AU733104B2 - The use of proteinase inhibitors for prevention or reduction of bone resorption - Google Patents

Description

I

WO 98/04287 PCT/EP97/04110 THE USE OF PROTEINASE INHIBITORS FOR PREVENTION OR REDUCTION OF BONE RESORPTION The present invention relates to the reduction of the rate of bone resorption by targeting the action or production of proteases.

Human bone is constantly undergoing remodelling. The fine balance between bone formation and bone resorption is regulated by local and systemic factors and by physical forces acting on various cells including, in the bone environment, osteoblasts and osteoclasts. However, in several bone metabolic diseases including most importantly osteoporosis and osteolytic bone metastasis, the balance is disturbed resulting in a sustained pathological net bone resorption.

Osteoporosis is a systemic skeletal disease characterised by low bone mass and microarchitectural deterioration of bone tissue, with a subsequent increase in bone fragility and susceptibility to fracture. Postmenopausal osteoporosis is a chronic disease which affects millions of women throughout the world and it has an enormous economical and social impact on society.

Reduction of bone resorption is believed to be an appropriate way to prevent and treat several metabolic bone diseases, including osteoporosis and osteolytic bone metastasis. Agents such as steroid hormones (especially oestrogen), calcitonin and bisphosphonates are able to suppress bone resorption and have been used for prevention and treatment of osteoporosis and/or osteolytic bone metastasis. However, these therapeutic agents fail to achieve satisfactory effects in some cases, due to subject limitation or uncertain efficacy. There is therefore need of a new prophylactic/ therapeutic method for preventing or treating accentuated bone resorption.

Removal of the mineralised osseous substance, i.e.

organic matrix embedded in deposits of calcium phosphate CONFIRMATION

COPY

WO 98/04287 PCT/EP97/04110 2 salts, is a complicated process. Though still a controversial subject, it seems probable that osteoclasts are the only cells capable of bone resorption. The progressing bone loss in patients with osteoporosis is caused by an increase in the activity of osteoclasts.

The expected life cycle of osteoclasts involve the following major phases: 1. recruitment of haematopoietic stem cells, the early precursor of osteoclasts, 2. proliferation and differentiation, 3. fusion into multinuclearity, 4. attachment to the resorptive bone surface, polarisation and removal of mineralised osseous substance, and 6. death by apoptosis, necrosis or a more random process.

These phases are, however, not necessarily separate events, thus, e.g. differentiation might take place during migration to the resorptive surface and fusion might take place on the bone surface. All these phases represent possibilities for intervention in order to regulate the level of bone resorption.

Traditionally, proteolytic enzymes have been known to play a role in degradation of the organic matrix of bone.

The knowledge about proteolytic enzymes involved in bone resorption mainly comes from in vitro and in vivo studies of the effects of natural and particularly synthetic enzyme inhibitors. Furthermore, histochemical and immunocytochemical characterisation of enzymes in bone cells and tissues as well as more recently identification of enzymeencoding mRNA in osteoclasts and other bone cells has increased the information about proteolytic enzymes involved in bone resorption. The proteolytic enzymes of major relevance to osteoclastic bone resorption seem to be members of the families of cysteine proteinases and matrix metalloproteinases (MMPs).

WO 98/04287 PCT/EP97/04110 3 The use of proteinase inhibitors in disease control has been suggested in several scientific publications and in patents and patent applications. For MMP inhibitors the main focus has been the potential of inhibitors in treatment of cancer and tumour metastasis, but also diseases such as arthritis, ulcers, periodontal and bone diseases,

HIV

infection, corneal and other eye diseases, diabetes and myocardial infarction have been the target of these speculations and ensuing early experiments (reviewed by Birkedal-Hansen et al, 1993 2).

In some particular cases, however, the studies have been emphatic leading to particularly important conclusions and products of relevance to the use of proteinase inhibitors in disease control. Selected peptidyl derivatives were shown to be effective inhibitors of metalloproteinases reaching Ki-values down to 5 pM for MMP-2 by kinetic studies based on a fluorogenic synthetic peptide substrate incubated with MMP-1, -2 or -3 and the substances were orally active and non-toxic in mice at suitable doses (W094/25434).

Membrane-type matrix metalloproteinases (MT-MMPs) were originally identified in cancer cells and have been implicated with the migration of these cells (Sato et al 19941 Based on this disclosure, its seems that the use of MT-MMP inhibitors will be appropriate for the reduction of the spread of tumours. No studies have, however, yet described inhibitors of MT-MMPs and thus no data are available on the use of MT-MMP inhibitors as agents in the treatment of diseases. From the usually low selectivity of synthetic MMP-inhibitors, it seems probable that some established MMP-inhibitors will inhibit MT-MMPs.

Furthermore, cDNA encoding MT1-MMP (also referred to in the literature as MT-MMP-1 and as MMP-14) as well as anti-MTl- MMP antibodies have been suggested, though rather unspecifically, as useful for application not only in the diagnostic area but also in other medical fields (EP-A- 0685557 and W095/25171).

The inhibition of cathepsins is considered another possible way of reducing bone resorption by using proteinase inhibitors. Several cathepsins are produced by osteoclas:s and though still somewhat controversial, they are apparently involved in the degradation of organic matrix in the acidic environment of the sub-osteoclastic resorption zone.

Recently a novel cathepsin named cathepsin K, cathepsin O or OC2 was cloned from osteoclasts and osteociast-like cells by several independent groups. It was suggested that development of anrisense probes or synthetic inhibios to hi s protenase could be of value in the -reatment of several diseases including osteoporosis. or ca theosi: i several compounds have been produced for use as soecific inhibitors in the treatment and orevention of osteoporosis (EP-A-0611756).

The general use of hybrid molecules for conferring specificity to cell- and tissue-interacting agents has been proposed in several modifications including hybrids consisting of three parts including not only a cell-binding 6 S 2r0 ligand and a chemical entity to be introduced into the target cell but also an intermediate part constituting a translocation domain for enabling the entrance of the chemical entity into the cell. Another approach to resist clearance and degradation and ease the uptake in 2 S cells of peptides and proteinase inhibitors is by administering them as lipid conjugates (W093/01828) Speculations about the biological roles of osteoclastic proteinases have been almost entirely focused on their potential ability as mediators of degradation of organic bone matrix in the sub-osteoclastic resorptive zone.

However, our recent findings have shown that proteolytic enzymes are also very important for the migration and attachment of osteoclasts to the resorptive surface (Blavier Delaiss6, 19953) Furthermore, the proteinasedependent migration of immature osteoclasts seems to be associated with the maturation into active bone-resorbing WO 98/04287 PCT/EP97/04110 osteoclasts as well as of importance for the events leading to fusion into multi-nuclearity, i.e. osteoclast differentiation processes.

Being an earlier phase of the osteoclast life cycle, interference by an inhibitor of a proteolytic enzyme involved in osteoclast migration and/or attachment might be more effective than inhibition of an enzyme involved directly in the resorptive process. This type of interference will also be easier to accomplish since the secreted enzymes of the migrating cells are not protected from inhibition as they are when secreted into the tightly sealed resorption zone which is formed when the active polarised osteoclasts attach to bone.

We have now discovered that an MT-MMP closely related to or identical to MT1-MMP, previously identified in cancer cells not related to bone, is expressed by osteoclasts. It may be expected that this osteoclast MT1-MMP plays an important role in the action of osteoclasts, probably being implicated in their migration to their site of action at which to degrade bone (see Examples 1, 2, 3-2 and 3-3 and Figures 1 to This finding indicates that also other membrane-associated metalloproteinases such as other MT-MMPs or members belonging to families of non-matrix type of membrane metalloproteinases meltrins and "A disintegrin and metalloproteinase"'s, ADAMs)) could be produced by osteoclasts.

Furthermore, we have identified and characterised the full length gene and the encoded protein of osteoclast metalloelastase MMP-12, a proteinase hitherto believed to be almost specifically expressed in macrophages, where it is obligatory for the invasion of these cells through basement membranes. Since macrophages and osteoclasts are closely related cell types both originating from the haematopoietic stem cell and differentiating late in its development, a similar role of MMP-12 in osteoclast invasion and migration is likely (see Example 3-4 and Figures 4 to 6).

6 The present invention provides the use of an agent in the manufacture of a medicament for the treatment of bone metabolic disease, characterised in that the agent acts by inhibition of the production or action of a membrane associated protease or the matrix metalloprotease MMP-12 involved in the resorptive activity of osteoclasts. More preferably. the invention provides the use of an agent in the manufacture of a medicament for the treatment of bone metabolic disease by inhibition of the production or action of a metalloproteinase involved in the resorptive activity of osteoclasts. Particularly, inhibition of the production or action of an MT-MMP but also of other membraneassociated metalloproteinases such as a meltrin or an ADAM as well as a secreted MMP in such as MMP-12.

Thus, according to a first embodiment of the invention, there is provided the use of an agent which acts by inhibition of the production or action of a membrane associated protease or the matrix metalloprotease MMP-12 involved in the resorptive activity of osteoclasts for the manufacture of a medicament for the treatment of bone metabolic 15 disease.

o According to a second embodiment of the invention, there is provided the use of an agent in the manufacture of a medicament for the treatment of bone metabolic disease by inhibition of the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration. fusion, attachment, polarisation, or survival of 0 osteoclasts.

According to a third embodiment of the invention, there is provided a method for the treatment of bone metabolic disease in a mammal requiring said treatment, which method includes or consists of administering to said mammal an effective amount of at least one agent which acts by inhibition of the production or action of a membrane S. associated protease or the matrix metalloprotease MMP-12 involved in the resorptive activity of osteoclasts.

According to a fourth embodiment of the invention, there is provided a method for the treatment of bone metabolic disease, which method includes or consists of administering to said mammal an effective amount of an agent mediating inhibition of the 'o recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, fusion, attachment, polarisation, or survival of osteoclasts.

According to a fifth embodiment of the invention, there is provided an agent which acts by inhibition of the production or action of a membrane associated protease or the matrix metalloprotease MMP-12 involved in the resorptive activity of osteoclasts, when i sed in the treatment of bone metabolic disease.

IR:\LIBA]03722.doc:mrr According to a sixth embodiment of the invention, there is provided an agent which i*hibits the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, ftision, attachment, polarisation, or survival of osteoclasts, when used for the treatment of bone metabolic disease.

The treatment may be for prevention or for cure of such diseases.

Preferably, the metalloproteinase is involved in the recruitment. proliferation, differentiation, or migration of osteoclast precursor cells or in the migration. fiision.

:imachment. polarisation, activity in removal of mineralised osseous substance, or survival Hl ofosteoclasts.

Though M/IT-MMP and MMP-12 produced by osteoclasts and osteoclast precursors is a major target for the inhibitory agent of the invention, the invention also includes regulation of bone metabolism by inhibition of non-osteoclastic proteinases which inIluences the life cycle of osteoclasts. Other bone cells such as osteoblasts and i chondrocytes are able to produce both latent and active forms of NIMMPs. cathepsins and plasminogen activator as well as natural inhibitors of some of these enzymes. These ciizymes might be important for the initial degradation of the bone surface exposing the inderlyin mineralised matrix to subsequent osteoclastic action (Delaisse Vaes 1992 and they might be involved in the degradation of collagen Fibres either released from the 2 bone by the action of osteoclasts or still remaining in the resorption pit after the osteoclast has left (Foged I al, 19966). Furthermore. latent pro-lormns of osteoblastic enzymes stored *i*b iii bone [R:\LIBA]03722.doc:mrr might be activated during osteoclast resorption. Finally, proteolytic enzymes of non-osteoclastic origin might have a chemotactic role in regulating the migration and maturation of osteoclasts.

The agent may be selectively inhibitory of MT1-MMP or MT-MMPs broadly, of MMP-12 or MMPs broadly, or of membraneassociated metalloproteinases or metalloproteinases broadly.

The agent may be an antibody selectively immunoreactive with an MT-MMP. Such an agent may alternatively be an I0 antisense oligo-nucleotide or oligo-nucleotide analogue directed aaainst a gene involved ir :he oroduction of an MT- MMP or an agent regulating MT-MMP activity. It mav be an MT-MMP substrate mimic inhibitor. It may be a broad spectrum matrix metalloproteinase (MMP) inhibitor or a broad spectrum membrane-associated metalloproteinase inhibitor.

It may also be a peptide, peptide analogue or other peptide S. mimicking agent obtained by screening an appropriate library for compounds reactive with an MT-MMP, an MMP or a membraneassociated metalloproteinase.

2 A preferred inhibitor provided by :he invention is the peptide S-K-Y-P-J-A-L-F-F-K (SEQ ID No.l) (J being the single letter code of hydroxyproline) and inhibitory variants thereof such as the peptide analogue S-K-Y (NO 2

P-J-

e:g A-L-F-F-K(Abz) (SEQ ID No.2).

25 In an alternative aspect, the invention includes the use of an agent in the manufacture of a medicament for the treatment of bone metabolic disease by inhibition of the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, fusion, attachment, polarisation, or death of osteoclasts.

Preferably, said agent produces said inhibition by inhibiting the production or action of a proteinase.

Also disclosed is an anti-bone resporption agent comprising a proteinase inhibitor active against a proteinase involved in bone resorption operatively linked to a ligand having binding specificity targeting the inhibitor to said proteinase or to the environment of the proteinase.

WO 98/04287 PCTIEP97/04110 8 The invention includes a new protease termed rabbit osteoclast MT1-MMP having the amino acid sequence given in Figure 1 and Figure 2, as well as an isolated nucleic acid coding for such a protein, e.g. one having the sequence set out in Figure 1. Proteins having high e.g. more than 75% eg more than 90% or 96% homology to the said rabbit osteoclast MT1-MMP are included also, as is human osteoclast MT1-MMP and isolated nucleic acid sequences encoding it.

The invention also includes a new protease termed rabbit osteoclast MMP-12 having the amino acid sequence given in Figure 4 and Figure 5, as well as an isolated nucleic acid coding for such a protein, e.g. one having the sequence set out in Figure 4. Human osteoclast MMP-12 and isolated nucleic acid sequences encoding it as well as other proteins and nucleic acid sequences with a high homology at least 50%, preferably at least 70, 80 or 90%) to rabbit osteoclast MMP-12 are also included in the invention.

Inhibition of proteolytic activity can be obtained in several ways and by several classes of agents. The inhibition could be direct, i.e. by an agent acting directly either on the proteinase in its active form(s) inhibiting its proteolytic activity or substrate recognition or on the latent form of the proteinase inhibiting its conversion into active proteinase. The most relevant directly acting inhibitors of proteinases include: 1. natural inhibitors which form specific complexes with an active proteinase and in some cases even with its latent pro-enzyme tissue inhibitors of metalloproteinases, TIMPs); 2. antibodies or antibody fragments which e.g. neutralise the active site or block the substrate recognition site; 3. synthetic pseudo-substrates which specifically interact at the catalytic site synthetic peptides linked to a chelating group) or the natural substrate recognition site; and WO 98/04287 PCT/EP97/04110 9 4. so-called entrapping reagents which are cleavable substrates which when cleaved undergo a conformational change which leads to entrapment of the proteinase a-macroglobulins).

The inhibition, however, could also be indirect i.e. by an agent regulating either the expression and/or production 'of the proteinase a natural transcription factor or its naturally regulating systemic or local factor, or a synthetic antisense probe specifically binding to and blocking the mRNA encoding the proteinase) or by an agent influencing the level or activity of a natural regulator of the proteinase an inhibitor of an enzyme responsible for catalytic activation of the target proteinase).

The development of many types of proteinase inhibitor is assisted by having the proteinase itself available. The production of proteinases may be performed either directly in cultures of isolated osteoclasts or indirectly by transfection of an expression plasmid containing proteinase encoding cDNA into a recipient cell line. For proteinase production in osteoclasts, the majority of e.g. MMP-9 is produced in its latent proform (pro-MMP-9) and therefore needs a subsequent activation process if the active form is required. The amount of proteinase obtained from production in osteoclast is severely restricted by: a) the non-proliferative nature of osteoclasts in culture and b) the technical difficulties in isolation of native osteoclasts in high numbers and purity.

For illustration, the production, purification and activation of osteoclastic pro-MMP-9 is described in Example 3-1. In contrast, both latent and active proteinase can be produced directly by recombinant techniques depending on whether the expression plasmid-transfected into the recipient cell is designed to contain the complete cDNA or a cDNA devoid of the region encoding the propeptide moiety of WO 98/04287 PCT/EP97/04110 the latent enzyme. Since active proteinases are generally less stable than their corresponding latent pro-enzymes and particularly under cell culture conditions might be degraded, production of latent proteinases is often preferable. For illustration, the identification and cloning of cDNA encoding several osteoclastic MMPs or parts thereof, including MMP-9, MMP-12 and MT1-MMP is described in Examples 1, 2 and 3-4.

Apart from natural regulators of metalloproteinase and particularly MMP production and activity, agents inhibiting metalloproteinases (including MMPs and especially MT-MMPs and MMP-12) involved in one or more phases of the osteoclast life cycle can include: 1. a substance which interacts at a specific site of the metalloproteinase or MMP thereby reducing its proteolytic activity to recognise a natural substrate, e.g. anti-MMP antibodies and fragments thereof as well as synthetic, peptide-mimicking proteinase inhibitors; 2. substances which influence the transcription or translation of metalloproteinase or MMP; 3. substances stimulating the level or activity of a natural inhibitor of metalloproteinase or MMP; and 4. substances reducing the level or activity of a natural activator of metalloproteinase or MMP, e.g. a substance analogous to the description in 1. and 2. but regulating a proteolytic enzyme responsible for activation of latent MMP.

Examples 5 and 6 below describe the development of inhibitory agents; the production and use of anti-proteinase antibodies (Example the production, identification and characterisation of synthetic, peptide-mimicking proteinase inhibitors (Example 6 and the design and use of antisense probes to proteinase mRNA (Example 6f).

Anti-proteinase antibodies are central tools for the development of proteinase inhibitors and under appropriate conditions can be used as inhibitors themselves (see Example WO 98/04287 PCT/EP97/04110 11 and Figure Thus, the applications for antiproteinase antibodies and parts thereof are several and in particular anti-MMP antibodies and antibody fragments will be useful: 1. In the production of recombinant MMP by use in immunoblotting or a similar immunodetection method for identification of clones expressing recombinant proteinases.

2. In affinity chromatographical purification of native or recombinant MMPs by immobilisation on activated resins produced for affinity columns such as e.g. divinyl sulfone agarose.

3. In immunoassays such as ELISA or RIA for quantitative determination of the specific MMP concentration in samples for diagnostic analysis e.g. tissue extracts, sera or urine samples, and in samples for research analysis e.g. cell culture medium.

4. In immunocytochemical identification of MMP-expression on the protein level by incubation with bone cells or tissue sections. As shown in Example 5, this can also lead to a demonstration of a particular cellular localisation of a MMP and thereby aid in the clarification of its biological role.

5. In the characterisation of MMP-activity by use as specific inhibitory agents. Antibodies have shown the highest specificity as MMP inhibitors in test tubes selectivity for a particular MMP and not others) and therefore will be important tools for characterisation of individual proteinases (Birkedal- Hansen et al, 19932). Especially, antibodies raised by immunisation with peptides mimicking a region comprising the catalytic site of a particular MMP could be expected to interfere with the proteolytic activity of this member but not other members of the MMP-family and thereby be of importance for the demonstration of the specific role of particular proteinase in bone metabolism.

WO 98/04287 PCT/EP97/041 12 6. In the manufacture of a medicament for the treatment of bone metabolic disease by use as direct MMP inhibitors or as constituents of hybrid MMP inhibitors. Two general principles for using anti-MMP antibodies or fragments thereof for treatment of bone metabolic disease are relevant: as direct inhibitors of proteinase activity or as site-directing agents merely assuring that another inhibitory agent is transported to the right target cell or tissue, e.g. by hybridisation on the protein or gene level of the antibody or a fragment thereof to a peptide-mimicking synthetic inhibitor. In both cases the use of antibodies in treatment of a bone metabolic disease requires its administration to animal or man in a proper pharmaceutical composition to avoid degradation and to ensure a beneficial effect.

Synthetic peptide and peptide-mimicking inhibitors of proteinases are promising agents for use for treatment of bone metabolic disease by inhibition of the action of proteinases involved in the recruitment, proliferation, differentiation, or migration or osteoclast precursor cells or in the migration, fusion, attachment, polarisation, removal of mineralised osseous substance, or death of osteoclasts. Several methods for production of peptide and peptide mimicking inhibitory agents are available, two of which are described in Example 6 One is based on a recently developed beaded polyethylene glycol cross-linked polyamide (PEGA) resin designed for peptide synthesis and with an open structure permitting biologically active proteins into the interior (Meldal et al 1 1994: Meldal Svendsen, 199512) The PEGA bead peptide library was developed for the complete characterisation of the specificity of proteinases in general and can be used for identification of first synthetic peptide substrates of osteoclast proteinases and subsequently inhibitors after a well-functioning substrate has been identified. In the first step of this procedure WO 98/04287 PCT/EP97/04110 13 millions of randomly synthesised fluorogenic peptides are screened for their ability to become hydrolysed during incubation with an osteoclast proteinase. The major purpose of this step is to identify a synthetic peptide substrate suitable for use in the second step of the procedure, i.e.

the identification of inhibitors of the same proteinase.

However, the identification of substrates might lead directly to inhibitory agents, since substrates with high affinity for the proteinase but little ability to become hydrolysed pseudo-substrates) can act as reversible inhibitors. In Example 6b, we report the finding of a peptide-mimicking molecule (CL-1) identified by incubation of MMP-9 with a PEGA bead substrate library, which has a low Km (3.4 M) but also a low kcat/Km (<500 M-'s suggesting its potential use as an inhibitor of osteoclastic MMP-9. Even better inhibitory characteristics of pseudo-substrates can be expected after modification of the originally identified substrates, e.g. either by linking peptide-mimicking substrates to chelating groups such as hydroxamates, thiols, phosphonamidates, phosphinates and phosphoramidates (reviewed by Birkedal-Hansen et al, 19932) or by designing pseudo-substrates which easily forms acyl-proteinase complexes but which hydrolyse slowly due to interaction with the binding site on the enzyme for the leaving group (Baggio et al 19961).

In the more regular cases where the identification of an appropriate synthetic substrate showing a low Km and a high Kcat/Km by incubation with the proteinase) is obtained either by the first step of the PEGA bead procedure or by simply being already commonly available, synthetic peptide inhibitors can be identified among millions of randomly designed peptides in a PEGA bead synthetic peptide inhibitor library (Meldal and Svendsen, 199512; Meldal et al, 199721) The screening is based on the rare ability of some peptides to inhibit the hydrolysis of the established synthetic peptide-mimicking substrate. Inhibitors of MMPs, MT-MMPs and membrane-associated metalloproteinases can be found by this method also.

WO 98/04287 PCT/EP97/04110 14 A novel modification of the original PEGA bead inhibitor technology was developed in order to optimise the synthesis of MMP inhibitors. It has previously been shown (Galardy et al, 199219) that substituting the cleavable peptide bond in a peptide substrate of fibroblast collagenase by a phosphorus-containing bond a phosphinate

(-PO

2 phosphonamidate (-P0 2 or phosphonate bond can cause inhibition of the proteolytic activity. For the first time, this knowledge has been used in combination with the PEGA bead technology by extending the group of building blocks used for synthesis of putative inhibitory peptide analogues on the PEGA-beads from just natural amino acids (including hydroxyproline) and their corresponding D-forms to also including pseudo dipeptides such as NH 2 -PlP/CPl'-COOH,

NH

2 -P1P/NPl'-COOH or NH 2 P1i/c'?'-COOH, where the two normal amino acids (P1 and PI') instead of being linked through the peptide bond are linked through the phosphinate, phosphonamidate or phosphonate bond P/N or P/ 0 This allows the synthesis of random PEGAbead inhibitor libraries with a structure such as: X1-X2- P1P/?1'-X3-X4-"linker"-PEGA, where X1 to X4 are natural amino acids and P 1 P/CP1 is a phosphinate pseudo dipeptide (as described in Example 6c and Figures 12-15).

By employment of the PEGA-bead substrate library technology, it has been possible to identify peptide sequences which are of use in the design of novel highly specific MMP-substrates (see Example 6 a and These substrates facilitate the design and use of PEGA-bead inhibitor libraries both through the use of one of these selective substrates in the library and through the use of the substrate sequence data for the design of the structure of the randomised inhibitors in the library (Meldal and Svendsen, 199512; Meldal et al, 199721). Particularly in the design of PEGA-bead inhibitor libraries based on inhibitors with a phosphorous containing bond, the substrate data were used for determination of the two amino acid R-groups around the phosphinate, phosphonamidate or phosphonate of the pseudo dipeptide (see Example 6c). Furthermore, the design WO 98/04287 PCT/EP97/04110 of selective inhibitors based on the characteristics of the novel MMP-substrate specificities will be facilitated (see data for CL-1, CL-21, CL-25 and CL-29 in Example 6b).

Finally, the specific substrates could become important tools for selective detection and quantification of MMPs in tissue samples in diagnosis and research.

The other method for identification of peptide and peptide mimicking inhibitory agents is based on the use of positional combinatorial peptide inhibitor libraries. A few members of these libraries of randomly synthesised peptides having in a single amino acid position an abnormal amino acid, such as a D-amino acid instead of an L-amino acid, in some case will act in an inhibitory way to a particular enzyme, probably due to a pseudo-substrate effect. If an inhibitory signal is obtained by incubation of a positional combinatorial peptide inhibitor library with a proteinase or a biological model system including essential proteinase activity, the peptide(s) in the library responsible for this inhibition must be subsequently identified by systematic segmentation of the library as described in Example 6 (d-e) for incubation of positional combinatorial peptide inhibitor libraries with murine foetal metatarsal cultures. Some preferred inhibitory libraries and peptide structures provided by the invention are the libraries X-X-w-X-X, X-X- 1-X-X and X-X-w-Y-X and the peptides C-L-w-Y-L, C-L-w-Y-M, C-Y-w-Y-L, V-Y-w-Y-M and L-F-w-Y-L, where X are natural amino acids including hydroxyproline, and w and 1 are Dtryptophan and D-leucine, respectively (see Example 6e).

Comparing the two methods, the major advantage and disadvantage of the PEGA bead library are the immediate identification of inhibitors and the need for incubation with a preferably purified proteinase preparation in a test tube, respectively. The major advantage and disadvantage of the positional combinatorial peptide inhibitor library is the possibility to screen directly for an inhibitory effect in a biological test system and the need for several cumbersome segmentations of the initial library to identify the agent originally causing the inhibition, respectively.

WO 98/04287 PCT/EP97/04110 16 Finally, one feature of the positional combinatorial peptide inhibitor library can be seen as both favourable and nonfavourable, since the functional background for an inhibitory response induced in the biological system by this type of library is uncertain i.e. the inhibitory peptides might not be proteinase inhibitors but have other regulatory functions.

A review by Eggleston and Mutter of methods for producing inhibitors mimicking inhibiting peptides appears in "Chemistry in Britain" May 1996, pages 39-4118. The techniques reviewed may be applied to peptides identified by the methods discussed above.

The benefits of using antisense probes to proteinases can be divided into two major aspects, an early aspect and a later aspect. The antisense probes are important tools for evaluation of the role of the corresponding proteinase in a biological process, because they can be used at an early stage of a study when anything else than the oligonucleotide sequence of this proteinase is unknown, and this even with usually high specificities i.e. with only a minor risk of cross-reaction to other proteinases if the design of the antisense probe and the experimental conditions are appropriate. Antisense probes were used successfully for inhibition of MMP synthesis by fibroblasts (Lin et al, 19959), and interfered with the proton pump activity of osteoclasts when assessed in both cell and tissue cultures (Laitala and Vaananen, 19948). Another major aspect of using antisense probes is their possible application in the treatment of diseases caused by over-expression of particular genes. For specific reduction of proteinase levels, gene therapeutic use of antisense probes to MMPs may be expected to be effective.

The identification of an antibody-derived or synthetic peptide-mimicking inhibitor of an osteoclast proteinase may be followed by appropriate modification of this compound to assure its use as a medicament for the treatment of bone metabolic disease. Several characteristics are necessary, particularly sufficient uptake and stability in the living WO98/04287 PCT/EP97/04110 17 organism to assure a beneficial effect, sufficient tissue or cell specific action to assure maximal effects at the target site of the organism relative to effects at non-target sites including acceptable levels of side effects, and a pharmacologically acceptable dose- and time-response to the treatment.

Administration of proteins, peptides and peptide-like substances to animals and humans requires protective routes of administration and/or protective formulation of the peptide in order to avoid degradation of the compound.

Though protective encapsulation for oral administration of peptides and peptide-like agents is a technology currently undergoing significant improvement, stabilisation of the agent itself prior to administration is advantageous. For peptide-mimicking MMP-inhibitors this has been possible by chemical modification of an initially identified compound apparently without important changes in its inhibitory capacity (Brown Giovazzi, 19954 and P. D. Brown personal communications June 1996).

Targeting of a proteinase inhibitor to e.g. osteoclasts and osteoclast precursors, can be obtained by two general means. One, is if the inhibitor due to its intrinsic specificity selectively reacts with the proteinase present on these cells either because the proteinase at this target cell is particularly available to the inhibitor (due to e.g.

the localisation of the cell, the localisation of the proteinase in the cell or simply by a local high concentration of the proteinase) or because the proteinase when produced by these cells is different from the corresponding proteinase as it is expressed in other cells and tissues (due to e.g. post-translational modifications).

The other way to obtain a specificity is by making hybrid molecules or conjugates combining one part of the agent having proteinase-inhibitory characteristics with another part having antibody or ligand specificity for the particular cells or tissue. These hybrids can be made by recombinant expression of fusion-proteins after cloning of a hybrid cDNA. E.g. a piece of cDNA encoding the osteoclast- WO 98/04287 PCT/EP97/04110 18 specific ligand calcitonin (or a receptor-binding part thereof) can be ligated to another piece of cDNA encoding a peptide inhibitor for an osteoclast proteinase. Hybrids can also be conjugates of two compounds e.g. by chemically linking an amino-bisphosphonate, which has high affinity for hydroxyapatite in bone, or an antibody specific for a component exposed in the osteoclast membrane, such as the calcitonin receptor with a peptide or peptide-mimicking proteinase inhibitor.

The invention will be further described and illustrated with reference to the examples which follow and the appended drawings in which: Figure 1 shows the nucleotide (SEQ ID No.3) and deduced amino acid sequence (SEQ ID No.4) of the MT1-MMP or MT1-MMP analogue identified in rabbit osteoclasts; Figure 2 shows a comparison between the amino acid sequence of the novel MT-MMP identified in rabbit osteoclasts (Rabbit) (SEQ ID No.4) and the previously reported amino acid sequences of Human (SEQ ID.No.5), Rat (SEQ ID No.6) and Mouse MT1-MMP (SEQ ID No. Positions with an amino acid identical in all 4 proteins are indicated Figure 3 shows schematically the structure of three MT1-MMP cDNA constructs and the corresponding control construct used in Example 3-2; Figure 4 shows the nucleotide (SEQ ID No.8) and deduced amino acid sequence (SEQ ID No.9) of the MMP-12 or MMP-12 analogue identified in rabbit osteoclasts; Figure 5 shows a comparison between the amino acid sequence of the novel MMP-12 identified in rabbit osteoclasts (Rabbit) (SEQ ID No.9) and the previously reported amino acid sequences of Human (SEQ ID No.10), Rat (SEQ ID No. 11) and Mouse MMP-12 (SEQ ID No.12). Positions with an amino acid identical in all 4 proteins are indicated WO 98/04287 PCT/EP97/04ll 0 19 Figure 6 shows schematically the structure of a MMP-12 cDNA construct and the corresponding control construct used in Example 3-4; Figure 7 shows the effect of various proteinase inhibitors on the migration of purified osteoclasts through collagen coated membranes. The values are relative to the number of migrations observed in the absence of proteinase inhibitor.

Figure 8 shows the effect of an MMP-inhibitor on pit formation by purified osteoclast seeded on dentine slices which were either not coated or coated with collagen. The values are relative to pit formation in the absence of collagen coating and MMP-inhibitor; Figure 9 shows the dose dependent inhibitory effect on MMP-9 proteolytic activity of sera from mice immunised with the conjugated femta-peptide RSGAPVDQMFPGVPL (SEQ ID No.13) (peptide B, mimicking a region of the rabbit MMP-9 hemopexin domain) alone or together with purified intact rabbit osteoclast pro-MMP-9. No inhibitory effect was observed for sera from non-immunised mice and for mice immunised with another non-related femta-peptide (peptide A) The values are relative to the average relative fluorescence generated during 30 minutes of incubation of the synthetic quenched fluorogenic substrate Mca-PLGL-Dpa-AR-NH 2 (Bachem) (SEQ ID No. 14) with a pre-incubated mixture of purified activated MMP-9 and the appropriate dilutions of 9 different control sera (non-immunised or immunised with non-relevant femtapeptides); Figure 10 shows the relationship between the initial velocity of enzymatic hydrolysis and the substrate concentration determined by continuous fluorometric assay of MMP-9 or subtilisin with either MR2: Abz-G-P-L-G-L-Lnor-A-R-Y(NO2)NH2) (SEQ ID No.15) or CL1: Abz-S-K-Y-P-J-A-L-F-Y(NO 2 (SEQ ID No.16). Assays were WO 98/04287 PCT/EP97/04110 performed at 370C, pH 7.5 and fluorescence read at Xex 320 nm and kem 425 nm. Peptide origin and kinetic parameters are reported in Table 1; Figure 11 shows inhibition of hydrolysis of CL1 by the MMP-inhibitor RP59794, but not the cysteine proteinase inhibitor E-64. MMP-9 (80 pmol) or subtilisin (3.4 pmol) were pre-incubated with either RP59794 or E-64 in a total volume of 40 il for 5 min at 370C. Subsequently, 1 ml of 2.8 tM CL-1 was added and the incubation continued for 2 to hrs. Inhibitor is listed in final concentrations; Figure 12 shows the synthesis of the phosphinate analogue to hydroxyproline for use as a building block in the subsequent generation of a hydroxyproline-methionine phosphinate pseudo dipeptide (see also Figure 13). The phosphinic acid analogue to trans-hydroxyproline is synthesised from potassium D- or L-erythronate. After bromination at the 2 and 4 position the acid is transformed into the methyl ester by methanol quenching. The 2-position is reduced and the ester converted into the alcohol by sodium borohydride reduction.

The primary alcohol is oxidized by sodium hypochlorite to the aldehyde and condensed with tritylamine. The imine formed is reacted with bis-trimethylsilyloxyphosphine to yield the phosphinate. Upon acid hydrolysis and intramolecular substitution of the bromine the free hydroxyproline is obtained; Figure 13 shows the synthesis of the hydroxyprolinemethionine phosphinate pseudo dipeptide for use in preparation of the PEGA bead phosphinate inhibitor library IIa (see Example 6c). The phosphinic acid analogue of hydroxyproline (see Figure 12) is derivatised with benzyloxycarbonyl chloride. 2-methylene-4-methyl mercaptobutanoic acid ethyl ester was synthesised from diethylmalonate sodiation and reaction with methyl mercaptoethyl chloride followed by selective basic ester hydrolysis, acid decarboxylation and reaction with WO 98/04287 PCT/EP97/04110 21 formaldehyde in the presence of piperidine. These reactions can be performed on a large scale. Reaction with the phosphinic acid analogue of hydroxyproline gives the dipeptide isosteric phosphinate. The phosphinate is protected by reaction with adamantylbromide followed by ester hydrolysis with sodium hydroxide. The Cbz group is cleaved hydrogenolytically and the free amine protected by reaction with FmocCl and sodium carbonate; Figure 14 shows the synthesis of the glycine-leucine phosphinate pseudo dipeptide for use in preparation of the PEGA bead phosphinate inhibitor library IIb (see Example 6c). The phosphinic acid analogue of glycine is synthesised from tritylamine and formaldehyde to give the imine which is reacted with bis-trimethylsilyloxyphosphine obtained from ammoniumphosphinate and hexamethyl disilazane. The product is deprotected by acid hydrolysis and is derivatised with benzyloxycarbonyl chloride. 2- Methylene-4-methyl pentanoic acid ethyl ester was synthesised from diethylmalonate sodiation and reaction with isobutylbromide followed by selective basic ester hydrolysis, acid decarboxylation and reaction with formaldehyde in the presence of piperidine. Reaction with the phosphinic acid analogue of glycine gives the dipeptide isosteric phosphinate. The phosphinate is protected by reaction with adamantylbromide followed by ester hydrolysis with sodium hydroxide. The Cbz group is cleaved hydrogenolytically and the free amine protected by reaction with FmocCl and sodium carbonate; Figure 15 shows the development and structure of the PEGA bead phosphinate inhibitor library (IIa) based on the hydroxyproline-methionine phosphinate pseudo dipeptide. The invariable quenched fluorescent substrate (here: Ac-

Y(NO

2 )PLJMKGK(Abz)G-"Linker"-) (SEQ ID No.17) and the randomly variable phosphinate inhibitor (here: XiX 2

JP/CMX

3

X

4 "Linker"-) are independently associated to the PEGA bead.

Alternatively an FmocLys(Aloc) residue can be used to WO 98/04287 22 PCT/EP97/04110 obtain orthogonal protection and incorporation of the two compounds and the order of synthesis of the library and the substrate may be reversed. This gives the possibility to use the same library with several substrates. The analogous library (IIb) was prepared similarly by using an invariable substrate corresponding to MR1 (see Table 3) and a randomly variable phosphinate inhibitor XlX 2

GP/CLX

3

X

4 -"Linker"-; Figure 16 shows inhibition of the 4Ca2+-release from foetal murine metatarsals cultured for 4 days in the presence of positional combinatorial pentapeptide inhibitor libraries.

The results for 5 selected libraries with the sequence X-X- D-X-X are shown. In these 5 cases D was either D-isoleucine, D-leucine, D-lysine, D-serine or D-tryptophan, and X were randomly varying L-amino acids. In contrast to the libraries with D-lys and D-ser, the pentapeptide libraries with a D-ile, D-leu or D-trp at the third position induced a significant reduction of bone resorption. The MMP-inhibitor RP59794 was included as a positive control.

Example 1 Isolation of cDNA encoding fragments of osteoclastic proteinases.

The use in PCR of degenerate nucleotide primer sets (designed from existing data describing the amino acid sequences of proteinases) for cloning of osteoclastic proteinases was exemplified by the studies described below leading to the identification of MMP-9, MMP-12 and MT1-MMP mRNA in rabbit osteoclasts: a. Isolation and purification of osteoclasts Osteoclasts were isolated from 10-day-old rabbits (125- 150 g) according to a method described previously (Tezuka et al, 19921) but with some modifications. Briefly, bone cells were released from marrow-depleted long bones and shoulder blades by mincing and mechanical agitation. A preparation WO 98/04287 PCT/EP97/04110 23 of unfractionated bone cells rich in osteoclasts was isolated by centrifugation (30 x g, 5 min) and seeded into tissue culture dishes. After a settling period of minutes, non-adhering cells were removed, and cultivation continued for 20 hrs at 37 0 C and 5 to 7.5% CO 2 in a-MEM (pH 7.3) supplemented with 5% foetal calf serum. The cells were washed with PBS and then treated with 0.001% pronase E and 0.02% EDTA for approximately 10 min. to release all nonosteoclastic cells. The purified osteoclasts were cultured for another 2 hrs before isolation of mRNA.

b. Amplification of MMP cDNA fragments by PCR, molecular cloning and homology analyses To identify possible MMP gene expression by rabbit osteoclasts, cDNA reverse-transcribed from mRNA from the purified osteoclasts was subjected to PCR with degenerate primers designed from conserved regions of MMP genes.

Briefly, the poly(A)+RNA from purified osteoclasts was prepared using a mRNA purification kit (Pharmacia Biotech, Uppsala, Sweden); single strand cDNA was synthesised from mRNA by use of a cDNA synthesis kit (Pharmacia); and aliquots of the synthesised cDNA were amplified by PCR with degenerate primers corresponding to the conserved amino-acid sequences in either the cysteine switch region (PRCGVPD (SEQ ID No.18)) or the region resembling a cleavage site for furin (RRKRYA (SEQ ID No.19)) in combination with the catalytic domain (GDXHFDXXE (SEQ ID No.20), where X is a variable amino acid) present in most members of the MMPfamily. The PCR reactions were cycled 45 times through the following steps: 1 min at 940C, 1 at 55 0 C, 1 min at 74 0

C.

Three cDNA bands 330-340, 380-390 bp and 560-570 bp in length were identified by electrophoresis in a 1% agarose gel. The cDNAs were purified and cloned into a pCRII vector (Invitrogen, San Diego, CA) according to the instruction manual and subsequently characterised by nucleotide sequencing.

WO 98/04287 PCT/EP97/04110 24 The high expression of MMP-9 mRNA by rabbit osteoclasts is well-known and from previous characterisation of the MMP- 9 gene the expected size of MMP-9 cDNA fragments amplified with degenerate primers used in this PCR would be 336 bp.

Our cloning and subsequent nucleotide sequencing confirmed that the isolated 330-340 bp cDNA originated from MMP-9.

The cloning of isolated 560-570 bp cDNA, resulted in a clone, B4 with a length of 567 bp which by nucleotide sequencing was found to share more than 80% similarity with a segment of the human metalloelastase (MMP-12) gene. The presence of mRNA encoding MMP-12 has previously been preliminarily identified in rabbit osteoclasts by partially sequencing randomly chosen cDNAs of an osteoclast cDNA library (Sakai et al, 199512) (see also Example 3-4).

The cloning of isolated 380-390 bp cDNA, resulted in another clone, A3 with a length of 387 bp, which shared more than 90% similarity with the human MT1-MMP cDNA sequence previously reported in cancer cells (Sato et al, 199413).

Since neither MT-MMPs nor any other membrane-associated proteinases have been previously identified in osteoclasts, the remaining part of this example as well as Example 2 describes studies of A3 and MT-MMP in osteoclasts.

c. Isolation of MTI-MMP cDNA from an osteoclast cDNA library A rabbit cDNA library (Tezuka et al, 199415) was screened by colony hybridisation, using the random-primed 32P-labelled PCR product of A3 as a probe. By screening 1x10 5 clones, one positive clone was identified and made into the plasmid form according to the instruction manual (Stratagene, lambda ZAP vector). This positive clone contained a cDNA insert of 1,842 bp which was isolated and sequenced. An open reading frame consisting of 1716 bp initiated with an ATG codon at nucleotide position 127 was found. According to gene bank searches, an identical nucleotide sequence did not exist and the highest similarity was 91% to the human MT1-MMP gene. Figure 1 shows the nucleotide sequence of the cloned insert. The deduced WO 98/04287 PCT/E1PQ7/fA11 n amino-acid sequence of the insert showed 96% similarity with human MT1-MMP (Figure There were no additions or deletions of specific sequences when compared to MT1-MMP of other species. Based on further comparisons of amino acid sequences of other MMPs, we concluded that the isolated novel cDNA encoded the rabbit homologue of MT1-MMP or of a closely related but previously unreported human osteoclast

MT-MMP.

d. Nucleotide sequence analysis The nucleotide sequence analysis of the A3 PCR fragment and of the rabbit MT1-MMP cDNA clone from the cDNA library was determined from both strands by the dideoxy chaintermination method using the Qiagen-purified plasmid DNA (Qiagen, USA), the Sequenase kit USA), and either pBluescript SK primers (Stratagene, USA) or synthetic oligonucleotide primers.

Example 2 Identification of MT1-MMP in osteoclasts.

The novel identification of MT1-MMP in osteoclasts was further substantiated by the studies described in the following examples: a. Cells and organs for RNA preparation Brain, kidney, liver, lung, calvaria, spleen and alveolar macrophages were isolated from 10-day-old rabbit.

Bone stromal cells were obtained from a culture of unfractionated rabbit bone cells (Tezuka et al, 199215) in alpha-MEM containing 10% FBS until confluence, and then subcultured 4 times. In all cases total RNA was prepared as reported previously (Tezuka et al, 199215) b. Northern blotting To investigate the mRNA expression of MT1-MMP in purified osteoclasts and to compare its level with that in WO 98/04287 PCT/EP97/04110 26 other tissues and cells, we performed Northern blotting.

Five micrograms of total RNA isolated from various organs and cells were blotted on nylon membranes after formaldehyde agarose gel electrophoresis, and hybridised with radioactive probes. The A3 PCR fragment and a fragment of human MT1-MMP cDNA (position 1647-2880, Sato et al, 199413) as well as (for quantitative normalisation) a synthetic oligonucleotide corresponding to 28 S ribosomal RNA were used as probes.

The cDNA probes were radiolabelled with a multiprime DNA labelling system (Amersham International plc., Buckinghamshire, England) using [alpha- 32 P]dCTP and the oligonucleotide probe was radiolabelled with a labelling kit (Amersham) using [gamma-32 P]ATP. Hybridisation was performed as described previously (Tezuka et al, 199215) and visualised by a Phosphorimager SF (Stratagene, La Jolla, CA). For both MT1-MMP probes, we found the same pattern of distribution-. as those reported previously for adult human tissues (Takino et al, 199514; Will and Hinzmann, 199517), and in addition a prominent expression of MT1-MMP in purified osteoclasts. It was noteworthy that expression was not detectable in liver and brain and low expressions were found in bone stromal cells and alveolar macrophages.

c. In situ hybridisation The expression of MT1-MMP in osteoclasts in vivo was examined by in situ hybridisation on sections of rabbit metatarsals. Consecutive paraffin sections of metacarpal bones of new-born rabbits were prepared as previously described (Blavier and Delaisse, 19953) A fragment of rabbit MT1-MMP cDNA (position 1-318, corresponding to 126 nucleotides in the non-coding 5'-region and 192 in the region encoding the N-terminal part of MT1-MMP) was used for probe synthesis. Digoxygenin-labelled antisense or sense RNA probes were prepared by use of a DIG RNA labelling kit (Boehringer Mannheim) according to the instruction manual and compared to paraffin sections stained for tartrateresistant acid phosphatase (Blavier and Delaiss6, 19953).

Many tartrate-resistant acid phosphatase-positive multi- WO 98/04287 PCT/EP97/04110 27 nucleated cells were positive for MT1-MMP, whether they were attached to calcified cartilage or to bone.

d. Immunocytochemistry An important property of the MT1-MMP in previous investigated non-osteoclastic cells is its localisation in their plasma membrane. The expression of MT1-MMP at the protein level and its cellular localisation in osteoclasts was investigated by immunocytochemistry. Unfractionated 1C rabbit bone cells were seeded on glass coverslips. After hr cultivation the non-adherent cells were discarded and the remaining cells were cultured for 1 to 18 hr, fixed and processed for immunocytochemistry. They were incubated for min in the presence of 1-3 (g/ml of the monoclonal MT1- SMMP antibody 113-5BT (Fuji Chemical Industries, Ltd.

Takoaka, Japan). This antibody was raised against a synthetic peptide corresponding to an amino acid sequence (CDGNFDTVAMLRGEM) (SEQ ID No.21) which differs by 1 amino acid from the corresponding rabbit sequence (V in rabbit instead of M in human at position 10). Rhodamine-labelled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) was used as secondary antibody at 200 times dilution. When incubating osteoclasts with an antibody against MT1-MMP we found fluorescence at specific points of its plasma membrane. Fluorescence did not appear when the MT1-MMP antibody was replaced by non-immune IgG.

All bright signals were in the focal plane where the cells were seen in contact with their substrate. In moving cells, mainly the extremities of the lamellopodia were illuminated.

In spread cells, the signals were arranged in a ring of small dots at the cell periphery. This pattern is reminiscent of podosomes. These are small extensions of the plasma membrane, that become abundant and organise in this particular way when the osteoclast is attaching. To investigate whether MT1-MMP is associated to podosomes, we stained the cell simultaneously for actin by addition of mg/ml fluorescein-labelled phalloidin (Sigma, Saint Louis, MO) during the incubation with the secondary antibody.

WO 98/04287 PCT/EP97/04110 28 Actin staining which is widely used to identify podosomes revealed the same ring of bright dots as shown with the anti MT1-MMP antibody. Therefore MT1-MMP appears to be localised on the podosomes. MT1-MMP staining was however somewhat more diffuse as compared to the sharp actin staining, probably because the sharp actin dots are due to bundles of actin filaments in the core of the podosome and oriented perpendicularly to the attachment surface, while MT1-MMP might be on the surface of the podosome. As expected, staining for actin illuminated also the extremities of the lamellopodia, as did the anti-MT1-MMP antibody. Similar localisations of MT-MMP were found when the osteoclast was cultured on bone slices. Thus these observations do not only demonstrate the presence on the protein level of MT1- MMP in the plasma membrane of the osteoclast, but provide new information on where exactly on the plasma membrane MT1- MMP is localised, i.e. at the level of lamellopodia and of podosomes.

Example 3 3-1 Production, purification and activation of osteoclast proteinases.

As noted in the summary of the invention, the production of osteoclast proteinases can be performed in cultures of osteoclasts or in cell lines transfected with cDNA encoding the osteoclast proteinase or a part thereof.

In all cases a purification of the product is needed and in those cases where the production leads to a latent pro-form of the proteinase a subsequent activation is also needed for some purposes. Exemplifying this process, the production, purification and activation of osteoclastic pro-MMP-9 was performed according to the following descriptions: WO 98/04287 PCT/EP97/04110 29 a. Osteoclast production of pro-MMP-9 When cultured at 370C and 5% CO2, under serum-free conditions to avoid contamination with serum-derived proteinases and natural inhibitors of proteinases, rabbit osteoclasts secreted 92 kDa pro-MMP-9 into the culture medium. According to studies by gelatinase-zymography, addition of 40 nM of phorbol 12-myristate 13-acetate

(PMA)

to the cell culture increased the yield of pro-MMP-9 at least 3-fold.

b. Purification of osteoclastic pro-MMP-9 The osteoclast conditioned medium was concentrated by kDa cut-off filtration (Amicon) and subsequently diluted in 2.5 mM sodium phosphate containing 0.04% Triton X-100 before application to an affinity column comprising hydroxyapatite (Bio-Rad, Hercules, CA). By this novel method for purification of MMPs, pro-gelatinases including pro-MMP-9 and pro-MMP-2 were observed to bind efficiently to the hydroxyapatite column. However, pro-MMP-9 was eluted from the column already by increasing the phosphate concentration to 5-10 mM, whereas higher concentrations (above 20 mM) of phosphate were needed to elute other progelatinases and gelatinases from the column.

c. Activation of osteoclastic pro-MMP-9 The purified latent pro-MMP-9 was activated either by a traditional method based on incubation with 1 mM (4-aminophenyl)mercuric acetate (APMA) for 2-8 hrs at 370C or by a method based on the activation of gelatinases as it is observed during analytical zymography. In the latter method the purified pro-MMP-9 was run into a slab gel by preparative SDS-PAGE. The SDS was substituted by Triton X- 100 during subsequent incubation of the gel for 16 hrs in a buffer containing 50 mM Tris-HC1, pH 7.5, 5 mM CaCl2, 1 4M ZnCl 2 and 1% Triton X-100. A part of the gel corresponding to an electrophoretic migration distance of compounds with an approximate molecular weight of 92±5 kDa (but including the by now activated approximate 68 kDa form of MMP-9) was WO 98/04287 PCT/EP97/04110 excised. The active MMP-9 was electrophoretically eluted from the excised gel.

3-2 Expression and characterisation of MT1-MMP fusion proteins The MT1-MMP cDNA fragment encoding amino acid residues Gln 40 -Glu 5 31 Ecl (containing the propeptide, catalytic, hinge and hemopexin, but not the signal peptide, transmembrane and cytoplasmic domains of rabbit osteoclast MT1-MMP, see Fig.

was PCR amplified using a 5'primer with an extra SnaBI site and a 3'primer with an extra NotI site. This fragment was inserted between the SmaI and NotI sites of the pGEX-6P- 2 vector (Pharmacia). The MT1-MMP cDNA fragments encoding 40 322 amino acid residues Gln -Asn 2 Ec2 (containing the propeptide, catalytic, and hinge, but not the signal peptide, hemopexin, transmembrane and cytoplasmic domains of rabbit osteoclast MT1-MMP, see Fig. 3) and Gln °-Leu282, Ec3 (containing the propeptide and catalytic but not the signal peptide, hinge, hemopexin, transmembrane and cytoplasmic domains of rabbit osteoclast MT1-MMP, see Fig. 3) were PCR amplified using 5'primers with an extra BamHI site and 3'primers with an extra XhoI site. These fragments were inserted between the BamHI and XhoI sites of pGEX-6P-2 vector (Pharmacia). The three corresponding constructs were used to express glutathione S-transferase (GST) fusion proteins in E.coli BL21 (Pharmacia).

Four overnight cultures of E.coli BL21 transformed with the three PGEX-MT1-MMP expression vectors and the PGEX vector alone (without any insert), were diluted 1:100 in 500 ml 2X YTA medium (Pharmacia). The cultures were grown at 37 0 C to an OD600= 1.0 before adding isopropyl-P-Dthiogalactopyranoside (IPTG) to a final concentration of 0.1 mM to induce expression. After induction for 3.5 hours at 30 0 C, the cells were pelleted and resuspended in 25 ml of ice-cold 1X PBS. All subsequent steps were carried out at 4 0 C or on ice. E.coli cells were lysed by sonication bursts of 10 seconds/burst). Cellular debris was pelleted WO 98/04287 PCT/EP97/04110 31 by centrifugation at 3000 rpm after incubation with 1% Triton X-100 for 30 minutes.

The purifications were carried out by affinity chromatography using Glutathione Sepharose 4B contained in the GST Purifications Modules, according to the manufacturer's instructions (Pharmacia). The supernatants obtained after the centrifugation of the sonicated samples were absorbed on 1 ml of the 50% slurry of Glutathione Sepharose 4B equilibrated with PBS by incubation at room temperature for 30 minutes. After washing several times with 1X PBS, the fusion proteins were eluted with 900 ul of Glutathione Elution buffer (10 mM reduced glutathione in mM Tris-HC1, pH The eluates were stored at -20 0 C until use.

The three fusion proteins migrated in SDS-PAGE as proteins of approx. 85, 60 and 55 kDa corresponding to their cDNA-deduced sizes of 87, 61 and 57 kDa, respectively. The fusion proteins were confirmed to be GST-MT1-MMP fusion proteins by Western-blotting using an anti-GST antibody reacting with all three proteins and an antibody to the hemopexin domain of MT1-MMP reacting with the large but not the two smaller proteins. Finally, amino acid sequencing of their propeptide domains further demonstrated that these.

proteins were truncated forms of MT1-MMP.

3-3 Proteolytic activity of GST-MT1-MMP fusion proteins after activation by trypsin or plasmin In order to obtain truncated MT1-MMP in active form, Ecl, Ec2 and Ec3 were incubated with trypsin or plasmin leading to removal of the GST-part and the propeptide domain of the fusion proteins.

a. Trypsin activation Eighty pl (20 pg approximately) of the eluted Ecl, Ec2, Ec3 and the GST tag alone were incubated at 25 0 C with 5 pg/ml trypsin (Promega) for 15-60 min in a final volume of 100 pl.

The reactions were stopped by the addition of 50 pg/ml SBTI.

WO 98/04287 PCT/EP97/04110 32 b. Plasmin activation Twenty-five pi (7 pg approximately) of the eluted Ecl, Ec2, Ec3 and the GST tag alone, were incubated with 2.7 pmol of human plasmin (Boehringer) at 25 0 C for 30 minutes in a final volume of 45 pl. The reactions were stopped by the .addition of 10 pM aprotinin.

c. Enzymatic assay The proteolytic activities were evaluated by fluorescence measurements (excitation wavelength: 320 nm, emission wavelength: 387 nm) of the hydrolysis of the quenched fluorescent peptide substrate Mca-PLGL-Dpa-AR-NH 2 (Bachem) (SEQ ID No.14) after incubation at 37 0 C for 180 minutes in 150 mM NaC1, 10 mM CaC12, 0.05 Brij-35 in mM Tris-HCl, pH 7.5 (see Table 1) d. Effect of inhibitors of MMPs Samples treated either with trypsin or plasmin in the conditions described above were preincubated for 30 minutes at 37 0 C in the absence or presence of the endogenous MMP inhibitors TIMP-1 (16.7 pg/ml) or TIMP-2 (16.7 pg/ml) or the synthetic MMP-inhibitor BB-94 (0.8x10 5 M, British Biotech).

The hydrolysis of the fluorescent substrate was evaluated afterwards as described above (See Table 1).

WO 98/04287 PCT/EP97/04110 33 Table 1. Hydrolysis in relative fluorescence units (RFUs) per 180 min of a synthetic substrate in the presence or absence of MMPinhibitors by truncated forms of recombinant osteoclast MT1-MMP activated by trypsin or plasmin.

RFU/ Trypsin activated Plasmin activated 180 min TIMP-1 TIMP-2 BB94 BB94 Inhibitor Inhibitor Ecl 139.4 ND ND 6.7 27.5 2.9 Ec2 172.4 148 7.1 6.0 104 3.2 Ec3 9.6 ND ND 6.9 4.1 3.8 pGEX 8.6 ND ND 6.8 3.8 ND: not done 3-4 The cloning, recombinant expression, activation and characterisation of rabbit osteoclast MMP-12.

Due to the expression and use in cell invasion of MMP- 12 in macrophages as well as the common hematopoieitic stem cell origin of osteoclasts and macrophages, we investigated whether MMP-12 was also expressed in osteoclasts. As indicated in Example lb and shown in the present example, this was indeed the case, and we therefore expect that MMP- 12 plays a similar role in osteoclast invasion and migration as it does in macrophages.

The isolation and sequencing of MMP-12 cDNA from the rabbit osteoclast cDNA library, and the subsequent steps of expression, characterisation and recombinant production of the MMP-12 fusion protein was done essentially as described for MT1-MMP cDNA (see Examples 1, 3-2 and Briefly, the osteoclast preparations were obtained from rabbit long bones and the reverse transcribed mRNA from these osteoclasts was amplified by PCR using degenerate primers based on regions conserved in the MMP family (see Example Ib). Among several PCR fragments of the predicted sizes, one (B4) presented WO 98/04287 PCT/EP97/04110 34 homology with a sequence of human MMP-12. When a randomprimed 32P-labelled probe based on the PCR product of clone B4 was used to screen a cDNA library of rabbit osteoclasts several positive clones were identified. One of these contained a cDNA insert of 1,792 bp including an open reading frame encoding a polypeptide of 464 amino acids sharing 74 66 and 65 identity to human, rat and mouse MMP-12, respectively (see Figures 4 and Based on this and further comparisons to other available protein sequences, we concluded that the isolated novel cDNA encoded the rabbit homologue of MMP-12 or of a closely related but previously unreported human MMP. The nucleotide sequence analysis of the B4 PCR fragment and rabbit MMP-12 cDNA clones from the cDNA library was done as described for MT1- MMP (see Example Id). Using this cDNA as a probe for northern blotting, we compared the levels of expression of MMP-12 in various cells and tissues from rabbits, including calvaria, brain, placenta, lung, liver, spleen, kidney, bone stromal cells, alveolar macrophages, and purified osteoclasts. Interestingly, the level of expression in purified osteoclasts was as high as in macrophages, while expression was almost not detectable in the other cells and tissues. To investigate whether MMP-12 is also expressed in osteoclasts in vivo, we performed in situ hybridisations on sections of metacarpals of new-born rabbits, and clearly identified MMP-12 in typical osteoclasts.

For expression and characterisation of a MMP-12 fusion protein, rabbit MMP-12 cDNA containing the open reading frame (bp 58-1437, see Figure 4) was amplified by PCR using primers sense 5'-CGGGATCCCTGTGGGTCACTTCTTCT-3' (SEQ ID No.22) and antisense 5'-CCGCTCGAGCTGGCACCATTACTAGC-3' (SEQ ID No.23) The cDNA fragment was inserted into the BamHI and XhoI sites of the pGEX-6P-2 vector as described for MT1- MMP. The cDNA was shown by direct sequence analysis to lie just 5' to the GST-encoding moiety of the vector and in proper reading frame with the plasmid translation initiation site (Figure 6).

WO 98/04287 PCT/EP97/04110 E.coli strain BL-21, transformed with pGEX-6P-2 alone (control vector) and pGEX-6P-2/MMP-12, were plated on Luria Broth (LB) agar plates with 50 pg/ml ampicillin at 37°C overnight. Single colonies were grown overnight in 50 ml of LB containing 50 pg/ml ampicillin in a shaking incubator at 300C. Subsequently, the overnight cultures were diluted 1:100 in 400 ml of LB containing 50 pg/ml ampicillin and grown at 300C to an OD 600 IPTG (Sigma) was added to a final concentration of 0.1 mM to induce production of fusion protein, and cells were maintained in culture for an additional 3 h.

Cell pellets were resuspended in 20 ml of a Tris-HCl buffer (2 mM CaC12 in 25 mM Tris-HCl, pH 7.6) containing 2 mg/ml of lysozyme and then lysed by sonication for 1 min in ice (6 bursts of 8 sec/burst). After sonication, 1 ml of Triton X-100 was added and extraction continued for minutes at 40C. After centrifugation for 10 min at 20,000 x g, the fusion protein according to SDS-PAGE was localised in the pellet (estimated molecular weight approx. 75 kDa corresponding well to the cDNA-deduced size of 83 kDa).

The pellet was solubilized in 20 ml of buffer containing 8 M urea and then stirred for 1 h at 40C. The sample was clarified by centrifugation at 40,000 x g for minutes at 4°C. Subsequently, the urea was removed completely by stepwise dialysis of the supernatant against the Tris-HCl buffer. The supernatant was subjected to SDS- PAGE and proteins stained by Coomassie Brilliant Blue R250.

Fusion protein expression was confirmed by Western blot using an antibody against the GST moiety. The presence of recombinant rabbit MMP-12 protein was ensured by fragmentation and subsequent amino acid sequence analysis. The elastolytic activity of the truncated recombinant MMP-12 was confirmed by elastin and gelatine zymography.

WO 98/04287 PCT/EP97/04110 36 Example 4 Assessment of the role of osteoclast MMPs in osteoclast migration.

In bone tissue cultures, we previously showed that MMPs are very important for the recruitment of osteoclasts to future resorption sites (Blavier and Delaiss6, 1995), but until now osteoclast purification techniques did not allow the demonstration of whether these MMPs were from osteoclasts or other cells. We therefore developed an experimental model in order to address the latter question.

Briefly, we seeded purified or non-purified osteoclasts on membranes (12 pm pore size) coated with type I collagen, and followed their migration to the lower surface of the membranes after an overnight culture in the absence or presence of MMP inhibitors. We found that not only when using non-purified osteoclast preparations, but also when using purified preparations, osteoclasts could extend cell processes into the pores of the membranes and spread over the lower surface of the membranes. This migration process was inhibited by MMP inhibitors of both the synthetic pseudo-substrate type (RP59794 and BB94) and the natural type (TIMP-2) (Figure This indicates that osteoclasts themselves can overcome a collagen barrier by migrating through it via an MMP dependent pathway, without the participation of other cells.

In order to evaluate how important MMPs are for this migration as compared to other proteinases, we also tested inhibitors of other classes of proteinases on this migration. Cysteine proteinase inhibitors that are potent inhibitors of the degradation of bone matrix in the subosteoclastic resorption zone, affected only slightly the migrations, whereas a serine proteinase inhibitor was without any effect (Figure Thus MMPs play a unique role in osteoclast migration as compared to other proteinases.

WO 98/04287 PCT/EP97/04110 37 In order to confirm the role of MMPs in an overall migration/resorption sequence, we seeded purified osteoclasts on dentine slices that were coated or not with type I collagen, cultured them overnight in the presence and absence of MMP inhibitor and followed the formation of pits in the dentine slices. We found that the MMP inhibitor inhibited pit formation only in the collagen coated dentine slices (Figure This indicates clearly that the role of MMPs is on the migration of the osteoclasts to their future resorption site, and not on resorption itself.

Example Preparation, characterisation and application of antibodies to MMPs Two approaches were used for the production of anti-MMP antibodies. In one approach, intact or truncated, native or recombinant MMP was used as an immunogen (see a, below) and in the other approach synthetic peptide mimicking a specific MMP-region was used as an immunogen after having been conjugated to a larger carrier protein (see b-d, below): a. Preparation and use of intact or truncated MMP immunogens As an example of the first approach, pro-MMP-9 purified from osteoclast cultures as described in Example 3-1 was used for immunisation either in its latent form or after activation by APMA or by in-gel treatment with SDS/Triton X- 100. The preparations of pro-MMP-9 and MMP-9 were injected intra-peritoneally every third week in female BALB/c-CFl murine hybrids. A final booster immunisation of the protein without adjuvant was given 3 days prior to splenectomy. The spleen cells were fused with P3-X-63-Ag8.653 myeloma cells in the presence of 50% polyethylene glycol 4000 and the resulting hybridoma cells propagated and cloned according to WO 98/04287 PCT/EP97/04110 38 standard procedures. Monoclonal antibody was purified from the conditioned medium of hybridoma cultures by using protein A affinity chromatography.

b. Preparation of MMP-mimicking conjugated peptide immunogens Based on the amino acid sequence of osteoclastic MT1- MMP (Figures 1 and 2) and sequences available for other members of the MMP family, such as MMP-9 and MMP-12, femtameric sequences polypeptide sequences of 15 amino acids) were selected due to: 1. their specificity for one member of the MMP family when compared to other members; 2. their putative properties as immunogens according to computer-based algorithms used for analyses of their hydrophilicity, their position and their expected secondary structure in the intact MMP; and 3. their conservation i.e. their possible sequence identity or similarity in corresponding regions of the same MMP in the human, rabbit and mouse species.

Corresponding to the selected femtameric sequences, femta-peptides were synthesised by using Fmoc-amino-acids-Opentafluorophenyl-esters in the presence of catalytic amounts of 3,4-dihydro-4-oxo,1,2,3-benzotriazin-3-yl in a fully automated custom made peptide synthesiser.

The femta-peptides were coupled to a proteinaceous carrier molecular (thyroglobulin). Briefly, thyroglobulin and glutaric anhydride (1:2 w/w) were incubated for 2 hrs at 0 C in 0.1 M sodium borate, pH 9.0 and subsequently desalted on a Nap 10/Sephadex G-25 column (Pharmacia) and dried by vacuum centrifugation. The carrier was resolubilized in 0.01 M sodium phosphate, pH 5.0 and incubated for 3 min at 200C with equal volumes of 5 mg/ml freshly prepared 1-ethyl- 3-(3-dimethylaminopropyl)-carbodiimide (CDI). The CDIactivated thyroglobulin was incubated 4 hrs at 20 0 C in equal volumes and amounts with the femta-peptide in 0.2 M WO 98/04287 PCT/EP97/04110 39 sodium phosphate, pH 9.0. The thyroglobulin/CDI/femtapeptide conjugates were dialysed and their protein content determined.

c. Production of polyclonal antibodies by use of conjugated peptide immunogens The thyroglobulin/CDI/femta-peptide conjugates were mixed with Freunds incomplete adjutant and injected intramuscularly once per month in female New Zealand White rabbits. Blood was collected and the immunoglobulin fraction purified from the corresponding serum by ammonium sulphate precipitation.

d. Production of monoclonal antibodies by use of conjugated peptide immunogens The thyroglobulin/CDI/femta-peptide conjugates were mixed with Freunds incomplete adjuvant and injected intraperitoneally every third week in female BALB/c-CF1 murine hybrids. A final booster immunisation of the conjugate without adjuvant was given 3 days prior to splenectomy. The spleen cells were fused with P3-X-63- Ag8.653 myeloma cells in the presence of 50% polyethylene glycol 4000 and the resulting hybridoma cells propagated and cloned according to standard procedures. Monoclonal antibody was purified from the conditioned medium of hybridoma cultures by using protein A affinity chromatography.

e. Characterisation and application of specific anti-MMP antibodies The antisera and monoclonal antibodies were selected and initially characterised by enzyme-linked immunosorbent assay (ELISA) based on 96-well polystyrene plates coated with either purified intact or truncated MMPs or homologous or heterologous conjugated femta-peptides. As indicated above, antisera and monoclonal antibodies showing MMP- WO 98/04287 PCT/EP97/04110 specificity according to the initial characterisation by ELISA have several applications. One example is their use in immunohistochemical identification of MMP-expression on the protein level by incubation of an anti-MMP antibody with bone cells or tissues. As described in Example 2d, the binding of a monoclonal antibody raised by immunisation with a MT1-MMP mimicking peptide to the actin-rich membranous areas of an osteoclast shows that MMP-antibodies not only are tools of central importance to the identification of the cells which produce a particular MMP, but also can demonstrate the cellular localisation of a MMP and thereby aid in the clarification of its biological role.

Sera from mice immunised with the thyroglobulinconjugated femta-peptide RSGAPVDQMFPGVPL (SEQ ID No.13) corresponding to a region in the hemopexin domain of rabbit MMP-9 and either boosted with the same conjugated peptide or with purified native osteoclast proMMP-9 showed inhibitory effects to activated MMP-9. The analysis was done by a fluorometric enzymatic assay based on pre-incubation of diluted sera with MMP-9 for 30 min at 370 before incubation with the synthetic peptide-like substrate Mca-PLGL-Dpa-AR-NH 2 (Bachem) (SEQ ID No.14) for 30 minutes at 370 in 150 mM NaC1, mM CaCl 2 0.05 Brij-35 in 50 mM Tris-HCl, pH (see Figure 9).

Example 6 Production of non-immunoglobulin inhibitors of osteoclast proteinases.

Production of non-immunoglobulin inhibitors of osteoclast proteinase aimed at two main type of agents, one being peptide or peptide-mimicking proteinase inhibitors another being antisense probes specifically binding to osteoclast proteinase mRNA. The peptide and peptide mimicking agents were produced by two methods: a technology based on PEGA bead peptide substrate and inhibitor libraries WO 98/04287 PCT/EP97/04110 41 (see a-c, below), the other being based on positional combinatorial peptide inhibitor libraries (see d-e, below).

The design and use of antisense probes is described in f (see below): a. Identification of MMP substrates by PEGA bead libraries According to previous descriptions (Meldal et al, 199410) two PEGA bead peptide substrate libraries were generated consisting each of approx. 106 different beads.

Each bead contained many copies of a single sequence: NX1-X2-

Y(NO

2 -X3-X4-X5-X6-X7-X8-K(Abz)c-PEGA (PEGA bead substrate library A) or X1-X2-Y(NO 2 )-X3-X4-X5-X6-K(Abz)C-PEGA (PEGA bead substrate library where X1 to X8 are amino acids varying randomly from bead to bead, and Y(N0 2 and K(Abz) is a quenching 3-nitrotyrosine and a fluorogenic lysine(2aminobenzoic acid), respectively. The libraries were incubated at 37°C with purified and activated osteoclast proMMP-9 (approx. 0.1 uM) and fluorogenic beads subsequently isolated by a micropipette under fluorescence microscopy.

The isolated beads were analysed by an amino acid sequencer.

The incubation of the randomised PEGA-bead substrate libraries lead to identification of 15 clearly fluorescent beads, indicating a specific cleavage of their corresponding peptide in contrast to the millions of other structures in the libraries. The amino acid sequences of the cleaved substrates showed some consistency (see Table In particular a proline at the third position (P3) towards the N-terminal from the cleavage site was highly conserved.

WO 98/04287 PCT/EP97/04110 42 Table 2. Amino acid sequences and cleavage site of quenched fluorogenic peptide substrates identified on PEGA bead libraries and Cleavage site P7 P6 P5 P4 P3 P2 P1 PI' P2' P3' P4' Bead A2 S K Y' P J A L F F K' A3 S R Y' P J G L? T K' W G Y' E A J G F T K' B1 A R Y' P K K V K' B2 N J Y' P J J Y K' B3 Y I Y' P J M L K' R P Y' P Y K K' B6 L K Y' P K L K' B7 F A Y' J M R K' B8 P A Y' M K K M K' B9 P L Y' M S J K' P V Y' M R G J K' Bl V R Y' L H G J K' b. Synthesis and characterisation of soluble peptides analogous to peptides identified by the PEGA bead substrate library technology To further evaluate the results observed for peptide substrates bound to PEGA beads, a series of soluble peptide substrates was synthesised by multiple column peptide synthesis (Meldal et al, 199411). The amino acid sequences of these putative soluble substrates were based on either single peptide substrate sequences or consensus sequences from the PEGA bead studies. The hydrolysis by MMP-9 and other MMPs of the soluble peptides was analysed by a standard fluorometric assay (excitation: 320 nm, emission: WO 98/04287 PCT/EP97/04110 43 425 nm) As an example, one of the fluorescent beads (A2 in Table 2) isolated from PEGA bead peptide substrate library contained two similar peptides with the sequences S-K- Y(N0 2 )-P-J-A-L-F-F-K(Abz)-PEGA (SEQ ID No.2) and L-F-F- K(Abz)-PEGA (SEQ ID No.24) indicating hydrolysis by .osteoclastic MMP-9 of the novel peptide-mimicking substrate

S-K-Y(NO

2 )-P-J-A-L-F-F-K(Abz) (SEQ ID No.2) at the P1-P1' position: A-L. Based on this information several soluble quenched fluorogenic peptides were synthesised CL-1 and CL-6, see Table 3 and Figures 10 and 11). By a similar strategy for the other amino acid sequences of substrates identified in the PEGA bead substrate libraries A and B, the first 30 soluble quenched fluorogenic peptide substrate candidates for MMP-9 (named CL-1 to CL-30) were synthesised by multiple column peptide synthesis. Their individual kinetic properties (kcat and Km) were determined by incubation at 37 0 C -with MMP-9 and recombinant truncated MT1-MMP of osteoclast origin, and as controls recombinant truncated MMP-1 and the osteoclast cysteine proteinase, cathepsin K; and the broad-reacting proteinase, subtilisin. Several of the hitherto produced 30 synthetic substrates showed a high selectivity for one or more MMPs; no or very low reactivity with cathepsin K; and kcat/Km ratios up to higher for MMP-9 than for subtilisin. This was particularly clear for the peptide substrates CL-21, CL-25 and CL-29 (see Table Further peptide substrate designing based on the sequence information obtained from both those of the peptides which were cleaved specifically by MMP-9 and those that were not, can be expected to lead to other even more selective synthetic MMP substrates.

For some of the 30 soluble putative peptide substrates, the kinetic behaviour was different from what was expected according to the hydrolysis of the corresponding peptide 3 immnobilised on the PEGA bead. the putative substrate, CL-1, was inhibitory to MMP-9 as would have been expected for a pseudo-substrate, i.e. with a low Km(3.4 and a low k-at/Km (250 M-s (see Table 3).

Table 3. Kinetic parameters for the hydrolysis of three established soluble MMP-9 substrates MR1, MR2) and two soluble substrates (CLl, CL6) designed according to results from PEGA bead library Name Sequence K1, (jiM) Kcat/Km 5-1 (and MMP-9 cleavage site: MMP-9 Subtilisin MMp-9a Subtilisin B Mca-P-L-G*LDpaA-RNH2(1 7.4 21.3 9.1-105 1. 1.107 MR1') Abz-G-P-LG*LY(NO) A-RNH, 7.7 1.6 3.1.105 2.1*106 MR2b N2 H 7.3 4 .8 9.0.104 9.6*106 CLV' 3.4 7.5 2. 5 *102 1.6*103 CL6C N2 PJALFFK(b)- 20.0 9.5 3.1*102 6.4*104 a Due to the lack of a proper MMP-9 standard, the estimation of for MMP-9 was not exact.

b Analogues of peptide B (Bachem M-1895) c Based on the isolated fluorogenic bead: A2 (see Table 2).

1. SEQ ID No.14 2. SEQ ID 3. SEQ ID 4. SEQ ID No.16 SEQ ID No.2 Table 4. Kinetic parameters for the hydrolysis of three soluble selective MMP-9 substrates (CL-21, and CL-29) designed according to results from PEGA bead substrate library The kinetic parameters are in pM-' x min'' and relatively to the corresponding value for MMP-9.

Peptide Sequence Bead MMP-9 Subtilisin Cathepsin K MMP-1 MMP-3 MT1-MMPb CL-21 Y'PLJMKGK'G B8/B9 5.5 100% 0.09 2% 0 0% 0.28 5% 0.05 1% 0 0% NJY'PJJYK'G B2 0.08 100% 0 0 0 0 0 <6% CL-29 Y'PJJMK'GJG B2/B10 0.38 100% 0.01 3% 0 0 0.01 3% 0.01 3% a The synthetic peptides were designed according to amino acid sequences of peptides from those beads of the PEGAbead substrate libraries that became fluorescent upon incubation with MMP-9 (Bead see Table 2).

b Represented by the trypsin-activated form of the truncated recombinant MT1-MMP, Ec2.

CL-21 SEQ ID No.26 SEQ ID No.27 CL-26 SEQ ID No.28 WO 98/04287 PCT/EP97/04110 46 c. Identification of MMP inhibitors by PEGA bead libraries According to previous descriptions (Meldal et al, 19941", Meldal Svendsen, 199511, Meldal et al, 1997 a PEGA bead peptide inhibitor library was generated consisting of approx. 106 different beads, each containing many copies of a single well-defined substrate sequence as well as many copies of a randomly generated putative inhibitor sequence:

N

X1-X2-X3- D-X4-X5-X6-VC-PEGA, where Xl to X6 are L-amino acids varying randomly from bead to bead, and D is a D-amino acid varying randomly from bead to bead. The library was incubated at 370C with active MMP-9 and beads remaining quenched dark compared to the majority of brightly fluorescent beads) were isolated by a micropipette under fluorescence microscopy. The isolated beads were analysed by an amino acid sequencer and since the substrate sequence was not degraded by the Edman degradation due to prior acylation at the N-terminus, the sequences obtained corresponded to potential peptide-mimicking MMP-9 inhibitors.

A novel type of PEGA bead inhibitor library was developed in order to identify peptide substrate mimicking MMP-inhibitors with a phosphinate instead of a peptide bond at the susceptible cleavage site between the expected P1 and P1' sites of the corresponding substrate). Two PEGA bead phosphinate inhibitor libraries (IIa and IIb) were generated. Each library consisted of approx. 106 different beads, and each PEGA bead contained many copies of a single well-defined substrate sequence as well as many copies of a randomly generated putative inhibitor sequence: NX1-X2-JP/CM-X3-X4-"Linker"-EGA (in IIa) or NX1-X2-GP/CLX-X3X4-"Linker"-PEGA (in IIb), where Xl to X4 are L-amino acids varying randomly from bead to bead, and JP/cM and GP/CL is the phosphinate pseudo dipeptide used in library IIa and IIb, respectively (see Figures 12-15). The design of the first two phosphinate pseudo dipeptides was based on the identity of suitable P1 and P1' amino acids in newly developed and existing MMP-9 substrates. Other combinations of pseudo amino acids around the phosphinate bond will also be WO 98/04287 PCT/EP97/04110 47 investigated according to the findings of MMP selective peptide substrates by use of e.g. PEGA bead substrate libraries.

d. Positional combinatorial peptide inhibitor libraries As an alternative to using the PEGA bead peptide libraries for identification of potential MMP inhibitors, 20 different positional combinatorial peptide inhibitor libraries (Houghten et al, 19917) were produced using pentapeptides constructs X-X- D-X-X, where D is the D-form of one of the 20 common amino acids (except glycine) or hydroxyproline, and X is a randomly varying natural L-form of one of the 20 common amino acids or hydroxyproline. The peptide libraries were purified by high performance liquid chromatography in order to remove salts and other substances which were toxic to bone tissue cultures before being tested for inhibitory effects on osteoclast migration and bone resorption in murine foetal metatarsal cultures. Each of the 20 libraries contained 30 pmol pentapeptides composed of up to 214(194,481) different structures.

e. Murine foetal metatarsal cultures for studying osteoclast migration and resorption in vitro 45Ca2+ pre-labelled metatarsals isolated from 17 day old NRM1 mouse foetuses were used as an organ culture model (Blavier Delaisse, 19953). Briefly, foetal bones were labelled by subcutaneous injection of 4Ca2 into pregnant mice at day 16 of gestation. Foetal metatarsals isolated on the following day thereby comprised 4 5 Ca-labelled calcified matrix developed in uteri between day 16 and 17. In the periosteum surrounding the calcified matrix numerous osteoclast precursors cells were present. Corresponding to the development of bone and bone marrow in metatarsals in vivo, subsequent cultivation of the isolated metatarsals in BGJb medium containing 30 nM dihydroxy-vitamin D3 and 0.1% Albumax for 1 to 7 days resulted in differentiation, fusion and migration of the osteoclast precursor cells leading to the presence of mature osteoclasts in the central calcified matrix where they resorbed bone and formed the primitive marrow cavity. The development WO 98/04287 PCT/EP97/04110 48 and bone resorbing activity of the osteoclasts was estimated by measurement of the release of 45Ca 2 into the culture medium at various time points and by microscopic inspection of the positioning in the cultured metatarsals of osteoclasts stained for tartrate-resistant acid phosphatase. The general MMP inhibitor, RP59794 which has been shown previously to inhibit the migration of osteoclasts and thereby reduce the release of 4Ca in the metatarsal culture model (Blavier Delaiss6, 19953) was included as a positive control in all experiments.

The effect of the 20 X-X-D-X-X combinatorial libraries on bone resorption was evaluated by measuring the change in accumulated 4Ca-release into the conditioned medium of the treated metatarsal culture relatively to the 4Ca-release of the corresponding non-treated metatarsal culture originating from the other leg of the same foetus at Day 1, 2 and 4. Each library was tested in 4 independent metatarsal cultures in the same experiment and in some cases the experiment was repeated.

Each of the 20 libraries was used in a concentration of 3 mM total peptide corresponding to a concentration of approx. nM for each of the 194,481 structures in a library. The majority of the 20 libraries did not significantly affect the bone resorption, whereas 1 of the 20 libraries (D=ile) showed significant reductions in the 4Ca-release at Day 4 (see Figure 16), and most importantly 2 of the 20 libraries (D=leu and D=trp) showed significant inhibitions at both Day 2 and Day 4 (see Figure 16 and Table Table 5 Change (in of 4Ca-release due to the addition of a X-X-D- X-X combinatorial library to 4-day metatarsal cultures Library Day 0-1 Day 0-2 Day 0-4 X-X-trp-X-X 0% (ns) -20% (0.02) -40% (0.05) X-X-leu-X-X 0% (ns) -34% (0.0001) -48% (0.0005) The p-values express the level of significance of the changes between the treated and corresponding non-treated group (n=4 for each).

WO 98/04287 PCT/EP97/04110 49 Further investigations of the X-X-trp-X-X library was done by performing a second screening of 28 libraries with a selected variation at one of the 4 X-positions. The following conformations were used U-X-trp-X-X, X-U-trp-X-X, X-X-trp-U-X and X-X-trp-X-U, where U is a random mixture of L-amino acids belonging to a specific undergroup: Ul: K and R U2: H, Y, F and W U3: E and Q U4: T, D, S and N C, V, L, I and M U6: P and J and U7: A, G Each of the 28 libraries was used in a concentration of 1.6-4.0 mM total peptide, corresponding to approx. 85 nM for each of the 18,522 to 46,305 structures in a library. The majority of the 28 libraries did not significantly affect the bone resorption, whereas 5 of the 28 libraries showed significant and/or marginally significant reductions in the 45Ca-release at Day 1, 2 and/or 4 (see Table 6).

Table 6. Change (in of 4 Ca-release due to the addition of a U-Xtrp-X-X, X-U-trp-X-X, X-X-trp-U-X or X-X-trp-X-U combinatorial library to 4-day metatarsal cultures Library Day 0-1 Day 0-2 Day 0-4 US-X-w-X-X -20% (0.28) -21% (0.12) -15% (0.11) X-U2-w-X-X -15% (0.35) -23% (0.05) -18% (0.15) -43% (0.007) -28% (0.04) -11% (0.05) X-X-w-U2-X -23% (0.21) -20% (0.003) -16% (0.001) -23% (0.15) -24% (0.0008) -15% (0.07) Further investigations of the U5-X-trp-X-X, X, X-X-trp-U2-X and X-X-trp-X-U5 libraries was done by performing a third screening of 23 libraries with a single variation at one of the 4 X-positions. The following conformations were used Z5-X-trp-X-X, X-Z2/5-trp-X-X, X-X-trp- Z2-X and X-X-trp-X-Z5, where Z2, Z5 or Z2/5 is a single L-amino acid belonging to undergroup(s) U2, U5 or U2 and respectively. With a few exceptions, each of the 23 libraries was used in concentration of 3.2 mM total peptide, corresponding to approx. 340 nM for each of the 9,261 WO 98/04287 PCT/EP97/04110 structures in a library. More than half of the 23 libraries did not significantly affect the bone resorption, whereas 11 of the 23 libraries showed significant and/or marginally significant reductions in the 4Ca-release at Day 1, 2 and/or 4 (see Table 7).

Table 7 Change (in of 45 Ca-release due to the addition of a trp-X-X, X-Z2/5-trp-X-X, X-X-trp-Z2-X library to 4-day metatarsal cultures or X-X-trp-X-Z5 combinatorial Library (Conc) Day 0-1 Day 0-2 Day 0-4 C-X-trp-X-X (3.2 mM) -22% (0.09) -38% (0.03) -34% (0.006) V-X-trp-X-X (3.2 mM) 6% (0.33) -30% (0.17) -23% (0.11) L-X-trp-X-X (3.2 mM) -23% (0.07) -32% (0.01) -20% (0.06) X-W-trp-X-X (3.2 mM) -19% (0.22) -26% (0.003) -22% (0.08) X-Y-trp-X-X (3.2 mM) -26% (0.04) -27% (0.06) -18% (0.17) X-F-trp-X-X (3.2 mM) -20% (0.19) -33% (0.06) -23% (0.09) X-C-trp-X-X (3.2 mM) -39% (0.02) -18% (0.14) -10% (0.13) X-L-trp-X-X (3.2 mM) -19% (0.39) -26% (0.07) -24% (0.20) X-X-trp-Y-X (3.2 mM) -25% (0.05) -48% (0.0003) -38% (0.0006) (0.8 mM) -45% (0.07) -26% (0.14) 8% (0.22) (0.8 mM) -39% (0.003) -30% (0.02) -18% (0.03) X-X-trp-X-L (3.2 mM) -12% (0.34) -34% (0.21) -17% (0.18) X-X-trp-X-M (3.2 mM) -26% (0.21) -26% (0.14) -18% (0.03) In an early attempt to identify single peptide inhibitory structures a fourth screening was performed on 20 peptides of the structure C/V/L-Y/F/W/C/L-trp-Y-M/L considered to be likely candidates according to the results in the 3 r d screening. Each of the 20 single structure peptides was used in a concentration of 13 pM. The majority of the 20 peptides did not significantly affect the bone resorption, whereas 5 of the 20 structures showed significant and/or marginally significant reductions in the 5Ca-release at Day 1, 2 and/or 4 (see Table Even better single peptide inhibitors will be obtained upon further investigations based on the data from the first 4 screenings.

Particularly further investigations of X-X-trp-Y-X WO 98/04287 PCT/EP97/04110 51 combinatorial libraries and a similar screening programme for X-X-leu-X-X seem promising.

Table 8. Change (in of 5Ca-release due to the addition of a single peptide structure with the sequence C/V/L-Y/F/W/C/L-trp-Y-M/L to 4day metatarsal cultures Structure Day 0-1 Day 0-2 Day 0-4 C-L-w-Y-L -30% (0.02) -22% (0.03) -15% (0.005) C-L-w-Y-M -29% (0.06) -26% (0.05) -13% (0.40) C-Y-w-Y-L -17% (0.008) -18% (0.009) -12% (0.11) V-Y-w-Y-M -17% (0.21) -21% (0.04) -15% (0.02) L-F-w-Y-L -34% (0.003) -37% (0.007) -26% (0.04) f. Design and use of antisense probes to MMPs.

Antisense oligonucleotide probes against various MMPs were produced in order to study their influence on bone metabolism and osteoclast biology in bone cell and tissue cultures as well as in animal models. The antisense oligonucleotide probes were designed by choosing sequences which were specific to a particular MMP and showing as little as possible similarity to any predictably relevant mammalian genes. In all cases a sense probe and/or a so-called scrambled probe was used as negative controls for comparison to the antisense probe. In order to stabilise the probes, some were produced in a partially phosphorthiolated form to protect them against degradation by nucleases (phosphate bonds which are phosphorthioate bonds instead of normal phosphordiesters are marked with a in the diagram below). In order to make the delivery of the probes to the interior of osteoclasts some of the probes were included in liposomes before application to the cell or tissue cultures.

The strategy in this type of experiments is exemplified by results from design, synthesis and testing of antisense probes to mouse and rabbit MMP-9.

wn 98/n42a7 T rt' <ft 4« fk 52 rLLLrYIIU4jqLu Two sets of probes (17-mers) to murine MMP-9 are shown in the Table below: Table 9: Selected probes for use in experiments with MMP-9 expression in murine cells and tissues: Scrambled (SEQ ID No.29) First set 5'-T*G*TGGTTCAGTTGTG*G*T Antisense (SEQ ID Sense (SEQ ID No.31) Second set 5'-G*GAC*T*CA*TGG*TGAG*G*A*C Antisense (SEQ ID No.32) Sense (SEQ ID No.33) The probes were used in the murine metatarsal system described in Example 6e and in a murine pre-osteoclast culture system. The latter was based on unfractionated bone cells isolated from 12 day old mice and cultured for 7 days in the presence of 5% fettle calf serum in order to eradicate all multinucleated osteoclast leaving only stromal cells and osteoclast precursors. Upon subsequent culture of approximately 10 days in the 'presence of 2 g/ml PGE 2 new mature osteoclasts were formed. The continuous differentiation of pre-osteoclasts to mature osteoclasts in this culture system correlated well to production of pro-MMP-9 according to gelatinase zymographical studies of the corresponding conditioned medium. For both test systems, the probe was added to the culture medium in a concentration varying between 1 and 4g/ml and the medium was renewed every day.

Seven antisense probes (14- to 18-mers) to rabbit MMP-9 were constructed as shown in the Table below: WO 98/04287 PCT/EP97/04110 53 Table 10: Selected probes for use in experiments with MMP-9 expression in rabbit cells and tissues: Probe 1 G*T*C*TGG*GGC*T*CA*TGG*T*G*A (start codon) (SEQ ID No.34) Probe 2 G*G*CT*CA*TGG*TGA*G*G (start codon) (SEQ ID Probe 3 G*G*GC*T*CA*TGG*TG*AGG*G*G*A (start codon) (SEQ ID NO.36) Probe 4 C*T*CA*TGG*TG*AGG*GGA*G*C*A (start codon) (SEQ ID No.37) Probe 5 A*T*GG*TG*AGG*GGAG*CA*G*C*G (start codon) (SEQ ID No.38) Probe 6 A*G*GT*GAG*TGG*CGT*CA*C*C*G (stem loop) (SEQ ID No.39) Probe 7 G*C*TGT*CA*AAG*T*TGGA*A*G*T (stem loop) (SEQ ID Scrambled 1 G*G*CC*T*C*TAC*CG*CAACT*G*C (SEQ ID No.41) Scrambled 2 G*G*C*C*T*C*TAGG*GGAAC*T*G*C (SEQ ID No.42) Five of the antisense probes spanned the start codon of the mRNA and two targeted single stranded loops (identified by mRNA secondary structure prediction algorithms) within the translated region.

Testing of the effects of the antisense and scrambled probes to rabbit MMP-9 was performed in osteoclasts isolated from long bones of 8 to 10 days old rabbits. The osteoclasts were cultured on bovine bone slices in 5 foetal calf serum, with renewal of media and oligonucleotides every day. The results were evaluated by quantification of MMP-9 in gelatinase zymography and by studies of osteoclast morphology and numbers as well as quantification of the secretion of tartrate-resistant acid phosphatase into the conditioned medium of the osteoclast cultures by enzymatic assay.

WO 98/04287 PCT/EP97/04110 54 References 1. Baggio R, Shi Y, Wu Y, Abeles R H. From poor substrates to good inhibitors: Design of inhibitors for serine and thiol proteases. Biochem 35:3351-3353. 1996.

2. Birkedal-Hansen H, Moore W G I, Bodden M K, Windsor L J, Birkedal-Hansen B, DeCarlo A, Engler J A. Matrix metalloproteinases: A review. Crit Rev Oral Biol Med 4:197-250. 1993.

3. Blavier L, Delaisse J-M. Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primitive long bones. J Cell Sci 108:3649-3659. 1995.

4. Brown P D, Giavazzi R. Matrix metalloproteinase inhibition: a review of anti-tumour activity. Annals of Oncology 6:967-974. 1995.

Delaisse J-M, Vaes G. Mechanism of mineral solubilization and matrix degradation in osteoclastic bone resorption.

In: Biology and physiology of the osteoclast (eds. Rifkin B R, Gay-G pp 289-314. Boca Raton, CRC Press. 1992.

6. Foged N T, Delaisse J-M, Hou P, Lou H, Sato T, Winding B, Bonde M. Quantification of the collagenolytic activity of isolated osteoclasts by enzyme-linked immunosorbent assay. J. Bone Miner Res 11:226-237. 1996.

7. Houghten R A, Pinilla C, Blondelle S E, Apell J R, Dooley C T, Cuervo J H. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354: 84-86. 1991.

8. Laitala T, Vaananen H K. Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H'- ATPase. J Clin Invest 93:2311-2318. 1994.

9. Lin M, Hultquist K L, Oh D H, Bauer E A, Hoeffler W K.

Inhibition of collagenase type I expression by psoralen antisense oligonucleotides in dermal fibroblasts. FASEB 9:1371-1377. 1995.

Meldal M, Svendsen I, Breddam K, Auzanneau F-I. Portionmixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity. Proc Natl Acad Sci 91:3314-3318. 1994.

11. Meldal M, Svendsen I. Direct visualization of enzyme inhibitors using a portion mixing inhibitor library containing a quenched fluorogenic substrate. J Chem Soc Perkin Trans: 1591-1596. 1995.

12. Sa kai D, Tong H-S, Minkin C. Osteoclast molecular WO 98/04287 55 PCT/EP97/04110 phenotyping by random cDNA sequencing. Bone 17:111-119.

1995.

13. Sato H, Takino T, Okada Y, Cao J, Shinegawa A, Yamamoto E, Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370: 61-65.

1994.

14. Takino T, Sato H, Yamamoto E, Seiki M. Cloning of a human gene potentially encoding a novel matrix metalloproteinase having a C-terminal transmembrane domain. Gene 155:293-298. 1995.

Tezuka K, Sato T, Kamioka H, Nijweide P J, Tanaka K, Matsuo T, Ohta M, Kurihara N, Hakeda Y, Kumegawa M.

Identification of osteopontin in isolated rabbit osteoclasts. Biochem Biophys Res Commun 186:911-917.

1992.

16. Tezuka K, Nemoto K, Tezuka Y, Sato T, Ikeda Y Kobori M, Kawashima H, Eguchi H, Hakeda Y, Kumegawa M.

Identification of matrix metalloproteinase 9 in rabbit osteoclasts. J Biol Chem 269:15006-15009. 1994.

17. Will H, Hinzmann B. cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment. Eur J Biochem 231:602-608. 1995.

18. Eggleston Z M, Mutter M. Shaping up to Proteins.

Chemistry in Britain:39-41. May 1996.

19. Galardy R E, Grobelny D, Kortylewicz Z P, Poncz L.

Inhibition of human skin fibroblast collagenase by phosphorous-containing peptides. Matrix Suppl 1: 259-262.

1992.

Shapiro S D, Griffin G L, Gilbert D J, Jenkins N A, Copeland N G Welgus H G, Senior R M, Ley T J.

Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase. J. Biol. Chem. 267:4664-4671. 1992.

21. Meldal M, Svendsen I, Juliano L, Juliano M A, Del Nery E, Scharfstein J. Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor library.

D-Amino acid inhibitors for cruzipain, cathepsin B and cathepsin L. J Pept Sci: in press, 1997.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Center for Clinical Basic Research STREET: Ballerup Byvej 222, CITY: Ballerup COUNTRY: Denmark POSTAL CODE (ZIP): DK-2750 (ii) TITLE OF INVENTION: The Use of Proteinase Inhibitors for the Prevention or Reduction of Bone Resorption (iii) NUMBER OF SEQUENCES: 42 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: GB 9615976.9 FILING DATE: 30-JUL-1996 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/product= "x is hydroxyproline" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Ser Lys Tyr Pro Xaa Ala Leu Phe Phe Lys 1 5 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site LOCATION:3 OTHER INFORMATION:/pioduct= "X is Y(N02)" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/product= 1X is hydroxyproline" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/product= "X is K(Abz)" SEQUENCE DESCRIPTION: SEQ ID NO: 2: Ser Lys Xaa Pro Xaa Ala Leu Phe Phe Xaa 1 5 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 2546 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA enomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Oryctolagus cuniculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TAACGCAGAG TTACATATAC ATACCTGGGG GGGGGGGGGG

CATATAAGAT

AGATTTTTAT

TTAGCACACG

TTACCTATTA

GTTTTACCTT

AATGTTAGCC

AGATGGCGGC

CCGACGGTCG

CTGCTCACAC

GCCCCGAAGC

TCTACTACTT

TTATTTCCTA

CAAACTTACA

GTTACCTTAT

ATTAGTTTTA

GCTAGGAATC

GCGACCCCTA

CGGACCATGT

TCGGCACCGC

CTGGCTGCAG

TCTCCTCAGT

ATTCATGTAG

TATACTTTAC

ACACAGAGTT

TAGTTACCTA

CCTATTAGTT

CAAAGTCGGT

GGCGAGGGCC

CTCCCGCCCC

ACTCGCCTCC

CAGTATGGCT

CACTGTCAGC

CGATCACTAA

TTATTATTTA

CTATCCTATC

TTAGTTTTAC

TTAAACTACT

GCCTCCGGAA

CCGCCGCGGA

ACGACCCTCC

CTCGGCTCGG

ACCTGCCTCC

TGCCATTGCT

GGTTCTACTA

TGTAGATTTT

TTTCTTTGAT

CCTATTAGTT

CTTATTAGTT

AATGTAGCGA

GACAAAGGCG

ACCGCCCAGC

CGCAGGCTCC

CCAAAAGCAA

AGGGAAGACC

AAGCCATGCA

ATGTAGCGTA

TCTAAATGTT

TATATTATCA

ACCTTATTAG

TTACCTATTA

GAATCTACTA

CCCCCGAGGG

CCGGCTGACC

TGAACTCCCC

AACAGCTTCA

TACGCACCCA

GAGGTTCTAC

GGTTTGCGAG

GCTGCGGTGT

CTACGCCATC

TACACCCCCA

CAAGGCATTC

CGCTTCCGCG

TCATGATCTT

TGGCTTCCTG

AGAATCTACT

GAATGAGGAC

GCCCTGGGCA

GGATGAAGAC

TAATATGGGA

CCCGGACTTT

CGGGAACTTT

TrGGTTCTGGA

AGTTCTGGCG

ATTCGTCTTC

GGCTACCCCA

ACCGACAAGA

AGAACCTACT

TGGACAGCGA

AGGGTCGTTC

TGGAAATTCA

GGGACTGGAT

GGAGGTGATC

GTGGTGCTGC

TCTTCAGGCG

CAAGGTCTGA

GATTGTATCC

TGACAGGCAA

TCCAGACAAG

CAGGGCCTCA

AGGTGGGCGA

CGCGTGTGGG

AGGTGCACTA

CTTTGCCGAG

GCCCACGCCT

AAATGTTAGG

CTGAAAACGG

ACTGGAGCAC

ACAGAGAACT

GCCAGTCGGG

TATCCCCGAT

GACACTGTGG

GGGTGAGGAA

GGGCCTGCCT

TTCAAAGGAG

AGCACATCAA

TCGATGCCGC

TCTTCCGGGG

GTACCCCAAG

ATGGGCAGTG

ACAACCAGAA

GGGCTGCCCG

ATCATCGAGG

CCGTGCTGCT

CCACGGGACT

CCCCCACCGC

AATAAAAAAT

GGCCGATACA

TTTGGGGCTG

AATGGCAGAA

ATATAAAATC

AGAGCGCCAC

TGCCTACATC

GGCTTCCATG

ACTTCCCGGG

GGGACACCCA

GAATGACATC

TCCAATGACC

TCGTGCTGCC

GTCCCCCACA

AAGCCCAGGA

CCGTGCTCCG

CAACCAAGTG

GCTTCCATCA

ATAAGCACTG

GGAGCTGGGC

TCTCTTCTGG

AAACAAGTAC

AACATCAAAG

ATGAAGTCTT

GCTGAAGGTG

GCTGGGGGCC

TGGACGAGGA

GCTACTCCTG

CCGAAGCGAA

TGGCCAACAC

AAGCATCAGC

GACACCAAAT

AGAAATCAAG

CATAATGAGA

TAAATGTTAG

ACCGAAATCT

CGCGATGGCC

GCGACAGCAC

CCCCAACATT

CTTTGACTCC

TTCCTGGTGG

CCTCAGCCAT

TGATGATGAC

AAGATGCCTC

ACCCCACCTA

AGGAGAGATG

ATGGACGGCT

ACACCGCCTA

GGTGTTTAAG

CGAAACTCTA

ATGCCCAATG

TACCGATTCA

TGTGGGAAGG

CACTTACTTC

GAGCCCGGCT

GTCCGGATGA

GGGCAGCAAG

GTGAACTGGC

ACTGCTCTAC

CCACTCCCAC

GAAGGCCATG

GCCAATGTCC

TCACTTTCTG

GCCACATTCG

ACTAAATGTA

GTGAGAAGCA

GCCCTTCAAG

GGAAAACTCT

GCGGAGCCCT

CTGTGCATGA

CATGGCACCG

CGCCGGGGCA

CTCCACCCAG

CGGGCCCAAC

TTTGTCTTCA

ACCCAAAATG

CAAGAGAGGA

ACGAGGCTTC

CTAAATGTTA

GAAAGAATCT

ACGAGGAGCT

CATCCCCGAG

TACAAGGGGA

ACCCCAAAAG

GAAGGGACTG

GAGCCGTGAG

CGTGGGCCTG

TGCCAGCGTT

CGCAAGGACT

AGGCGCCCCC

GAAGGAAGCG

CATCCAGAAT

AGGCCATTCG

GCGTAGACTG

GAAGCCGACA

ATGGCGAGGG

ACTAAATGTT

GGACTGTCCG

GCTGGGCCAT

TTTTACCAAT

TCCAACAGCT

GACAACCAAT

ATCAATGTGA

AAAGGAGCGC

CCCATCGGCC

AGGATGGCAA

CCTGGAGCCT

GGGGCTTCCC

ACTAAATGTT

CAGGGCAAAG

TCTAACCCAG

AAACAAATAC

TCCGCCCTGC

AGGAAGAGAC

CGCGGCCGCC

GCGGTCTTCT

CCCTGCTGGA

TTGCTCTTCC

780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2546 AAAAAAA AAAAAA ATAGAATCTA CTAAATGTTA GAACTACTAA TGTAGA INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 582 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: ORGANISM: Oryctolagus cuniculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Ser Pro Ala Pro Arg Pro Ser Arg Arg Thr Phe Asp Ile Asp Asp Ala Gln Arg 145 Glu Ile Asp Ile Leu Ser Leu Ala Thr Lys Ile Asn 130 Lys Val Met Gly Gly 210 Gly Pro Arg Ala Asp Phe Gin 115 Tyr Ala His Ile Glu 195 Gly Thr Glu Thr Met Thr Gly 100 Gly Thr Phe Tyr Phe 180 Gly Asp Ala Ala His Gin Met Ala Leu Pro Arg Ala 165 Phe Gly Thr Ala Leu Gin Phe Ala Ile Trp Val 135 Trp Ile Glu Leu Phe 215 Ser Gin 40 Arg Tyr Met Lys Gin 120 Gly Glu Arg Gly Ala 200 Asp Leu Gin Ser Gly Arg Ala 105 His Glu Ser Asp Phe 185 His Ser Leu Ser Gly Gin Arg 75 Pro Val Glu Ala Thr 155 Arg Gly Tyr Glu Leu Lys Leu Leu Thr Cys Arg Thr 125 Phe Leu Lys Ser Pro 205 Trp Leu Asn Pro Ala Lys Val Arg Cys Ala Phe Ala 175 Pro Pro Val Leu Ser Gly Ala Ala Pro Tyr Ile Ile Arg 160 Asp Phe Asn Arg Asn Glu Asp Leu Asn Gly Asn Asp Ile Phe Leu Val Ala Val His Glu 225 230 23S Leu Gly His Ala Leu Gly Leu Glu His Ser Asn Asp Pro Ser Ala Ile 245 250 255 Met Ala Pro Phe Tyr Gin Trp Met Asp Thr Glu Asn Phe Val Leu Pro 260 265 270 Asp Asp Asp Arg Arg Gly Ile Gin Gin Leu Tyr Gly Ser Gin Ser Gly 275 280 285 Ser Pro Thr Lys Met Pro Pro Pro Pro Arg Thr Thr Ser Arg Thr Phe 290 295 300 Ile Pro Asp Lys Pro Arg Asn Pro Thr Tyr Gly Pro Asn Ile Cys Asp 305 310 315 320 Gly Asn Phe Asp Thr Val Ala Val Leu Arg Gly Glu Met Phe Val Phe 325 330 335 Lys Glu Arg Trp Phe Trp Arg Val Arg Asn Asn Gin Val Met Asp Gly 340 345 350 Tyr Pro Met Pro Ile Gly Gin Phe Trp Arg Gly Leu Pro Ala Ser Ile 355 360 365 Asn Thr Ala Tyr Glu Arg Lys Asp Gly Lys Phe Val Phe Phe Lys Gly 370 375 380 Asp Lys His Trp Val Phe Asp Glu Ala Ser Leu Glu Pro Gly Tyr Pro 385 390 395 400 Lys His Ile Lys Glu Leu Gly Arg Gly Leu Pro Thr Asp Lys Ile Asp 405 410 415 Ala Ala Leu Phe Trp Met Pro Asn Gly Lys Thr Tyr Phe Phe Arg Gly 420 425 430 Asn Lys Tyr Tyr Arg Phe Asn Glu Glu Leu Arg Ala Val Asp Ser Glu 435 440 445 Tyr Pro Lys Asn Ile Lys Val Trp Glu Gly Ile Pro Glu Ser Pro Arg 450 455 460 Gly Ser Phe Met Gly Ser Asp Glu Val Phe Thr Tyr Phe Tyr Lys Gly 465 470 475 480 Asn Lys Tyr Trp Lys Phe Asn Asn Gin Lys Leu Lys Val Glu Pro Gly 485 490 495 Tyr Pro Lys Ser Ala Leu Arg Asp Trp Met Gly Cys Pro Ala Gly Gly 500 505 510 Arg Pro Asp Glu Gly Thr Glu Glu Glu Thr Glu Val Ile Ile Ile Glu 515 520 525 Val Asp Glu Glu Gly Ser Gly Ala Val Ser Ala Ala Ala Val Val Leu 530 535 540 Pro Val Leu Leu Leu Leu Leu Val Leu Ala Val Gly Leu Ala Val Phe 545 550 555 560 Phe Phe Arg Arg His Gly Thr Pro Lys Arg Leu Leu Tyr Cys Gin Arg 565 570 575 Ser Leu Leu Asp Lys Val 580 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 582 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ser Pro Ala Pro Arg Pro Ser Arg Cys Leu Leu Leu Pro Leu Leu 1 5 10 Thr Leu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala Gln Ser Ser Ser 25 Phe Ser Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr Leu Pro Pro Gly 40 Asp Leu Arg Thr His Thr Gln Arg Ser Pro Gln Ser Leu Ser Ala Ala 55 Ile Ala Ala Met Gln Lys Phe Tyr Gly Leu Gin Val Thr Gly Lys Ala 70 75 Asp Ala Asp Thr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 90 Asp Lys Phe Gly Ala Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Tyr 100 105 110 Ala Ile Gin Gly Leu Lys Trp Gin His Asn Glu Ile Thr Phe Cys Ile 115 120 125 Gin Asn Tyr Thr Pro Lys Val Gly Glu Tyr Ala Thr Tyr Glu Ala Ile 130 135 140 Arg Lys Ala Phe Arg Val Trp Glu Ser Ala Thr Pro Leu Arg Phe Arg 145 150 155 160 Glu Val Pro Tyr Ala Tyr Ile Arg Glu Gly His Glu Lys Gln Ala Asp 165 170 175 Ile Met Ile Phe Phe Ala Glu Gly Phe His Gly Asp Ser Thr Pro Phe 180 185 190 Asp Gly Glu Gly Gly Phe Leu Ala His Ala Tyr Phe Pro Gly Pro Asn 195 200 205 Ile Gly Gly Asp Thr His Asn 225 Leu Met Asp Phe Val1 305 CGlv Lys Tyr Asn Asp 38S Lys Al a Asn Tvr Gly 465 Asn Tyr Arg Val1 210 Giu Gly Ala Asp Pro 290 Pro Asn Glu Pro Thr 370 Lys His Ala Lys Pro 450 Ser Lys Pro Pro Asp 530 Asp His Pro Asp 275 Thr Asp Phe Arg Met 355 Ala His Ile Leu Tyr 435 Lys Phe Tyr Lys Asp 515 Glu Leu Ala Phe 260 Arg Lys Lys Asp Trp 340 Pro Tyr Trp Lys Phe 420 Tyr Asn Met Trp Ser 500 Giu Glu Asn Leu 245 Tyr Arg Met Pro Thr 325 Phe Ile Giu Val1 Glu 405 Trp Arg Ile Gly Lys 485 Ala Gly Gly Gly 230 Gly Gin Gly Pro Lys 310 Val1 Trp Gly Arg Phe 390 Leu Met Phe Lys Ser 470 Phe Leu Thr Gly Phe Asp Ser 215 Asn Asp Ile Leu Glu His Trp Met Asp 265 Ile Gin Gin 280 Pro Gin Pro 295 Asn Pro Thr Ala Met Leu Arg Val Arg 345 Gin Phe Trp 360 Lys Asp Gly 375 Asp Glu Ala Gly Arg Gly Pro Asn Gly 425 Asn Glu Glu 440 Val Trp Glu 455 Asp Glu Val Asn Asn Gin Arg Asp Trp 505 Glu Glu Glu 520 Gly Ala Val 535 Giu Leu 235 Ser Glu Tyr Thr Gly 315 Gly Asn Gly Phe Leu 395 Pro Thr Arg Ile Thr 475 Leu Gly Glu Ala Pro 220 Val1 Asp Asn Gly Thr 300 Pro Gl1u Gin Leu Val 380 Glu Thr Tyr Al a Pro 460 Tyr Lys Cys Val1 Ala 540 Trp Al a Pro Phe Gly 285 Ser Asn Met Val1 Pro 365 Phe Pro Asp Phe Val1 445 Glu Phe Val1 Pro Ile 525 Ala Thr Val1 Ser Val1 27 0 Glu Arg Ile Phe Met 350 Al a Phe Gly Lys Phe 430 Asp Ser Tyr Giu Ser 510 Ile Val1 Val1 His Al a 255 Leu Ser Pro Cys Val1 335 Asp Ser Lys Tyr Ile 415 Arg Ser Pro Lys Pro 495 Gly Ile Val1 Arg Glu 240 Ile Pro Gly Ser Asp 320 Phe Gly Ile Gly Pro 400 Asp Gly- Glu Arg Gly 480 Gly Gly Giu Leu 63 Pro Val Leu Leu Leu Leu Leu Val Leu Ala Val Gly Leu Ala Val Phe 545 550 555 560 Phe Phe Arg Arg His Gly Thr Pro Arg Arg Leu Leu Tyr Cys Gin Arg 565 570 575 Ser Leu Leu Asp Lys Val 580 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 582 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Rattus norvegicus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Ser Pro Ala Pro Arg Pro Ser Arg Ser Leu Leu Leu Pro Leu Leu 1 5 10 Thr Leu Gly Thr Thr Leu Ala Ser Leu Gly Trp Ala Gin Ser Ser Asn 25 Phe Ser Pro Glu Ala Trp Leu Gin Gin Tyr Gly Tyr Leu Pro Pro Gly 40 Asp Leu Arg Thr His Thr Gin Arg Ser Pro Gin Ser Leu Ser Ala Ala 55 Ile Ala Ala Ile Gin Arg Phe Tyr Gly Leu Gin Val Thr Gly Lys Ala 70 75 Asp Ser Asp Thr Met Lys Ala Met Arg Arg Pro Arg Cys Gly Val Pro 90 Asp Lys Phe Gly Thr Glu Ile Lys Ala Asn Val Arg Arg Lys Arg Tyr 100 105 110 Ala Ile Gin Gly Leu Lys Trp Gin His Asn Glu Ile Thr Phe Cys Ile 115 120 125 Gin Asn Tyr Thr Pro Lys Val Gly Glu Tyr Ala Thr Phe Glu Ala Ile 130 135 140 Arg Lys Ala Phe Arg Val Trp Glu Ser Ala Thr Pro Leu Arg Phe Arg 145 150 155 160 Glu Val Pro Tyr Ala Tyr Ile Arg Glu Gly His Glu Lys Gin Ala Asp 165 170 175 Ile Asp Ile Asn 225 Leu Met: Asp Ser Val1 305 Gly Lys Tyr Asn Asp 385 Lys Ala Asn Tyr Gly 465 Asn Tyr Met Gly Gly 210 Giu Gly Al a Asp Pro 290 Pro Asn Glu Pro Thr 370 Lys His Ala Lys Pro 450 Ser Lys Pro Ile Glu 195 Gly Asp His Pro Asp 275 Thr Asp Phe Arg Met 355 Ala His Ile Leu Tyr 435 Lys Phe Tyr Lys Leu 180 Gly Asp Leu Ala Phe 260 Arg Lys Lys Asp Trp 340 Pro Tyr Trp Lys Phe 420 Tyr Asn Met Trp Ser 500 Phe Gly Thr As n Leu 245 Tyr Arg Met Pro Thr 325 Phe Ile Glu Val1 Glu 405 T rp Arg Ile Gly Lys 485 Ala Giu Leu Phe 215 Asn Leu Trp Ile Pro 295 As n Ala Arg Gin Lys 375 Asp Gly Pro Asn Val1 455 Asp Asn Arg Gly Al a 200 Asp Asp Giu Met Gin 280 Gin Pro Met Val1 Phe 360 Asp Glu Arg Asn Glu 440 Trp Glu Asn Asp His Ala Ala Phe Ser 250 Thr Leu Arg Tyr Arg 330 Asn Arg Lys Ser Leu 410 Lys Phe Giy Phe Lys 490 Met Ser Pro 205 Trp Ala Pro Phe Ser 285 Ser Asn Met Val1 Pro 365 Phe Pro Asp Phe Val1 445 Giu Phe Val1 Pro Thr 190 Gly Thr Val1 Ser Vali 2 Lys Arg Ile Phe Met 350 Ala Phe Gly Lys Phe 430 Asp Ser Tyr Glu Ser 510 Pro Pro Val His Asp 255 Leu Ser Pro cys Val1 335 Asp Ser Lys Tyr Ile 415 Arg Ser Pro Lys Pro 495 Gly Phe Asn Gin Giu 240 Ile Pro Gly Ser Asp 320 Phe Gly Ile Gly Pro 400 Asp Gly Giu Arg Gly 480 Gly Gly Arg Pro Asp Glu Gly Thr Glu Glu 515 520 Val Asp Glu Glu Gly Ser Gly Ala 530 535 Pro Val Leu Leu Leu Leu Leu Val 545 550 Phe Phe Arg Arg His Gly Thr Pro 565 Ser Leu Leu Asp Lys Val 580 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 582 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Mus cookii Glu Thr Glu Val lie Ile lie Glu 525 Val Ser Ala Ala Ala Val Val Leu 540 Leu Ala Val Gly Leu Ala Val Phe 555 560 Lys Arg Leu Leu Tyr Cys Gin Arg 570 575 (xi) Met 1 Thr Phe Asp Ile Tyr Asp Ala Gin SEQUENCE DESCRIPTION: SEQ ID NO: 7: Ser Pro Ala Pro Arg Pro Ser Arg Ser 5 10 Leu Gly Thr Ala Leu Ala Ser Leu Gly 25 Ser Pro Glu Ala Trp Leu Gin Gin Phe 40 Leu Arg Thr His Thr Gin Arg Ser Pro 55 Ala Ala Ile Gin Lys Phe Tyr Gly Leu 70 Ser Glu Thr Met Lys Ala Met Arg Arg 90 Lys Phe Gly Thr Glu Ile Lys Ala Asn 100 105 Ile Gin Gly Leu Lys Trp Gin His Asn 115 120 Asn Tyr Thr Pro Lys Val Gly Glu Tyr 130 135 Leu Ala Tyr Thr Val Arg Arg Ile Thr 140 Leu Gin Leu Leu Thr Cys Arg Thr 125 Phe Pro Gly Pro Ser Gly Gly Lys 110 Phe Glu Leu Ser Arg Val Lys Val Arg Cys Ala Leu Asn Gly Asp Ala Pro Tyr Ile Ile Arg 145 Glu Ile Asp Ile Asn 225 Leu Met Asp Ser Val 305 Gly Lys Tyr Asn Asp 385 Asn Thr Asn Tyr Gly 465 Lys Val Met Gly Gly 210 Glu Gly Ser Asp Pro 290 Pro Asn Glu Pro Thr 370 Lys His Ala Lys Pro 450 Ser Ala Pro Ile Glu 195 Gly Asp His Pro Asp 275 Thr Asp Phe Arg Met 355 Ala His Ile Leu Tyr 435 Lys Phe Phe Tyr Leu 180 Gly Asp Leu Ala Phe 260 Arg Lys Lys Asp Trp 340 Pro Tyr Trp Lys Phe 420 Tyr Asn Met Arg Ala 165 Phe Gly Thr Asn Leu 245 Tyr Arg Met Pro Thr 325 Leu Ile Glu Val Glu 405 Trp Arg Ile Gly Val 150 Tyr Pro Phe His Gly 230 Gly Gin Gly Pro Lys 310 Val Trp Gly Arg Cys 390 Leu Met Phe Lys Ser 470 Trp Ile Glu Leu Phe 215 Asn Leu Trp Ile Pro 295 Asn Ala Arg Gin Lys 375 Val Val Pro Asn Val 455 Asp Glu Arg Gly Ala 200 Asp Asp Glu Met Gin 280 Gin Pro Met Val Phe 360 Asp Glu Arg Asn Glu 440 Trp Glu Ser Glu Leu 185 His Ser Ile His Asp 265 Gin Pro Ala Leu Arg 345 Trp Gly Ala Gly Gly 425 Glu Glu Val Ala Thr 155 Gly His 170 His Gly Ala Tyr Ala Glu Phe Leu 235 Ser Asn 250 Thr Glu Leu Tyr Arg Thr Tyr Gly 315 Arg Gly 330 Asn Asn Arg Gly Thr Phe Ser Leu 395 Leu Pro 410 Lys Thr Phe Arg Gly Ile Phe Thr 475 Pro Glu Asp Phe Pro 220 Val Asp Asn Gly Thr 300 Pro Glu Gln Leu Val 380 Glu Ser Tyr Ala Pro 460 Tyr Leu Lys Ser Pro 205 Trp Ala Pro Phe Ser 285 Ser Asn Met Val Pro 365 Phe Pro Asp Phe Val 445 Glu Phe Arg Gin Thr 190 Gly Thr Va 1 Ser Val 270 Lys Arg Ile Phe Me t 350 Ala Phe Gly Lys Phe 430 Asp Ser Tyr Phe Ala 175 Pro Pro Val His Asp 255 Leu Ser Pro Cys Val 335 Asp Ser Lys Tyr Ile 415, Arg Ser Pro Lys Arg 160 Asp Phe Asn Gln Glu 240 Ile Pro Gly Ser Asp 320 Phe Gly Ile Gly Ala 400 Asp Gly Glu Arg Gly 480 Asn Lys Tyr Trp Asn Ls Ty Trp Phe Asn Asn Gin LeLsVaGl Leu Lys Val Glu Pro Gly 495 Tyr Pro Lys Arg Pro Asp 515 Val Asp Glu Ser 500 Glu Leu Arg Asp Gly Cys Pro Gly Thr Glu Glu 520 Ala Thr Giu Val Ser Gly Gly 510 Ile Ile Glu Val Val Leu Glu Gly Ser 530 Pro Val Gly S35 Leu Val Ser Ala Al a 540 Gly Leu Leu Leu Val Leu Ala Val1 555 Leu Leu Ala Val Phe 560 Arg Phe Arg Arg Thr Pro Lys Leu Tyr Cys Ser Leu Leu Asp Lys Val 580 ()INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 1792 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genornic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Oryctolagus cuniculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CTGAATTGAA CCATTCAGGA GAAGTTCGCA ATGAAGTTTC TTCTATTGAT

TGGGTCACTT

TACTTGGAAA

AACAGGAACT

ACTGGGCAAC

GATGTTTATC

TACAGAATCA

AAAGCTTTTC

AAGGCTGACA

GGCAGAGGTG

CTTCTGGAGC

ACTTTTATGG

TCATAGAGGA

TGGACACATC

ATTTCAAAAC

AAAATTACAC

AAGTATGGAG

TCATGATACT

GTGTCATAGC

TGATCCTCTG

CCTTAAGGTG

AAAAGTCCAG

TACTTTGGAA

CATGCCAGGG

TCCAGACATG

CGATGTGACC

TTTTGCTAGT

CCATGCTTTT

AAGGAAAACG

GAGAGAATTC

GAAATGCAGC

ATGATGCACA

AGACCAGTAT

AAGCGTGAGG

CCCCTGAAAT

GGAGCTCATG

GGGCCTGGAC

ATATGCTATT

CAATGACAAA

AATTCTTGGG

AGCCTCGATG

GGAGGAAACA

ATGTTGAGTA

TCAGAAAGAT

GAGACTATGG

CTGGTATTGG

ACTGACCCTG

TGCTGAAAAC

AATGAAAACT

GCTAAATGTG

TGGAGTGCCT

TTACATCACC

TGCCATTCAG

TACGACAGGC

TGCTTTTGAT

AGGAGATACA

CATTTTGATG

GCTGTCCATG

ATGTTTCCCA

CGTGGCATTC

AATCCGGAAC

AATAAA-ATAT

ACCAGTGTCC

TATGAAATTG

AGCCATCTAA

TTTGTGAAAA

CTGGATAATC

AAGCTGATCA

CAAAGATACT

CGTGTCACCA

ACTTCCACTT

TGATGTGTAT

ATATAAGTTT

ATTTGGAAAT

TGGTTTCTAC

AGGATGAAAT

AGCTTGGCCA

CCTATGGTTA

AGTCCCTTTA

CAACTGCCTG

TTTTCTTTAA

GTTTAATTTC

GAGACAGACA

GACTACAACC

AAATTGATGC

TGTACTGGAG

CCAAGCACTT

ACTATTTCTT

AGAAGCTGAA

AATAAGTATT

CATAAAATAA

TTCAATTTTG

AGATGCTTTC

CATTGTTTGA

CTGGAGTAAA

TGCCTTGGGA

TATTGATCTC

TGGAGGCCCA

TGACCACAAT

AGACAGCTTT

TTCCTTATGG

TCAAGTATTC

AAACTATCCC

AGCTGTCTTT

ATACGATGAA

CCCAGGAATT

CCAGGGACCT

AAGCAATAGC

TATTGCATAC

AGTAAAATAT

AAAACCCTTA

AGAGGCCAAG

GAAGTTACAA

AGTTATAAAG

CTTGATCATT

AACACATTTC

GAGCAGCATC

TTGAAATTTG

TTCTGGTGGA

CCAACCTTGC

CTTTTTAAAG

AAGAGCATAC

AACCCCAGTC

AGGAGAGAGG

GGGCCGAAAA

AAC CAACTTG

TGGTTTGATT

ATACTATGTG

ATAGATCATA.

TTGTACATTT

AGAGTATCTT

TTATATATTA

GCACAAACTT

CAAATGATCC

ACCTCTCTGC

AACCCATGCC

ATGCAGTTAC

AGATTCCTAA

CTTCAGGCAT

GTGACAAGTT

ATTCCCTGGG

TCCGGAAGAC

TCATGGATGC

TTGACGCAGT

AATATGACAC

GCTAGTAATG

ATCAATGTAA

GAGAAGTGAT

TTGCTTAACT

TTGTAGAATG

TTCAAATAAA

GTTCCTTGTT

AAAGGCCATA

TGATGACATA

AAAACCTGAC

TACAGTGGGA

GAGTTCAACG

TGAGGCTGCT

CTGGTTAATT

CTTCCCTGAC

CTACTTCTTT

TGGTTATCCC

CTTCTATTTC

ATTTTCCAGT

GTGCCAGTTG

CACTACATGG

TGTACCAAAT

CTACTATTAA

CTTTGTGAGT

AAATTCAAAT

660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1792 AAATTATATA TTATTCAAAT AAAAACTTTG AAGAAAAAAA AAAAAAAAAA AA INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 464 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Oryctolagus cuniculus (xi) Met 1 SEQUENCE DESCRIPTION: SEQ ID NO: 9: Lys Phe Leu Leu Leu Ile Leu Thr Leu Trp Val Thr Ser Ser Gly 5 10 Ala Asp Pro Leu Lys Glu Asn Asp Giu Lys Phe Met Thr Ile Ile Arg 145 Gly Al a Asp Leu Asn 225 Asn Tyr Giu Val1 Ile 305 Pro Asn Thr Leu Met Met Lys Gin 130 Lys Ala His Giu Val1 210 Asp Thr Gly Pro Gly 290 Pro Thr Phe Asn Gly His Pro Asn 115 Lys Ile His Ala Asp 195 Ala Pro Phe Gly Thr 275 Asn Lys Leu Tyr Arg Leu Lys Gly 100 Tyr Ala Thr Gly Phe 180 Giu Val1 Lys His Pro 260 Ala Lys Ser Pro Gly Asn Asn Pro Arg Thr Phe Thr Asp 165 Gly Ile His Ala Leu 245 Giu Cys Ile Ser Ser 325 Leu Phe Val1 70 Arg Pro Pro Gin Gly 150 Tyr Pro Trp Giu Ile 230 Ser Gin Asp Phe Thr 310 Gly Lys Ile 55 Thr Cys Val1 Asp Val1 135 Lys Gly Gly Ser Leu 215 Met Al a His His Phe 295 Thr Ile Val1 40 Giu Gly Gly Trp Met 120 Trp Al a Ala Pro Lys 200 Gly Phe Asp Gin Asn 280 Phe Ser Glu Met 25 Giu Giu Gin Val1 Arg 105 Lys Ser Asp Phe Gly 185 S er His Pro Asp Pro 265 Leu Lys Val Al a Leu Arg Lys Leu Pro 90 Lys Arg Asp Ile Asp 170 Ile Tyr Ala Thr Ile 250 Met Lys Asp Arg Ala 330 Phe Ile Val Asp 75 Asp His Giu Val1 Met 155 Gly Gly Lys Leu Tyr 235 Arg Pro Phe Ser Leu 315 Tyr Ala Pro Gin Thr Val1 Tyr Asp Tim 140 Ile Arg Gly Gly Gly 220 Gly Gly Lys Asp Phe 300 Ile Giu Giu Met Giu Ser Tyr Ile Val1 125 Pro Leu Gly Asp Thr 205 Leu Tyr Ile Pro Al a 285 Phe Ser Ile Asn Tim Met Thr His Thr 110 Giu Leu Phe Gly Thr 190 Asn Asp Ile Gin Asp 270 Val1 Trp Ser Gly Tyr Lys Gin Leu Phe Tyr Tyr Lys Al a Val1 175 His Leu His Asp Ser Asn Thr Trp Leu Asp 335 Leu Met Gin Glu Lys Arg Ala Phe Ser 160 Ile Phe Phe Ser Leu 240 Leu Pro Thr Lys Trp 320 Arg His Gin Val Phe Leu Phe Lys Gly Asp Lys Phe Trp Leu Ile Ser His 350 Leu Arg Leu Gin Pro Asn Tyr Pro Lys Ser Ile His Ser Leu Gly Phe 355 360 365 Pro Asp Phe Val Lys Lys Ile Asp Ala Ala Val Phe Asn Pro Ser Leu 370 375 380 Arg Lys Thr Tyr Phe Phe Val Asp Asn Leu Tyr Trp Arg Tyr Asp Glu 385 390 395 400 Arg Arg Glu Val Met Asp Ala Gly Tyr Pro Lys Leu Ile Thr Lys His 405 410 415 Phe Pro Gly Ile Gly Pro Lys Ile Asp Ala Val Phe Tyr Phe Gin Arg 420 425 430 Tyr Tyr Tyr Phe Phe Gin Gly Pro Asn Gin Leu Glu Tyr Asp Thr Phe 435 440 445 Ser Ser Arg Val Thr Lys Lys Leu Lys Ser Asn Ser Trp Phe Asp Cys 450 455 460 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 470 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Lys Phe Leu Leu Ile Leu Leu Leu Gin Ala Thr Ala Ser Gly Ala 1 5 10 Leu Pro Leu Asn Ser Ser Thr Ser Leu Glu Lys Asn Asn Val Leu Phe 25 Gly Glu Arg Tyr Leu Glu Lys Phe Tyr Gly Leu Glu Ile Asn Lys Leu 40 Pro Val Thr Lys Met Lys Tyr Ser Gly Asn Leu Met Lys Glu Lys Ile 55 Gin Glu Met Gin His Phe Leu Gly Leu Lys Val Thr Gly Gin Leu Asp 70 75 Thr Ser Thr Leu Glu Met Met His Ala Pro Arg Cys Gly Val Pro Asp 90 Leu His His Phe Arg Glu Met Pro Gly Gly Pro Val Trp Arg Lys His 100 105 110 Tyr Asp Thr 145 Val1 Lys Gly Gly Gly 225 Lys Gly Asn Asp Phe 305 Ile Giu Trp His Phe 385 Trp Leu Phe Ile Val1 130 Pro Val1 Gly Asp Thr 210 Leu Tyr Ile Pro Al a 290 Phe Ser Ile Leu Ser 370 Asn Arg Ile Tyr Thr 115 Asp Leu Phe Gly Al a 195 Asn Gly Val1 Gin Asp 275 Val1 Trp Ser Giu Ile 355 Phe Pro Tyr Thr Ser 435 Tyr Tyr Lys Aia Ile 180 His Leu His Asp Ser 260 Asn Thr Leu Leu Ala 340 Ser Giy Arg Asp Lys 420 Lys Arg Ala Phe Arg 165 Leu Phe Phe Ser Ile 245 Leu Ser Thr Lys Trp 325 Arg Asn Phe Phe Giu 405 Asn Asn Ile Ile Ser 150 Gly Ala Asp Leu Ser 230 Asn Tyr Giu Val1 Val1 310 Pro Asn Leu Pro Tyr 390 Arg Phe Lys Asn Arg 135 Lys Ala His Giu Thr 215 Asp Thr Gly Pro Gly 295 Ser Thr Gin Arg Asn 375 Arg Arg Gin Tyr Asn 120 Lys Ile His Ala Asp 200 Ala Pro Phe Asp Ala 280 Asn Giu Leu Val1 Pro 360 Phe Thr Gin Gly Tyr 440 Tyr Ala Asn Gly Phe 185 Glu Val1 Lys Arg Pro 265 Leu Lys Arg Pro Phe 345 Glu Val1 Tyr Met Ile 425 Tyr Pro Gin Gly 155 Phe Pro Trp Glu Val1 235 Ser Giu Asp Phe Lys 315 Gly Phe Asn Lys Phe 395 Asp Pro Phe Met 125 Trp Ala Al a Ser Thr 205 Gly Phe Asp Gin Asn 285 Phe Ser Giu Asp Pro 365 Asp Asp Gly Ile Gly 445 Asn Ser Asp Phe Gly 190 His His Pro Asp Arg 270 Leu Lys Val1 Al a Asp 350 Lys Al a Asn Tyr Asp 430 Ser Arg Glu Asn Val le -Leu 160 Asp Gly 175 Ile Gly Ser Gly Ser Leu Thr Tyr 240 Ile Arg 255 Leu Pro Ser Phe Asp Arg Asn Leu 320 Ala Tyr 335 Lys Tyr Ser Ile Ala Vai Gin Tyr 400 Pro Lys 415 Ala Val Asn Gin 72 Phe Glu Tyr Asp Phe Leu Leu Gin Arg Ile Thr Lys Thr Leu Lys Ser 450 455 460 Asn Ser Trp Phe Gly Cys 465 470 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 465 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Rattus norvegicus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Lys Phe Leu Leu Val Leu Val Leu Leu Val Ser Leu Gln Val Ser 1 5 10 Ala Cys Gly Ala Ala Pro Met Asn Glu Ser Glu Phe Ala Glu Trp Tyr 25 Leu Ser Arg Phe Phe Asp Tyr Gin Gly Asp Arg Ile Pro Met Thr Lys 40 Thr Lys Thr Asn Arg Asn Leu Leu Glu Glu Lys Leu Gin Glu Met Gin 55 Gin Phe Phe Gly Leu Glu Val Thr Gly Gin Leu Asp Thr Ser Thr Leu 70 75 Lys Ile Met His Thr Ser Arg Cys Gly Val Pro Asp Val Gin His Leu 90 Arg Ala Val Pro Gin Arg Ser Arg Trp Met Lys Arg Tyr Leu Thr Tyr 100 105 110 Arg Ile Tyr Asn Tyr Thr Pro Asp Met Lys Arg Ala Asp Val Asp Tyr 115 120 125 Ile Phe Gin Lys Ala Phe Gin Val Trp Ser Asp Val Thr Pro Leu Arg 130 135 140 Phe Arg Lys Ile His Lys Gly Glu Ala Asp Ile Thr Ile Leu Phe Ala 145 150 155 160 Phe Gly Asp His Gly Asp Phe Tyr Asp Phe Asp Gly Lys Gly Gly Thr 165 170 175 Leu Ala His Ala Phe Tyr Pro Gly Pro Gly Ile Gin Gly Asp Ala His 180 185 190 73 Phe Asp Glu Ala Glu Thr Trp Thr Lys Ser Phe Gin Gly Thr Asn Leu Phe Ser 225 Pro Leu Pro Thr Arg 305 Trp Arg Asn Phe Arg 385 Val His Arg Leu 210 Asn Asn Tyr Pro Val 290 Leu Pro Asn Leu Pro 370 Gin Arg Phe His Val Asn Thr Gly Ser 275 Gly Pro Thr Gin Val 355 Ala Lys Gin Pro Tyr Ala Pro Phe Ala 260 Thr Asp Gly Ile Leu 340 Pro Ser Val Glu Gly 420 Tyr Val Lys Arg 245 Pro Val Lys Ser Pro 325 Phe Glu Val Tyr Leu 405 Ile Ile His Ser 230 Leu Val Cys Ile Pro 310 Ser Leu Pro Lys Phe 390 Met Arg Phe Glu Leu 215 Ile Met Ser Ala Lys Asn His Gin 280 Phe Phe 295 Ala Thr Gly Ile Phe Lys His Tyr 360 Lys Ile 375 Phe Val Asp Ala Pro Lys Gin Gly 440 Lys Thr 455 Gly Tyr Asp Pro 265 Ser Phe Asn Gin Asp 345 Pro Asp Asp Ala Ile 425 Ala Leu His Pro Asp 250 Ser Leu Lys Ile Ala 330 Glu Arg Ala Lys Tyr 410 Asp Tyr Ser Ser Thr 235 Ile Leu Ser Asp Thr 315 Ala Lys Ser Ala Gin 395 Pro Ala Gin Ser Leu 220 Tyr His Thr Phe Trp 300 Ser Tyr Tyr Ile Val 380 Tyr Lys Val Leu Thr 460 205 Gly Arg Ser Asn Asp 285 Phe Ile Glu Trp His 365 Phe Trp Leu Leu Glu 445 Ser Leu Tyr Ile Pro 270 Ala Phe Ser Ile Leu 350 Ser Asp Arg Ile Tyr 430 Tyr Trp Arg Leu Gin 255 Gly Val Trp Ser Gly 335 Ile Leu Pro Tyr Ser 415 Phe Asp Phe His His 240 Ser Ser Thr Trp Met 320 Gly Asn Gly Leu Asp 400 Thr Lys Pro Gly 435 Leu Leu Asp Arg Val Thr INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 462 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 74 (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Met Lys Phe Leu Met Met Ile Val Phe Leu Gin Val Ser Ala Cys Gly 1 5 10 Ala Ala Pro Met Asn Asp Ser Glu Phe Ala Glu Trp Tyr Leu Ser Arg 25 Phe Tyr Asp Tyr Gly Lys Asp Arg Ile Pro Met Thr Lys Thr Lys Thr 40 Asn Arg Asn Phe Leu Lys Glu Lys Leu Gin Glu Met Gin Gin Phe Phe 55 Gly Leu Glu Ala Thr Gly Gin Leu Asp Asn Ser Thr Leu Ala Ile Met 70 75 His Ile Pro Arg Cys Gly Val Pro Asp Val Gin His Leu Arg Ala Val 90 Pro Gin Arg Ser Arg Trp Met Lys Arg Tyr Leu Thr Tyr Arg Ile Tyr 100 105 110 Asn Tyr Thr Pro Asp Met Lys Arg Glu Asp Val Asp Tyr Ile Phe Gin 115 120 125 Lys Ala Phe Gin Val Trp Ser Asp Val Thr Pro Leu Arg Phe Arg Lys 130 135 140 Leu His Lys Asp Glu Ala Asp Ile Met Ile Leu Phe Ala Phe Gly Ala 145 150 155 160 His Gly Asp Phe Asn Tyr Phe Asp Gly Lys Gly Gly Thr Leu Ala His 165 170 175 Val Phe Tyr Pro Gly Pro Gly Ile Gin Gly Asp Ala His Phe Asp Glu 180 185 190 Ala Glu Thr Trp Thr Lys Ser Phe Gin Gly Thr Asn Leu Phe Leu Val 195 200 205 Ala Val His Glu Leu Gly His Ser Leu Gly Leu Gin His Ser Asn Asn 210 215 220 Pro Lys Ser Ile Met Tyr Pro Thr Tyr Arg Tyr Leu Asn Pro Ser Thr 225 230 235 240 Phe Arg Leu Ser Ala Asp Asp Ile Arg Asn Ile Gin Ser Leu Tyr Gly 245 250 255 Ala Pro Val Lys Pro Pro Ser Leu Thr Lys Pro Ser Ser Pro Pro Ser 260 265 270 Thr Phe Cys His Gin Ser Leu Ser Phe Asp Ala Val Thr Thr Val Gly 275 280 285 Glu Lys Ile Leu Phe Phe Lys Asp Trp Phe Phe Trp Trp Lys Leu Pro 290 295 300 Gly Ser Pro Ala Thr Asn Ile Thr Ser Ile Ser Ser Ile Trp Pro Ser 305 310 315 320 Ile Pro Ser Ala Ile Gin Ala Ala Tyr Glu Ile Glu Ser Arg Asn Gin 325 330 335 Leu Phe Leu Phe Lys Asp Glu Lys Tyr Trp Leu Ile Asn Asn Leu Val 340 345 350 Pro Glu Pro His Tyr Pro Arg Ser Ile Tyr Ser Leu Gly Phe Ser Ala 355 360 365 Ser Val Lys Lys Val Asp Ala Ala Val Phe Asp Pro Leu Arg Gin Lys 370 375 380 Val Tyr Phe Phe Val Asp Lys His Tyr Trp Arg Tyr Asp Val Arg Gin 385 390 395 400 Glu Leu Met Asp Pro Ala Tyr Pro Lys Leu Ile Ser Thr His Phe Pro 405 410 415 Gly Ile Lys Pro Lys Ile Asp Ala Val Leu Tyr Phe Lys Arg His Tyr 420 425 430 Tyr Ile Phe Gin Gly Ala Tyr Gin Leu Glu Tyr Asp Pro Leu Phe Arg 435 440 445 Arg Val Thr Lys Thr Leu Lys Ser Thr Ser Trp Phe Gly Cys 450 455 460 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Oryctolagus cuniculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Arg Ser Gly Ala Pro Val Asp Gin Met Phe Pro Gly Val Pro Leu 1 5 10 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:1 OTHER INFORMATION:/product= "X is Mca-P" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/product= "X is Dpa-A" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Xaa Leu Gly Leu Xaa Arg 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:1 OTHER INFORMATION:/product= "X is Abz-G" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:6 OTHER INFORMATION:/product= "X is Lnor" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:9 OTHER INFORMATION:/product= "X is Y(NO2)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Xaa Pro Leu Gly Leu Xaa Ala Arg Xaa INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:1 OTHER INFORMATION:/product= "X is Abz-S" (ix) FEATURE: NAME/KEY: Modified-site OTHER INFORMATION:/product= "X is hydroxyproline" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:9 OTHER INFORMATION:/product= "X is Y (NO2)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Xaa Lys Tyr Pro Xaa Ala Leu Phe Xaa Asp 1 5 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:1 OTHER INFORMATION:/product= "x is Y(NO2)" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:4 OTHER INFORMATION:/product= "x is J" (ix) FEATURE: NAME/KEY: Modified-site LOCATION:8 OTHER INFORMATION:/product= "x is K(Abz)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Xaa Pro Leu Xaa Met Lys Gly Xaa Gly 1 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS.: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Pro Arg Cys Gly Val Pro Asp 1 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Arg Arg Lys Arg Tyr Ala 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:3..8 OTHER INFORMATION:/product= "each x is variable" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gly Asp Xaa His Phe Asp Xaa Xaa'Glu 1 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21': Cys Asp Gly Asn Phe Asp Thr Val Ala Met Leu Arg Gly Glu Met 1 5 10 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Oryctolagus cuniculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2f: CGGGATCCCT GTGGGTCACT TCTTCT 26 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: ORGANISM: Oryctolagus cuniculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CCGCTCGAGC TGGCACCATT ACTAGC 26 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:4 OTHER INFORMATION:/product= "X is K (Abz)-PEGA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Leu Phe Phe Xaa 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:1 OTHER INFORMATION:/product= "X is Abz-G" (ix) FEATURE: NAME/KEY: Cleavage-site (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Xaa Pro Leu Gly Leu Xaa Ala Arg 1 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:4 OTHER INFORMATION:/product= "X J" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26': Tyr Pro Leu Xaa Met Lys Gly Lys Gly 1 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:2..6 82 OTHER INFORMATION:/product= "each X =J" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Asn Xaa Tyr Pro Xaa Xaa Tyr Lys Gly 1 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: Modified-site LOCATION:3..8 OTHER INFORMATION:/product= "each X J" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Tyr Pro Xaa Xaa Met Lys Gly Xaa Gly 1 INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29;: TGGTATGTGG TCTGTGT INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 83 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGTGGTTCAG TTGTGGT 17 INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: ACCACAACTG AACCACA 17 INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GGACTCATGG TGAGGAC 17 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 84 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CGGATACAGG TGTCGGA 17 INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: GTCTGGGGCT CATGGTGA 18 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGCTCATGGT GAGG 14 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GGGCTCATGG TGAGGGA 17 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: CTCATGGTGA GGGGAGCA 18 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: ATGGTGAGGG GAGCAGCG 18 INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: AGGTGAGTGG CGTCACCG 18 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCTGTCAAAG TTGGAAGT 18 INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: GGCCTCTACC GCAACTGC 18 INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 87 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: GGCCTCTAGG GGAACTGC 18

Claims (56)

1. The use of an agent which acts by inhibition of the production or action of a membrane associated protease or the matrix metalloprotease MMP-12 involved in the resorptive activity of osteoclasts for the manufacture of a medicament for the treatment of bone metabolic disease.

2. The use claimed in claim 1, wherein the agent acts by inhibition of the production or action of a membrane-type matrix metalloproteinase (MT-MMP) or the matrix metalloproteinase MMP-12 involved in the resorptive activity of osteoclasts.

3. The use claimed in claim 2, wherein a protease is inhibited which is involved in the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, fusion, attachment, polarisation, activity in removal of mineralised osseous substance, or survival of osteoclasts.

4. The use claimed in any preceding claim, wherein the agent is an antibody selectively immunoreactive with a said protease. The use claimed in any one of claims 1 to 3, wherein the agent is an antisense oligonucleotide or oligonucleotide analogue directed against a gene involved in the production of a said protease.

6. The use claimed in any one of claims 1 to 5, wherein said protease is encoded by a gene which comprises a nucleotide sequence encoding an amino acid sequence having more than homology with the amino acid sequence set forth in SEQ ID NO. 4. 20 7. The use claimed in claim 6, wherein the gene comprises a nucleotide sequence encoding an amino acid sequence having more than 90% homology with the amino acid sequence set forth in SEQ ID NO. 4.

8. The use claimed in any one of claims 1 to 5, wherein said protease is encoded by a gene Swhich comprises a nucleotide sequence having more than 50% homology with the nucleotide 25 sequence set forth in SEQ ID NO. 8.

9. The use claimed in claim 8, wherein the gene comprises a nucleotide sequence having more than 90% homology with the nucleotide sequence set forth in SEQ ID NO. 8.

10. The use claimed in any one of claims 1 to 5, wherein the protease which is the subject of :i the inhibition has the amino acid sequence as set forth in SEQ ID NO.

11. The use claimed in any one of claims 1 to 5, wherein the protease which is the subject of the inhibition has the amino acid sequence as set forth in SEQ ID NO.

12. The use claimed in any one of claims 1 to 3, wherein the agent is a protease substrate mimic inhibitor.

13. The use claimed in any one of claims 1 to 3, wherein the agent is a broad spectrum matrix metalloproteinase (MMP) inhibitor or a broad spectrum membrane-associated metalloproteinase inhibitor.

14. The use claimed in any one of claims 1 to 3, wherein the agent is a selective inhibitor of MT1-MMP or MMP-12. The use claimed in any one of claims 1 to 3, wherein the agent is a selective inhibitor of 0, a protease having more than 75% homology with the amino acid sequence set forth in SEQ ID NO.4. C00281#.doc 89

16. The use claimed in claim 15, wherein said protease has more than 90% homology with the amino acid sequence set forth in SEQ ID NO. 4.

17. The use claimed in any one of claims 1 to 3, wherein the agent is a selective inhibitor of a protease encoded by a nucleotide sequence having more than 50% homology with the nucleotide sequence set forth in SEQ ID NO. 8.

18. The use claimed in claim 17, wherein the agent is a selective inhibitor of a protease comprising an amino acid sequence having more than 90% homology with the nucleotide sequence set forth in SEQ ID NO. 8.

19. The use claimed in any one of claims 1 to 3, wherein the agent is a selective inhibitor of a protease having the amino acid sequence as set forth in SEQ ID NO. The use claimed in any one of claims 1 to 3, wherein the agent is a selective inhibitor of a protease having the amino acid sequence as set forth in SEQ ID NO.

21. The use claimed in any one of claims 1 to 3, wherein the agent is a selective inhibitor of a specific member of one of the families of membrane-associated metalloproteinase.

22. The use claimed in claim 21, wherein the metalloproteinase is a meltrin or an ADAM. .i 23. The use claimed in any one of claims 1 to 3, wherein the agent is a peptide or peptide analogue obtained by screening a peptide library for peptides reactive with a said protease.

24. The use of an agent in the manufacture of a medicament for the treatment of bone metabolic disease by inhibition of the recruitment, proliferation, differentiation, or migration of 20 osteoclast precursor cells or in the migration, fusion, attachment, polarisation, or survival of osteoclasts. The use claimed in claim 24, wherein said agent produces said inhibition by inhibiting the production or action of a proteinase.

26. A method for the treatment of bone metabolic disease in a mammal requiring said treatment, which method includes or consists of administering to said mammal an effective amount of at least one agent which acts by inhibition of the production or action of a membrane associated protease or the matrix metalloprotease MMP-12 involved in the resorptive activity of osteoclasts.

27. The method claimed in claim 26, wherein the agent acts by inhibition of the production or action of a membrane-type matrix metalloproteinase (MT-MMP) or the matrix metalloproteinase 30 MMP-12 involved in the resorptive activity of osteoclasts.

28. The method claimed in claim 27, wherein a protease is inhibited which is involved in the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, fusion, attachment, polarisation, activity in removal of mineralised osseous substance, or survival of osteoclasts.

29. The method claimed in any one of claims 26 to 28, wherein the agent is an antibody selectively immunoreactive with a said protease. The method claimed in any one of claims 26 to 28, wherein the agent is an antisense oligonucleotide or oligonucleotide analogue directed against a gene involved in the production of a said protease. 40 31. The method claimed in any one of claims 26 to 30, wherein said protease is encoded by -b A"^a gene which comprises a nucleotide sequence encoding an amino acid sequence having more than "Y 5% homology with the amino acid sequence set forth in SEQ ID NO. 4. C00281#.doc

32. The method claimed in claim 31, wherein the gene comprises a nucleotide sequence encoding an amino acid sequence having more than 90% homology with the amino acid sequence set forth in SEQ ID NO. 4.

33. The method claimed in any one of claims 26 to 30, wherein said protease is encoded by a gene which comprises a nucleotide sequence having more than 50% homology with the nucleotide sequence set forth in SEQ ID NO. 8.

34. The method claimed in claim 33, wherein the gene comprises a nucleotide sequence having more than 90% homology with the nucleotide sequence set forth in SEQ ID NO. 8. The method claimed in any one of claims 26 to 30, wherein the protease which is the subject of the inhibition has the amino acid sequence as set forth in SEQ ID NO.

36. The method claimed in any one of claims 26 to 30, wherein the protease which is the subject of the inhibition has the amino acid sequence as set forth in SEQ ID NO.

37. The method claimed in any one of claims 26 to 28, wherein the agent is a protease substrate mimic inhibitor.

38. The method claimed in any one of claims 26 to 28, wherein the agent is a broad spectrum matrix metalloproteinase (MMP) inhibitor or a broad spectrum membrane-associated metalloproteinase inhibitor.

39. The method claimed in any one of claims 26 to 28, wherein the agent is a selective :o inhibitor of MT1-MMP or MMP-12. 20 40. The method claimed in any one of claims 26 to 28, wherein the agent is a selective inhibitor of a protease having more than 75% homology with the amino acid sequence set forth in SEQ ID NO.4.

41. The method claimed in claim 40, wherein said protease has more than 90% homology with the amino acid sequence set forth in SEQ ID NO. 4. 25 42. The method claimed in any one of claims 26 to 28, wherein the agent is a selective inhibitor of a protease encoded by a nucleotide sequence having more than 50% homology with the nucleotide sequence set forth in SEQ ID NO. 8.

43. The method claimed in claim 42, wherein the agent is a selective inhibitor of a protease comprising an amino acid sequence having more than 90% homology with the nucleotide sequence 30 set forth in SEQ ID NO. 8.

44. The method claimed in any one of claims 26 to 28, wherein the agent is a selective inhibitor of a protease having the amino acid sequence as set forth in SEQ ID NO. The method claimed in any one of claims 26 to 28, wherein the agent is a selective inhibitor of a protease having the amino acid sequence as set forth in SEQ ID NO.

46. The method claimed in any one of claims 26 to 28, wherein the agent is a selective inhibitor of a specific member of one of the families of membrane-associated metalloproteinase.

47. The method claimed in claim 46, wherein the metalloproteinase is a meltrin or an ADAM.

48. The method claimed in any one of claims 26 to 28, wherein the agent is a peptide or peptide analogue obtained by screening a peptide library for peptides reactive with a said protease.

49. A method for the treatment of bone metabolic disease, which method includes or A ."nsists of administering to said mammal an effective amount of an agent mediating inhibition of the C00281#.doc 91 recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, fusion, attachment, polarisation, or survival of osteoclasts. The method claimed in claim 49, wherein said agent produces said inhibition by inhibiting the production or action of a proteinase.

51. An agent which acts by inhibition of the production or action of a membrane associated protease or the matrix metalloprotease MMP-12 involved in the resorptive activity of osteoclasts, when used in the treatment of bone metabolic disease.

52. The agent when used as claimed in claim 51, wherein the agent acts by inhibition of the production or action of a membrane-type matrix metalloproteinase (MT-MMP) or the matrix o1 metalloproteinase MMP-12 involved in the resorptive activity of osteoclasts.

53. The agent when used as claimed in claim 52, wherein a protease is inhibited which is involved in the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, fusion, attachment, polarisation, activity in removal of mineralised osseous substance, or survival of osteoclasts.

54. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is an antibody selectively immunoreactive with a said protease.

55. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is an antisense oligonucleotide or oligonucleotide analogue directed against a gene involved in the production of a said protease. 20 56. The agent when used as claimed in any one of claims 51 to 55, wherein said protease is encoded by a gene which comprises a nucleotide sequence encoding an amino acid sequence having more than 75% homology with the amino acid sequence set forth in SEQ ID NO. 4.

57. The agent when used as claimed in claim 56, wherein the gene comprises a nucleotide sequence encoding an amino acid sequence having more than 90% homology with the amino acid 25 sequence set forth in SEQ ID NO. 4.

58. The agent when used as claimed in any one of claims 51 to 55, wherein said protease is encoded by a gene which comprises a nucleotide sequence having more than 50% homology with the nucleotide sequence set forth in SEQ ID NO. 8.

59. The agent when used as claimed in claim 58, wherein the gene comprises a nucleotide 0 sequence having more than 90% homology with the nucleotide sequence set forth in SEQ ID NO. 8. The agent when used as claimed in any one of claims 51 to 55, wherein the protease which is the subject of the inhibition has the amino acid sequence as set forth in SEQ ID NO.

61. The agent when used as claimed in any one of claims 51 to 55, wherein the protease which is the subject of the inhibition has the amino acid sequence as set forth in SEQ ID NO.

62. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a protease substrate mimic inhibitor.

63. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a broad spectrum matrix metalloproteinase (MMP) inhibitor or a broad spectrum membrane-associated metalloproteinase inhibitor.

64. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a N. selective inhibitor of MT1-MMP or MMP-12. C00281#.doc 92 The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a selective inhibitor of a protease having more than 75% homology with the amino acid sequence set forth in SEQ ID NO.4.

66. The agent when used as claimed in claim 65, wherein said protease has more than homology with the amino acid sequence set forth in SEQ ID NO. 4.

67. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a selective inhibitor of a protease encoded by a nucleotide sequence having more than 50% homology with the nucleotide sequence set forth in SEQ ID NO. 8.

68. The agent when used as claimed in claim 67, wherein the agent is a selective inhibitor of a protease comprising an amino acid sequence having more than 90% homology with the nucleotide sequence set forth in SEQ ID NO. 8.

69. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a selective inhibitor of a protease having the amino acid sequence as set forth in SEQ ID NO. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a selective inhibitor of a protease having the amino acid sequence as set forth in SEQ ID NO.

71. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a selective inhibitor of a specific member of one of the families of membrane-associated metalloproteinase. 2 72. The agent when used as claimed in claim 71, wherein the metalloproteinase is a meltrin or an ADAM.

73. The agent when used as claimed in any one of claims 51 to 53, wherein the agent is a peptide or peptide analogue obtained by screening a peptide library for peptides reactive with a said protease. •74. An agent which inhibits the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, fusion, attachment, polarisation, or survival of osteoclasts, when used for the treatment of bone metabolic disease.

75. The agent when used as claimed in claim 69, wherein said agent produces said inhibition :by inhibiting the production or action of a proteinase. 30 Dated 20 February 2001 CENTER FOR CLINICAL AND BASIC RESEARCH Patent Attorneys for the Applicant/Nominated Person SPRUSON&FERGUSON C00281#.doc