US20160304622A1

US20160304622A1 - Human antibody against aggrecanase-type adamts species for therapeutics of aggrecanase-related diseases

Info

Publication number: US20160304622A1
Application number: US15/029,823
Authority: US
Inventors: Akira Miyakoshi; Mikiko NAKAMURA; Kanehisa KOJOH; Satsuki Mochizuki; Yasunori Okada
Original assignee: Keio University; Genefrontier Corp
Current assignee: Keio University; Genefrontier Corp
Priority date: 2013-10-15
Filing date: 2014-10-14
Publication date: 2016-10-20
Also published as: AU2014335251B2; WO2015056808A1; US20190233541A1; JP2016538839A; ZA201603098B; JP6454893B2; EP3057992B1; CN105849128A; EP3057992A1; CN105849128B; AU2014335251A1; CA2927525A1; CA2927525C; EP3057992A4; US10640573B2

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

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

BACKGROUND ART

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].
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

[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.
[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.
[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.
[4] Porter S, Clark I M, Kevorkian L, Edwards D R. The ADAMTS metalloproteinases. Biochem J. 2005; 386: 15-27.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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
[15] Rothe, C. et al. J. Mol. Biol. 2008; 376:1182-1200

SUMMARY OF INVENTION

Technical Problem

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

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.
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

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

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 μg) for 16 h at 37° C. Aggrecanase activity of ADAMTS4 was evaluated by immunoblotting with anti-aggrecan neoepitope (NITEGE³⁹²) antibody. TS(−), buffer alone; Fab(−), ADAMTS4 incubated without Fab; Control, control Fab.

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.

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).

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-1α-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-1α-stimulated chondrocytes. Osteoarthritic chondrocytes were cultured in the presence and absence of IL-1α 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

The present invention provides an antibody which has a specific binding activity to human aggrecanase, and inhibits the aggrecanase activity of the aggrecanase.
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.
The antibody of the present invention has a specific binding activity to human aggrecanase.
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.
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)).
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° C. for 16 hr, deglycosylating them with chondroitinase ABC and keratanase, and analyzing the obtained reaction product by immunoblotting using an anti-NITEGE³⁹²aggrecan 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.
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.
The antibody of this embodiment preferably also inhibits, in addition to the aggrecanase activity of human ADAMTS4, the aggrecanase activity of human ADAMTS5.
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_Hherein) and a heavy chain constant region. The heavy chain constant region is constituted by 3 domains of C _H1, C _H2 and C _H3. Each light chain is constituted by a light chain variable region (to be abbreviated as V_Lherein) and a light chain constant region. The light chain constant region is constituted by a single domain C_L. V_Hand V_Lregions 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_Hand V_Lis 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.
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_L, V_H, C_Land C_H1domains, (ii) F(ab′)₂fragment, 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_Hand C_H1domains, (v) Fv fragment constituted by V_Land V_Hdomains in a single arm of an antibody, (vi) dAb fragment constituted by V_Hdomain (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_Land V_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_Land V_Hregions 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.
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.
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.
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.
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).
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.
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×His consisting of six His (histidine) residues, 10×His, human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-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.
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).
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%).
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.
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.
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.
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.
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.
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.
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).
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.
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, Cγ1, Cγ2, Cγ3, Cγ4, Cδ, Cα1, Cα2, Cε can be used in H chain, and Cκ, Cλ can be used in L chain.
In the antibodies of (1) and (2), the constant region of Cκ of human antibody is preferably used for the light chain, and the constant region of Cγ1 of human antibody is preferably used for the heavy chain.
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.
The antibody of the above-mentioned (1′) corresponds to a preferable embodiment of the antibody of the above-mentioned (1).
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.
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.
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.
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.
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.
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.
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%).
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.
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.
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.
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.
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.
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, SRα promoter and the like are preferable.
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.
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^r), neomycin resistance gene (sometimes to be abbreviated as Neo^r, G418 resistance) and the like.
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.
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° 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° 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° C.-35° 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° 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° C.-40° C. for about 15-60 hr. In any culture, aeration and stirring may be performed as necessary.
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.
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.
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.
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.
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.
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.
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.
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.).
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.
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.
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

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

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-β-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.

Expression vectors containing cDNA fragments encoding the residues Phe²¹³-Cys⁶⁸⁵of 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 Ser²⁶²-Pro⁶²²of ADAMTS5) was purchased from R&D Systems Inc.

Immunoblotting of Human Anti-ADAMTS Antibodies.

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 μg/ml; clones 237-1, 237-5, 237-21, 237-43 and 237-53) at 4° 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).
Recombinant ADAMTS4 (180 ng), and ADAMTS5 (180 ng) were incubated for 30 min at 37° 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 μg) for 16 h at 37° C. After deglycosylation of aggrecan with chondroitinase ABC and keratanase (Seikagaku Corporation, Tokyo, Japan), aggrecanase activity was monitored by immunoblotting using the anti-NITEGE³⁹²aggrecan neoepitope antibody (1.2 μg/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).

Recombinant FLAG and dihydrofolic acid reductase (DHFR)-tagged proteins of each domain of ADAMTS4 and the thrombospondin domain with NH₂- 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 μg/ml) or human anti-ADAMTS antibody (clone 237-53; 2 μg/ml).
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_D(the affinity) was calculated from the determined K_aand K_dvalues.
Inhibition of Aggrecanase Activity in Cultured Chondrocytes with Anti-ADAMTS Antibody (Clone 237-53).
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 μg/ml of ascorbic acid, and treated with or without interleukin-1α (IL-1α (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 μg/ml, clone 237-53) or human control IgG (5 μg/ml; Invitrogen, Carlsbad, Calif.) for 1 h, and then incubated in the presence of aggrecan (100 μg) 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-NITEGE³⁹²neoepitope antibody (1.2 μg/ml). Informed consent was obtained from the patients with osteoarthritis for the experimental use of the surgical samples according to the hospital ethics guidelines.
To examine the mRNA expression of ADAMTS4 and 5, total RNA was prepared from the chondrocytes treated with or without IL-1α (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

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 (NITEGE³⁹²)-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
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

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.
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 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)

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.
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 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)

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

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

Aggrecanase activity of ADAMTS4 and ADAMTS5 was assayed by immunoblotting demonstration of the 65-kDa aggrecan fragments with the COOH-terminal sequence of NITEGE³⁹²using 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_Dvalues of 1.17×10⁻⁸M and 1.46×10⁻⁹M for ADAMTS4 and ADAMTS5, respectively.
Cultured chondrocytes from osteoarthritic cartilage expressed ADAMTS5, but not ADAMTS4 (FIG. 4B, left). When the chondrocytes were treated with IL-1α, 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-1α (FIG. 4B, right). When IL-1α-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

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

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

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.