A CONJUGATE OF A COLLAGEN II-BINDING FRAGMENT AND AN ARTHRITIS-AFFECTING PHARMACEUTICAL SUBSTANCE

Abstract: The invention provides a conjugate of a collagen II-binding chondroadherin fragment and an arthritis-affecting pharmaceutical substance, such as a cytokine, a cytokine antagonist and/or a protease inhibitor. It has turned out that chondroadherin specifically interacts with collagen II thereby forming a complex. Release of cartilage extracellular matrix components, such as chondroadherin and collagen II is a symptom of arthitis. By providing a conjugate according to the present invention it is therefore possible to direct arthitis-affecting pharmaceutical substances directly to the pathogenic tissue. The inventive conjugate can be manufactured by chemical crosslinking. In case the arthitis-affecting pharmaceutical substance is a protein or a peptide, the conjugate can be a fusion protein and be produced by genetic engineering techniques.

Description: A conj ugate of a collagen II-binding fragment and an arthrit is- affect ing pharmaceutical substanc e The present invention relates to a novel conjugate consisting of a collagen-binding fragment of chondroadherin covalently linked to a pharmaceutically interesting substance such as cytokines, cytokine-inhibiting substances and protease inhibitors. The invention also relates to pharmaceutical compositions containing the new conjugates. Technical background The major constituents of the extracellular matrix of cartilage are type II collagen, proteoglycans and a number of proteins including some of the minor collagens. The matrix macromolecules have very different structural and functional characteristics and each component contributes differently to the organisation and properties of the tissue. The matrix are assembled in multicomponent networks where the interactions between the matrix constituents are essential for the maintenance of tissue integrity (Heinegard et al., editors. Osteoarthritis. 1 ed. Oxford University Press; 1998; 7.2.1, Biochemistry and metabolism of normal and osteoarthritic cartilage, p. 74 - 84). One of the non-collagenous proteins in cartilage is chondroadherin. It was first isolated from bovine tracheal cartilage (Larsson et al., J. Biol. Chem. 1991; 266:20428-33). The protein and cDNA sequence of bovine chondroadherin (Neame et al., J. Biol. Chem. 1994;269:21547-54) showed that it belongs to the family of leucine-rich repeat (LRR) proteins (Patthy, J. Mol. Biol. 1987; 198:567-77). A subgroup of LRR proteins have several members in extracellular matrices and are characterised by a conserved sequence xLxxLxLxx(N,C,T)x(L,I) followed by 9 to 14 less conserved residues. This sequence may be repeated up to 30 times, while in the extracellular proteins it usually occurs 10 to 11 times and in other subgroups 5 - 6 times. The repeat domain is flanked by two cysteine-bonded loops and usually short extensions in the N-terminal and C-terminal regions of the protein. The crystal structure of one member of the LRR protein family, ribonuclease inhibitor, has been solved and refined at 2.5 A (Kobe et al., Nature 1993; 366:751-6). It shows that each LRR is composed of a β-sheet and an α-helix. The β-sheet that forms the consensus part of the LRR and the less conserved helices are exposed at different faces of the protein. Although the repeats are larger and higher in number than in the matrix proteins, it is likely that at least some features are represented in other members of this family. A common property of the LRR proteins is that their 3- dimensional structure seems to facilitate protein-protein interactions. In chondroadherin the LRR sequence is repeated 11 times. This protein differs from other members of this ECM family by having two cysteine-bonded loops in the C- terminal region and also by lacking any N-terminal extensions. Its different geneorganisation defines its own subfamily (Landgren et al, Genomics 1998; 47:84-91). Chondroadherin has, as compared to other LRR proteins exemplified by decorin and fibromodulin, a rather restricted distribution being confined to cartilage and tendon. Studies of the developing femoral head of rats showed that chondroadherin is localised mainly in the territorial matrix in somewhat later stages of articular cartilage development (Shen et al, Biochem. J. 1998; 330: 549-57). Fibromodulin and decorin on the other hand are part of the structural, interterritorial matrix in cartilage (Hedlund et al, Matrix Biol. 1994, 14, 227-32; Miosge et al., Histochem. J. 1994, 26, 939-45). The precise function of chondroadherin is unknown. It has been shown to bind cells via the α 2 βι integrin, without stimulating cell spreading (Camper et al., J. Cell. Biol. 1997, 138, 1159-67). The collagen family of proteins includes several members which are restricted to cartilage extracellular matrix. The collagens can be classified on the basis of their molecular shape and properties. Collagen type II is the major collagen found in cartilage. It is a fibril -forming collagen, composed of three identical polypeptide chains. These are folded into a unique triple helix with a characteristic amino acid sequence with repeating triplets of GL Y-X- Y, where X often is a proline and Y often is a hydroxyproline (Bornstein et al., editors: The Proteins, Academic Press (New York 1979), p. 411 - 632). The helix has surface characteristics promoting interactions with other matrix constituents. In the tissue, collagen associate to form fibrils which in turn are linked in interactions via cross- bridging molecules. These include other collagens such as collagen type IX (Eyre et al., FEBS Lett. 1987, 220: 337-41), as well as non-collagenous molecules such as decorin, fibromodulin (Hedbom et al., J. Biol. Chem. 1993, 268: 27307-12), lumican (Rada et al., Exp. Eye. Res. 1993, 56: 635-48), the latter also being members of the LRR family of proteins. Recently it has been shown that another abundant cartilage protein, cartilage oligomeric matrix protein, COMP, also can bind collagen type II (Rosenberg et al., J. Biol. Chem. 1998, 273: 20397-403). It is known that cytokines mediates and induces inflammatory reactions. They are also key components of the immune system. The change of cartilage matrix turnover in arthritic diseases is thought to be driven by cytokines. They induce production of proteases and activates them via unknown mechanisms. It would therefore be possible to therapeutically treat and/or alleviate the symptoms of arthritic diseases by administrating a therapeutically effective amount of cytokine antagonists and/or protease inhibitors in order to counteract the change of cartilage matrix turnover. A major problem relating to therapeutic use of these compounds is to provide a suitable way of administrating them, so that they can reach their target, e.g. an arthritis-affected joint, in a sufficiently efficient way. Summary of the invention The above mentioned problem can be overcome by providing a conjugate of a collagen II-binding chondroadherin fragment and an arthritis-affecting pharmaceutical substance, such as a cytokine, a cytokine antagonist and/or a protease inhibitor. It has turned out that chondroadherin specifically interacts with collagen II thereby forming a complex. Release of cartilage extracellular matrix components, such as chondroadherin and collagen II is a symptom of arthitis. By providing a conjugate according to the present invention it is therefore possible to direct arthitis-affecting pharmaceutical substances directly to the pathogenic tissue. The inventive conjugate can be manufactured by chemical crosslinking. In case the arthitis-affecting pharmaceutical substance is a protein or a peptide, the conjugate can be a fusion protein and be produced by genetic engineering techniques. Definitions As disclosed herein, the term "arthritis-affecting pharmaceutical substance" can be a cytokine, a cytokine antagonist and/or a protease inhibitor. Examples of cytokines are interleukins, such as II- 1, tumour necrosis factor alfa (TNFα), αjAT, PR3, and MPO. Preferably, the arthritis-affecting substance is a protease inhibitor capable of inhibiting metalloproteases that might occur in cartillage tissue. Examples of such protease inhibitors are oti -antitrypsin, cti -antichymotrypsin, inter-α-trypsin inhibitor, α 2 macroglobulin and tissue inhibitors of metalloproteinases (TIMP-1 to TIMP-4). Figures The present invention will be described with references to the enclosed figures, in which: Fig. 1 discloses SDS-PAGE of collagen II and recombinant chondroadherin that are used in the surface plasmon resonance assay. Figures indicate that the main part of collagen type II and chondroadherin used are monomers; Fig. 2 describes zonal rate sedimentation of chondroadherin containing complexes on glycerol gradients. The upper panel shows SDS-PAGE and Coomassie-staining of fractions from gradient centrifugation in glycerol of flow through from ion exchange chromatography of medium from APMA-treated bovine cartilage. Bottom fractions are to the left and top fractions to the right. Collagen type II is mainly present in the upper fractions. The lower panel shows Western blot analysis of chomdroadherin in the same fractions as above. Chondroadherin is mainly present in the upper fractions; Fig. 3 relates to electron microscopy of collagen II - chondroadherin complexes. Fig. 3 A shows electron microscopy of collagen type II - chondroadherin complexes in flow through from ion exchange chromatography of medium from APMA-treated bovine cartilage. Complexes are indicated by arrows. The polarity of collagen is defined by antibody F4, indicated by arrow head. Fig. 3B shows the same thing as fig. 3 A but with addition of gold-labeled affinity-purified antibodies raised against bovine chondroadherin showing that the protein interacting with collagen type II is chondroadherin. Complexes are indicated by an arrow. The polarity of collagen is defined by antibody F4, indicated by an arrow head. Fig. 3C describes electron microscopy of reconstituted complexes of recombinant chondroadherin and collagen type II. Complexes are indicated by an arrow. The polarity of collagen is defined by antibody F4, indicated by an arrow head; Fig. 4 relates to distribution of bound chondroadherin along monomeric collagen II. The upper panel (A) shows the distribution of chondroadherin along collagen type II observed in the complexes isolated from cartilage. The relative frequency denotes the fraction of chondroadherin molecules which are bound to a given site on collagen II. The C-terminus is at 0 nm as determined by using an antibody against the C-terminal end of collagen type II. The lower panel (B) shows the binding sites of the in vitro reconstituted collagen II - chondroadherin complexes; Fig. 5 shows characteristics of the chondroadherin - collagen II interaction. Surface plasmon resonance assay (BIAcore) showing interaction of collagen type II and chondroadherin. The figures show the association phase (0 - 60 seconds) and the dissociation phase (0 - 60 seconds). Panel A shows interaction of collagen type II on immobilised chondroadherin. Panel B shows interaction of chondroadherin on immobilised collagen type II; and Fig. 6 discloses the cDNA sequence for human chondroadherin and its deduced amino acid sequence. The signal sequence is indicated by bolded letters. Stop codons are indicated by dots. Detailed description of the invention The change of cartilage matrix turnover in arthritic diseases is thought to be driven by cytokines. Among all, they induce production of proteinases. These en2ymes degrade matrix constituents, thereby affecting tissue integrity. To study the release of cartilage components, particularly chondroadherin, under influence of activated endogenous proteinases, cartilage was treated with APMA to activate matrix metalloproteinases (Ganu et al, Arthritis Rheum. 1998, 41: 2143-51). Chondroadherin was among the molecules released to the medium in buffers that show little extraction on their own. Zonal rate centrifugation of the release material in glycerol gradients shows that collagen and chondroadherin cofractionates. This is unexpected in view of their widely different masses. It therefore appears that the two molecules occurred in the same complexes, which indicates interaction. To further identify and define the binding site (sites), negative staining and electron microscopy were performed of material from tissue extracts as well as from in vitro recombined collagen and chondroadherin. The combined analyses showed that chondroadherin and collagen indeed interact and that the binding sites are identical in material released from tissue and in complexes formed by pure components. In both systems one major and one minor binding site, 185 nm and 267 run, respectively from the C-terminus of collagen, was identified. To study the quality of the interaction, pure components were analysed using surface plasmon resonance in the BIAcore. The K D of the interaction was calculated, both for the interaction when chondroadherin was immobilised and when collagen was immobilised. The K D , in the nanomolar range, differs somewhat depending on which component in the interaction pair that was immobilised. The tighter binding of collagen to immobilised chondroadherin may result from the two binding sites for chondroadherin on collagen enhancing the binding strength as compared to chondroadherin interacting with immobilised collagen. This assumption is further strengthened by the difference in K D :s using models allowing for 1 and 2 binding sites on the analyte, respectively. It is also possible that microenvironment on the chip becomes somewhat dependent on which ligand was immobilised. The complexes released from the cartilage and characterised by electron microscopy consist of collagen type II with one or two chondroadherin molecules attached. Neither collagen nor chondroadherin seem to be degraded indicating that their release is dependent on cleavage of their anchorage in the tissue. The complexes consist of intact chondroadherin and intact collagen, which further strengthen the assumption that these complexes occur in vivo. The collagen in the complexes is monomeric but in a mature form without peptides. This is particularly interesting in view of the very low abundance of non-aggregated collagen molecules being highly interactive. Since collagen was not in its proform, it appears to have been processed in the tissue. Whether this was a result of the induced proteolysis is not clear. However, all collagen was processed, at the same time as fragmentation of other proteins like chondroadherin and COMP was limited, indicating that processing had occurred prior to APMA treatment. In articular cartilage chondroadherin is located territorially, while most other LRR proteins also known to interact with collagen such as decorin and fibromodulin are located interterritorially, where they have structural functions in the network. It is reasonable to speculate that proteins located more close to the cells have a regulatory role, communicating between cells and matrix. Both collagen type II and chondroadherin have been shown to bind chondrocytes (Sommarin et al., Exp. Cell Res. 1989, 184: 181-92). These cells are known to express several different integrins on their surface, including the αiβi and α 2 βι integrins (Durr et al., Exp. Cell Res. 1993, 207: 235-44; Woods et al., Arthritis Rheum. 1994, 37: 537-44). The α2β 1 integrin has been shown to be a receptor for chondroadherin (Camper et al., J. Cell. Biol. 1997, 138: 1159-67) whereas both these integrins have been shown to bind collagen type II. Even though they in part interact with the same integrin only collagen is able to induce spreading of the cells when grown on collagen and chondroadherin, respectively (Sommarin et al., Exp. Cell Res. 1989, 184: 181-92). By partly sharing the same receptor on the chondrocyte and by interacting with each other, a complex system is created. This potentially includes crossbridging of a number of receptors. Future work should sort out the consequences of interactions of individual components in comparison to that of the complexes, e.g. with regard to ensuing intracellular signalling and cell function. An obvious function of collagen type II in articular cartilage is to form highly organised fibrils that are of fundamental importance for the mechanical properties of the tissue. Several studies have shown that the assembly of collagen fibres is influenced by many other molecules like collagen type IX as well as by matrix proteins in the LRR protein family such as decorin, fibromodulin and lumican. They all bind to fibrillar collagens type I and II (Hedbom et al., J. Biol. Chem. 1993, 268: 27307-12; Rada et al., Exp. Eye Res. 1993, 56: 635-48; van der Rest et al., J. Biol. Chem. 1988, 263: 1615-8). A consequence of this interaction is that they are able to modulate fibril formation in vitro and affect the dimensions of the fibrils (Vogel et al., Biochem. J. 1984, 223: 587-97). Many connective tissue disorders have been linked to alterations in collagen assemblies and thus function. This could be due to either defects in collagen itself or in alterations in proteins involved in the assembly of collagen fibrils. Genes for several members of the LRR protein family have been functionally deleted in mice. A major phenotype in the null mutants of lumican (Chakravarti et al., J. Cell. Biol. 1998, 141 : 1277-86), decorin (Danielson et al., J. Cell. Biol. 1997, 136: 729-43), and fibromodulin (Svensson et al., J. Biol. Chem. 1999, 274: 9636-47), is dysregulation of the collagen fibril formation. The result is irregular, thick or thin collagen fibrils. These studies verify that in vitro observations of interactions between LRR proteins and collagen are of biological importance. Chondroadherin, also being a member of this LRR protein family, is now shown to interact with collagen. It is likely that also this protein infuences the assembly ant thereby the function of fibrillar collagen. Since chondroadherin is abundant in the territorial matrix it may have a role particularly in the early events in collagen assembly. Indeed, the observation of monomeric soluble collagen with bound chondroadherin indicates a role in regulating collagen assembly. The role may particularly involve preventing premature associations of the collagen molecules into fibrils. Molecular complexes released from cartilage upon APMA treatment where separated into two chondroadherin-containing classes show major differencies in ionic charge. One fraction was now shown to represent monomeric collagen chondroadherin complexes. The other fraction was much more anionic. Since chondroadherin, at neutral pH, does not bind to DEAE cellulose used for separation, it appears that chondroadherin is bound to an anionic "carrier" protein. In view of the presence of biglycan in this fraction, it is possible that there are complexes containing biglycan and chondroadherin and poosibly other molecules. This fraction might thus represent yet another interaction with chondroadherin of potential interest. Accordingly, this interaction between chondroadherin and collagen II can be utilised for medical purposes, such as for treating and/or alleviating the symptoms of arthritic diseases such as rheumatism and rhematoid arthritis. A symptom of such diseases is that both chondroadherin and collagen II are released in the synovial fluid and in serum in connection with degradation of matrix constituents. As already mentioned above, cytokines are believed to cause this degradation by inducing production of proteinases. The present invention provides conjugates comprising a) a chondroadherin fragment having the ability to specifically bind to collagen II, and b) and an arthritis-affecting pharmaceutical substance, such as a cytokine, a cytokine antagonist and/or a protease inhibitor. Examples of suitable cytokines are interleukins, such as II- 1, tumour necrosis factor alfa (TNFα), αi AT, PR3, and MPO. As disclosed above, symptoms of arthritis diseases, such as rheumatism and rhematoid arthritis, are characterised by degradation of matrix constituents. This degradation is caused by proteases and in particular metalloproteinases. Accordingly, the tissue inhibitors of metalloproteinases (TIMP-1 to TIMP -4) [Brew et al, Biochim. Biophys. Acta 1477: 267-83 (2000)] are particularly preferred as an arthritis-affecting pharmaceutical substance. Other protease inhibitors, such as αi-antitrypsin, α antichymotropsin, inter- α-tryp sin inhibitor, α 2 - macroglobulin and anti-trombin III could also be used as an arthritis-affecting pharamaceutical substance. A conjugate according to the present invention comprising chondroadhein connected to a protease, such as TIMP-1, TIMP-2, TIMP-3 or TIMP-4 could be used to protect cartillage tissue from proteolytic degradation. The chondroasherin part of the conjugate binds to collagen II, and accordingly the conjugate will be located in the cartillage tissue. The protease part is therefor capable of inhibiting proteases directly in the cartillage tissue. The conjugate according to the present invention can be manufactured by chemically cross-linking a) chondroadherin or a collagen II-binding fragment thereof, and b) the arthritis-affecting pharmaceutical substance. This can for instance be carried out by reactions involving common and well-known chemical crosslinkers, such as glutaraldehyde. The conjugates of the present invention can of course also be produced using recombinant DNA techniques. Nucleic acid sequences encoding a) chondroadherin or a collagen II-binding fragment thereof, and b) the arthritis-affecting pharmaceutical substance, are ligated together in order to obtain a fused nucleic acid encoding a fusion protein comprising a functionally efficient a) chondroadherin or a collagen II-binding fragment thereof, and b) a functionally efficient arthritis- affecting pharmaceutical substance. Suitable techniques that can be used for obtaining such a fusion protein can be found in Sambrook: Molecular Cloning, A Laboratory Manual. Second Edition. The chondroadherin part of the conjugate according to the present invention can be the complete condroadherin as disclosed in Fig. 6, or alternatively it can be a truncated derivative comprising at least 10 amino acids. However, such a truncated chondroadherin derivative must be able to specifically bind to collagen type II. The present invention also provides pharmaceutical compositions comprising the conjugate according to the invention together with a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition of the invention is intended for parenteral administration, preferably directly into a tissue affected with an arthritic disease, such as an affected joint. In such a case the composition contains a therapeutically effective amount of conjugate in sterile physiological saline. Experimental section: Materials and Methods: Cloning and sequencing of human chondroadherin cDNA: A λZAP II cDNA library made from human chondrocytes (provided by Dr. Michael Bayliss) was screened using a 450 bp bovine chondroadherin cDNA probe. A 1444 bp clone was isolated and purified with Plasmid Midi Kit (Qiagen). DNA sequencing was performed in full on both strands by standard double-strand dideoxy termination sequencing using T3, T7 and synthetic internal specific primers. Expression of recombinant chondroadherin and generation of antibodies against human chondroadherin: A polyclonal antiserum against human chondroadherin was raised by immunising a rabbit with a peptide from the C-terminal region of the protein (GQHIRDTDAFRS, SEQ. ID. NO. 2) coupled to keyhole limpet hemocyanin using glutaraldehyde (Collawn et al., editors. Current protocols in molecular biology. John Wiley & Sons, Inc. 1989, 11.15, Production of antipeptide antibodies, p. 2 - 3). This antiserum has been shown to react only with chondroadherin in crude tissue extracts. A 1.4 kbp human chondroadherin cDNA Not I-fragment was ligated to the Epstein Barr Virus-based eucaryotic expression vector pCEP4 (Invitrogene, California, USA). This vector is maintained extrachromosomally in eucaryotic cells and expresses high levels of recombinant protein (Young et al., Gene 1988, 62, 171 - 85). The transcription is driven by the cytomegalovirus promoter included in the pCEP4 vector. The endogenous signal peptide of human chondroadherin was used. The construct in pCE04 was transfected to human EBNA cells by using lipofectamine (Gibco, BRL). After transfection the cells were grown in Dulbecco's modified Eagle's medium (DMEM)(Gibco, BRL) containing 10 % fetal calf serum. After 48 hours, hygromycin (Calbiochem)(160 μg/ml) was added to the culture medium as a selector. The cells were expanded and when left confluent the media were changed to DMEM without fetal calf serum. The cells were left for 48 hours after which the medium was harvested and centrifuged for 5 minutes at 3000xg. Phenyl methyl sulfonylfluoride (1 mM, final concentration) was added as a protease inhibitor. One liter of collected medium was diluted 4 times to a final concentration of 20 mM Tris, pH 7.3 and applied to a CM52 column previously equilibrated in 20 mM Tris, pH 7. The column was washed with 20 mM Tris and eluted with a gradient of from 0 to 0.5 M NaCl in 20 mM Tris, pH 7. The purity and identity of the protein was confirmed by SDS-PAGE and Western blotting using antibodies raised against the C-terminal region of human chondroadherin. Isolation of chondroadherin complexes from cartilage Fresh calf articular cartilage was dissected into 2 - 3 mm pieces, washed twice in PBS followed by two washes in Hepes-buffered saline supplemented with CaCl 2 (HBSCa)(50 mM Hepes, 0.15 M NaCl, 1 mM CaCl 2 , pH 7.4). The cartilage was incubated at 37 °C over night in 15 volumes (w/v) HBSCa, supplemented with 1 mM APMA (p-amino-phenyl mercuric acetate, Sigma) and 10 μM cycloheximide. The supernatant was collected by brief centrifugation to remove the coarser cartilage pieces followed by centrifugation at 17700xg for 30 minutes. Proteinases activated by APMA were inhibited by adding EDTA to a final concentration of 9.8 mM. The supernatant was finally made particle-free by centrifugation in a Beckman Ti50.2 rotor at 43000 rpm (100,000 xg) for one hour at 4 °C. Ion exchange chromatography 200 ml of the supernatant from the cartilage incubate was applied to a 32 ml column of DE52 (Whatman) equilibrated in Hepes-buffered saline containing 9.8 mM EDTA, pH 7.4. The flow through was kept and further analysed. Eluation was made with a gradient of sodium acetate (0 - 1.5 M, pH 7.4). Zonal rate centrifugation Gradients (4 ml), 10 - 30 % glycerol in Hepes-buffered saline with EDTA (49 mM Hepes, 0.15 M NaCl, 9.8 mM EDTA, pH 7.4) were prepared. The sample in the same buffer (200 μl) was applied on top and then centrifuged at 50,000 rpm (-257,000 xg) in a Beckman Sw60.1 swinging bucket rotor for 15 h at 18 °C. The gradient was emptied from the bottom in 200 μl fractions. SDS-PAGE and Western blot: Samples of the fractions from the zonal rate centrifugation were prepared for SDS- PAGE by precipitation with 9 volumes 95 % ethanol containing 50 mM sodium acetate and dissolved in sample buffer (Paulsson et al., Biochem. J. 1983, 212:659- 67). Samples were separated on 4 - 16 % polyacrylamide gels. Proteins were either stained directly or transferred, using a tank blot system (Mini Trans-Blot, BioRad), to PVDF membranes (fluorotrans transfer membranes, Pall Filtron). Blotted membranes were blocked for additional protein binding with 3 % (W/v) milk powder in 10 mM Tris, 0.15 M NaCl, 0.2 % Tween 20, pH 7.4. Primary antibody, raised against bovine chondroadherin, was applied in the same buffer solvent. Bound antibodies were detected after incubation with secondary antibodies conjugated to horseradish peroxidase by chemiluminescence using the ECL system (Amersham Life Science). Isolation and purification of collagen II: Collagen II was purified from pepsin-digested bovine articular cartilage as previously described (Miller, Biochemistry 1972, 11, 4903-9; Vogel et al., Biochem. J. 1984, 223, 587-97). The purity of collagen II was confirmed by SDS- PAGE (fig. 1). Electron microscopy: Chondroadherin and collagen-containing samples from the flow through of the DE52 column (~ 10 μg/ml) were prepared for electron microscopy either directly or after incubation with gold-labeled affinity-purified antiserum against bovine chondroadherin. Recombinant chondroadherin and collagen type II were preincubated and treated in the same manner as samples isolated from tissue extract. Samples were adsorbed to 400 mesh carbon-coated copper grid which was previously rendered hydrophilic by glow-discharge at low pressure in air. The grid was immediately blotted, washed with two drops of water and stained with 0.75 % uranyl formate for 15 seconds. When used, antibodies were labeled with colloidal gold of 3 nm size as previously described (Baschong et al., J. Electron. Microsc. Tech. 1990, 14:313-23). Samples were observed in a Jeol 1200 EX transmission electron microscope operated at 60 kV accelerating voltage and 75,000x magnification. Images were recorded on Kodak ESTAR Thick Base 4489 plates without preirridiation at a dose of typically 2000 electrons/nm . Evaluation of the data from electron micrographs was done as previously described (Engel et al., Methods Enzymol. 1987, 145:3-78). An antibody against the C-terminal region of collagen type II (a gift from Dr. Rikard Holmdahl) was used to determine the polarity of the type II collagen molecules. Interactions studied by Surface Plasmon Resonance: The BOAcore™ 20 system (BIAcore AB, Sweden) was used to further characterise the interaction and to determine the binding characteristics between chondroadherin and type II collagen. In one set of experiments pure recombinant chondroadherin was concentrated and the buffer was changed to 10 mM Hepes, 0.15 M NaCl, 0.05% Tween, pH 7.4. The carboxymethylated dextran surface on the chip (CM5 sensor chip, BIAcore AB, Uppsala Sweden) was activated with 35 μl of 50 nM N-hydroxysuccineimide and 35 μl 200 mM N-ethyl-N' -(dimethyl aminopropyl) carbodiimide at 25 °C at a flow rate of 5 μl/ml. Chondroadherin (35 μl at 100 μg/ml) was immobilised at 25 °C at a flow rate of 5 μl/min. One surface was used as negative control and contained no coupled protein. Remaining activated groups were blocked with 40 μl of 1M ethanolamine, pH 8.5. The immobilisation of recombinant chondroadherin resulted in 4000 resonance units (RU)(1000 RU = 1 ng/mm 2 ). The binding assay was performed at 25 °C by using pepsinised collagen II at different concentations ranging from 0.09 to 6 μg/ml in 10 mM Hepes, 0.15 M NaCl, 0.05% Tween, pH 7.4 applied at 40 μl/min. The surface was regenerated by using 0.5 M acetic acid, 2 M NaCl followed by 0.1 M NaHC0 3 , 2 M NaCl, pH 9.2. In another set of experiments pepsinised collagen type II (2.1 mg/ml in 0.1 M acetic acid) was diluted to 100 μg/ml in 10 mM sodium citrate, pH 3.2 and immediately immobilised under the same conditions as above (running buffer: 10 mM Hepes, 0.15 M NaCl, 0.05% tween, pH 7.4). The immobilisation of collagen type II resulted in 3000 RU. The binding assay was performed at 25 °C by using recombinant chondroadherin at different concentrations ranging from 0.03 to 4 μg/ml in lOmM Hepes, 0.15 M NaCl, 0.05% Tween, pH 7.4 applied at 40 μl/ml. The surface was regenerated by using 10 mM Hepes, 2M NaCl, pH 7.4 followed by 0.1 M NaHC0 3 , 2 M NaCl, pH 9.2. lo The BIAevaluation software (version 3.0) was used to calculate the different binding constants (K D )(Fagerstam et al., J. Chromatogr. 1992, 597:397-410). Results: Nucleotide and deduced amino acid sequence: The screening of a human cDNA library resulted in the isolation of a 1444 bp clone. The deduced amino acid sequence is similar to the one published (Grover et al., Genomics 1997, 45:379-85). However, certain differences were found. At position 114 in the sequence leucine was found instead of valine, and at position 166 alanine was found instead of proline. Our sequence at these positions was found to be in agreement with the sequences from mouse (Landgren et al., Genomics 1998, 47: 84- 91), rat (Shen et al., Biochem. J. 1998, 330:549-57), and cow (Neame et al., J. Biol. Chem. 1994, 269:21547-54). Expression of native recombinant protein: The recombinant chondroadherin was eluted from the CM52 column at an ionic strength of 0.18 - 0.22 M NaCl. The yield of native recombinant chondroadherin was between 1 and 2 μg/ml culture medium. The protein appears pure as shown by SDS-PAGE (fig. 1) and wastern blotting using antibodies raised against the C- terminal region of the protein. Chondroadherin exists in two forms that differ by nine amino acids in the C-terminal. Both forms are expressed by EBNA cells and upon purification on a CM52 ion exchanger they elute differently, in partly overlapping fractions. Cartilage extraction and preparation of supernatant: Chondroadherin was released from cartilage using APMA in Hepes-buffered saline. Release without APMA was 10 - 15 % of that with APMA and was not further analysed. Gel filtration on Superose & of the supernatant representing trleased proteins showed that chondroadherin eluted in early fractions, indicating that it occured in complexes of considerable size (data not shown). Ultracentrifugation at 100,000 g for one hour showed that chondroadherin remained in the supernatant, thus not being present in particles. To further study the complexes, the supernatant was subjected to anion exchange chromatography. The chondroadherin pool then separated into two fractions, one passing through and one being retained on the column. The latter was eluted together with several proteins, including biglycan as a major component, at a sodium acetate concentration of 0.7 - 0.8 M. The pool passing through the DE column contained collagen II αl -chains (as deduced from migration on SDS-PAGE under non-reduced and reduced conditions) as well as chondroadherin (as detected by Western blot) and a few other components. Among those other components as a prominent band was identified as the C-terminal propeptide of collagen II by western blot (data not shown). The pool containing collagen and chondroadherin, as well as other components, was further fractionated by zonal rate centrifugation and analysed by electron microscopy. Zonal rate centrifugation: Samples of the flow through from the DE52 column, containing several different molecular entities was further fractionated by zonal centrifugation in glycerol gradients. The composition of the various fractions was studied by SDS-PAGE electrophoresis and Western blotting of non-reduced samples. Collagen type II was detected in the upper part of the gradient with the slowest sedimentation (fig. 2). The same fractions from an identical gradient also contained chondroadherin demonstrated by Western blot (fig. 2). Electron microscopy: Electron microscopy showed filaments of intact monomeric collagen molecules with bound globules having the typical three-lobe appearance of the LRR proteins at distinct locations (Fig. 3A). To verify their nature, the complexes were incubated with gold-labeled affinity-purified antibodies to chondroadherin before electron microscopy. The picture then showed the typical IgG structure of three globes associated with the presumptive chondroadherin globules bound to collagen (fig. 3B). Thus the complexes isolated represent collagen with bound chondroadherin. No other molecules appeared to bind at these sites on collagen. Similar complexes could be formed by recombinant chondroadherin and isolated pepsinised collagen II. Equimolar amounts of collagen II molecules was defined by an antibody recognising an epitope close to their C-terminal end (kindly provided by Dr. Rikard Holmdahl, Lund). The binding sites were defined by measuring the distance from the C-terminal end of collagen to the site of chondroadherin binding. The relative frequency of chondroadherin binding at different sites on the collagen are shown in figure 4. These data demonstrate that there are two different binding sites, 185 nm and 267 nm from the C-terminal end of collagen and that both these sites show similar occupancy in vivo (fig. 4A) and in vitro (fig. 4B). Interaction between chondroadherin and collagen II, in vitro: In a first set of experiments the interaction of collagen type II with immobilised chondroadherin was studied using different concentrations of collagen, resulting in the association and dissociation curves shown in fig. 5A. Evaluation using the Langmuir model predicting a 1 : 1 interaction unraveled a dissociation constant of 0.6 nM. Using a model allowing for a bivalent analyte (collagen II) gave a significantly lower K D for the strongest of the two putative binding sites. In another set of experiments collagen type II was immobilised and the interaction with chondroadherin studied (fig. 5B). Evaluation of these data showed that the dissociation constant of the interaction in this system was 40 nM.

Claims: Claims: 1. A conjugate comprising a) chondroadherin or a collagen II-binding fragment thereof, and b) an arthritis-affecting pharmaceutical substance. 2. A conjugate according to claim 1, characterised in that arthritis-affecting pharmaceutical substance is chosen from the group of cytokines, cytokine antagonists and protease inhibitors. 3. A conjugate according to claim 2, characterised in that the arthritis-affecting substance is chosen from the group of tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3.TIMP-4), αi -antitrypsin, rxi -antichymotrypsin, inter-α-trypsin inhibitor, α 2 -macro globulin and anti-trombin III. 4. A conjugate according to anyone of claims 1-3, characterised in that the chondroadherin part and the arthritis-affecting pharmaceutical substance have conjugated by means of chemical cross-linking. 5. A conjugate according to anyone of claims 1-3, which is a fusion protein comprising a) chondroadherin or a collagen II-binding fragment thereof, and b) an arthritis-affecting pharmaceutical substance. 6. A nucleic acid sequence encoding the fusion protein of claim 5. 7. A conjugate according to anyone of claims 1-5, characterised in that the chondroadherin part comprises at least 10 consecutive amino acid residues from the amino acid sequence disclosed in fig. 6. 8. A conjugate according to anyone of claims 1 -5 or 7, for medical use. 9. Use of a conjugate according to claim 8, for preparing a pharmaceutical composition for treating and/or alleviating the symptoms of arthritic diseases such as rheumatism and rhematoid arthritis. 10. A pharmaceutical composition comprising a conjugate according to anyone of claims 1 - 5 or 7, together with a pharmaceutically acceptable carrier.

Inventors: HEINEGAARD DICK, HEINEGAARD, DICK
Applicants: ANAMAR MEDICAL AB, HEINEGAARD DICK, HEINEGAARD, DICK
Application year: 2000
Main classification: A61K38
IPC codes: A61K38, A61K45, A61P19, A61P29, A61P43, C07K14, C07K19, C12N15