HUMAN ANTIBODY AGAINST AGGRECANASE-TYPE ADAMTS SPECIES FOR THERAPEUTICS OF AGGRECANASE-RELATED DISEASES

Abstract

The present invention provides an antibody that specifically binds to human aggrecanase, and inhibits enzymatic activity of the human aggrecanase. In one embodiment, aggrecanase is ADAMTS4. In one embodiment, the antibody recognizes a particular epitope in human ADAMTS4, and inhibits not only aggrecanase activity of human ADAMTS4 but also aggrecanase activity of human ADAMTS5. In addition, the present invention also provides use of said antibody in the prophylaxis or treatment of the progression of arthritis.

Description

TECHNICAL FIELD

[0001] The present invention relates to an anti-human aggrecanase antibody, and pharmaceutical use thereof.

BACKGROUND ART

[0002] Aggrecan degradation and subsequent digestion of collagen fibrils are the central pathway for the destruction of cartilage in human joint diseases including osteoarthritis (OA) and rheumatoid arthritis (RA). Collagen degradation is carried out principally by collagen-degrading matrix metalloproteinases (MMPs) such as MMP-1, MMP-8 and MMP-13 [1-3]. On the other hand, aggrecan-degrading metalloproteinases called aggrecanases are considered to play a key role in the aggrecan degradation [4, 5]. Aggrecanases belong to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, and ADAMTS1, 4, 5, 8, 9 and 15 are known to have aggrecanase activity [4, 6]. Recent studies using ADAMTS4 and ADAMTS5 knockout mice have indicated that ADAMTS5, but not ADAMTS4, plays an essential role in aggrecan degradation in mouse arthritides [7, 8]. However, because there is little information about the biochemical character, expression patterns or gene promoter structures of mouse ADAMTS4 and ADAMTS5, the data from knockout mice must be interpreted carefully and should not be extrapolated to the human disease OA and RA [9, 10]. In human chondrocytes, ADAMTS4 is inducible by treatment with cytokines such as interleukin-1 (IL-1), but the expression of ADAMTS5 is constitutive [9, 11-13]. Our recent study also showed that among aggrecanase-type ADAMTS species, ADAMTS4 is selectively overexpressed in human osteoarthritic cartilage with a direct correlation to the degree of cartilage destruction, while ADAMTS5 is constitutively expressed in both normal and osteoarthritic cartilage [10]. These suggest that ADAMTS4 is a major aggrecanase in human osteoarthritic cartilage. ADAMTS4 is also overexpressed by synovial cells and articular chondrocytes in RA, suggesting the involvement of this proteinase in cartilage destruction of RA joints. ADAMTS4 and ADAMTS5 can digest not only aggrecan but also other members of the proteoglycan lectican family including versican and brevican. Since versican is a major proteoglycan in the skin and blood vessel wall, its degradation by ADAMTS4 and ADAMTS5 is also implicated in tissue destruction and repair of the skin and blood vessels under pathological conditions such as chronic ulcer and fibrosis of the skin and various vasculitides, respectively. In addition, tumor cells in glioblastoma multiforme are known to overexpress ADAMTS5 and tumor cell-derived ADAMTS5 is suggested to play a role in invasion by cleavage of brevican [14].

[0003] The phage display method is one of the display techniques that have realized an in vitro high-speed selection by forming a one-to-one correspondence in the form of phage particle between a functional peptide or protein and a DNA encoding same. This phage display method is applied to antibody selection, and many antibodies obtained by this method have been developed as pharmaceutical products [15]. Furthermore, a method of obtaining a specific antibody by a combination of a human artificial antibody library and a phage display method has been established, and such methods have been practicalized by plural companies, as evidenced by HuCAL (Human Combinatorial Antibody Library) of MorphoSys.

DOCUMENT LIST

Non-Patent Documents



[0004] [1] Dahlberg L, Billinghurst R C, Manner P, Nelson F, Webb G, Ionescu M, et al. Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1). Arthritis Rheum. 2000; 43: 673-82.

[0005] [2] Tortorella M D, Malfait A M, Deccico C, Arner E. The role of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage degradation. Osteoarthritis Cartilage. 2001; 9: 539-52.

[0006] [3] Pratta M A, Yao W, Decicco C, Tortorella M D, Liu R Q, Copeland R A, et al. Aggrecan protects cartilage collagen from proteolytic cleavage. J Biol Chem. 2003; 278: 45539-45.

[0007] [4] Porter S, Clark I M, Kevorkian L, Edwards D R. The ADAMTS metalloproteinases. Biochem J. 2005; 386: 15-27.

[0008] [5] Struglics A, Larsson S, Pratta M A, Kumar S, Lark M W, Lohmander L S. Human osteoarthritis synovial fluid and joint cartilage contain both aggrecanase- and matrix metalloproteinase-generated aggrecan fragments. Osteoarthritis Cartilage. 2006; 14:101-13.

[0009] [6] Okada Y. Proteinases and matrix degradation. In: JHarris E D, Budd R C, Genovese M C, Firestein G S and Sargent J S (ed) Kelley's textbook of Rheumatology Philadelphia: 8th edition, Elsevier Saunders 2008, in press.

[0010] [7] Glasson S S, Askew R, Sheppard B, Carito B, Blanchet T, Ma H L, et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature. 2005; 434: 644-8.

[0011] [8] Stanton H, Rogerson F M, East C J, Golub S B, Lawlor K E, Meeker C T, et al. ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature. 2005; 434: 648-52.

[0012] [9] Song R H, Tortorella M D, Malfait A M, Alston J T, Yang Z, Arner E C, et al. Aggrecan degradation in human articular cartilage explants is mediated by both ADAMTS-4 and ADAMTS-5. Arthritis Rheum. 2007; 56: 575-85.

[0013] [10] Naito S, Shiomi T, Okada A, Kimura T, Chijiiwa M, Fujita Y, et al. Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilage. Pathol Int. 2007; 57: 703-11.

[0014] [11] Bau B, Gebhard P M, Haag J, Knorr T, Bartnik E, Aigner T. Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro. Arthritis Rheum. 2002; 46: 2648-57.

[0015] [12] Moulharat N, Lesur C, Thomas M, Rolland-Valognes G, Pastoureau P, Anract P, et al. Effects of transforming growth factor-beta on aggrecanase production and proteoglycans degradation by human chondrocytes in vitro. Osteoarthritis Cartilage. 2004; 12: 296-305.

[0016] [13] Hui W, Barksby E, Young D A, Cawston T E, McKie N, Rowan A D. Oncostatin M in combination with tumour necrosis factor alpha induces a chondrocyte membrane-associated aggrecanase that is distinct from ADAMTS aggrecanase-1 or -2. Ann Rheum Dis. 2005; 64: 1624-32.

[0017] [14] Nakada M, Miyamori H, Kita D, Takahashi T, Yamashita J, Sato H, Miura R, Yamaguchi Y, Okada Y. Acta Neuropathol 110:239-246, 2005

[0018] [15] Rothe, C. et al. J. Mol. Biol. 2008; 376:1182-1200

SUMMARY OF INVENTION

Technical Problem

[0019] An object of the present invention is to provide an anti-human aggrecanase antibody (particularly, anti-human ADAMTS4 antibody) useful for the prophylaxis or treatment of the progression of various diseases represented by arthritis wherein Lectican family molecule, which is a proteoglycan, is degraded.

Solution to Problem

[0020] To solve the above-mentioned problem, the present inventors produced plural anti-human aggrecanase antibodies that bind to human aggrecanase. As a result, they have found that the produced anti-human ADAMTS4 antibodies inhibit enzymatic activity of human ADAMTS4, and can prevent aggrecan degradation by articular chondrocytes that occurs in arthritis. Furthermore, they have found that an antibody that recognizes a particular epitope also shows cross-reactivity with aggrecanases other than human ADAMTS4, and can also inhibit their activity. Based on the above-mentioned findings, they have conducted further studies in an attempt to develop a therapeutic drug for the diseases represented by arthritis, wherein aggrecanase acts on the tissue destruction, which resulted in the completion of the present invention.

[0021] Accordingly, the present invention relates to the following.

[1] An antibody which specifically binds to a human aggrecanase and inhibits aggrecanase activity of said aggrecanase. [2] The antibody according to [1], wherein the human aggrecanase is human ADAMTS4. [3] The antibody according to [2], which further inhibits aggrecanase activity of human ADAMTS5. [4] The antibody according to [2] or [3], which binds to human ADAMTS4 at an epitope comprising the amino acid sequence depicted in SEQ ID NO: 9. [5] The antibody according to any one of [2] to [4], which comprises a light chain variable region and a heavy chain variable region, wherein (1) the light chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 4, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 6; or (2) the light chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 4, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 6, except that 1 to 3 amino acids are substituted, deleted, inserted, or added in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 3, and/or 1 to 3 amino acids are substituted, deleted, inserted, or added in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 4 to 6. [6] The antibody according to [5], wherein the light chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 7 and the heavy chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 8. [7] The antibody according to any one of [1] to [6], which is a human antibody. [8] A pharmaceutical composition which comprises the antibody according to any one of [1] to [7]. [9] A polynucleotide which encodes the antibody according to any one of [1] to [7]. [10] A vector which comprises the polynucleotide according to [9]. [11] A transformant which comprises the vector according to [10]. [12] An agent for preventing or treating arthritis, which comprises an antibody which specifically binds to a human aggrecanase and inhibits aggrecanase activity of said aggrecanase. [13] The agent according to [12], wherein the human aggrecanase is human ADAMTS4. [14] The agent according to [12] or [13], wherein the antibody is the antibody according to any one of [1] to [7]. [15] A method of preventing or treating arthritis in a mammal, which comprises administering effective amount of an antibody which specifically binds to a human aggrecanase and inhibits aggrecanase activity of said aggrecanase to the mammal. [16] The method according to [15], wherein the human aggrecanase is human ADAMTS4. [17] The method according to [15] or [16], wherein the antibody is the antibody according to any one of [1] to [7]. [18] An antibody which specifically binds to a human aggrecanase and inhibits aggrecanase activity of said aggrecanase, for use in prophylaxis or treatment of arthritis. [19] The antibody according to [18], wherein the human aggrecanase is human ADAMTS4. [20] The antibody according to [18] or [19], which is the antibody according to any one of [1] to [7]. [21] Use of an antibody which specifically binds to a human aggrecanase and inhibits aggrecanase activity of said aggrecanase, for producing an agent for preventing or treating arthritis. [22] The use according to [21], wherein the human aggrecanase is human ADAMTS4. [23] The use according to [21] or [22], wherein the antibody is the antibody according to any one of [1] to [7].

Advantageous Effect of Invention

[0022] According to the present invention, an anti-human aggrecanase antibody (particularly, anti-human ADAMTS4 antibody) useful for the prophylaxis or treatment of the progression of arthritis is provided.

BRIEF DESCRIPTION OF DRAWINGS

[0023] FIG. 1. Immunoreactivity of candidate Fabs with ADAMTS4 and ADAMTS5, and their inhibition of ADAMTS4 aggrecanase activity. (A) Recombinant ADAMTS4 (left) and ADAMTS5 (right) (100 ng/lane each) transferred onto the PVDF membranes were reacted with each candidate Fab (clone 237-1, 237-5, 237-21, 237-43 or 237-53), followed by immunoblotting. (B) Inhibition of ADAMTS4 activity by the Fabs. Recombinant ADAMTS4 (180 ng) was reacted with each Fab (clone 237-1, 237-5, 237-21, 237-43 or 237-53) or control Fab in a 1:1 molar ratio, and then incubated with aggrecan (100 .mu.g) for 16 h at 37.degree. C. Aggrecanase activity of ADAMTS4 was evaluated by immunoblotting with anti-aggrecan neoepitope (NITEGE.sup.392) antibody. TS(-), buffer alone; Fab(-), ADAMTS4 incubated without Fab; Control, control Fab.

[0024] FIG. 2. Immunoreactivity of anti-ADAMTS antibody (clone 237-53) with ADAMTS, ADAM and MMP species. (A) Silver-stained gels of ADAMTS4, 5 and 1, ADAM10, 12 and 17, and MMP1, 2, 3, 9 and 13. The samples (100 ng/lane) were subjected to SDS-PAGE, and the gels were stained with silver stain kit. (B) Immunoreactivity of the antibody (clone 237-53) with the ADAMTS, ADAM and MMP species. The samples transferred on PVDF membranes were immunoblotted with anti-ADAMTS antibody clone 237-53. Note that the antibody reacts with ADAMTS4 and ADAMTS5, but not with other ADAMTS, ADAM and MMP species.

[0025] FIG. 3. Domain mapping analysis of anti-ADAMTS antibody (clone 237-53). (A and B) Immunoreactivity of the antibody with each domain of ADAMTS4. Recombinant FLAG/DHFR-tagged proteins corresponding to the metalloproteinase (Met) domain, disintegrin and thrombospondin domains (Dis/TSP), disintegrin (Dis) domain or thrombospondin (TSP) domain of ADAMTS4 were prepared by PUREfrex. These proteins were immunoblotted with anti-FLAG antibody (positive controls; left) or the antibody clone 237-53 (right).

[0026] FIG. 4. Inhibition of aggrecanase activity of ADAMTS4 and ADAMTS5 by anti-ADAMTS antibody (clone 237-53), and effect of the antibody on the expression of the ADAMTS species and aggrecanase activity in IL-1.alpha.-stimulated chondrocytes. (A) Inhibition of aggrecanase activity of ADAMTS4 and ADAMTS5 by anti-ADAMTS antibody (clone 237-53). Recombinant ADAMTS proteins were reacted with anti-ADAMTS antibody in molar ratios of 1:0.2-5 (enzyme:antibody) or control normal IgG (Control; 1:5 molar ratio), and then incubated with aggrecan. The aggrecan digestion was monitored by immunoblotting with anti-aggrecan neoepitope antibody (upper). Inhibition was evaluated by densitometric analysis of the immunoblots (lower). (B) Effect of the antibody (clone 237-53) on the mRNA expression of ADAMTS4 and ADAMTS5 and aggrecanase activity in the IL-1.alpha.-stimulated chondrocytes. Osteoarthritic chondrocytes were cultured in the presence and absence of IL-1.alpha. and anti-ADAMTS antibody or control normal IgG (Control). Then, the mRNA expression of these ADAMTS species (left) and aggrecanase activity in the conditioned media (right) were examined by RT-PCR and immunoblotting with anti-aggrecan neoepitope antibody, respectively. GAPDH, a control for loaded samples.

DESCRIPTION OF EMBODIMENTS

[0027] The present invention provides an antibody which has a specific binding activity to human aggrecanase, and inhibits the aggrecanase activity of the aggrecanase.

[0028] Aggrecanase is a known protease which is a member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) protein family, and acts on and degrades a proteoglycan known as aggrecan. Aggrecanase encompasses ADAMTS4, ADAMTS5, ADAMTS1, ADAMTS8, ADAMTS9, ADAMTS15 and the like.

A representative amino acid sequence of human ADAMTS4 is shown in SEQ ID NO: 15, a representative cDNA sequence of human ADAMTS4 is shown in SEQ ID NO: 14, a representative amino acid sequence of human ADAMTS5 is shown in SEQ ID NO: 17, and a representative cDNA sequence of human ADAMTS5 is shown in SEQ ID NO: 16.

[0029] The antibody of the present invention has a specific binding activity to human aggrecanase.

[0030] The "human aggrecanase" means that the amino acid sequence or nucleotide sequence of aggrecanase has an amino acid sequence or nucleotide sequence which is the same as or substantially the same as the amino acid sequence or nucleotide sequence of aggrecanase naturally expressed in human. The "substantially the same" means that the amino acid sequence or nucleotide sequence of interest has 70% or more (preferably 80% or more, more preferably 90% or more, further preferably 95% or more, most preferably 99% or more) identity with the amino acid sequence or nucleotide sequence of a particular aggrecanase naturally expressed in human, and has the function of the particular human aggrecanase. Biological species other than human, proteins other than aggrecanase, gene and fragments thereof are also interpreted in the same manner.

[0031] The "specific binding" of an antibody to antigen X means that the binding affinity of an antibody to antigen X in an antigen-antibody reaction is higher than the binding affinity to a non-specific antigen (e.g., bovine serum albumin (BSA)).

[0032] The antibody of the present invention has an activity to inhibit the enzymatic activity of human aggrecanase. The enzymatic activity of human aggrecanase specifically means an activity of human aggrecanase to cleave aggrecan (e.g., human or swine aggrecan). The activity of human aggrecanase to cleave aggrecan can be evaluated by incubating swine aggrecan and human aggrecanase at 37.degree. C. for 16 hr, deglycosylating them with chondroitinase ABC and keratanase, and analyzing the obtained reaction product by immunoblotting using an anti-NITEGE.sup.392 aggrecan neo-epitope antibody according to, for example, the methods described in Yatabe T, et. al. Ann Rheum Dis. 2009; 68:1051-8 and Hashimoto G, et al. J Biol Chem. 2004; 279:32483-91.

[0033] In a preferable embodiment, the antibody of the present invention has a specific binding activity to human ADAMTS4, and inhibits the aggrecanase activity of human ADAMTS4.

[0034] The antibody of this embodiment preferably also inhibits, in addition to the aggrecanase activity of human ADAMTS4, the aggrecanase activity of human ADAMTS5.

[0035] In the present specification, the "antibody" is used as one encompassing a full-length antibody and any antigen-binding fragment (i.e., "antigen-binding portion") thereof or a single chain thereof. The "antibody" refers to a glycoprotein containing at least two heavy chains (H) and two light chains (L), which are linked by a disulfide bond, or an antigen-binding portion thereof. Each heavy chain is constituted by a heavy chain variable region (to be abbreviated as V.sub.H herein) and a heavy chain constant region. The heavy chain constant region is constituted by 3 domains of C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain is constituted by a light chain variable region (to be abbreviated as V.sub.L herein) and a light chain constant region. The light chain constant region is constituted by a single domain C.sub.L. V.sub.H and V.sub.L regions are further subdivided into regions with higher variability called complementarity determining regions (CDRs), which contain more highly conservative regions called framework regions (FRs) scattered therein. Each V.sub.H and V.sub.L is constituted by 3 CDRs and 4 FRs, which are aligned in the following order, i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxy terminal. The variable regions of said heavy chain and light chain contain binding domains that interact with an antigen. The constant region of an antibody can mediate the binding of immunoglobulin to host tissues or factors, including various cells (e.g., effector cells) of the immune system and the first component (C1q) of the conventional complement system.

[0036] In the present specification, the "antigen-binding portion" of an antibody is used to refer to one or more fragments of an antibody retaining an ability to specifically bind to an antigen (e.g., human ADAMTS4). It has been clarified that the antigen binding function of an antibody is performed by a fragment of a full-length antibody. Examples of the binding fragment included in the term "antigen binding portion" of an antibody include (i) Fab fragment, a monovalent fragment constituted by V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains, (ii) F(ab').sub.2 fragment, a divalent fragment containing two Fab fragments linked by disulfide bond in the hinge region, (iii) Fab' fragment, an inherent Fab having a hinge region portion (see FUNDAMENTAL IMMUNOLOGY, Paul ed., 3. sup. rd ed. 1993), (iv) Fd fragment constituted by V.sub.H and C.sub.H1 domains, (v) Fv fragment constituted by V.sub.L and V.sub.H domains in a single arm of an antibody, (vi) dAb fragment constituted by V.sub.H domain (Ward et al., (1989) Nature 341:544-546), (vii) isolated complementarity determining region (CDR) and (viii) nanobody which is a heavy chain variable region containing single variable domain and two constant regions. While V.sub.L and V.sub.H, which are the two domains of Fv fragment, are encoded by different genes, they can be linked by a synthetic linker to produce a single protein chain from them by recombinant techniques, wherein, in this chain, V.sub.L and V.sub.H regions pair with each other to form a monovalent molecule (known as a single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibody is also encompassed in the "antigen-binding portion" of an antibody. Such antibody fragments are obtained by those of ordinary skill in the art by known conventional techniques, and screened for usefulness in the same manner as with unmodified antibody.

[0037] The antibody of the present invention is preferably a monoclonal antibody. The "monoclonal antibody" refers to a preparation of an antibody molecule of a single molecule composition. The monoclonal antibody composition shows single binding-specificity and affinity for a particular epitope.

[0038] The antibody of the present invention is preferably a human antibody or a humanized antibody. The "human antibody" refers to an antibody having variable regions derived from a human germline immunoglobulin sequence in both the framework and CDR regions. Furthermore, when an antibody contains a constant region, the constant region also derives from a human germline immunoglobulin sequence. In the present specification, the "human antibody" also encompasses even an embodiment including an amino acid residue not encoded by a human germline immunoglobulin sequence (e.g., mutation introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). In the present specification, moreover, the "humanized antibody" refers to an antibody wherein a CDR sequence derived from the germline of an animal species other than human, such as mouse, is fused on the human framework sequence.

[0039] In the present specification, the human antibody encompasses a "reconstituted human antibody". The reconstituted human antibody refers to a modified antibody wherein at least one CDR contained in the first human donor antibody is used in the second human acceptor antibody, instead of CDR of the second human acceptor antibody. Preferably, all 6 CDRs are substituted. More preferably, the whole antigen binding region (e.g., Fv, Fab or F(ab')2) of the first human donor antibody is used instead of the corresponding region in the second human acceptor antibody. More preferably, the Fab region of the first human donor antibody is operably linked to an appropriate constant region of the second human acceptor antibody to form a full-length antibody.

[0040] The reconstituted human antibody can be produced by conventional gene recombinant techniques disclosed in, for example, EP125023, WO96/02576, the above-mentioned document 15 and the like. To be specific, for example, a DNA sequence designed to link a desired CDR in a donor human antibody and a desired framework region (FR) in an acceptor human antibody is synthesized by PCR method using, as primers, several oligonucleotides produced to have a region overlapping with the terminal regions of both CDR and FR (see the method described in WO98/13388). The obtained DNA is linked to a DNA encoding a human antibody constant region or a human antibody constant region mutant, which is incorporated into a expression vector and the vector is introduced into a host to allow for production, whereby a reconstituted human antibody can be obtained (see EP125023, WO96/02576).

[0041] In the present specification, moreover, the human antibody encompasses an "artificial human antibody". The artificial human antibody can be produced by conventional gene recombinant techniques disclosed in, for example, the above-mentioned document 15 and the like.

[0042] The antibody of the present invention also includes a fusion protein wherein the aforementioned antibody and other peptide or protein are fused. The production method of a fusion protein includes linking a polynucleotide encoding the antibody of the present invention and a polynucleotide encoding other peptide or polypeptide to match the frame, introducing same into an expression vector, and allowing expression thereof in a host, and techniques known to those of ordinary skill in the art can be used. As other peptide to be fused with the antibody of the present invention, known peptides such as FLAG (Hopp, T. P. et al., BioTechnology (1988) 6, 1204-1210), 6.times.His consisting of six His (histidine) residues, 10.times.His, human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, .alpha.-tubulin fragment, B-tag, Protein C fragment and the like can be used. Examples of other polypeptide to be fused with the antibody of the present invention include GST (glutathione-S-transferase), HA (influenza hemagglutinin), immunoglobulin constant region, (3-galactosidase, MBP (maltose binding protein) and the like. A commercially available polynucleotide encoding such peptide or polypeptide is fused with a polynucleotide encoding the antibody of the present invention, and a fusion polynucleotide prepared thereby is expressed, whereby a fusion polypeptide can be prepared.

[0043] The antibody of the present invention may be a conjugate antibody bound with various molecules, for example, polymer substances such as polyethylene glycol (PEG), hyaluronic acid and the like, radioactive substance, fluorescent substance, luminescence substance, enzyme, toxin and the like. Such conjugate antibody can be obtained by chemically modifying the obtained antibody. The modification method of antibody has already been established in this field (e.g., U.S. Pat. No. 5,057,313, U.S. Pat. No. 5,156,840).

[0044] The antibody of the present invention is preferably isolated or purified. Being "isolated or purified" means that an operation to remove components other than the component of interest has been applied to the state of natural presence. The purity of the isolated or purified antibody of the present invention (ratio of the weight of the antibody of the present invention to the total protein weight) is generally 50% or more, preferably 70% or more, more preferably 90% or more, most preferably 95% or more (for example, substantially 100%).

[0045] In a particular embodiment, the antibody of the present invention specifically binds to human ADAMTS4 in an epitope containing the amino acid sequence depicted in SEQ ID NO: 9 (YCEGRRTRF), and inhibits the aggrecanase activity of human ADAMTS4.

[0046] The epitope containing the amino acid sequence depicted in SEQ ID NO: 9 includes, for example, an epitope consisting of a continuous partial sequence of the amino acid sequence depicted in SEQ ID NO: 15, which contains the amino acid sequence depicted in SEQ ID NO: 9, and preferably has an amino acid length of 20 or less, more preferably 12 or less. As the epitope containing the amino acid sequence depicted in SEQ ID NO: 9, specifically,

an epitope consisting of the amino acid sequence depicted in SEQ ID NO: 9, an epitope consisting of the amino acid sequence depicted in SEQ ID NO: 10 (GGKYCEGRRTRF), an epitope consisting of the amino acid sequence depicted in SEQ ID NO: 11 (GKYCEGRRTRFR), an epitope consisting of the amino acid sequence depicted in SEQ ID NO: 12 (KYCEGRRTRFRS), and an epitope consisting of the amino acid sequence depicted in SEQ ID NO: 13 (YCEGRRTRFRSC) can be mentioned.

[0047] The amino acid sequence depicted in SEQ ID NO: 9 is a partial amino acid sequence of human ADAMTS4, and does not show very high identity with the corresponding partial sequence of human ADAMTS5. Surprisingly, however, an antibody that specifically binds to human ADAMTS4 in an epitope containing the amino acid sequence depicted in SEQ ID NO: 9 can also inhibit the aggrecanase activity of human ADAMTS5 in addition to the aggrecanase activity of human ADAMTS4.

[0048] Specific examples of the antibody that specifically binds to human ADAMTS4 and inhibits the aggrecanase activity of human ADAMTS4 include the antibodies described in (1) or (2) below:

(1) an antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 4, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 6; and (2) an antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 4, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 6, except that 1 to 3 amino acids are substituted, deleted, inserted, and/or added in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 3, and/or 1 to 3 amino acids are substituted, deleted, inserted, and/or added in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 4 to 6.

[0049] In the embodiment of (2), 1-3 (preferably 1 or 2, more preferably 1) amino acids are preferably substituted, deleted, inserted, and/or added only in the amino acid sequence of CDR3 in the light chain variable region.

[0050] Examples of the method for substituting one or plural amino acid residues with other desired amino acid include site-directed mutagenesis method (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, M J, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, H W, Kramer, B, Pflugfelder, M, and Fritz, H J(1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz H J (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367, Kunkel, T A (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 82, 488-492). Using these methods, desired amino acid in an antibody can be substituted by other amino acid of interest. Also, using the library technique such as framework shuffling (Mol Immunol. 2007 April; 44(11):3049-60) and CDR repair (US2006/0122377) and the like, an amino acid in a framework or CDR can also be substituted by other appropriate amino acid.

[0051] In the antibody of the present invention, as a framework region (FR) of the antibody to be linked to a CDR, a framework which enables the CDR to form a good antigen binding site is selected. While FR to be used for the antibody of the present invention is not particularly limited and any FR can be used, FR of a human antibody is preferably used. As the FR of a human antibody, one having a natural sequence may be used, or one or plural amino acids in the framework region having a natural sequence may be substituted, deleted, added and/or inserted and the like as necessary, so that CDR will form an appropriate antigen binding site. For example, a mutant FR sequence having desired properties can be selected by measuring and evaluating the binding activity of an antibody having FR with substituted amino acid to an antigen (Sato, K. et al., Cancer Res. (1993)53, 851-856).

[0052] In the antibodies of (1) and (2), FR of Vk4 (Kabat database) of human antibody is preferably used for the light chain, and FR of VH1a (Kabat database) of human antibody is preferably used for the heavy chain.

[0053] The constant region used for the antibody of the present invention is not particularly limited, and any constant region may be used. Preferable examples of the constant region used for the antibody of the present invention include constant regions of human antibody (constant regions derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM and the like). For example, C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4, C.delta., C.alpha.1, C.alpha.2, C.epsilon. can be used in H chain, and C.kappa., C.lamda. can be used in L chain.

[0054] In the antibodies of (1) and (2), the constant region of C.kappa. of human antibody is preferably used for the light chain, and the constant region of C.gamma.1 of human antibody is preferably used for the heavy chain.

[0055] Preferable antibody of the present invention includes the following:

(1') An antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 7 and the heavy chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 8.

[0056] The antibody of the above-mentioned (1') corresponds to a preferable embodiment of the antibody of the above-mentioned (1).

[0057] The antibodies of the above-mentioned (1) and (2) preferably also inhibit aggrecanase activity of human ADAMTS5 in addition to the aggrecanase activity of human ADAMTS4.

[0058] In a particular embodiment, the antibodies of the above-mentioned (1) and (2) specifically bind to human ADAMTS4 in an epitope containing the amino acid sequence depicted in SEQ ID NO: 9 and inhibit aggrecanase activity of human ADAMTS4. Said antibodies can also inhibit aggrecanase activity of human ADAMTS5 in addition to the aggrecanase activity of human ADAMTS4.

[0059] The present invention provides a polynucleotide containing a nucleotide sequence encoding the above-mentioned antibody of the present invention. The polynucleotide may be a DNA or RNA, or a DNA/RNA chimera. The polynucleotide may be double stranded or single stranded. When the polynucleotide is double stranded, it may be a double stranded DNA, a double stranded RNA or a DNA:RNA hybrid.

[0060] The polynucleotide of the present invention encompasses a polynucleotide containing a nucleotide sequence encoding both the heavy chain variable region and the light chain variable region of the antibody of the present invention, and a combination of a polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of the antibody of the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain variable region of the antibody of the present invention.

[0061] The polynucleotide of the present invention can be easily produced based on the information of the amino acid sequence of the antibody of the present invention, known sequence information and sequence information described in the Sequence Listing in the present specification, and by utilizing known gene recombination techniques. For example, suitable primers are designed based on the sequence information, a DNA encoding the elements constituting the antibody of the present invention is amplified by the PCR reaction, DNA fragments are ligated by appropriate enzymes such as ligase and the like, whereby the polynucleotide of the present invention can be produced. Alternatively, a polynucleotide encoding each element may be synthesized by a polynucleotide synthesizer, based on the information of the amino acid sequence of the antibody of the present invention.

[0062] The obtained polynucleotide encoding the antibody of the present invention may be, depending on the object, directly used, or used after digestion with a restriction enzyme when desired, or addition of a linker. The polynucleotide may have ATG as a translation initiation codon on the 5' terminal side, and may have TAA, TGA or TAG as a translation stop codon on the 3' terminal side. These translation initiation codon and translation stop codon can be added using a suitable synthesized DNA adapter.

[0063] The polynucleotide of the present invention is preferably isolated or purified. The isolated or purified polynucleotide of the present invention has a purity (ratio of the weight of the polynucleotide of the present invention to the total polynucleotide weight) of generally 50% or more, preferably 70% or more, more preferably 90% or more, most preferably 95% or more (for example, substantially 100%).

[0064] The present invention provides a vector comprising the above-mentioned polynucleotide of the present invention. The vector of the present invention encompasses a vector comprising a polynucleotide comprising a nucleotide sequence encoding both the heavy chain variable region and the light chain variable region of the antibody of the present invention, and a combination of a vector comprising a polynucleotide comprising a nucleotide sequence encoding the heavy chain variable region of the antibody of the present invention and a vector comprising a polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the antibody of the present invention. The vector is preferably isolated or purified. Examples of the vector include expression vector, cloning vector and the like, which can be selected according to the object. Preferably, the vector is an expression vector. The expression vector can express the antibody of the present invention. The expression vector can be produced by operably linking the polynucleotide of the present invention to the downstream of a promoter in a suitable expression vector. The kind of the vector includes, for example, plasmid vector, virus vector and the like, which can be appropriately selected according to the host to be used.

[0065] As the host, the genus Escherichia (Escherichia coli etc.), the genus Bacillus (Bacillus subtilis etc.), yeast (Saccharomyces cerevisiae etc.), insect cell (established cell line derived from larva of Mamestra brassicae (Spodoptera frugiperda cell; Sfcell) etc.), insect (larva of Bombyx mori etc.), mammalian cells (rat nerve cell, monkey cell (COS-7 etc.), Chinese hamster cell (CHO cell etc.) etc.) and the like are used.

[0066] Examples of the mammal include, but are not limited to, experiment animals such as rodents such as mouse, rat, hamster and guinea pig and the like, rabbit and the like, domestic animals such as swine, bovine, goat, horse, sheep, mink and the like, companion animals such as dog, cat and the like, primates such as human, monkey, Macaca fascicularis, Macaca mulatta, marmoset, orangutan, chimpanzee and the like, and the like.

[0067] Examples of the plasmid vector include plasmid vectors derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmid vectors derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), plasmid vectors derived from yeast (e.g., pSH19, pSH15) and the like, which can be appropriately selected according to the kind of the host to be used and the object of use.

[0068] The kind of the virus vector can be appropriately selected according to the kind of the host to be used and object of use. For example, when an insect cell is used as a host, baculovirus vector and the like can be used. When a mammalian cell is used as a host, retrovirus vectors such as moloney murine leukemia virus vector, lentivirus vector, sindbis virus vector and the like, adenovirus vector, herpes virus vector, adeno-associated virus vector, parvovirus vector, vaccinia virus vector, sendai virus vector and the like can be used.

[0069] The promoter can be selected according to the kind of the host to be used, and one capable of initiating transcription in the host can be selected. For example, when the host is the genus Escherichia, trp promoter, lac promoter, T7 promoter and the like are preferable. When the host is the genus Bacillus, SPOT promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable. When the host is a mammalian cell, subgenomic(26S) promoter, CMV promoter, SR.alpha. promoter and the like are preferable.

[0070] The vector of the present invention may contain a signal sequence for antibody secretion. As the signal sequence for antibody secretion when it is produced in the periplasm of Escherichia coli, pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used.

[0071] When desired, the vector of the present invention may contain enhancer, splicing signal, polyA addition signal, selection marker, SV40 replication origin (hereinafter sometimes to be abbreviated as SV40ori) and the like each in an operable manner. Examples of the selection marker include dihydrofolate reductase (hereinafter sometimes to be abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (sometimes to be abbreviated as Amp.sup.r), neomycin resistance gene (sometimes to be abbreviated as Neo.sup.r, G418 resistance) and the like.

[0072] By introducing the above-mentioned vector of the present invention into the above-mentioned host by gene transfer methods known per se (e.g., lipofection method, calcium phosphate method, microinjection method, proplast fusion method, electroporation method, DEAE dextran method, gene transfer method by Gene Gun etc.), a transformant with the vector introduced thereinto (transformant of the present invention) can be produced. When an expression vector is used as the vector to be introduced, the transformant can express the antibody of the present invention. The transformant of the present invention is useful for the production of the antibody of the present invention and the like.

[0073] The antibody of the present invention can be produced by culturing the transformant of the present invention by a method known per se according to the kind of the host, and isolating the antibody of the present invention from the culture. When the host is the genus Escherichia, the transformant is cultured in an appropriate medium such as LB medium, M9 medium and the like at generally about 15-43.degree. C. for about 3-24 hr. When the host is the genus Bacillus, the transformant is cultured in an appropriate medium generally at about 30-40.degree. C. for about 6-24 hr. When the host is yeast, the transformant is cultured in an appropriate medium such as Burkholder's medium and the like generally at about 20.degree. C.-35.degree. C. for about 24-72 hr. When the host is an insect cell or insect, the transformant is cultured in an appropriate medium such as Grace's Insect medium added with about 10% of bovine serum and the like generally at about 27.degree. C. for about 3-5 days. When the host is an animal cell, the transformant is cultured in an appropriate medium such as MEM medium added with about 10% of bovine serum and the like generally at about 30.degree. C.-40.degree. C. for about 15-60 hr. In any culture, aeration and stirring may be performed as necessary.

[0074] As for the production method of antibody by genetic engineering, for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137 and the like can be referred to.

[0075] The separation and purification of the antibody of the present invention from a culture is not limited in any manner, and the separation and purification methods generally used for purification of antibody can be employed. For example, antibody can be separated and purified by appropriately selecting and combining chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization and the like.

[0076] Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gelfiltration, reversed-phase chromatography, adsorption chromatography and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed by using liquid phase chromatography, for example, liquid phase chromatography such as HPLC, FPLC and the like. Examples of the column to be used for affinity chromatography include protein A column and protein G column. For example, as a column using protein A, Hyper D, POROS, Sepharose FF (manufactured by GE Amersham Biosciences) and the like can be mentioned. The present invention also encompasses an antibody highly purified by these purification methods.

[0077] In addition, the present invention provides a pharmaceutical composition containing the above-mentioned antibody of the present invention as an active ingredient. Aggrecanases (particularly, ADAMTS4 and 5) degrade aggrecan and contribute to the cartilage destruction in arthritis such as osteoarthritis, rheumatoid arthritis and the like. Therefore, administration of the antibody of the present invention inhibits aggrecanase activity, suppresses aggrecan degradation, suppresses cartilage destruction and, as a result, can prevent or treat progression of arthritis. Accordingly, the antibody of the present invention and the pharmaceutical composition of the present invention are useful as prophylactic or therapeutic agents for the progression of arthritis and the like. Particularly, the antibody of the present invention in the embodiment wherein the antibody inhibits not only the aggrecanase activity of human ADAMTS4 but also the aggrecanase activity of human ADAMTS5 can simultaneously inhibit plural kinds of aggrecanases. Therefore, a superior cartilage denaturation or destruction suppressive effect, and a superior prophylactic or therapeutic effect on arthritis can be expected. The kind of arthritis is not particularly limited as long as it accompanies cartilage destruction or denaturation due to aggrecan degradation by aggrecanase (particularly, ADAMTS4 and 5), and the antibody of the present invention provides a prophylactic or therapeutic effect. Examples thereof include, but are not limited to, articular cartilage denaturation or destruction due to aggrecan degradation in, for example, osteoarthritis, rheumatoid arthritis, ankylosing arthritis, psoriatic arthritis and the like, denaturation and destruction of intervertebral disc in disc hernia and the like.

[0078] Furthermore, since the involvement of ADAMTS4 and 5 in the infiltration of brain tumor cells due to the Brevican degradation in brain tumor (glioblastoma multiforme), vascular destruction due to Versican degradation in intractable vasculitis, skin tissue destruction, excess repair action and the like due to Versican degradation and the product thereof in skin chronic ulcer, keloid and the like has been pointed out, the antibody of the present invention and the pharmaceutical composition of the present invention are also useful as prophylactic or therapeutic agents for the progression of these diseases and the like.

[0079] When the antibody of the present invention is "contained as an active ingredient", it means that the antibody of the present invention is contained as at least one of the active ingredients, and does not limit the content thereof. The pharmaceutical composition of the present invention may contain other active ingredient(s) together with the antibody of the present invention.

[0080] The antibody of the present invention can be formulated according to a conventional method (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A). Where necessary, moreover, it may contain a pharmaceutically acceptable carrier and/or additive. For example, it can contain surfactant (PEG, Tween etc.), excipient, antioxidant (ascorbic acid etc.), colorant, flavor, preservative, stabilizer, buffering agent (phosphate, citrate, other organic acid etc.), chelating agent (EDTA etc.), suspending agent, isotonizing agent, binder, disintegrant, lubricant, glidant, corrigent and the like. Not being limited to these, the pharmaceutical composition of the present invention may contain other conventional carriers as appropriate. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, cornstarch, inorganic salts and the like. It may also contain other low-molecular-weight polypeptide, serum albumin, gelatin and protein such as immunoglobulin and the like, as well as amino acid. When an aqueous solution for injection is formulated, the antibody of the present invention is dissolved in, for example, isotonic solution containing saline, glucose or other auxiliary agent. Examples of the auxiliary agent include D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be used in combination with suitable solubilizing agents, for example, alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG etc.), non-ionic surfactant (polysorbate80, HCO-50) and the like.

[0081] Where necessary, polypeptide may also be included in a microcapsule (microcapsules made of hydroxymethylcellulose, gelatin, poly[methylmethacrylate] and the like), or formulated as a colloid drug delivery system (liposome, albumin microsphere, microemulsion, nanoparticles and nanocapsule etc.) (see Remington's Pharmaceutical Science 16th edition &, Oslo Ed. (1980) etc.). Furthermore, a method of formulating a drug as a sustained-release medicament is also known, and applicable to polypeptide (Langer et al., J. Biomed. Mater. Res. (1981)15: 167-277; Langer, Chem. Tech. (1982)12: 98-105; U.S. Pat. No. 3,773,919; EP-A-58,481; Sidman et al., Biopolymers (1983) 22: 547-56; EP No. 133,988). Furthermore, it is also possible to increase the liquid amount to be subcutaneously administered by adding or blending hyaluronidase to or with the present agent (e.g., WO 2004/078140 etc.).

[0082] The content of the antibody of the present invention in a pharmaceutical composition is, for example, about 0.01-100 wt %, preferably 0.1-99.9%, of the whole pharmaceutical composition.

[0083] While the pharmaceutical composition of the present invention can be administered both orally and parenterally, it is preferably administered parenterally. Specifically, it is administered to patients by injection or transdermal administration. As an example of the dosage form of injection, it can be administered systemically or topically by intravenously injection, intramuscular injection, subcutaneous injection and the like. It may also be administered to the treatment site or in the vicinity thereof by topical injection, particularly intramuscular injection. Examples of the dosage form of transdermal administration include ointment, gel, cream, plaster, patch and the like, which can be administered systemically or topically. In addition, the administration method can be appropriately selected according to the age and symptom of the patients. The dose can be selected from, for example, the range of 0.5 mg-10 mg/kg body weight as the antibody of the present invention. However, the pharmaceutical composition of the present invention is not limited by these doses.

[0084] All references cited in the present specification, including publication, patent document and the like, are hereby incorporated individually and specifically by reference, to the extent that the entireties thereof have been specifically disclosed herein.

EXAMPLES

[0085] The present invention is explained in more detail in the following by referring to Examples, which are not to be construed as limitative. Various gene manipulations in the Examples followed the method described in Molecular cloning third. ed. (Cold Spring Harbor Lab. Press, 2001).

Materials and Methods

Phage Display Library Panning and Fab Generation

[0086] Production of monoclonal Fab antibodies specific to ADAMTS4 and ADAMTS5 were generated using the Human Combinatorial Antibody Library (HuCAL; MorphoSys AG, Martinried, Germany). Recombinant ADAMTS4 and ADAMTS5 (R&D Systems Inc., Minneapolis, Minn.) were biotinylated and incubated with HuCAL. Bound Fab expressing phages were enriched in three consecutive panning rounds. The pool of Fab genes was isolated from phagemids and inserted into Escherichia coli expression vectors that lead to functional periplasmic expression of Fab equipped with Strep-tag II. After transformation, individual colonies were picked up and grown in microtiter plates. After induction of antibody expression by incubation with isopropyl-.beta.-thiogalactopyranoside overnight, the cells were enzymatically lysed and the crude extracts were tested by enzyme-linked immunosorbent assay (ELISA). The DNA sequences of the antibody VH CDR regions were determined for clones that gave strong signals on the antigens in the ELISA. Colonies containing Fabs were chosen for subsequent purification, and some of the Fabs were reformatted into whole human IgG1 for further experiments.

Recombinant Human ADAMTS4 and ADAMTS5.

[0087] Expression vectors containing cDNA fragments encoding the residues Phe.sup.213-Cys.sup.685 of human ADAMTS4, which correspond to the metalloproteinase, disintegrin, thrombospondin and cysteine-rich domains of ADAMTS4, with the Strep-tag II at the C-terminal were transfected to HEK293T cells using Lipofectamine (Life Technologies, Rockville, Md.). The culture media were harvested at 2 days after the transfection, and recombinant human ADAMTS4 was purified by using the Strep-Tactin Sepharose according to manufacturer's instructions (IBA Biotechnica, Hanover, Germany). Recombinant ADAMTS5 protein containing the metalloproteinase, disintegrin and thrombospondin domains (residues of Ser.sup.262-Pro.sup.622 of ADAMTS5) was purchased from R&D Systems Inc.

Immunoblotting of Human Anti-ADAMTS Antibodies.

[0088] Recombinant proteins of human ADAMTS1 (R&D Systems), ADAMTS4, ADAMTS5 (R&D Systems), ADAMTS15 (R&D Systems), ADAM10 (R&D Systems), ADAM12 (Mochida Pharmaceutical Co., Ltd., Tokyo, Japan), ADAM17 (R&D Systems) and MMP-13 (Millipore, Billerica, Mass.) and purified human MMP-1, MMP-2, MMP-3 and MMP-9 (Daiichi Fine Chemical, Co., Ltd., Toyama, Japan) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and the samples resolved on the gels were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with candidate Fabs against ADAMTS species (5 .mu.g/ml; clones 237-1, 237-5, 237-21, 237-43 and 237-53) at 4.degree. C. for 16 h. After washing with phosphate buffered saline containing 0.1% Tween 20, the membranes were reacted with horseradish peroxidase-conjugated secondary antibody against human IgG (Invitrogen, Carlsbad, Calif.) for 1 h at room temperature. A chemiluminescence reagent (Pierce ECL western blotting substrate; Thermo Fisher Scientific, Waltham, Mass.) was used to make the labeled protein bands visible. All the samples were also examined on silver-stained gels, which were prepared by silver stain kit (Cosmo Bio Co., Ltd, Carlsbad, Calif.).

Inhibition of Aggrecanase Activity of ADAMTS4 and 5 with Human Anti-ADAMTS Antibody (Clone 237-53).

[0089] Recombinant ADAMTS4 (180 ng), and ADAMTS5 (180 ng) were incubated for 30 min at 37.degree. C. with human anti-ADAMTS antibody (IgG1; clone 237-53) in molar ratios of 1:0.2-5 (enzyme:antibody) or human control normal IgG1 (R&D Systems), and then reacted with porcine aggrecan (100 .mu.g) for 16 h at 37.degree. C. After deglycosylation of aggrecan with chondroitinase ABC and keratanase (Seikagaku Corporation, Tokyo, Japan), aggrecanase activity was monitored by immunoblotting using the anti-NITEGE.sup.392 aggrecan neoepitope antibody (1.2 .mu.g/ml) (Hashimoto G, et al. J Biol Chem. 2004; 279:32483-91). Density of the protein band was evaluated by densitometry using Image J analysis software (National Institute of Health, Bethesda, Md.).

Domain Mapping of Anti-ADAMTS Antibody (Clone 237-53).

[0090] Recombinant FLAG and dihydrofolic acid reductase (DHFR)-tagged proteins of each domain of ADAMTS4 and the thrombospondin domain with NH.sub.2- or COOH-terminal deletion were synthesized using cell-free translation system (PUREfrex) (Gene Frontier Corporation, Chiba, Japan). These samples were subjected to SDS-PAGE and then immunoblotted with anti-FLAG antibody (Sigma-Aldrich, St Louis, Mo.; 2 .mu.g/ml) or human anti-ADAMTS antibody (clone 237-53; 2 .mu.g/ml).

[0091] Surface plasma resonance interaction (BIAcore) analysis Recombinant ADAMTS species were covalently immobilized via amine coupling on CM5 sensor chip flow chambers (GE Healthcare Life Sciences, Buckinghamshire, UK). IgG1 of clone 237-53 was injected to the chambers using BIAcore 3000 (GE Healthcare Life Sciences). The K.sub.D (the affinity) was calculated from the determined K.sub.a and K.sub.d values.

Inhibition of Aggrecanase Activity in Cultured Chondrocytes with Anti-ADAMTS Antibody (Clone 237-53).

[0092] Chondrocytes isolated by enzymatic dissociation from human osteoarthritic cartilage were cultured in Dulbecco's modified Eagle medium/Ham's F-12 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum and 25 .mu.g/ml of ascorbic acid, and treated with or without interleukin-1.alpha. (IL-1.alpha. (1 ng/ml; Dainippon Sumitomo Pharmaceutical Company Ltd., Okada, Japan) for 24 h after serum-starvation by culturing in the medium containing 0.2% lactalbumin hydrolysate. They were treated with ADAMTS antibody (5 .mu.g/ml, clone 237-53) or human control IgG (5 .mu.g/ml; Invitrogen, Carlsbad, Calif.) for 1 h, and then incubated in the presence of aggrecan (100 .mu.g) for 16 h. The concentrated media were subjected to SDS-PAGE after deglycosylation and transferred onto PVDF membranes. Aggrecanase activity was evaluated by immunoblotting with anti-NITEGE.sup.392 neoepitope antibody (1.2 .mu.g/ml). Informed consent was obtained from the patients with osteoarthritis for the experimental use of the surgical samples according to the hospital ethics guidelines.

[0093] To examine the mRNA expression of ADAMTS4 and 5, total RNA was prepared from the chondrocytes treated with or without IL-1.alpha. (1 ng/ml) and human anti-ADAMTS antibody (clone 237-53) for 18 h, and reversed-transcribed to cDNAs using SuperScript II reverse transcriptase (Life Technologies, Rockville, Md.). The cDNAs were amplified by PCR with primers specific to ADAMTS4 and 5 and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (Naito S, et. al. Pathol Int. 2007; 57:703-11).

Results

Screening of Human Antibodies Against ADAMTS4 and ADAMTS5

[0094] By screening human antibody library (HuCAL) using the phage display method, a total of 5 clones (237-1, 237-5, 237-21, 237-43 and 237-53) that were reactive with both ADAMTS4 and ADAMTS5 were obtained by ELISA. Immunoblotting analysis indicated that all the clones recognize recombinant ADAMTS4 and ADAMTS5, although the reactivity to ADAMTS5 was different among the clones (FIG. 1A). To examine whether the candidate clones inhibit aggrecanase activity of ADAMTS4, Fab species of the clones were incubated with ADAMTS4 in a molar ratio of 1:1, and then the activity was monitored by immunoblotting using the neo-epitope (NITEGE.sup.392)-specific antibody. As shown in FIG. 1B, clone 237-53 inhibited the aggrecanase activity among the five candidate clones.

Immunoreactivity of Clone 237-53 with ADAMTS4 and ADAMTS5

[0095] Since clone 237-53 showed inhibitory activity to ADAMTS4, this antibody was focused. When cross-reactivity of the antibody to ADAMTS, ADAM and MMP species was examined by immunoblotting, clone 237-53 reacted with ADAMTS4 and ADAMTS5. However, no immunoreactivity was obtained with ADAMTS1, ADAMTS15, ADAM10, ADAM12, ADAM17, MMP-1, MMP-2, MMP-3, MMP-9 or MMP-13 (FIG. 2). The data suggest that clone 237-53 reacts with some region commonly present in ADAMTS4 and ADAMTS5.

Determination of the Epitope of ADAMTS4 Recognized by Clone 237-53

[0096] To determine the immunoreactive domain of ADAMTS4 by the antibody clone 237-53, we first examined reactivity to the recombinant proteins of metalloproteinase domain alone or disintegrin and thrombospondin domains of ADAMTS4 generated by the PUREfrex. As shown in FIG. 3A, the antibody recognized only the protein of the disintegrin and thrombospondin domains. Thus, we further examined the immunoreactivity with disintegrin or thrombospondin domain, and found that the antibody recognizes only the thrombospondin domain (FIG. 3B), indicating that the thrombospondin domain of ADAMTS4 contains the epitope for the antibody.

[0097] To identify the epitope in more detail, a peptide array with immobilized partial peptides of human ADAMTS4 was used for epitope mapping of the antibody clone 237-53. To be specific, as shown in the following Table, a peptide array consisting of peptides having the residue number of 12 amino acid residues and an offset of 3 amino acid residues was produced relative to a sequence covering the thrombospondin domain of human ADAMTS4. HRP-labeled antibody clone 237-53 was reacted with the peptide array.

TABLE-US-00001 TABLE 1 1 AGGWGPWGPWGD (SEQ ID NO: 18) 2 GGWGPWGPWGDC (SEQ ID NO: 19) 3 GWGPWGPWGDCS (SEQ ID NO: 20) 4 WGPWGPWGDCSR (SEQ ID NO: 21) 5 GPWGPWGDCSRT (SEQ ID NO: 22) 6 PWGPWGDCSRTC (SEQ ID NO: 23) 7 WGPWGDCSRTCG (SEQ ID NO: 24) 8 GPWGDCSRTCGG (SEQ ID NO: 25) 9 PWGDCSRTCGGG (SEQ ID NO: 26) 10 WGDCSRTCGGGV (SEQ ID NO: 27) 11 GDCSRTCGGGVQ (SEQ ID NO: 28) 12 DCSRTCGGGVQF (SEQ ID NO: 29) 13 CSRTCGGGVQFS (SEQ ID NO: 30) 14 SRTCGGGVQFSS (SEQ ID NO: 31) 15 RTCGGGVQFSSR (SEQ ID NO: 32) 16 TCGGGVQFSSRD (SEQ ID NO: 33) 17 CGGGVQFSSRDC (SEQ ID NO: 34) 18 GGGVQFSSRDCT (SEQ ID NO: 35) 19 GGVQFSSRDCTR (SEQ ID NO: 36) 20 GVQFSSRDCTRP (SEQ ID NO: 37) 21 VQFSSRDCTRPV (SEQ ID NO: 38) 22 QFSSRDCTRPVP (SEQ ID NO: 39) 23 FSSRDCTRPVPR (SEQ ID NO: 40) 24 SSRDCTRPVPRN (SEQ ID NO: 41) 25 SRDCTRPVPRNG (SEQ ID NO: 42) 26 RDCTRPVPRNGG (SEQ ID NO: 43) 27 DCTRPVPRNGGK (SEQ ID NO: 44) 28 CTRPVPRNGGKY (SEQ ID NO: 45) 29 TRPVPRNGGKYC (SEQ ID NO: 46) 30 RPVPRNGGKYCE (SEQ ID NO: 47) 31 PVPRNGGKYCEG (SEQ ID NO: 48) 32 VPRNGGKYCEGR (SEQ ID NO: 49) 33 PRNGGKYCEGRR (SEQ ID NO: 50) 34 RNGGKYCEGRRT (SEQ ID NO: 51) 35 NGGKYCEGRRTR (SEQ ID NO: 52) 36 GGKYCEGRRTRF (SEQ ID NO: 10) 37 GKYCEGRRTRFR (SEQ ID NO: 11) 38 KYCEGRRTRFRS (SEQ ID NO: 12) 39 YCEGRRTRFRSC (SEQ ID NO: 13) 40 CEGRRTRFRSCN (SEQ ID NO: 53) 41 EGRRTRFRSCNT (SEQ ID NO: 54) 42 GRRTRFRSCNTE (SEQ ID NO: 55) 43 RRTRFRSCNTED (SEQ ID NO: 56) 44 RTRFRSCNTEDC (SEQ ID NO: 57) 45 TRFRSCNTEDCP (SEQ ID NO: 58)

[0098] As a result, 237-53 specifically bound to the above-mentioned peptides #36-#39. The results suggest that the epitope of 237-53 contains the amino acid sequence depicted in SEQ ID NO: 9 (YCEGRRTRF) which is common to peptides #36-#39.

[0099] Escherichia coli of the obtained clone 237-53 was cultured, and plasmid was recovered (QIAprep Spin MiniPrep kit: manufactured by QIAGEN) and used for the DNA sequence analysis. Table 2 shows the amino acid sequences of CDRs (complementarity determining regions) of 237-53 H chain and L chain.

TABLE-US-00002 TABLE 2 light chain LCDR1 LCDR2 LCDR3 237- RSSQSILYSSNNNYLA HTASARES QQYYSVSI 53 (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) heavy chain HCDR1 HCDR2 HCDR3 237- GTFSSFAIS GIFPIFGQANYAQKFQG FSDWWEWQMDY 53 (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6)

[0100] The full-length amino acid sequences of the variable regions of H chain and L chain of 237-53 were as follows.

TABLE-US-00003 L chain VLk4 (SEQ ID NO: 7) DIVMTQSPDSLAVSLGERATINCRSSQSILYSSNNNYLAWYQQKPGQPPK LLIHTASARESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSVS ITFGQGTKVEIKRT H chain VH1a (SEQ ID NO: 8) QVQLVQSGAEVKKPGSSVKVSCKASGGIFSSFAISWVRQAPGQGLEWMGG IFPIFGQANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARFS DWWEWQMDYWGQGTLVTVSS

Inhibition of Aggrecanase Activity of ADAMTS4 and ADAMTS5 by the Antibody Clone 237-53

[0101] Aggrecanase activity of ADAMTS4 and ADAMTS5 was assayed by immunoblotting demonstration of the 65-kDa aggrecan fragments with the COOH-terminal sequence of NITEGE.sup.392 using the aggrecan neoepitope-specific antibody. As shown in FIG. 4A, the antibody clone 237-53 blocked the activity of ADAMTS4 to less than 20% of the original activity, while the ADAMTS5 activity was slightly inhibited to approximately 70% of the original activity. No inhibition was observed with normal control IgG (FIG. 4A). Kinetic analysis using BIAcore demonstrated high affinity binding of this antibody to ADAMTS species, showing K.sub.D values of 1.17.times.10.sup.-8 M and 1.46.times.10.sup.-9 M for ADAMTS4 and ADAMTS5, respectively.

[0102] Cultured chondrocytes from osteoarthritic cartilage expressed ADAMTS5, but not ADAMTS4 (FIG. 4B, left). When the chondrocytes were treated with IL-1.alpha., ADAMTS4 was induced, but the expression of ADAMTS5 was unchanged (FIG. 4B, left). Aggrecanase activity of the untreated chondrocytes was minimal, but it was increased after stimulation with IL-1.alpha. (FIG. 4B, right). When IL-1.alpha.-stimulated chondrocytes were treated with the antibody clone 237-53, the aggrecanase activity was substantially reduced to the control level, but no inhibition was observed by treatment with normal control IgG (FIG. 4B, right).

INDUSTRIAL APPLICABILITY

[0103] According to the present invention, an anti-human aggrecanase antibody useful for the prophylaxis or treatment of arthritis is provided.

[0104] This application is based on U.S. provisional patent application Ser. No. 61/891,087 (filing date: Oct. 15, 2013), the contents of which are incorporated in full herein by this reference.

TABLE-US-00004 Table 3-1 SEQ ID NO: 1 237-53 LCDR1 RSSQSILYSSNNNYLA SEQ ID NO: 2 237-53 LCDR2 HTASARES SEQ ID NO: 3 237-53 LCDR3 QQYYSVSI SEQ ID NO: 4 237-53 HCDR1 GTFSSFAIS SEQ ID NO: 5 237-53 HCDR2 GIFPIFGQANYAQKFQG SEQ ID NO: 6 237-53 HCDR3 FSDWWEWQMDY SEQ ID NO: 7 237-53 VL (kappa4) DIVMTQSPDSLAVSLGERATINCRSSQSILYSSNNNYLAWYQQKPGQPPKLLIHTASARESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYYSVSITFGCGTKVEIKRT SEQ ID NO: 6 237-53 VH (VH1a) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSFAISWVRQAPGQGLEWMGGIFPIFGQANYAQKFQGRVTIT ADESTSTAYMELSSLRSEDTAVYYCARFSDWWEWQMDYWGQGTLVTVSS Table 3-2 SEQ ID NO: 9 237-53 epitope YCEGRRTRF SEQ ID NO: 10 237-53 epitope GGKYCEGRRTRF SEQ ID NO: 11 237-53 epitope GKYCEGRRTRFR SEQ ID NO: 12 237-53 epitope KYCEGRRTRFRS SEQ ID NO: 13 237-53 epitope YCEGRRTRFRSC SEQ ID NO: 14 human ADAMTS4 cDNA sequence GGGGAGAACCCACAGGGAGACCCACAGACACATATGCACGAGAGAGACAGAGGAGGAAAGAGACAGAGAC AAAGGCACAGCGGAAGAAGGCAGAGACAGGGCAGGCACAGAAGCGGCCCAGACAGAGTCCTACAGAGGGA GAGGCCAGAGAAGCTGCAGAAGACACAGGCAGGGAGAGACAAAGATCCAGGAAAGGAGGGCTCAGGAGGA GAGTTTGGAGAAGCCAGACCCCTGGGCACCTCTCCCAAGCCCAAGGACTAAGTTTTCTCCATTTCCTTTA ACGGTCCTCAGCCCTTCTGAAAACTTTGCCTCTGACCTTGGCAGGAGTCCAAGCCCCCAGGCTACAGAGA GGAGCTTTCCAAAGCTAGGGTGTGGAGGACTTGGTGCCCTAGACGGCCTCAGTCCCTCCCAGCTGCAGTA CCAGTGCCATGTCCCAGACAGGCTCGCATCCCGGGAGGGGCTTGGCAGGGCGCTGGCTGTGGGGAGCCCA ACCCTGCCTCCTGCTCCCCATTGTGCCGCTCTCCTGGCTGGTGTGGCTGCTTCTGCTACTGCTGGCCTCT CTCCTGCCCTCAGCCCGGCTGGCCAGCCCCCTCCCCCGGGAGGAGGAGATCGTGTTTCCAGAGAAGCTCA ACGGCAGCGTCCTGCCTGGCTCGGGCGCCCCTGCCAGGCTGTTGTGCCGCTTGCAGGCCTTTGGGGAGAC GCTGCTACTAGAGCTGGAGCAGGACTCCGGTGTGCAGGTCGAGGGGCTGACAGTGCAGTACCTGGGCCAG GCGCCTGAGCTGCTGGGTGGAGCAGAGCCTGGCACCTACCTGACTGGCACCATCAATGGAGATCCGGAGT Table 3-3 CGGTGGCATCTCTGCACTGGGATGGGGGAGCCCTGTTAGGCGTGTTACAATATCGGGGGGCTGAACTCCA CCTCCAGCCCCTGGAGGGAGGCACCCCTAACTCTGCTGGGGGACCTGGGGCTCACATCCTACGCCGGAAG AGTCCTGCCAGCGGTCAAGGTCCCATGTGCAACGTCAAGGCTCCTCTTGGAAGCCCCAGCCCCAGACCCC GAAGAGCCAAGCGCTTTGCTTCACTGAGTAGATTTGTGGAGACACTGGTGGTGGCAGATGACAAGATGGC CGCATTCCACGGTGCGGGGCTAAAGCGCTACCTGCTAACAGTGATGGCAGCAGCAGCCAAGGCCTTCAAG CACCCAAGCATCCGCAATCCTGTCAGCTTGGTGGTGACTCGGCTAGTGATCCTGGGGTCAGGCGAGGAGG GGCCCCAAGTGGGGCCCAGTGCTGCCCAGACCCTGCGCAGCTTCTGTGCCTGGCAGCGGGGCCTCAACAC CCCTGAGGACTCGGACCCTGACCACTTTGACACAGCCATTCTGTTTACCCGTCAGGACCTGTGTGGAGTC TCCACTTGCGACACGCTGGGTATGGCTGATGTGGGCACCGTCTGTGACCCGGCTCGGAGCTGTGCCATTG TGGAGGATGATGGGCTCCAGTCAGCCTTCACTGCTGCTCATGAACrGGGTCATGTCTTCAACATGCTCCA TGACAACTCCAAGCCATGCATCAGTTTGAATGGGCCTTTGAGCACCTCTCGCCATGTCATGGCCCCTGTG ATGGCTCATGTGGATCCTGAGGAGCCCTGGTCCCCCTGCAGTGCCCGCTTCATCACTGACTTCCTGGACA ATGGCTATGGGCACTGTCTCTTAGACAAACCAGAGGCTCCATTGCATCTGCCTGTGACTTTCCCTGGCAA GGACTATGATGCTGACCGCCAGTGCCAGCTGACCTTCGGGCCCGACTCACGCCATTGTCCACAGCTGCCG CCGCCCTGTCCTGCCCTCTGGTGCTCTGGCCACCTCAATGGCCATGCCATGTGCCAGACCAAACACTCGC CCTGGGCCGATGGCACACCCTGCGGGCCCGCACAGGCCTGCATGGGTGGTCGCTGCCTCCACATGGACCA GCTCCAGGACTTCAATATTCCACAGGCTGGTGGCTGGGGTCCTTGGGGACCATGGGGTGACTGCTCTCGG ACCTGTGGGGGTGGTGTCCAGTTCXCCTCCCGAGACTGCACGAGGCCTGTCCCCCGGAATGGTGGCAAGT ACTGTGAGGGCCGCCGTACCCGCTTCCGCTCCTGCAACACTGAGGACTGCCCAACTGGCTCAGCCCTGAC CTTCCGCGAGGAGCAGTGTGCTGCCTACAACCACCGCACCGACCTCTTCAAGAGCTTCCCAGGGCCCATG GACTGGGTTCCTCGCTACACAGGCGTGGCCCCCCAGGACCAGTGCAAACTCACCTGCCAGGCCCAGGCAC TGGGCTACTACTATGTGCTGGAGCCACGGGTGGTAGATGGGACCCCCTGTTCCCCGGACAGCTCCTCGGT CTGTGTCCAGGGCCGATGCATCCATGCTGGCTGTGATCGCATCATTGGCTCCAAGAAGAAGTTTGACAAG TGCATGGTGTGCGGAGGGGACGGTTCTGGTTGCAGCAAGCAGTCAGGCTCCTTCAGGAAATTCAGGTACG GATACAACAATGTGGTCACTATCCCCGCGGGGGCCACCCACATTCTTGTCCGGCAGCAGGGAAACCCTGG CCACCGGAGCATCTACTTGGCCCTGAAGCTGCCAGATGGCTCCTATGCCCTCAATGGTGAATACACGCTG ATGCCCTCCCCCACAGATGTGGTACTGCCTGGGGCAGTCAGCTTGCGCTACAGCGGGGCCACTGCAGCCT CAGAGACACTGTCAGGCCATGGGCCACTGGCCCAGCCTTTGACACTGCAAGTCCTAGTGGCTGGCAACCC CCAGGACACACGCCTCCGATACAGCTTCTTCGTGCCCCGGCCGACCCCTTCAACGCCACGCCCCACTCCC CAGGACTGGCTGCACCGAAGAGCACAGATTCTGGAGATCCTTCGGCGGCGCCCCTGGGCGGGCAGGAAAT AACCTCACTATCCCGGCTGCCCTTTCTGGGCACCGGGGCCTCGGACTTAGCTGGGAGAAAGAGAGAGCTT CTGTTGCTGCCTCATGCTAAGACTCAGTGGGGAGGGGCTGTGGGCGTGAGACCTGCCCCTCCTCTCTGCC CTAATGCGCAGGCTGGCCCTGCCCTGGTTTCCTGCCCTGGGAGGCAGTGATGGGTTAGTGGATGGAAGGG GCTGACAGACAGCCCTCCATCTAAACTGCCCCCTCTGCCCTGCGGGTCACAGGAGGGAGGGGGAAGGCAG GGAGGGCCTGGGCCCCAGTTGTATTTATTTAGTATTTATTCACTTTTATTTAGCACCAGGGAAGGGGACA Table 3-4 AGGACTAGGGTCCTGGGGAACCTGACCCCTGACCCCTCATAGCCCTCACCCTGGGGCTAGGAAATCCAGG GTGGTGGTGATAGGTATAAGTGGTGTGTGTATGCGTGTGTGTGTGTGTGAAAATGTGTGTGTGCTTATGT ATGAGGTACAACCTGTTCTGCTTTCCTCTTCCTGAATTTTATTTTTTGGGAAAAGAAAAGTCAAGGGTAG GGTGGGCCTTCAGGGAGTGAGGGATTATCTTTTTTTTTTTTTCTTTCTTTCTTTCTTTTTTTTTTTTGAG ACAGAATCTCGCTCTGTCGCCCAGGCTGGAGTGCAATGGCACAATCTCGGCTCACTGCATCCTCCGCCTC CCGGGTTCAAGTGATTCTCATGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCTCCTGCCACCACGCCC GGCTAATTTTTGTTTTGTTTTGTTTGGAGACAGAGTCTCGCTATTGTCACCAGGGCTGGAATGATTTCAG CTCACTGCAACCTTCGCCACCTGGGTTCCAGCAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGAGATTAT AGGCACCTACCACCACGCCCGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAG GCTGGTCTCGAACTCCTGACCTTAGGTGATCCACTCGCCTTCATCTCCCAAAGTGCTGGGATTACAGGCG TGAGCCACCGTGCCTGGCCACGCCCAACTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTG GCCAGGCTGCTCTTGAACTCCTGACCTCAGGTAATCGACCTGCCTCGGCCTCCCAAAGTGCTGGGATTAC AGGTGTGAGCCACCACGCCCGGTACATATTTTTTAAATTGAATTCTACTATTTATGTGATCCTTTTGGAG TCAGACAGATGTGGTTGCATCCTAACTCCATGTCTCTGAGCATTAGATTTCTCATTTGCCAATAATAATA CCTCCCTTAGAAGTTTGTTGTGAGGATTAAATAATGTAAATAAAGAACTAGCATAACACTCAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO: 15 human ADAMTS4 amino acid sequence MSQTGSHPGRGLAGRWLWGAQPCLLLPIVPLSWLVWLLLLLLASLLPSARLASPLPREEEIVFPEKLNGS VLPGSGAPARLLCRLQAFGETLLLELEQDSGVQVEGLTVQYLGQAPELLGGAEPGTYLTGTINGDPESVA SLHWDGGALLGVLQYRGAELHLQPLEGGTPNSAGGPGAHILRRKSPASGQGPMCNVKAPLGSPSPRPRRA KRFASLSRFVETLVVADDKMAAFHGAGLKRYLLTVMAAAAKAFKHPSIRNPVSLVVTRLVILGSGEEGPQ VGPSAAQTLRSFCAWQRGLNTPEDSDPDHFDTAILFTRQDLCGVSTCDTLGMADVGTVCDPARSCAIVED DGLQSAFTAAHELGHVFNMLHDNSKPCISLNGPLSTSRHVMAPVMAHVDPEEPWSPCSARFITDFLDNGY GHCLLDKPEAPLHLPVTFPGKDYDADRQCQLTFGPDSRHCPQLPPPCAALWCSGHLNGHAMCQTKHSPWA DGTPCGPAQACMGGRCLHMDQLQDFNIPQAGGWGPWGPWDGCSRTCGGGVQFSSRDCTRPVPRNGGKYCE GRRTRFRSCNTEDCPTGSALTFREEQCAAYNHRTDLFKSFPGPMDWVPRYTGVAPQDQCKLTCQAQALGY YYVLEPRVVDGTPCSPDSSSVCVQGRCIHAGGCDRIIGSKKKFDKCMVCGGDGSGCSKQSGSFRKFRYGN NVVTIPAGATHILVRQQGNPGHRSIYLALKLPDGSYALNGEYTLMPSPTDVVLPGAVSLRYSGATAASET LSGHGPLAQPLTLQVLVAGNPQDTRLRYSFFVPRPTPSTPRTPQDWLHRRAQILEILRRRPWAGRK SEQ ID NO: 16 human ADAMTS5 cDNA sequence ATAAATTCATTGTTCCACCTCCTCGCATCTTCACAGCGCTCGCGCTGCTCTCGGCGCTCGCAGCTGCCGA Table 3-5 CTGGGGATGACGGCGGGCAGGAGGAGACCGCAGCCGAAGGGACACAGACACGCCGCTTCACCAGCTCGCC TCAGGCTGCCCCCCTGCATTTTTGTTTTAATTTTTACGGCTTTTTCCCCTCTCTTTCTTCCCTTCCTCCT GGTCCCAGCAGAGCCAAGGAAACCCACAAAATAAGAAAGGAAGTGGGCCCCGGAGCTTGGAACCTCCACA GCCGGCTTGTCCAGCGCAGCGCGGGGGCGGGAGGCTGCGCGCACCAGTTGCCAGCCCGGTGCGCGGTACC TTTCCTTACTTTTCTTGAAACAGCGATCGTGCCTGCATTTGGTGGTTTTTTGGTTTTTGTTTTTTTCCTT TTCCCGTATTTGCTGAATCTCCACTATCCGACTTTTTTTTTTTAATCTTTTCTTTCCCCCCCCCCCCACC CCACCTCTTTCTGGAGCACGAATCCAAACATTTTCCCAAGCAACAAAGAAAAGTTCGCACGCTGGCACCG CAGCCCCGACAGGCTGGCGCTGCTCCCGGGCCCCCCTCCCTCCGACACTTGACTCAATCCTGCAAGCAAG TGTGTGTGTGTCCCCATCCCCCGCCCCGTTAACTTCATAGCAAATAACAAATACCCATAAAGTCCCAGTC GCGCAGCCCCTCCCCGCGGGCAGCGCACTATGCTGCTCGGGTGGGCGTCCCTGCTGCTGTGCGCGTTCCG CCTGCCCCTGGCCGCGGTCGGCCCCGCCGCGACACCTGCCCAGGATAAAGCCGGGCAGCCTCCGACTGCT GCAGCAGCCGCCCAGCCCCGCCGGCGGCAGGGGGAGGAGGTGCAGGAGCGAGCCGAGCCTCCCGGCCACC CGCACCCCCTGGCGCAGCGGCGCAGGAGCAAGGGGCTGGTGCAGAACATCGACCAACTCTACTCCGGCGG CGGCAAGGTGGGCTACCTCGTCTACGCGGGCGGCCCGAGGTTCCTCTTGGACCTGGAGCGAGATGGTTCG GTGGGCATTGCTGGCTTCGTGCCCGCAGGAGGCGGGACGAGTGCGCCCTGGCGCCACCGGAGCCACTGCT TCTATCGGGGCACAGTGGACGGTAGTCCCCGCTCTCTGGCTGTCTTTGACCTCTGTGGGGGTCTCGACGG CTTCTTCGCGGTCAAGCACGCGCGCTACACCCTAAAGCCACTGCTGCGCGGACCCTGGGCGGAGGAAGAA AAGGGGCGCGTGTACGGGGATGGGTCCGCACGGATCCTGCACGTCTACACCCGCGAGGGCTTCAGCTTCG AGGCCCTGCCGCCGCGCGCCAGCTGCGAAACCCCCGCGTCCACACCGGAGGCCCACGAGCATGCTCCGGC GCACAGCAACCCGAGCGGACGCGCAGCACTGGCCTCGCAGCTCTTGGACCAGTCCGCTCTCTCGCCCGCT GGGGGCTCAGGACCGCAGACGTGGTGGCGGCGGCGGCGCCGCTCCATCTCCCGGGCCCGCCAGGTGGAGC TGCTTCTGGTGGCTGACGCGTCCATGGCGCGGTTGTATGGCCGGGGCCTGCAGCATTACCTGCTGACCCT GGCCTCCATCGCCAATAGGCTGTACAGCCATGCTAGCATCGAGAACCACATCCGCCTGGCCGTGGTGAAG GTGGTGGTGCTAGGCGACAAGGACAAGAGCCTGGAAGTGAGCAAGAACGCTGCCACCACACTCAAGAACT TTTGCAAGTGGCAGCACCAACACAACCAGCTGGGAGATGACCATGAGGAGCACTACGATGCAGCTATCCT GTTTACTCGGGAGGATTTATGTGGGCATCATTCATGTGACACCCTGGGAATGGCAGACGTTGGGACCATA TGTTCTCCAGAGCGCAGCTGTGCTGTGATTGAAGACGATGGCCTCCACGCAGCCTTCACTGTGGCTCACG AAATCGGACATTTACTTGGCCTCTCCCATGACGATTCCAAATTCTGTGAAGAGACCTTTGGTTCCACAGA AGATAAGCGCTTAATGTCTTCCATCCTTACCAGCATTGATGCATCTAAGCCCTGGTCCAAATGCACTTCA GCCACCATCACAGAATTCCTGGATGATGGCCATGGTAACTGTTTGCTGGACCTACCACGAAAGCAGATCC TGGGCCCCGAAGAACTCCCAGGACAGACCTACGATGCCACCCAGCAGTGCAACCTGACATTCGGGCCTGA GTACTCCGTGTGTCCCGGCATGGATGTCTGTGCTCGCCTGTGGTGTGCTGTGGTACGCCAGGGCCAGATG GTCTGTCTGACCAAGAAGCTGCCTGCGGTGGAAGGGACGCCTTGTGGAAAGGGGAGAATCTGCCTGCAGG GCAAATGTGTGGACAAAACCAAGAAAAAATATTATTCAACGTCAAGCCATGGCAACTGGGCATCTTGGGG ATCCTGGGGCCAGTGTTCTCGCTCATGTGGAGGAGGAGTGCAGTTTGCCTATCGTCACTGTAATAACCCT Table 3-6 GCTCCCAGAAACAAC6GACGCTACTGCACAGGGAAGAGGGCCATCTACCGCTCCTGCAGTCTCATGCCCT GCCCACCCAATGGTAAATCATTTCGTCATGAACAGTGTGAGGCCAAAAATGGCTATCAGTCTGATGCAAA AGGAGTCAAAACTTTTGTGGAATGGGTTCCCAAATATGCAGGTGTCCTGCCAGCGGATGTGTGCAAGCTG ACCTGCAGAGCCAAGGGCACTGGCTACTATGTGGTATTTTCTCCAAAGGTGACCGATGGCACTGAATGTA GGCTGTACAGTAATTCCGTCTGCGTCCGGGGGAAGTGTGTGAGAACTGCCTGTGACGGCATCATTGGCTC AAAGCTGCAGTATGACAAGrGCGGAGTATGTGGAGGAGACAACTCCAGCTGTACAAAGATTGTTGGAACC TTTAATAAGAAAAGTAAGGGTTACACTGACGTGGTGAGGATTCCTGAAGGGGCAACCCACATAAAAGTTC GACAGTTCAAAGCCAAAGACCAGACTAGATTCACTGCCTATTTAGCCCTGAAAAAGAAAAACGGTGAGTA CCTTATCAATGGAAAGTACATGATCTCCACTTCAGAGACTATCATTGACATCAATGGAACAGTCATGAAC TATAGCGGTTGGAGCCACAGGGATGACTTCCTGCATGGCATGGGCTACTCTGCCACGAAGGAAATTCTAA TAGTGCAGATTCTTGCAACAGACCCCACTAAACCATTAGATGTCCGTTATAGCTTTTTTGTTCCCAAGAA GTCCACTCCAAAAGTAAACTCTGTCACTAGTCATGGCAGCAATAAAGTGGGATCACACACTTCGCAGCCG CAGTGGGTCACGGGCCCATGGCTCGCCTGCTCTAGGACCTGTGACACAGGTTGGCACACCAGAACGGTGC AGTGCCAGGATGGAAACCGGAAGTTAGCAAAAGGATGTCCTCTCTCCCAAAGGCCTTCTGCGTTTAAGCA ATGCTTGTTGAAGAAATGTTAGCCTGTGGTTATGATCTTATGCACAAAGATAACTGGAGGATTCAGCACT GATGCAGTCGTGGTGAACAGGAGGTCTACCTAACGCACAGAAAGTCATGCTTCAGTGACATTGTCAACAG GAGTCCAATTATGGGCAGAATCTGCTCTCTGTGACCAAAAGAGGATGTCCACTGCTTCACGTGACAGTGG TGACCTTGCAATATAGAAAAACTTGGGAGTTATTGAACATCCCCTGGGCTTACAAGAAACACTGATGAAT GTAAAATCAGGGGACATTTGAAGATGGCAGAACTGTCTCCCCCTTGTCACCTACCTCTGATAGAATGTCT TTAATGGTATCATAATCATTTTCACCCATAATACACAGTAGCTTCTTCTTACTGTTTGTAAATACATTCT CCCTTGGTATGTCACTTTATATCCCCTGGTTCTATTAAAATATCCATATATATTTCTATAAAAAAAGTGT TTGACCAAAGTAGGTCTGCAGCTATTTCAACTTCCTTCCGTTTCCAGAAAGAGCTGTGGATATTTTACTG GAAATTAAGAACTTGCTGCTGTTTTAATAAGATGTAGTATATTTTCTGACTACAGGAGATAAAATTTCAG TCAAAAAACCATTTTGACAGCAAGTATCTTCTGAGAAATTTTGAAAAGTAAATAGATCTCAGTGTATCTA GTCACTTAAATACATACACGGGTTCATTTACTTAAACCTTTGACTGCCTGTATTTTTTTCAGGTAGCTAG CCAAATTAATGCATAATTTCAGATGTAGAAGTAGGGTTTGCGTGTGTGTGTGTGATCATACTCAAGAGTC TAAAAACTAGTTTCCTTGTGTTGGAAATTTAAAAGGAAAAAAATCGTATTTCACTGTGTTTTCAATTTAT ATTTTCACAACTACTTTCTCTCTCCAGAGCTTTCATCTGATATCTCACAATGTATGATATACGTACAAAA CACACAGCAAGTTTTCTATCATGTCCAACACATTCAACACTGGTATACCTCCTACCAGCAAGCCTTTAAA ATGCATTTGTGTTTGCTTATTTGTTTTGTTCAAGGGTTCAGTAAGACCTACAATGTTTTGTATTTCTTGA CTTATTTTATTAGAAACATTAAAGATCACTTGGTAGTTAGCCACATTGAGAAGTGGTTATCATTGTTAAT GTGGTTAATGCCAAAAAGTGGTTAATATTAATAAGACTGTTTCCACACCATAGGCAATAATTTCTTAATT TAAAAAATCTAAGTATATTCCTATTGTACTAAATATTTTTCCCAACTGGAAAGCACTTGATTGTACCCGT AAGTGTTTGAGTGATGACATGTGATGATTTTCAGAAAGTTGTTGTTTTTGTTTCCATAGCCTGTTTAAGT AGGTTGTAAGTTTGAATAGTTAGACATGGAAATTATTTTATAAGCACACACCTAAAGATATCTTTTTAGA Table 3-7 TGATAAAATGTACACCCCCCCATCACCAACCTCACAACTTAGAAAATCTAAGTTGTTTGATTTCTTTGGG ATTTCTTTTGTTGTGAAACACTGCAAAGCCAATTTTTCTTTATAAAAATTCATAGTAATCCTGCCAAATG TGCCTATTGTTAAAGATTTGCATGTGAAGATCTTAGGGAACCACTGTTTGAGTTCTACAAGCTCATGAGA GTTTATTTTTATTATAAGATGTTRRTAATATAAAAGAATTATGTAACTGATCACTATATTACATCATTTC AGTGGGCCAGGAAAATAGATGTCTTGCTGTTTTCAGTATTTTCTTAAGAAATTGCTTTTAAAACAAATAA TTGTTTTACAAAACCAATAATTATCCTTTGAATTTTCATAGACTGACTTTGCTTTTGACGTAGAAATTTT TTTTCTCAATAAATTATCACTTTGAGAAATGAGGCCTGTACAAGGCTGATAACCTATATGTGATGGAGAT CACCCAATGCCAAGGGCAGAAAGCAAACCTAGTTAAATAGGTGAGAAAAAAAATAATAATCCCAGTGCCA TTTGTCTGTGCAAAGAGAATTAGGAGAGAGGTTAATGTTACTTTTTTCCATTTTGGAAATAATTTTAATC AAGTAACTCAAATGTGACAAAATTTATTTTTATTTTTTGTGGTTATATTCCCAACAACATTAAAAAATAC TCGAGGCATAAATGTAGTTGTCTCCTACTCTGCTTCTCTTACTATACTCATACATTTTTAATATGGTTTA TCAATGATTCATGTTTCCCTCAAATAGTGATGGTTTACACCTGTCATGGAAACAATCCTAGAGAGCTCAG AGCAATTAAACCACTATTCCATGCTTTTAAGTAGTTTTCTCCACCTTTTTCTTATGAGTCTCACTAGATT GACTGAGGAATGTATGTCTAAATTCCTGGAGAAGATGATATGGATTGGAAACTGAAATTCAGAGAAATGG AGTGTTCAATAGATACCACGAATTGTGAACAAAGGGAAAATTCTATACAACTCAATCTAAGTCAGTCCAC TTTGACTTCGTACTGTCTTTCACCTTTCCATTGTTGCATCTTGAATTTTTTAAAATGTCTAGAATTCAGG ATGCTAGGGGCTACTTCTTTAAAAAAAAAAAAAAAAAAGAATTCGTCTGAAAATGCTCAGGTTTGTAAGA ATCTAATCTCACTTACATAACTAAGCACTCCATAATAAGTTTTATTAAGTACAAAGGGAGCCAGAAAAAA TGACATTTATTTCTTCTAGATCAGAAAAATTTAAATTAAGCCCTGCCTTGCTGTTTAGAAATATGTGGGC

ATTGTTATAATTTATTCAATAAATTTATGTTCCTTTGCCTTCCTGTGGAAACAGTTTTATCCCACTAAAC TAGGAATTAGGGGATAAATCACAAACAAAAAAAAAGTTGCAGCACTGAAAAAAAGTAATTTATTGTTTTT GCAACTGGTATGTGAATTTGTGTGATAAAATTATTTATTCTTATTTAACAAAAATATGTTCAAATTTTTC TATATTTAAAATGTTTTGCTGTTGTCCTACTTTTTAATTTATGCTTCATGTTTGTGTATAAAGTACACTT TTACACTTTGTGAGTTTACATAATATACAGCACTGGTTGCTTTTGTATTTTTTTACAGAAAGCTTTCTGT GTGAAGCAGGTGTATATGTATATATTCCTCATGTATTCTTATTCTGATACTATCATTTTTCTTTCCAAGG AAATTTTAATCTGTCATGACCAATAGTGTTCATTACTTGTGCCTATGATAATAGGTTTTTTACATCACAT TAACACTATTTTTTCCAAGTCACAAATAAGAAAAACACTTATTCAATGAAACAAGGTGCAAGTTTTAAAT TTGGGTACACAAATAGCCTAGAAGCTTCCTACAGACGCTAAGACACAGCCAATAATCAGATCCTTTCACT TCATCGAGAAACTTGGACAAGTCGATATTGATGTATTAGATGAAAGTTGTCTACACACAACRRCTGAGGG ATACAAACGATAATAAAACCAAATGTTGTCTGTTTCTCCTTTAGAAACACCTCCTAAAATTAATATCATT TAGTCTCTAGTGTCTGTAGGATTCTACAGATGAGCACAAATAGATTGGGTTTGTATAACAAATGCTAATA GTCATAACTGTTTCTACAAATATGGGGTGTCCATTAAGAGAATGTGATGTTTTCCTACTGCTGTTGAATC CCATGGGGTGATTATAGGACTTGAAATAGGCAGAGTCACCTCTGATGACATCAGCTTGCCTCTGTGATTT CACAGTCTGATCCTGGCAACAAGACAAAGCACCCTTGGACACACAGCCAATCTCTGGTTGTGATATTTCC CCATTGATTCCTTCCTTGTTAACAAGGTCATTTTAATGGTTCAGGTGAGGACAGCAGCCAGATTCAAAGT Table 3-8 CCAGAATTTGTGCTGTTACATAGAGTTCACACTGTCAAATAACATTGAATTTAATAATGATCAAATTTTT CTAGTAGTCTTTGGCAGAGTGTATAATCTCATTGGCATGATTGGTGAATATTACTAATCTCTTTATAATG AAAGATGCTTTACAAATACCTTATATTTGGTAACATTTCAAAACTACTAAATAAATGAAATAGCCATGTG TACAGAAATGGTCATTTAAAGCTTTAATAGAACCAAATTCAAGACAATGTATCATTTAGACACACAGAAA ACGAACTTGTATGTTTTCCCTATTATTTTTCTCATTTGCCAACAATCTATAGTTTTAGGTTATCAAACAG ATAGATCAACTTAACTGGCTAGTACATTGAAAAATCTTCCTAAGAATCCTTTGTTAGCATAATCTATAGA GATAATTTCTCAAATTATATCATCATGATGCATATAAACTCTATAATGTATAATTGTGTTTCATTTATTT AATGTATGAGAACATATTGAAATACAAAACCATGCATTAGCCAAAAAATTGGAATACAGGTAGTGTTCAG ATCAGCAAAACATTCAGTCTGGTAAATGCCTGCCTGGGGCTATGATATCATTCTCAATGCAGGTTTTATC GAAAAACTAAAAGAATATGTTGTTAGATGATGTTGGTTTTGAAAAAAAAAAGACATTAACATACACATTA GTTAGCCCAGTTAATTGCATTCTACTAATATAGTTGCACATTAGCAATAATTTTGCTGTCTCTGGTCTTT ATTTTGTGGCTTCAACTAACTGGACCATGTGGACTGTAAAGGTCAAATGGAAAAAACGAGCAGTGGCCCC TCATCCTGTAAGGTACTGCTACATCAGAGTGACCTAAAAGTCTAACACTGTGAGGAAAACTGTGATTTGT AGGAAAAAAAAAAAAAACAAATAAAAAACAGGGCATGCTTTTTAATTTTTTTCCACTTTCCTTTGGCACA CCCAATGAACAATTCTAATTTTTATTGAGGTGCTAACATCTTTCGTGACCGACTGTCAAATGTGGTATTT TTGAGTTACTATTTTTCTACATGATTTTACAGTTTGCAAGAAAGACCTCTAAGCTTTGTGTCACGGTAGG GCACAACTTGATACTCAAAATTTGAAAAATAAGCACATCCAATGATTGTTTTGACCAACAGTGGTCAGTG ACGTAAACTGCATGTGCATCTGAGGACATTTAAGGGGTCATTAAAATTTGAGGAGCATCAGGCCGGAGTA GCAGACTTTTAGATGAGTCATATTTCAGCATTCACTAAGTCCTCAGCATTCCATTCAAACTGTCGTGTAT ATTTGGCCTGATTTTTTTTCAAGCTTTGCAATAATTTATGTTATTGGTAAACACTTGGTGACTATATCTC AGCCAATTTTTTAACAACTCACAATATATTAGAAACACGTCTACCTATACTGAGAGTATATTTACAATAG AAGAACATACTGTATGTGACTTTGTAAAGCTAGACTTTTGATTAAGAAATATATAATCTCTGGATGCTAT TTTTGCATTATACACTCAGGCACAACGTAAACCTTGATGGCTCATCTTGCTACAATTACGAGTTGAAAAA CACTACTTACGTATTTGTATGACCTATTAGTCAGAGGAAATCATACATATGCTTTGTAAATAGACTTTGC AGATAACTAAATAGACTGAAGAAATATGTTGCATTTGATAGAAGCAATTGCATAAATATTTGGTTTCTAT ATTAGAGTCTGTGAGTAAAGTCAAGTAATAAACCTAAGTAGGTATAACAGATTTTTAAACCTTGAAACTT GCTTTGATGGTAGAGAAAATCATTGAAGATTTACATACTGTATATAAGATGTAAAATGTACGCTGCTTAT TACCCTCAATTTTCCAGAAGCAATGGTATATAATGCAGTTGAAAAACCAAAAATCTTGGAAAACTAAGAC GGGTCTTGTTTAAAATGTCTCTCAGCTTTGGCAACCTTCAAATCTTAATCAACTATTTAAAGCATTACTG TGTCTTGTAGCCTGCATTCCACAACAGCTCTGTTATTCAGGTAAAAGACTTGAACTGAGCCGTTTGGGAC CTATACTGTAATATTTTCATTGAGGAACAATATCCTATTTTGTAAAGCATTTCCCTATGTGTGACTTTAA ACTGTAAAATTAAACACTGCTTTTGTGGGTTCAGTGGGCATAATAAATATAAATTGTAAACTAGGTTAAA GTA Table 3-9 SEQ ID NO: 17 human ADAMTS5 amino acid sequence MLLGWASLLLCAFRLPLAAVGPAATPAQDKAGQPPTAAAAAQPRRRQGEEVQERAEPPGHPHPLAQRRRS KGLVQNIDQLYSGGGKVGYLVYAGGRRFLLDLERDGSVGIAGFVPAGGGTSAPWRHRSHCFYRGTVDGSP RSLAVFDLCGGLDGFFAVKHARYTLKPLLRGPWAEEEKGRVYGDGSARILHVYTREGFSFEALPPRASCE TPASTPEAHEHAPAHSNPSGRAALASQLLDQSALSPAGGSGPQTWWRRRRRSISRARQVELLLVADASMA RLYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQ LGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSH DDSKFCEETFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQILGPEELPGQT YDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGEKGRICLQGKCVDTKKK YYSTSSHGNWGSWGSWGQCSRSCGGGVQFAYRHCNNPAPRNNGRYCTGKRAIYRSCSLMPCPPNGKSFRH EQCEAKNGYQSDAKGVKTFVEWVPKYAGVLPADVCKLTCRAKGTGYYVVFSPKVTDGTECRLYSNSVCVR GKCVRTGCDGIIGSKLQYDKCGVCGGDNSSCTKIVGTFNKKSKGYTDVVRIPEGATHIKVRQFKAKDQTR FTAYLALKKKNGEYLINGKYMISTSETIIDINGTVMNYSGWSHRDDFLHGMGYSATKEILIVQILATDPT KPLDVRYSFFVPKKSTPKVNSVTSHGSNKVGSHTSQPQWVTGPWLACSRTCDTGWHTRTVQCQDGNRKLA KGCPLSQRPSAFKQCLLKKC

Sequence CWU 1

1

58116PRTArtificial SequenceSynthetic Sequence 1Arg Ser Ser Gln Ser Ile Leu Tyr Ser Ser Asn Asn Asn Tyr Leu Ala 1 5 10 15 28PRTArtificial SequenceSynthetic Sequence 2His Thr Ala Ser Ala Arg Glu Ser 1 5 38PRTArtificial SequenceSynthetic Sequence 3Gln Gln Tyr Tyr Ser Val Ser Ile 1 5 49PRTArtificial SequenceSynthetic Sequence 4Gly Thr Phe Ser Ser Phe Ala Ile Ser 1 5 517PRTArtificial SequenceSynthetic Sequence 5Gly Ile Phe Pro Ile Phe Gly Gln Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 611PRTArtificial SequenceSynthetic Sequence 6Phe Ser Asp Trp Trp Glu Trp Gln Met Asp Tyr 1 5 10 7114PRTArtificial SequenceSynthetic Sequence 7Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile His Thr Ala Ser Ala Arg Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr 85 90 95 Tyr Ser Val Ser Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr 8120PRTArtificial SequenceSynthetic Sequence 8Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Phe 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Phe Pro Ile Phe Gly Gln Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser Asp Trp Trp Glu Trp Gln Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 99PRTArtificial SequenceSynthetic Sequence 9Tyr Cys Glu Gly Arg Arg Thr Arg Phe 1 5 1012PRTArtificial SequenceSynthetic Sequence 10Gly Gly Lys Tyr Cys Glu Gly Arg Arg Thr Arg Phe 1 5 10 1112PRTArtificial SequenceSynthetic Sequence 11Gly Lys Tyr Cys Glu Gly Arg Arg Thr Arg Phe Arg 1 5 10 1212PRTArtificial SequenceSynthetic Sequence 12Lys Tyr Cys Glu Gly Arg Arg Thr Arg Phe Arg Ser 1 5 10 1312PRTArtificial SequenceSynthetic Sequence 13Tyr Cys Glu Gly Arg Arg Thr Arg Phe Arg Ser Cys 1 5 10 144410DNAHomo sapiens 14ggggagaacc cacagggaga cccacagaca catatgcacg agagagacag aggaggaaag 60agacagagac aaaggcacag cggaagaagg cagagacagg gcaggcacag aagcggccca 120gacagagtcc tacagaggga gaggccagag aagctgcaga agacacaggc agggagagac 180aaagatccag gaaaggaggg ctcaggagga gagtttggag aagccagacc cctgggcacc 240tctcccaagc ccaaggacta agttttctcc atttccttta acggtcctca gcccttctga 300aaactttgcc tctgaccttg gcaggagtcc aagcccccag gctacagaga ggagctttcc 360aaagctaggg tgtggaggac ttggtgccct agacggcctc agtccctccc agctgcagta 420ccagtgccat gtcccagaca ggctcgcatc ccgggagggg cttggcaggg cgctggctgt 480ggggagccca accctgcctc ctgctcccca ttgtgccgct ctcctggctg gtgtggctgc 540ttctgctact gctggcctct ctcctgccct cagcccggct ggccagcccc ctcccccggg 600aggaggagat cgtgtttcca gagaagctca acggcagcgt cctgcctggc tcgggcgccc 660ctgccaggct gttgtgccgc ttgcaggcct ttggggagac gctgctacta gagctggagc 720aggactccgg tgtgcaggtc gaggggctga cagtgcagta cctgggccag gcgcctgagc 780tgctgggtgg agcagagcct ggcacctacc tgactggcac catcaatgga gatccggagt 840cggtggcatc tctgcactgg gatgggggag ccctgttagg cgtgttacaa tatcgggggg 900ctgaactcca cctccagccc ctggagggag gcacccctaa ctctgctggg ggacctgggg 960ctcacatcct acgccggaag agtcctgcca gcggtcaagg tcccatgtgc aacgtcaagg 1020ctcctcttgg aagccccagc cccagacccc gaagagccaa gcgctttgct tcactgagta 1080gatttgtgga gacactggtg gtggcagatg acaagatggc cgcattccac ggtgcggggc 1140taaagcgcta cctgctaaca gtgatggcag cagcagccaa ggccttcaag cacccaagca 1200tccgcaatcc tgtcagcttg gtggtgactc ggctagtgat cctggggtca ggcgaggagg 1260ggccccaagt ggggcccagt gctgcccaga ccctgcgcag cttctgtgcc tggcagcggg 1320gcctcaacac ccctgaggac tcggaccctg accactttga cacagccatt ctgtttaccc 1380gtcaggacct gtgtggagtc tccacttgcg acacgctggg tatggctgat gtgggcaccg 1440tctgtgaccc ggctcggagc tgtgccattg tggaggatga tgggctccag tcagccttca 1500ctgctgctca tgaactgggt catgtcttca acatgctcca tgacaactcc aagccatgca 1560tcagtttgaa tgggcctttg agcacctctc gccatgtcat ggcccctgtg atggctcatg 1620tggatcctga ggagccctgg tccccctgca gtgcccgctt catcactgac ttcctggaca 1680atggctatgg gcactgtctc ttagacaaac cagaggctcc attgcatctg cctgtgactt 1740tccctggcaa ggactatgat gctgaccgcc agtgccagct gaccttcggg cccgactcac 1800gccattgtcc acagctgccg ccgccctgtg ctgccctctg gtgctctggc cacctcaatg 1860gccatgccat gtgccagacc aaacactcgc cctgggccga tggcacaccc tgcgggcccg 1920cacaggcctg catgggtggt cgctgcctcc acatggacca gctccaggac ttcaatattc 1980cacaggctgg tggctggggt ccttggggac catggggtga ctgctctcgg acctgtgggg 2040gtggtgtcca gttctcctcc cgagactgca cgaggcctgt cccccggaat ggtggcaagt 2100actgtgaggg ccgccgtacc cgcttccgct cctgcaacac tgaggactgc ccaactggct 2160cagccctgac cttccgcgag gagcagtgtg ctgcctacaa ccaccgcacc gacctcttca 2220agagcttccc agggcccatg gactgggttc ctcgctacac aggcgtggcc ccccaggacc 2280agtgcaaact cacctgccag gcccaggcac tgggctacta ctatgtgctg gagccacggg 2340tggtagatgg gaccccctgt tccccggaca gctcctcggt ctgtgtccag ggccgatgca 2400tccatgctgg ctgtgatcgc atcattggct ccaagaagaa gtttgacaag tgcatggtgt 2460gcggagggga cggttctggt tgcagcaagc agtcaggctc cttcaggaaa ttcaggtacg 2520gatacaacaa tgtggtcact atccccgcgg gggccaccca cattcttgtc cggcagcagg 2580gaaaccctgg ccaccggagc atctacttgg ccctgaagct gccagatggc tcctatgccc 2640tcaatggtga atacacgctg atgccctccc ccacagatgt ggtactgcct ggggcagtca 2700gcttgcgcta cagcggggcc actgcagcct cagagacact gtcaggccat gggccactgg 2760cccagccttt gacactgcaa gtcctagtgg ctggcaaccc ccaggacaca cgcctccgat 2820acagcttctt cgtgccccgg ccgacccctt caacgccacg ccccactccc caggactggc 2880tgcaccgaag agcacagatt ctggagatcc ttcggcggcg cccctgggcg ggcaggaaat 2940aacctcacta tcccggctgc cctttctggg caccggggcc tcggacttag ctgggagaaa 3000gagagagctt ctgttgctgc ctcatgctaa gactcagtgg ggaggggctg tgggcgtgag 3060acctgcccct cctctctgcc ctaatgcgca ggctggccct gccctggttt cctgccctgg 3120gaggcagtga tgggttagtg gatggaaggg gctgacagac agccctccat ctaaactgcc 3180ccctctgccc tgcgggtcac aggagggagg gggaaggcag ggagggcctg ggccccagtt 3240gtatttattt agtatttatt cacttttatt tagcaccagg gaaggggaca aggactaggg 3300tcctggggaa cctgacccct gacccctcat agccctcacc ctggggctag gaaatccagg 3360gtggtggtga taggtataag tggtgtgtgt atgcgtgtgt gtgtgtgtga aaatgtgtgt 3420gtgcttatgt atgaggtaca acctgttctg ctttcctctt cctgaatttt attttttggg 3480aaaagaaaag tcaagggtag ggtgggcctt cagggagtga gggattatct tttttttttt 3540ttctttcttt ctttcttttt tttttttgag acagaatctc gctctgtcgc ccaggctgga 3600gtgcaatggc acaatctcgg ctcactgcat cctccgcctc ccgggttcaa gtgattctca 3660tgcctcagcc tcctgagtag ctgggattac aggctcctgc caccacgccc ggctaatttt 3720tgttttgttt tgtttggaga cagagtctcg ctattgtcac cagggctgga atgatttcag 3780ctcactgcaa ccttcgccac ctgggttcca gcaattctcc tgcctcagcc tcccgagtag 3840ctgagattat aggcacctac caccacgccc ggctaatttt tgtattttta gtagagacgg 3900ggtttcacca tgttggccag gctggtctcg aactcctgac cttaggtgat ccactcgcct 3960tcatctccca aagtgctggg attacaggcg tgagccaccg tgcctggcca cgcccaacta 4020atttttgtat ttttagtaga gacagggttt caccatgttg gccaggctgc tcttgaactc 4080ctgacctcag gtaatcgacc tgcctcggcc tcccaaagtg ctgggattac aggtgtgagc 4140caccacgccc ggtacatatt ttttaaattg aattctacta tttatgtgat ccttttggag 4200tcagacagat gtggttgcat cctaactcca tgtctctgag cattagattt ctcatttgcc 4260aataataata cctcccttag aagtttgttg tgaggattaa ataatgtaaa taaagaacta 4320gcataacact caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 441015837PRTHomo sapiens 15Met Ser Gln Thr Gly Ser His Pro Gly Arg Gly Leu Ala Gly Arg Trp 1 5 10 15 Leu Trp Gly Ala Gln Pro Cys Leu Leu Leu Pro Ile Val Pro Leu Ser 20 25 30 Trp Leu Val Trp Leu Leu Leu Leu Leu Leu Ala Ser Leu Leu Pro Ser 35 40 45 Ala Arg Leu Ala Ser Pro Leu Pro Arg Glu Glu Glu Ile Val Phe Pro 50 55 60 Glu Lys Leu Asn Gly Ser Val Leu Pro Gly Ser Gly Ala Pro Ala Arg 65 70 75 80 Leu Leu Cys Arg Leu Gln Ala Phe Gly Glu Thr Leu Leu Leu Glu Leu 85 90 95 Glu Gln Asp Ser Gly Val Gln Val Glu Gly Leu Thr Val Gln Tyr Leu 100 105 110 Gly Gln Ala Pro Glu Leu Leu Gly Gly Ala Glu Pro Gly Thr Tyr Leu 115 120 125 Thr Gly Thr Ile Asn Gly Asp Pro Glu Ser Val Ala Ser Leu His Trp 130 135 140 Asp Gly Gly Ala Leu Leu Gly Val Leu Gln Tyr Arg Gly Ala Glu Leu 145 150 155 160 His Leu Gln Pro Leu Glu Gly Gly Thr Pro Asn Ser Ala Gly Gly Pro 165 170 175 Gly Ala His Ile Leu Arg Arg Lys Ser Pro Ala Ser Gly Gln Gly Pro 180 185 190 Met Cys Asn Val Lys Ala Pro Leu Gly Ser Pro Ser Pro Arg Pro Arg 195 200 205 Arg Ala Lys Arg Phe Ala Ser Leu Ser Arg Phe Val Glu Thr Leu Val 210 215 220 Val Ala Asp Asp Lys Met Ala Ala Phe His Gly Ala Gly Leu Lys Arg 225 230 235 240 Tyr Leu Leu Thr Val Met Ala Ala Ala Ala Lys Ala Phe Lys His Pro 245 250 255 Ser Ile Arg Asn Pro Val Ser Leu Val Val Thr Arg Leu Val Ile Leu 260 265 270 Gly Ser Gly Glu Glu Gly Pro Gln Val Gly Pro Ser Ala Ala Gln Thr 275 280 285 Leu Arg Ser Phe Cys Ala Trp Gln Arg Gly Leu Asn Thr Pro Glu Asp 290 295 300 Ser Asp Pro Asp His Phe Asp Thr Ala Ile Leu Phe Thr Arg Gln Asp 305 310 315 320 Leu Cys Gly Val Ser Thr Cys Asp Thr Leu Gly Met Ala Asp Val Gly 325 330 335 Thr Val Cys Asp Pro Ala Arg Ser Cys Ala Ile Val Glu Asp Asp Gly 340 345 350 Leu Gln Ser Ala Phe Thr Ala Ala His Glu Leu Gly His Val Phe Asn 355 360 365 Met Leu His Asp Asn Ser Lys Pro Cys Ile Ser Leu Asn Gly Pro Leu 370 375 380 Ser Thr Ser Arg His Val Met Ala Pro Val Met Ala His Val Asp Pro 385 390 395 400 Glu Glu Pro Trp Ser Pro Cys Ser Ala Arg Phe Ile Thr Asp Phe Leu 405 410 415 Asp Asn Gly Tyr Gly His Cys Leu Leu Asp Lys Pro Glu Ala Pro Leu 420 425 430 His Leu Pro Val Thr Phe Pro Gly Lys Asp Tyr Asp Ala Asp Arg Gln 435 440 445 Cys Gln Leu Thr Phe Gly Pro Asp Ser Arg His Cys Pro Gln Leu Pro 450 455 460 Pro Pro Cys Ala Ala Leu Trp Cys Ser Gly His Leu Asn Gly His Ala 465 470 475 480 Met Cys Gln Thr Lys His Ser Pro Trp Ala Asp Gly Thr Pro Cys Gly 485 490 495 Pro Ala Gln Ala Cys Met Gly Gly Arg Cys Leu His Met Asp Gln Leu 500 505 510 Gln Asp Phe Asn Ile Pro Gln Ala Gly Gly Trp Gly Pro Trp Gly Pro 515 520 525 Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly Gly Val Gln Phe Ser Ser 530 535 540 Arg Asp Cys Thr Arg Pro Val Pro Arg Asn Gly Gly Lys Tyr Cys Glu 545 550 555 560 Gly Arg Arg Thr Arg Phe Arg Ser Cys Asn Thr Glu Asp Cys Pro Thr 565 570 575 Gly Ser Ala Leu Thr Phe Arg Glu Glu Gln Cys Ala Ala Tyr Asn His 580 585 590 Arg Thr Asp Leu Phe Lys Ser Phe Pro Gly Pro Met Asp Trp Val Pro 595 600 605 Arg Tyr Thr Gly Val Ala Pro Gln Asp Gln Cys Lys Leu Thr Cys Gln 610 615 620 Ala Gln Ala Leu Gly Tyr Tyr Tyr Val Leu Glu Pro Arg Val Val Asp 625 630 635 640 Gly Thr Pro Cys Ser Pro Asp Ser Ser Ser Val Cys Val Gln Gly Arg 645 650 655 Cys Ile His Ala Gly Cys Asp Arg Ile Ile Gly Ser Lys Lys Lys Phe 660 665 670 Asp Lys Cys Met Val Cys Gly Gly Asp Gly Ser Gly Cys Ser Lys Gln 675 680 685 Ser Gly Ser Phe Arg Lys Phe Arg Tyr Gly Tyr Asn Asn Val Val Thr 690 695 700 Ile Pro Ala Gly Ala Thr His Ile Leu Val Arg Gln Gln Gly Asn Pro 705 710 715 720 Gly His Arg Ser Ile Tyr Leu Ala Leu Lys Leu Pro Asp Gly Ser Tyr 725 730 735 Ala Leu Asn Gly Glu Tyr Thr Leu Met Pro Ser Pro Thr Asp Val Val 740 745 750 Leu Pro Gly Ala Val Ser Leu Arg Tyr Ser Gly Ala Thr Ala Ala Ser 755 760 765 Glu Thr Leu Ser Gly His Gly Pro Leu Ala Gln Pro Leu Thr Leu Gln 770 775 780 Val Leu Val Ala Gly Asn Pro Gln Asp Thr Arg Leu Arg Tyr Ser Phe 785 790 795 800 Phe Val Pro Arg Pro Thr Pro Ser Thr Pro Arg Pro Thr Pro Gln Asp 805 810 815 Trp Leu His Arg Arg Ala Gln Ile Leu Glu Ile Leu Arg Arg Arg Pro 820 825 830 Trp Ala Gly Arg Lys 835 169663DNAHomo sapiens 16ataaattcat tgttccacct cctcgcatct tcacagcgct cgcgctgctc tcggcgctcg 60cagctgccga ctggggatga cggcgggcag gaggagaccg cagccgaagg gacacagaca 120cgccgcttca ccagctcgcc tcaggctgcc cccctgcatt tttgttttaa tttttacggc 180tttttcccct ctctttcttc ccttcctcct ggtcccagca gagccaagga aacccacaaa 240ataagaaagg aagtgggccc cggagcttgg aacctccaca gccggcttgt ccagcgcagc 300gcgggggcgg gaggctgcgc gcaccagttg ccagcccggt gcgcggtacc tttccttact 360tttcttgaaa cagcgatcgt gcctgcattt ggtggttttt tggtttttgt ttttttcctt 420ttcccgtatt tgctgaatct ccactatccg actttttttt tttaatcttt tctttccccc 480cccccccacc ccacctcttt ctggagcacg aatccaaaca ttttcccaag caacaaagaa 540aagttcgcac gctggcaccg cagcccggac aggctggcgc tgctgccggg cccccctccc 600tccgacactt gactcaatcc tgcaagcaag tgtgtgtgtg tccccatccc ccgccccgtt 660aacttcatag caaataacaa atacccataa agtcccagtc gcgcagcccc tccccgcggg 720cagcgcacta tgctgctcgg gtgggcgtcc ctgctgctgt gcgcgttccg cctgcccctg 780gccgcggtcg gccccgccgc gacacctgcc caggataaag ccgggcagcc tccgactgct 840gcagcagccg cccagccccg ccggcggcag ggggaggagg tgcaggagcg agccgagcct 900cccggccacc cgcaccccct ggcgcagcgg cgcaggagca aggggctggt gcagaacatc 960gaccaactct actccggcgg cggcaaggtg ggctacctcg tctacgcggg cggccggagg 1020ttcctcttgg acctggagcg agatggttcg gtgggcattg ctggcttcgt gcccgcagga 1080ggcgggacga gtgcgccctg gcgccaccgg agccactgct tctatcgggg cacagtggac 1140ggtagtcccc gctctctggc tgtctttgac ctctgtgggg gtctcgacgg cttcttcgcg 1200gtcaagcacg cgcgctacac cctaaagcca ctgctgcgcg gaccctgggc ggaggaagaa 1260aaggggcgcg tgtacgggga tgggtccgca cggatcctgc acgtctacac ccgcgagggc 1320ttcagcttcg aggccctgcc gccgcgcgcc agctgcgaaa cccccgcgtc cacaccggag 1380gcccacgagc atgctccggc gcacagcaac ccgagcggac gcgcagcact ggcctcgcag 1440ctcttggacc agtccgctct ctcgcccgct gggggctcag gaccgcagac gtggtggcgg 1500cggcggcgcc gctccatctc ccgggcccgc caggtggagc tgcttctggt ggctgacgcg 1560tccatggcgc ggttgtatgg ccggggcctg cagcattacc tgctgaccct ggcctccatc 1620gccaataggc tgtacagcca tgctagcatc gagaaccaca tccgcctggc cgtggtgaag 1680gtggtggtgc taggcgacaa ggacaagagc ctggaagtga gcaagaacgc tgccaccaca 1740ctcaagaact tttgcaagtg gcagcaccaa cacaaccagc tgggagatga ccatgaggag 1800cactacgatg cagctatcct gtttactcgg gaggatttat gtgggcatca ttcatgtgac 1860accctgggaa tggcagacgt tgggaccata tgttctccag agcgcagctg tgctgtgatt 1920gaagacgatg gcctccacgc agccttcact gtggctcacg aaatcggaca tttacttggc 1980ctctcccatg acgattccaa attctgtgaa gagacctttg gttccacaga agataagcgc 2040ttaatgtctt ccatccttac cagcattgat gcatctaagc cctggtccaa atgcacttca 2100gccaccatca cagaattcct ggatgatggc catggtaact gtttgctgga cctaccacga 2160aagcagatcc tgggccccga agaactccca ggacagacct acgatgccac ccagcagtgc 2220aacctgacat

tcgggcctga gtactccgtg tgtcccggca tggatgtctg tgctcgcctg 2280tggtgtgctg tggtacgcca gggccagatg gtctgtctga ccaagaagct gcctgcggtg 2340gaagggacgc cttgtggaaa ggggagaatc tgcctgcagg gcaaatgtgt ggacaaaacc 2400aagaaaaaat attattcaac gtcaagccat ggcaactggg gatcttgggg atcctggggc 2460cagtgttctc gctcatgtgg aggaggagtg cagtttgcct atcgtcactg taataaccct 2520gctcccagaa acaacggacg ctactgcaca gggaagaggg ccatctaccg ctcctgcagt 2580ctcatgccct gcccacccaa tggtaaatca tttcgtcatg aacagtgtga ggccaaaaat 2640ggctatcagt ctgatgcaaa aggagtcaaa acttttgtgg aatgggttcc caaatatgca 2700ggtgtcctgc cagcggatgt gtgcaagctg acctgcagag ccaagggcac tggctactat 2760gtggtatttt ctccaaaggt gaccgatggc actgaatgta ggctgtacag taattccgtc 2820tgcgtccggg ggaagtgtgt gagaactggc tgtgacggca tcattggctc aaagctgcag 2880tatgacaagt gcggagtatg tggaggagac aactccagct gtacaaagat tgttggaacc 2940tttaataaga aaagtaaggg ttacactgac gtggtgagga ttcctgaagg ggcaacccac 3000ataaaagttc gacagttcaa agccaaagac cagactagat tcactgccta tttagccctg 3060aaaaagaaaa acggtgagta ccttatcaat ggaaagtaca tgatctccac ttcagagact 3120atcattgaca tcaatggaac agtcatgaac tatagcggtt ggagccacag ggatgacttc 3180ctgcatggca tgggctactc tgccacgaag gaaattctaa tagtgcagat tcttgcaaca 3240gaccccacta aaccattaga tgtccgttat agcttttttg ttcccaagaa gtccactcca 3300aaagtaaact ctgtcactag tcatggcagc aataaagtgg gatcacacac ttcgcagccg 3360cagtgggtca cgggcccatg gctcgcctgc tctaggacct gtgacacagg ttggcacacc 3420agaacggtgc agtgccagga tggaaaccgg aagttagcaa aaggatgtcc tctctcccaa 3480aggccttctg cgtttaagca atgcttgttg aagaaatgtt agcctgtggt tatgatctta 3540tgcacaaaga taactggagg attcagcact gatgcagtcg tggtgaacag gaggtctacc 3600taacgcacag aaagtcatgc ttcagtgaca ttgtcaacag gagtccaatt atgggcagaa 3660tctgctctct gtgaccaaaa gaggatgtgc actgcttcac gtgacagtgg tgaccttgca 3720atatagaaaa acttgggagt tattgaacat cccctgggct tacaagaaac actgatgaat 3780gtaaaatcag gggacatttg aagatggcag aactgtctcc cccttgtcac ctacctctga 3840tagaatgtct ttaatggtat cataatcatt ttcacccata atacacagta gcttcttctt 3900actgtttgta aatacattct cccttggtat gtcactttat atcccctggt tctattaaaa 3960tatccatata tatttctata aaaaaagtgt ttgaccaaag taggtctgca gctatttcaa 4020cttccttccg tttccagaaa gagctgtgga tattttactg gaaattaaga acttgctgct 4080gttttaataa gatgtagtat attttctgac tacaggagat aaaatttcag tcaaaaaacc 4140attttgacag caagtatctt ctgagaaatt ttgaaaagta aatagatctc agtgtatcta 4200gtcacttaaa tacatacacg ggttcattta cttaaacctt tgactgcctg tatttttttc 4260aggtagctag ccaaattaat gcataatttc agatgtagaa gtagggtttg cgtgtgtgtg 4320tgtgatcata ctcaagagtc taaaaactag tttccttgtg ttggaaattt aaaaggaaaa 4380aaatcgtatt tcactgtgtt ttcaatttat attttcacaa ctactttctc tctccagagc 4440tttcatctga tatctcacaa tgtatgatat acgtacaaaa cacacagcaa gttttctatc 4500atgtccaaca cattcaacac tggtatacct cctaccagca agcctttaaa atgcatttgt 4560gtttgcttat ttgttttgtt caagggttca gtaagaccta caatgttttg tatttcttga 4620cttattttat tagaaacatt aaagatcact tggtagttag ccacattgag aagtggttat 4680cattgttaat gtggttaatg ccaaaaagtg gttaatatta ataagactgt ttccacacca 4740taggcaataa tttcttaatt taaaaaatct aagtatattc ctattgtact aaatattttt 4800cccaactgga aagcacttga ttgtacccgt aagtgtttga gtgatgacat gtgatgattt 4860tcagaaagtt gttgtttttg tttccatagc ctgtttaagt aggttgtaag tttgaatagt 4920tagacatgga aattatttta taagcacaca cctaaagata tctttttaga tgataaaatg 4980tacacccccc catcaccaac ctcacaactt agaaaatcta agttgtttga tttctttggg 5040atttcttttg ttgtgaaaca ctgcaaagcc aatttttctt tataaaaatt catagtaatc 5100ctgccaaatg tgcctattgt taaagatttg catgtgaaga tcttagggaa ccactgtttg 5160agttctacaa gctcatgaga gtttattttt attataagat gtttttaata taaaagaatt 5220atgtaactga tcactatatt acatcatttc agtgggccag gaaaatagat gtcttgctgt 5280tttcagtatt ttcttaagaa attgctttta aaacaaataa ttgttttaca aaaccaataa 5340ttatcctttg aattttcata gactgacttt gcttttgacg tagaaatttt ttttctcaat 5400aaattatcac tttgagaaat gaggcctgta caaggctgat aacctatatg tgatggagat 5460cacccaatgc caagggcaga aagcaaacct agttaaatag gtgagaaaaa aaataataat 5520cccagtgcca tttgtctgtg caaagagaat taggagagag gttaatgtta cttttttcca 5580ttttggaaat aattttaatc aagtaactca aatgtgacaa aatttatttt tattttttgt 5640ggttatattc ccaacaacat taaaaaatac tcgaggcata aatgtagttg tctcctactc 5700tgcttctctt actatactca tacattttta atatggttta tcaatgattc atgtttccct 5760caaatagtga tggtttacac ctgtcatgga aacaatccta gagagctcag agcaattaaa 5820ccactattcc atgcttttaa gtagttttct ccaccttttt cttatgagtc tcactagatt 5880gactgaggaa tgtatgtcta aattcctgga gaagatgata tggattggaa actgaaattc 5940agagaaatgg agtgttcaat agataccacg aattgtgaac aaagggaaaa ttctatacaa 6000ctcaatctaa gtcagtccac tttgacttcg tactgtcttt cacctttcca ttgttgcatc 6060ttgaattttt taaaatgtct agaattcagg atgctagggg ctacttcttt aaaaaaaaaa 6120aaaaaaaaga attcgtctga aaatgctcag gtttgtaaga atctaatctc acttacataa 6180ctaagcactc cataataagt tttattaagt acaaagggag ccagaaaaaa tgacatttat 6240ttcttctaga tcagaaaaat ttaaattaag ccctgccttg ctgtttagaa atatgtgggc 6300attgttataa tttattcaat aaatttatgt tcctttgcct tcctgtggaa acagttttat 6360cccactaaac taggaattag gggataaatc acaaacaaaa aaaaagttgc agcactgaaa 6420aaaagtaatt tattgttttt gcaactggta tgtgaatttg tgtgataaaa ttatttattc 6480ttatttaaca aaaatatgtt caaatttttc tatatttaaa atgttttgct gttgtcctac 6540tttttaattt atgcttcatg tttgtgtata aagtacactt ttacactttg tgagtttaca 6600taatatacag cactggttgc ttttgtattt ttttacagaa agctttctgt gtgaagcagg 6660tgtatatgta tatattcctc atgtattctt attctgatac tatcattttt ctttccaagg 6720aaattttaat ctgtcatgac caatagtgtt cattacttgt gcctatgata ataggttttt 6780tacatcacat taacactatt ttttccaagt cacaaataag aaaaacactt attcaatgaa 6840acaaggtgca agttttaaat ttgggtacac aaatagccta gaagcttcct acagacgcta 6900agacacagcc aataatcaga tcctttcact tcatcgagaa acttggacaa gtcgatattg 6960atgtattaga tgaaagttgt ctacacacaa cttctgaggg atacaaacga taataaaacc 7020aaatgttgtc tgtttctcct ttagaaacac ctcctaaaat taatatcatt tagtctctag 7080tgtctgtagg attctacaga tgagcacaaa tagattgggt ttgtataaca aatgctaata 7140gtcataactg tttctacaaa tatggggtgt ccattaagag aatgtgatgt tttcctactg 7200ctgttgaatc ccatggggtg attataggac ttgaaatagg cagagtcacc tctgatgaca 7260tcagcttgcc tctgtgattt cacagtctga tcctggcaac aagacaaagc acccttggac 7320acacagccaa tctctggttg tgatatttcc ccattgattc cttccttgtt aacaaggtca 7380ttttaatggt tcaggtgagg acagcagcca gattcaaagt ccagaatttg tgctgttaca 7440tagagttcac actgtcaaat aacattgaat ttaataatga tcaaattttt ctagtagtct 7500ttggcagagt gtataatctc attggcatga ttggtgaata ttactaatct ctttataatg 7560aaagatgctt tacaaatacc ttatatttgc taacatttca aaactactaa ataaatgaaa 7620tagccatgtg tacagaaatg gtcatttaaa gctttaatag aaccaaattc aagacaatgt 7680atcatttaga cacacagaaa aggaacttgt atgttttccc tattattttt ctcatttgcc 7740aacaatctat agttttaggt tatcaaacag atagatcaac ttaactggct agtacattga 7800aaaatcttcc taagaatcct ttgttagcat aatctataga gataatttct caaattatat 7860catcatgatg catataaact ctataatgta taattgtgtt tcatttattt aatgtatgag 7920aacatattga aatacaaaac catgcattag ccaaaaaatt ggaatacagg tagtgttcag 7980atcagcaaaa cattcagtct ggtaaatgcc tgcctggggc tatgatatca ttctcaatgc 8040aggttttatg gaaaaactaa aagaatatgt tgttagatga tgttggtttt gaaaaaaaaa 8100agacattaac atacacatta gttagcccag ttaattgcat tctactaata tagttgcaca 8160ttagcaataa ttttgctgtc tctggtcttt attttgtggc ttcaactaac tggaccatgt 8220ggactgtaaa ggtcaaatgg aaaaaacgag cagtggcccc tcatcctgta aggtactgct 8280acatcagagt gacctaaaag tctaacactg tgaggaaaac tgtgatttgt aggaaaaaaa 8340aaaaaaacaa ataaaaaaca gggcatgctt tttaattttt ttccactttc ctttggcaca 8400cccaatgaac aattctaatt tttattgagg tgctaacatc tttcgtgacc gactgtcaaa 8460tgtggtattt ttgagttact atttttctac atgattttac agtttgcaag aaagacctct 8520aagctttgtg tcacggtagg gcacaacttg atactcaaaa tttgaaaaat aagcacatcc 8580aatgattgtt ttgaccaaca gtggtcagtg acgtaaactg catgtgcatc tgaggacatt 8640taaggggtca ttaaaatttg aggagcatca ggccggagta gcagactttt agatgagtca 8700tatttcagca ttcactaagt cctcagcatt ccattcaaac tgtcgtgtat atttggcctg 8760attttttttc aagctttgca ataatttatg ttattggtaa acacttggtg actatatctc 8820agccttttct ttaacaactc acaatatatt agaaacacgt ctacctatac tgagagtata 8880tttacaatag aagaacatac tgtatgtgac tttgtaaagc tagacttttg attaagaaat 8940atataatctc tggatgctat ttttgcatta tacactcagg cacaacgtaa accttgatgg 9000ctcatcttgc tacaattacg agttgaaaaa cactacttac gtatttgtat gacctattag 9060tcagaggaaa tcatacatat gctttgtaaa tagactttgc agataactaa atagactgaa 9120gaaatatgtt gcatttgata gaagcaattg cataaatatt tggtttctat attagagtct 9180gtgagtaaag tcaagtaata aacctaagta ggtataacag atttttaaac cttgaaactt 9240gctttgatgg tagagaaaat cattgaagat ttacatactg tatataagat gtaaaatgta 9300cgctgcttat taccctcaat tttccagaag caatggtata taatgcagtt gaaaaaccaa 9360aaatcttgga aaactaagac gggtcttgtt taaaatgtct ctcagctttg gcaaccttca 9420aatcttaatc aactatttaa agcattactg tgtcttgtag cctgcattcc acaacagctc 9480tgttattcag gtaaaagact tgaactgagc cgtttgggac ctatactgta atattttcat 9540tgaggaacaa tatcctattt tgtaaagcat ttccctatgt gtgactttaa actgtaaaat 9600taaacactgc ttttgtgggt tcagtgggca taataaatat aaattgtaaa ctaggttaaa 9660gta 966317930PRTHomo sapiens 17Met Leu Leu Gly Trp Ala Ser Leu Leu Leu Cys Ala Phe Arg Leu Pro 1 5 10 15 Leu Ala Ala Val Gly Pro Ala Ala Thr Pro Ala Gln Asp Lys Ala Gly 20 25 30 Gln Pro Pro Thr Ala Ala Ala Ala Ala Gln Pro Arg Arg Arg Gln Gly 35 40 45 Glu Glu Val Gln Glu Arg Ala Glu Pro Pro Gly His Pro His Pro Leu 50 55 60 Ala Gln Arg Arg Arg Ser Lys Gly Leu Val Gln Asn Ile Asp Gln Leu 65 70 75 80 Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly Arg 85 90 95 Arg Phe Leu Leu Asp Leu Glu Arg Asp Gly Ser Val Gly Ile Ala Gly 100 105 110 Phe Val Pro Ala Gly Gly Gly Thr Ser Ala Pro Trp Arg His Arg Ser 115 120 125 His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser Leu Ala 130 135 140 Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val Lys His 145 150 155 160 Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg Gly Pro Trp Ala Glu Glu 165 170 175 Glu Lys Gly Arg Val Tyr Gly Asp Gly Ser Ala Arg Ile Leu His Val 180 185 190 Tyr Thr Arg Glu Gly Phe Ser Phe Glu Ala Leu Pro Pro Arg Ala Ser 195 200 205 Cys Glu Thr Pro Ala Ser Thr Pro Glu Ala His Glu His Ala Pro Ala 210 215 220 His Ser Asn Pro Ser Gly Arg Ala Ala Leu Ala Ser Gln Leu Leu Asp 225 230 235 240 Gln Ser Ala Leu Ser Pro Ala Gly Gly Ser Gly Pro Gln Thr Trp Trp 245 250 255 Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu 260 265 270 Leu Val Ala Asp Ala Ser Met Ala Arg Leu Tyr Gly Arg Gly Leu Gln 275 280 285 His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His 290 295 300 Ala Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val 305 310 315 320 Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys Asn Ala Ala Thr 325 330 335 Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly 340 345 350 Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr Arg Glu 355 360 365 Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly Met Ala Asp Val 370 375 380 Gly Thr Ile Cys Ser Pro Glu Arg Ser Cys Ala Val Ile Glu Asp Asp 385 390 395 400 Gly Leu His Ala Ala Phe Thr Val Ala His Glu Ile Gly His Leu Leu 405 410 415 Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu Glu Thr Phe Gly Ser 420 425 430 Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu Thr Ser Ile Asp Ala 435 440 445 Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr Ile Thr Glu Phe Leu 450 455 460 Asp Asp Gly His Gly Asn Cys Leu Leu Asp Leu Pro Arg Lys Gln Ile 465 470 475 480 Leu Gly Pro Glu Glu Leu Pro Gly Gln Thr Tyr Asp Ala Thr Gln Gln 485 490 495 Cys Asn Leu Thr Phe Gly Pro Glu Tyr Ser Val Cys Pro Gly Met Asp 500 505 510 Val Cys Ala Arg Leu Trp Cys Ala Val Val Arg Gln Gly Gln Met Val 515 520 525 Cys Leu Thr Lys Lys Leu Pro Ala Val Glu Gly Thr Pro Cys Gly Lys 530 535 540 Gly Arg Ile Cys Leu Gln Gly Lys Cys Val Asp Lys Thr Lys Lys Lys 545 550 555 560 Tyr Tyr Ser Thr Ser Ser His Gly Asn Trp Gly Ser Trp Gly Ser Trp 565 570 575 Gly Gln Cys Ser Arg Ser Cys Gly Gly Gly Val Gln Phe Ala Tyr Arg 580 585 590 His Cys Asn Asn Pro Ala Pro Arg Asn Asn Gly Arg Tyr Cys Thr Gly 595 600 605 Lys Arg Ala Ile Tyr Arg Ser Cys Ser Leu Met Pro Cys Pro Pro Asn 610 615 620 Gly Lys Ser Phe Arg His Glu Gln Cys Glu Ala Lys Asn Gly Tyr Gln 625 630 635 640 Ser Asp Ala Lys Gly Val Lys Thr Phe Val Glu Trp Val Pro Lys Tyr 645 650 655 Ala Gly Val Leu Pro Ala Asp Val Cys Lys Leu Thr Cys Arg Ala Lys 660 665 670 Gly Thr Gly Tyr Tyr Val Val Phe Ser Pro Lys Val Thr Asp Gly Thr 675 680 685 Glu Cys Arg Leu Tyr Ser Asn Ser Val Cys Val Arg Gly Lys Cys Val 690 695 700 Arg Thr Gly Cys Asp Gly Ile Ile Gly Ser Lys Leu Gln Tyr Asp Lys 705 710 715 720 Cys Gly Val Cys Gly Gly Asp Asn Ser Ser Cys Thr Lys Ile Val Gly 725 730 735 Thr Phe Asn Lys Lys Ser Lys Gly Tyr Thr Asp Val Val Arg Ile Pro 740 745 750 Glu Gly Ala Thr His Ile Lys Val Arg Gln Phe Lys Ala Lys Asp Gln 755 760 765 Thr Arg Phe Thr Ala Tyr Leu Ala Leu Lys Lys Lys Asn Gly Glu Tyr 770 775 780 Leu Ile Asn Gly Lys Tyr Met Ile Ser Thr Ser Glu Thr Ile Ile Asp 785 790 795 800 Ile Asn Gly Thr Val Met Asn Tyr Ser Gly Trp Ser His Arg Asp Asp 805 810 815 Phe Leu His Gly Met Gly Tyr Ser Ala Thr Lys Glu Ile Leu Ile Val 820 825 830 Gln Ile Leu Ala Thr Asp Pro Thr Lys Pro Leu Asp Val Arg Tyr Ser 835 840 845 Phe Phe Val Pro Lys Lys Ser Thr Pro Lys Val Asn Ser Val Thr Ser 850 855 860 His Gly Ser Asn Lys Val Gly Ser His Thr Ser Gln Pro Gln Trp Val 865 870 875 880 Thr Gly Pro Trp Leu Ala Cys Ser Arg Thr Cys Asp Thr Gly Trp His 885 890 895 Thr Arg Thr Val Gln Cys Gln Asp Gly Asn Arg Lys Leu Ala Lys Gly 900 905 910 Cys Pro Leu Ser Gln Arg Pro Ser Ala Phe Lys Gln Cys Leu Leu Lys 915 920 925 Lys Cys 930 1812PRTArtificial SequenceSynthetic Sequence 18Ala Gly Gly Trp Gly Pro Trp Gly Pro Trp Gly Asp 1 5 10 1912PRTArtificial SequenceSynthetic Sequence 19Gly Gly Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys 1 5 10 2012PRTArtificial SequenceSynthetic Sequence 20Gly Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser 1 5 10 2112PRTArtificial SequenceSynthetic Sequence 21Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg 1 5 10 2212PRTArtificial SequenceSynthetic Sequence 22Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr 1 5 10 2312PRTArtificial SequenceSynthetic Sequence 23Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys 1 5 10 2412PRTArtificial SequenceSynthetic Sequence 24Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly 1 5 10 2512PRTArtificial SequenceSynthetic Sequence 25Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly 1 5 10 2612PRTArtificial SequenceSynthetic Sequence 26Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly Gly 1 5 10 2712PRTArtificial SequenceSynthetic Sequence 27Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly Gly Val 1 5 10 2812PRTArtificial SequenceSynthetic Sequence 28Gly Asp Cys Ser Arg Thr Cys Gly Gly Gly Val Gln 1 5 10 2912PRTArtificial SequenceSynthetic Sequence 29Asp Cys Ser Arg Thr Cys Gly Gly Gly Val Gln Phe 1 5 10 3012PRTArtificial SequenceSynthetic Sequence 30Cys Ser Arg

Thr Cys Gly Gly Gly Val Gln Phe Ser 1 5 10 3112PRTArtificial SequenceSynthetic Sequence 31Ser Arg Thr Cys Gly Gly Gly Val Gln Phe Ser Ser 1 5 10 3212PRTArtificial SequenceSynthetic Sequence 32Arg Thr Cys Gly Gly Gly Val Gln Phe Ser Ser Arg 1 5 10 3312PRTArtificial SequenceSynthetic Sequence 33Thr Cys Gly Gly Gly Val Gln Phe Ser Ser Arg Asp 1 5 10 3412PRTArtificial SequenceSynthetic Sequence 34Cys Gly Gly Gly Val Gln Phe Ser Ser Arg Asp Cys 1 5 10 3512PRTArtificial SequenceSynthetic Sequence 35Gly Gly Gly Val Gln Phe Ser Ser Arg Asp Cys Thr 1 5 10 3612PRTArtificial SequenceSynthetic Sequence 36Gly Gly Val Gln Phe Ser Ser Arg Asp Cys Thr Arg 1 5 10 3712PRTArtificial SequenceSynthetic Sequence 37Gly Val Gln Phe Ser Ser Arg Asp Cys Thr Arg Pro 1 5 10 3812PRTArtificial SequenceSynthetic Sequence 38Val Gln Phe Ser Ser Arg Asp Cys Thr Arg Pro Val 1 5 10 3912PRTArtificial SequenceSynthetic Sequence 39Gln Phe Ser Ser Arg Asp Cys Thr Arg Pro Val Pro 1 5 10 4012PRTArtificial SequenceSynthetic Sequence 40Phe Ser Ser Arg Asp Cys Thr Arg Pro Val Pro Arg 1 5 10 4112PRTArtificial SequenceSynthetic Sequence 41Ser Ser Arg Asp Cys Thr Arg Pro Val Pro Arg Asn 1 5 10 4212PRTArtificial SequenceSynthetic Sequence 42Ser Arg Asp Cys Thr Arg Pro Val Pro Arg Asn Gly 1 5 10 4312PRTArtificial SequenceSynthetic Sequence 43Arg Asp Cys Thr Arg Pro Val Pro Arg Asn Gly Gly 1 5 10 4412PRTArtificial SequenceSynthetic Sequence 44Asp Cys Thr Arg Pro Val Pro Arg Asn Gly Gly Lys 1 5 10 4512PRTArtificial SequenceSynthetic Sequence 45Cys Thr Arg Pro Val Pro Arg Asn Gly Gly Lys Tyr 1 5 10 4612PRTArtificial SequenceSynthetic Sequence 46Thr Arg Pro Val Pro Arg Asn Gly Gly Lys Tyr Cys 1 5 10 4712PRTArtificial SequenceSynthetic Sequence 47Arg Pro Val Pro Arg Asn Gly Gly Lys Tyr Cys Glu 1 5 10 4812PRTArtificial SequenceSynthetic Sequence 48Pro Val Pro Arg Asn Gly Gly Lys Tyr Cys Glu Gly 1 5 10 4912PRTArtificial SequenceSynthetic Sequence 49Val Pro Arg Asn Gly Gly Lys Tyr Cys Glu Gly Arg 1 5 10 5012PRTArtificial SequenceSynthetic Sequence 50Pro Arg Asn Gly Gly Lys Tyr Cys Glu Gly Arg Arg 1 5 10 5112PRTArtificial SequenceSynthetic Sequence 51Arg Asn Gly Gly Lys Tyr Cys Glu Gly Arg Arg Thr 1 5 10 5212PRTArtificial SequenceSynthetic Sequence 52Asn Gly Gly Lys Tyr Cys Glu Gly Arg Arg Thr Arg 1 5 10 5312PRTArtificial SequenceSynthetic Sequence 53Cys Glu Gly Arg Arg Thr Arg Phe Arg Ser Cys Asn 1 5 10 5412PRTArtificial SequenceSynthetic Sequence 54Glu Gly Arg Arg Thr Arg Phe Arg Ser Cys Asn Thr 1 5 10 5512PRTArtificial SequenceSynthetic Sequence 55Gly Arg Arg Thr Arg Phe Arg Ser Cys Asn Thr Glu 1 5 10 5612PRTArtificial SequenceSynthetic Sequence 56Arg Arg Thr Arg Phe Arg Ser Cys Asn Thr Glu Asp 1 5 10 5712PRTArtificial SequenceSynthetic Sequence 57Arg Thr Arg Phe Arg Ser Cys Asn Thr Glu Asp Cys 1 5 10 5812PRTArtificial SequenceSynthetic Sequence 58Thr Arg Phe Arg Ser Cys Asn Thr Glu Asp Cys Pro 1 5 10

Claims

1. An antibody which specifically binds to a human aggrecanase and inhibits aggrecanase activity of said aggrecanase.

2. The antibody according to claim 1, wherein the human aggrecanase is human ADAMTS4.

3. The antibody according to claim 2, which further inhibits aggrecanase activity of human ADAMTS5.

4. The antibody according to claim 2, which binds to human ADAMTS4 at an epitope comprising the amino acid sequence depicted in SEQ ID NO: 9.

5. The antibody according to claim 2, which comprises a light chain variable region and a heavy chain variable region, wherein (1) the light chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 4, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 6; or (2) the light chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 4, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 6, except that 1 to 3 amino acids are substituted, deleted, inserted, or added in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 3, and/or 1 to 3 amino acids are substituted, deleted, inserted, or added in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 4 to 6.

6. The antibody according to claim 5, wherein the light chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 7 and the heavy chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 8.

7. The antibody according to claim 1, which is a human antibody.

8. A pharmaceutical composition which comprises the antibody according to claim 1.

9. A polynucleotide which encodes the antibody according to claim 1.

10. A vector which comprises the polynucleotide according to claim 9.

11. A transformant which comprises the vector according to claim 10.

12.-14. (canceled)

15. A method of preventing or treating arthritis in a mammal, which comprises administering an effective amount of an antibody which specifically binds to a human aggrecanase and inhibits aggrecanase activity of said aggrecanase to the mammal.

16. The method according to claim 15, wherein the human aggrecanase is human ADAMTS4.

17.-23. (canceled)

24. The method according to claim 16, wherein the antibody further inhibits aggrecanase activity of human ADAMTS5.

25. The method according to claim 16, wherein the antibody binds to human ADAMTS4 at an epitope comprising the amino acid sequence depicted in SEQ ID NO: 9.

26. The method according to claim 16, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein (1) the light chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 4, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 6; or (2) the light chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, and the heavy chain variable region comprises CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 4, CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 5 and CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 6, except that 1 to 3 amino acids are substituted, deleted, inserted, or added in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 3, and/or 1 to 3 amino acids are substituted, deleted, inserted, or added in at least one amino acid sequence selected from the group consisting of SEQ ID NO: 4 to 6.

27. The method according to claim 26, wherein the light chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 7 and the heavy chain variable region comprises the amino acid sequence depicted in SEQ ID NO: 8.

28. The method according to claim 15, wherein the antibody is a human antibody.

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