitrocellulose replicas of the transformed colonies were hybridized to the ³²P labeled DNA probe in standard hybridization buffer (IX Blotto[25X Blotto = %5 nonfat dried milk,0.02% azide in dH2O] + 1% NP-40 + 6X SSC +0.05% Pyrophosphate) under high stringency conditions (65 °C for 2 hours). After 2 hours hybridization, the radioactively labeled DNA probe containing hybridization solution was removed and the filters were washed under high stringency conditions (3X SSC, 0.05% Pyrophosphate for 5 minutes at RT; followed by 2.2X SSC, 0.05% Pyrophosphate for 15 minutes at RT; followed by 2.2X SSC, 0.05% Pyrophosphate for 1-2 minutes shaking at 65°C. The filters were wrapped in Saran wrap and exposed to X-ray film for overnight. The autoradiographs were developed and positively hybridizing transformants of various signal intensities were identified. These positive clones were picked; grown for 12 hours in selective medium and plated at low density (approximately 100 colonies per plate). Nitrocellulose replicas of the colonies were hybridized to the ³²P labeled probe in standard hybridization buffer ((IX Blotto[25X Blotto = %5 nonfat dried milk, 0.02% azide in dH2O] + 1% NP-40 + 6X SSC +0.05% Pyrophosphate) under high stringency conditions (65 °C for 2 hours). After 2 hours hybridization, the radioactively labeled DNA probe containing hybridization solution was removed and the filters were washed under high stringency conditions (3X SSC, 0.05% Pyrophosphate for 5 minutes at RT standing; followed by 2.2X SSC, 0.05% Pyrophosphate for 15 minutes shaking at RT; followed by 2.2X SSC, 0.05% Pyrophosphate for 1-2 minutes shaking at 65°C. The filters were wrapped in Saran wrap and exposed to X-ray film for overnight. The autoradiographs were developed and positively hybridizing transformants were identified. Bacterial stocks of purified hybridization positive clones were made and plasmid DNA was isolated. The sequences of the cDNA inserts were determined and are set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO: 5.

Sequences have been deposited in the American Type Culture Collection 10801 University Blvd. Manassas, VA 20110-2209 USA as PTA -2283. The cDNA insert contained the sequences of the DNA probe used in the hybridization.

SEQ ID NO: 1 sets forth EST7.1 comprising nucleotides #1 to #1506. SEQ ID NO: 1 aligns with KIAA1312 from 195 to 330 amino acids. Nucleotides #1081 to # 1245 of SEQ ID NO: 1 correspond with nucleotides #1161 to #1456 of SEQ ID NO:6. SEQ ID NO: 2 sets forth EST 7.4 comprising nucleotides #1 to #1028. SEQ ID NO: 2 aligns with KIAA1312 from 47 to 330 amino acids. Nucleotides # 22 to #872 of SEQ ID NO 2 corresponds with nucleotides #606 to #1456 of SEQ ID NO:6. SEQ ID NO: 3 sets forth EST 7.8 comprising nucleotides #1 - #12054. SEQ ID NO: 3 aligns with KIAA1312 from 195 to 281 amino acids. SEQ ID NO: 4 sets forth EST 7.9 comprising nucleotides #1 to #687. EST 7.9 aligns with KIAA1312 from 149-330 amino acids. SEQ ID NO: 4 nucleotides #14 to #555 correspond with nucleotides # 915 to #1456 of SEQ ID NO: 6 .SEQ ID NO: 5 sets forth EST 7.8B comprising nucleotides #1 to #466 which extends the 5' sequence of KIAA1312. SEQ ID NO: 6 comprising nucleotides #1 to # 5605 sets forth the nucleotide sequence of SEQ ID NO: 5 (nucleotides #l-#466) and KIAA1312 (nucleotides # 467 to # 5605). SEQ ID NO:7 comprising amino acids #1 to #1610 sets forth the amino acid sequence of EST 7 .8B (amino acids #1-#139) and the amino acids of KIAA1312 (amino acids #140 to #1610).

This human Aggrecanase gene, full-length EST7, was isolated using a PCR strategy. Tissue sources were identified by a PCR screen of various phage and plasmid libraries using oligos designed to EST7. EST7 was found expressed in small intestine, brain and kidney libraries. Based on the publicly available sequence, PCR primers to the full-length EST7 sequence were designed. Three overlapping pieces of EST7 were amplified using the following primer sets. The first PCR primer set amplified from bp 1- 1864 of the full-length EST7 sequence; 5P primer sequence - ATAAGATTGCGGCCGCCACCATGCAGTTTGTATCCTGGGCCACAC (this primer incorporated an 8 bp tail (ATAAGATT), a NOTl sequence (GCGGCCGC) and a Kozak sequence (CCACC) upstream of the initiator Met (ATG)) and the 3P primer sequence - GTCTGTTGCACTCTCGAATGGCTGTT. The second primer set amplified from bp 1752-3485 of the full-length EST7 sequence; 5P primer sequence - AGAAATGGATGTCCCCGTGACAGATG and the 3P primer sequence - TGGGTATCAGTTGGTCTAGTTGCTGC. The third PCR primer set amplified from bp 3300-5054 of the full-length EST7 sequence; 5P primer sequence - GCCAACATCTATGCAGACTTGTCAGC and 3P primer sequence - GGAATTCTAGCTTGGGAAAGCTGAGGA. The Advantage-GC 2 PCR Kit from Clontech was used to amplify the full-length EST7 gene products. Reaction conditions were those recommended in the user manual; with the following exceptions: per 50 ul reaction the amount of GC Melt used was 5 ul; the amount of phage library (Clontech human kidney 5 ' -STRETCH PLUS cDNA library) used was 2 ul of a stock with titer > 10⁸ pfu/ml or lOng plasmid library DNA linearized with Notl, and the amount of each PCR primer used was lul of a 10 pmol/ul stock. Cycling conditions were as follows: 94°C for 1 in, one cycle; followed by 40 cycles consisting of 94^°C for 15 sec/68 °C for 3 min. The primer pairs were used in PCR amplification reactions containing each of the 3 tissue sources; kidney, small intestine or brain. PCR products resulting from the amplification of the 5P 1864 base pair product were digested with Notl and BamHl and ligated into the CS2+ vector (digested with the same) using standard digestion and ligation conditions. PCR products resulting from the amplification of the internal 1733 base pair product were digested with BamHl and Nsil and ligated into the CS2+ vector (digested with the same) using standard digestion and ligation conditions. PCR products resulting from the amplification of the 3P 1754 base pair product were digested with Nsil and EcoRl and ligated into the CS2+ vector (digested with the same) using standard digestion and ligation conditions. Ligated products were transformed into ElectroMAX DH10B cells from Life Technologies. Cloned PCR fragments of EST7 were sequenced to determine fidelity. The full-length sequence for EST7 was the consensus sequence derived from the KIAA1312 and the ADAMTS9 sequences. PCR products with the correct sequence were excised from the CS2+ vector using the appropriate enzyme pairs described above. A full-length version of EST7 was constructed by ligating these 3 PCR products, 5P (Notl BamHl), internal (BamHl/Nsil) and 3P (Nsil/EcoRl), into the Cos expression vector pED6-dpcl (digested with Notl and EcoRl).

The full length ADAMTS9 EST7 sequence in pED6-dpcl:Not 1 to EcoRl is set forth in SEQ ID NO:8. The peptide sequence is set forth in SEQ ID NO:9.

EXAMPLE 2

Expression of Aggrecanase

In order to produce murine, human or other mammalian aggrecanase-related

proteins, the DNA encoding it is transferred into an appropriate expression vector and

introduced into mammalian cells or other preferred eukaryotic or prokaryotic hosts

including insect host cell culture systems by conventional genetic engineering techniques. Expression system for biologically active recombinant human aggrecanase is

contemplated to be stably transformed mammalian cells, insect, yeast or bacterial cells.

One skilled in the art can construct mammalian expression vectors by employing

the sequence of SEQ ID NO; 8 or SEQ ID NO: 6 comprising nucleotide # 1 to # 466 or comprising nucleotide # 1 to #5605 of SEQ ID NO 6 or the DNA sequences of SEQ ID

NO: 1, SEQ ID NO: 2, SEQ ID NO;3, SEQ ID NO:4, or SEQ ID NO: 5 encoding

aggrecanase-related proteins or other modified sequences and known vectors, such as

pCD [Okayama et al., Mol. Cell Biol. 2:161-170 (1982)], pJL3, pJL4 [Gough et al., EMBO , 4:645-653 (1985)] and pMT2 CXM. The mammalian expression vector pMT2 CXM is a derivative of p91023(b)

(Wong et al., Science 228:810-815, 1985) differing from the latter in that it contains the

ampicillin resistance gene in place of the tetracycline resistance gene and further contains

a Xhol site for insertion of cDNA clones. The functional elements of pMT2 CXM have

been described (Kaufman, R.J., 1985, Proc. Natl. Acad. Sci. USA 82:689-693) and

include the adenovirus VA genes, the S V40 origin of replication including the 72 bp

enhancer, the adenovirus major late promoter including a 5' splice site and the majority of

the adenovirus tripartite leader sequence present on adenovirus late mRNAs, a 3' splice

acceptor site, a DHFR insert, the SV40 early polyadenylation site (SV40), and pBR322

sequences needed for propagation in R coli.

Plasmid pMT2 CXM is obtained by EcoRl digestion of pMT2-VWF, which has

been deposited with the American Type Culture Collection (ATCC), Rockville, MD

(USA) under accession number ATCC 67122. EcoRl digestion excises the cDNA insert

present in pMT2-VWF, yielding pMT2 in linear form which can be ligated and used to

transform E. coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2 DNA can be

prepared by conventional methods. pMT2 CXM is then constructed using loopout/in mutagenesis [Morinaga, et al. Biotechnology 84: 636 (1984). This removes bases 1075

to 1145 relative to the Hind HI site near the SV40 origin of replication and enhancer

sequences of pMT2. In addition it inserts the following sequence:

5' PO-CATGGGCAGCTCGAG-3'

at nucleotide 1145. This sequence contains the recognition site for the restriction

endonuclease Xho I. A derivative of pMT2CXM, termed pMT23, contains recognition sites for the restriction endonucleases Pstl, Eco RI, Sail and Xhol. Plasmid pMT2 CXM

and pMT23 DNA may be prepared by conventional methods.

pEMC2βl derived from pMT21 may also be suitable in practice of the invention.

pMT21 is derived from pMT2 which is derived from pMT2-VWF. As described above

EcoRI digestion excises the cDNA insert present in pMT-VWF, yielding pMT2 in linear

form which can be ligated and used to transform K Coli HB 101 or DH-5 to ampicillin

resistance. Plasmid pMT2 DNA can be prepared by conventional methods. pMT21 is derived from pMT2 through the following two modifications. First, 76

bp of the 5' untranslated region of the DHFR cDNA including a stretch of 19 G residues

from G/C tailing for cDNA cloning is deleted. In this process, a Xhol site is inserted to

obtain the following sequence immediately upstream from DHFR: 5' -

CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3'

Pstl Eco RI Xhol

Second, a unique Clal site is introduced by digestion with EcoRV and Xbal, treatment

with Klenow fragment of DNA polymerase I, and ligation to a Clal linker (CATCGATG).

This deletes a 250 bp segment from the adenovirus associated RNA (VAI) region but

does not interfere with VAI RNA gene expression or function. pMT21 is digested with

EcoRI and Xhol, and used to derive the vector pEMC2Bl.

A portion of the EMCV leader is obtained from pMT2-ECATl [S.K. Jung, et al, J_

Virol 63: 1651-1660 (1989)] by digestion with Eco RI and Pstl, resulting in a 2752 bp fragment. This fragment is digested with Taql yielding an Eco RI-Taql fragment of 508 bp which is purified by electrophoresis on low melting agarose gel. A 68 bp adapter and

its complementary strand are synthesized with a 5' Taql protruding end and a 3' Xhol

protruding end which has the following sequence:

5'-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT Taql

GAAAAACACGATTGC-3¹ Xhol

This sequence matches the EMC virus leader sequence from nucleotide 763 to 827. It

also changes the ATG at position 10 within the EMC virus leader to an ATT and is

followed by a Xhol site. A three way ligation of the pMT21 Eco RI-16hoI fragment, the

EMC virus EcoRI-Taql fragment, and the 68 bp

oligonucleotide adapter TaqI-16hoI adapter resulting in the vector pEMC2βl.

This vector contains the S V40 origin of replication and enhancer, the adenovirus

major late promoter, a cDNA copy of the majority of the adenovirus tripartite leader

sequence, a small hybrid intervening sequence, an SV40 polyadenylation signal and the

adenovirus VA I gene, DHFR and β-lactamase markers and an EMC sequence, in

appropriate relationships to direct the high level expression of the desired cDNA in

mammalian cells.

The construction of vectors may involve modification of the aggrecanase-related

DNA sequences. For instance, aggrecanase cDNA can be modified by removing the non- coding nucleotides on the 5' and 3' ends of the coding region. The deleted non-coding

nucleotides may or may not be replaced by other sequences known to be beneficial for

expression. These vectors are transformed into appropriate host cells for expression of

aggrecanase-related proteins. Additionally, the sequence of SEQ ID NO: 8 or the

sequence of SEQ ID NO: 6 comprising nucleotide # 1 to # 466 or comprising nucleotide

# 1 to #5605 of SEQ ID NO: 6 or other sequences encoding aggrecanase-related proteins

such as the sequences comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID

NO:4, or SEQ ID NO: 5 can be manipulated to express a mature aggrecanase-related

protein by deleting aggrecanase encoding propeptide sequences and replacing them with

sequences encoding the complete propeptides of other aggrecanase proteins.

One skilled in the art can manipulate the sequences of SEQ ID NO: 1 through

SEQ ID NO: 6 or SEQ ID NO: 8 by eliminating or replacing the mammalian regulatory

sequences flanking the coding sequence with bacterial sequences to create bacterial

vectors for intracellular or extracellular expression by bacterial cells. For example, the

coding sequences could be further manipulated (e.g. ligated to other known linkers or

modified by deleting non-coding sequences therefrom or altering nucleotides therein by

other known techniques). The modified aggrecanase-related coding sequence could then

be inserted into a known bacterial vector using procedures such as described in T.

Taniguchi et al., Proc. Natl Acad. Sci. USA. 77:5230-5233 (1980). This exemplary

bacterial vector could then be transformed into bacterial host cells and a aggrecanase-

related protein expressed thereby. For a strategy for producing extracellular expression of aggrecanase-related proteins in bacterial cells, see, e.g. European patent application EPA

177,343.

Similar manipulations can be performed for the construction of an insect vector

[See, e.g. procedures described in published European patent application 155,476] for

expression in insect cells. A yeast vector could also be constructed employing yeast regulatory sequences for intracellular or extracellular expression of the factors of the

present invention by yeast cells. [See, e.g., procedures described in published PCT

application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a aggrecanase-related protein of the

invention in mammalian, bacterial, yeast or insect host cell systems may involve the

construction of cells containing multiple copies of the heterologous Aggrecanase-related gene. The heterologous gene is linked to an amplifiable marker, e.g. the dihydrofolate

reductase (DHFR) gene for which cells containing increased gene copies can be selected

for propagation in increasing concentrations of methotrexate (MTX) according to the

procedures of Kaufman and Sharp, J. Mol. Biol, 159:601-629 (1982). This approach can be employed with a number of different cell types.

For example, a plasmid containing a DNA sequence for an aggrecanase-related

protein of the invention in operative association with other plasmid sequences enabling

expression thereof and the DHFR expression plasmid pAdA26SV(A)3 [Kaufman and

Sharp, Mol. Cell. Biol.. 2: 1304 (1982)] can be co-introduced into DHFR-deficient CHO

cells, DUKX-BΠ, by various methods including calcium phosphate coprecipitation and transfection, electroporation or protoplast fusion. DHFR expressing transformants are selected for growth in alpha media with dialyzed fetal calf serum, and subsequently

selected for amplification by growth in increasing concentrations of MTX (e.g. sequential

steps in 0.02, 0.2, 1.0 and 5uM MTX) as described in Kaufman et al, Mol Cell Biol.

5:1750 (1983). Transformants are cloned, and biologically active aggrecanase expression

is monitored by the assays described above. Aggrecanase protein expression should

increase with increasing levels of MTX resistance. Aggrecanase polypeptides are

characterized using standard techniques known in the art such as pulse labeling with

[35S] methionine or cysteine and polyacrylamide gel electrophoresis. Similar procedures

can be followed to produce other related aggrecanase-related proteins.

In one example the aggrecanase gene of the present invention set forth in SEQ ID

NO: 8 is cloned into the expression vector pED6 [Kaufman et al., Nucleic Acid Res.

19:44885-4490(1991)]. COS and CHO DUKX Bll cells are transiently transfected with

the aggrecanase sequence of the invention (+/- co-transfection of PACE on a separate

pED6 plasmid) by lipofection(LF2000, Invitrogen). Duplicate tranfections are performed

for each gene of interest: (a) one for harvesting conditioned media for activity assay and

(b) one for 35-S-methionine/cysteine metabolic labeling.

On day one media is changed to DME(COS) or alpha(CHO) media + 1% heat-

inactivated fetal calf serum+/- lOOμg/ml heparin on wells(a) to be harvested for activity

assay. After 48h (day4), conditioned media is harvested for activity assay. On day 3, the duplicate wells(b) were changed to MEM (methionine-free/cysteine

free) media + 1% heat-inactivated fetal calf serum +100μg/ml heparin + lOOμCi/ml 35S- methioine/cysteine (Redivue Pro mix, Amersham). Following 6h incubation at 37°C, conditioned media is harvested and run on SDS-PAGE gels under reducing conditions.

Proteins are visualized by autoradiography.

EXAMPLE 3

Biological Activity of Expressed Aggrecanase

To measure the biological activity of the expressed aggrecanase-related proteins

obtained in Example 2 above, the proteins are recovered from the cell culture and purified

by isolating the aggrecanase-related proteins from other proteinaceous materials with

which they are co-produced as well as from other contaminants. The purified protein may

be assayed in accordance with assays described above. Purification is carried out using standard techniques known to those skilled in the art.

Protein analysis is conducted using standard techniques such as SDS-PAGE

acrylamide [Laemmli, Nature 227:680 (1970)] stained with silver [Oakley, et al. Anal.

Biochem. 105:361 (1980)] and by immunoblot [Towbin, et al. Proc. Natl. Acad. Sci. USA

76:4350 (1979)]

The foregoing descriptions detail presently preferred embodiments of the present

invention. Numerous modifications and variations in practice thereof are expected to

occur to those skilled in the art upon consideration of these descriptions. Those

modifications and variations are believed to be encompassed within the claims appended

hereto.

Claims

What is claimed is:

1. An isolated DNA molecule comprising a DNA sequence set forth in SEQ ID

NO:8.

2. An isolated DNA molecule comprising a DNA sequence set forth in SEQ ID NO.

6.

3. An isolated DNA molecule comprising a DNA sequence set forth in SEQ ID NO.

5.

4. An isolated DNA molecule comprising a DNA sequence selected from the group

consisting of

a) the sequence of SEQ ID No. 1,

b) the sequence of SEQ ID NO : 2,

c) the sequence of SEQ ID NO: 3,

d) the sequence of SEQ ID NO : 4,

c) naturally occurring human allelic sequences and equivalent degenerative

codon sequences of a) through d).

5. A vector comprising a DNA molecule of claim 1 in operative association with an expression control sequence therefor.

6. A vector comprising a DNA molecule of claim 2 in operative association with an

expression control sequence therefor.

7. A host cell transformed with the DNA sequence of claim 1.

8. A host cell transformed with a DNA sequence of claim 2.

9. A method for producing a purified human aggrecanase protein, said method

comprising the steps of:

(a) culturing a host cell transformed with a DNA molecule according to claim

1; and

(b) recovering and purifying said aggrecanase protein comprising the amino

acid sequence of SEQ ID NO:9 from the culture medium.

10. A method for producing a purified human aggrecanase protein, said method

comprising the steps of:

(a) culturing a host cell transformed with a DNA molecule according to claim

2; and

(b) recovering and purifying said aggrecanase protein comprising amino acid

sequence of SEQ ID NO: 7 from the culture medium.

11. A purified aggrecanase polypeptide comprising the amino acid sequence set forth

in SEQ ID NO: 9.

12. A purified aggrecanase polypeptide comprising the amino acid sequence set forth

in SEQ ID NO 7.

13. A purified aggrecanase polypeptide produced by the steps of

(a) culturing a cell transformed with a DNA molecule according to claim 1;

and

(b) recovering and purifying from said culture medium a polypeptide

comprising the amino acid sequence set forth in SEQ ID NO. 97.

14. A purified aggrecanase polypeptide produced by the steps of

(a) culturing a cell transformed with a DNA molecule according to claim 2; and

(b) recovering and purifying from said culture medium a polypeptide

comprising the amino acid sequence set forth in SEQ ID NO. 7.

15. An antibody that binds to a purified aggrecanase protein of claim 11.

16. An antibody that binds to a purified aggrecanase protein of claim 11.

17. A method for developing inhibitors of aggrecanase comprising the use of

aggrecanase protein set forth in SEQ ID NO. 7 or a fragment thereof.

18. A method for developing inhibitors of aggrecanase comprising the use of the

amino acid sequence set forth in SEQ ID NO: 9 or a fragment thereof.

19. The method of claim 17 wherein said method comprises three dimensional

structural analysis.

20. The method of claim 18 wherein said method comprises computer aided drug

design.

20. A composition for inhibiting the proteolytic activity of aggrecanase comprising a

peptide molecule which binds to the aggrecanase inhibiting the proteolytic

degradation of aggrecan.

21. A method for inhibiting the cleavage of aggrecan in a mammal comprising

administering to said mammal an effective amount of a compound that inhibits

aggrecanase activity.

22. An isolated nucleotide sequence comprising the DNA insert of ATCC PTA-2283.

23. A protein composition comprising the amino acid sequence set forth in SEQ JD NO:7 from nucleotide #140 to #1610 or a fragment thereof for use in the

development of aggrecanase inhibitors.

24. A protein composition comprising the amino acid sequence set forth in SEQ ID NO: 9 or a fragment thereof for use in the development of aggrecanase inhibitors.

0/12