JP2003531109A - Conjugates of collagen II binding fragments and arthritically active pharmaceutical substances - Google Patents

Abstract

  (57) [Summary] The present invention provides a conjugate of a collagen II binding chondroadherin fragment and an arthritic pharmaceutical agent such as a cytokine, cytokine antagonist and / or protease inhibitor. It has been found that chondroadherin interacts specifically with collagen II and thus forms a complex. Release of cartilage extracellular matrix components such as chondroadherin and collagen II is one symptom of arthritis. Thus, by providing a conjugate according to the invention, it is possible to direct the arthritic pharmaceutical substance directly to the pathogenic tissue. The conjugate of the present invention can be manufactured by chemical crosslinking. If the arthritic pharmaceutical is a protein or peptide, the conjugate can be a fusion protein and can be produced by genetic engineering techniques.

Description

Detailed Description of the Invention

The present invention relates to novel conjugates consisting of collagen-binding fragments of chondroadherin covalently linked to pharmaceutically advantageous substances such as cytokines, cytokine inhibitors and protease inhibitors. The invention also relates to pharmaceutical compositions containing the novel conjugates.

Technical Background The major components of the extracellular matrix of cartilage are numerous proteins, including type II collagen, proteoglycans and some of the minor collagens. Substrate macromolecules have very different structural and functional characteristics, with each component contributing differently to the organization and properties of tissue. The matrix is organized into a multi-component network in which interactions between matrix components are essential for maintaining the integrity of the tissue. (Hein
egard et al., editors Osteoarthritis. 1 ed. Oxford University Press ； 1
998; 7.2.1, biochemistry and metabolism of normal and osteoarthritic cartilage, p74-84)
.

One of the non-collagenous proteins in cartilage is chondroadherin. It was first isolated from bovine tracheal cartilage (Larsson et al., J. Biol. Chem. 1
991; 266: 20428-33). Protein and DNA sequence of bovine chondroadherin (Neame et al., J. Biol. Chem. 1994; 269: 21547-
54) shows that it is a leucine-rich repeat (LRR) protein (Parthy, J. Mol. Bio
l.1987; 198: 567-77). LR
The R protein subgroup has multiple members in the extracellular matrix, and has a conserved sequence xLxxLxLxx (N, C, T) x (L, I) followed by 9 to 14 relatively low conserved residues. Characterized by a group. This sequence can be repeated up to 30 times, but is usually 10-11 times in extracellular proteins and 5-6 in the other subgroups.
It only happens once. The repeat domain is flanked by two cysteine-linked loops and usually a short extension within the N-terminal and C-terminal regions of the protein. LR
Crystal structure of one member of R protein family, ribonuclease inhibitor is 2
Elucidated at a level of .5Å (Kobe et al., Nature 1993; 366: 751
-6). This indicates that each LRR is composed of one β-sheet and one α-helix. The β-sheet and the relatively poorly conserved helix that form the consensus part of the LRR are exposed on different faces of the protein. Although the repeats are larger and more abundant than the substrate proteins, at least some features are likely to be represented in other members of this family. LRR
A common property of proteins is that their three-dimensional structure appears to facilitate protein-protein interactions. Within chondroadherin, the LRR sequence is repeated 11 times. This protein differs from other members of the ECM family by having two cysteine binding loops in the C-terminal region and also lacking any N-terminal extensions. Its different genetic makeup defines its own subfamily (Landgren et al., Genomics 1998; 47.
: 84-91). Chondroadherin has a rather restricted distribution restricted to cartilage and tendons compared to other LRR proteins, of which decorin and fibromodulin are examples. Studies of the developing femoral head in the rat have shown that chondroadherin is localized predominantly within the periluminal stroma at some later stage of articular cartilage development (Shen et al. Biochem. J. 1998; 330: 549.
-57). On the other hand, fibromodulin and decorin are part of the structural interluminal matrix in cartilage (Hedlund et al., Matrix Biol. 1994, 14, 227-.
32; Miosge et al., Histochem. J. 1994, 26, 939-45).

The exact function of chondroadherin is unknown. It is known to bind cells via the α ₂ β ₁ integrin without promoting cell spreading. (Camper et al., J. Cell. Biol. 1997, 138, 1159-67). The collagen family of proteins includes multiple members restricted to the extracellular matrix of cartilage. Collagen can be classified based on the shape and properties of its molecule. Collagen type II is the major collagen found in cartilage. It is 3
It is a myofibrillar collagen composed of two identical polypeptide chains. They fold into a unique triple helix with a characteristic amino acid sequence with a repeating triplet of GLY-XY, where X is often proline and Y is often hydroxyproline. Bomstein et al., Editors: The Proteins
Academic Press (New York 1979), pp. 411-632). The helix has surface features that facilitate its interaction with other substrate components. Within tissues, collagen associates to form myofibrils, which are themselves linked in interactions via bridging molecules. These include collagen type IX (Eyre et
al., FEBS Lett. 1987, 220: 337-41), as well 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 is also a member of the LRR protein family). Recently, another abundant cartilage protein, cartilage oligomer matrix protein, COM
It has also been shown that P can also bind to collagen type II. (Rosenberg et
al., J. Biol. Chem. 1998, 273: 20397-403) It is known that cytokines mediate and induce an inflammatory response. These are also
It is also an important component of the immune system. Changes in cartilage matrix turnover in joint diseases are thought to be driven by cytokines. Cytokines induce the production of proteases and activate them through unknown mechanisms. Therefore, by administering a therapeutically effective amount of a cytokine antagonist and / or a protease inhibitor for the purpose of neutralizing changes in cartilage matrix turnover, the symptoms of arthritic disease are treated therapeutically and / or It will be possible to mitigate. The main problem with the therapeutic use of these compounds is to provide them suitable administration methods in such a way that they can reach their target in a sufficiently efficient manner, for example in a joint suffering from arthritis. is there.

SUMMARY OF THE INVENTION The above problems can be overcome by providing a conjugate of collagen II-binding chondroadherin fragments and arthritic pharmaceutical agents such as cytokines, cytokine antagonists and / or protease inhibitors. it can. It was found that chondroadherin interacts specifically with collagen II and thus forms one complex. Chondroadherin and collagen I
Release of cartilage extracellular matrix components such as I is one symptom of arthritis. By providing the conjugate according to the invention, it is possible to direct the arthritic pharmaceutical substance directly to the pathogenic tissue.

The conjugate of the present invention can be manufactured by chemical crosslinking. If the arthritic drug is a protein or peptide, the conjugate can be a fusion protein and can be produced by genetic engineering techniques.

DEFINITIONS The term "arthritic drug" disclosed herein may be a cytokine, cytokine antagonist and / or protease inhibitor. Examples of cytokines are interleukins such as II-1, tumor necrosis factor alpha (TNFα), α ₁ AT, PR3 and MPO. Preferably, the substance that affects arthritis is a protease inhibitor that has the ability to inhibit metalloproteases that may occur in cartilage tissue. Examples of such protease inhibitors include α ₁ -antitrypsin, α ₁ -antichymotrypsin, inter α-trypsin inhibitor, α ₂ -macroglobulin, and tissue inhibitor of metalloproteinase (TIM).
P-1 to TIMP-4).

[0008] change in the turnover of cartilage matrix in Description articular disorders of the present invention is believed to be driven by cytokines. Among these, they induce the production of proteinases. These enzymes degrade substrate components and thus affect the integrity of the tissue.
To study the release of cartilage components, particularly chondroadherin, under the influence of activated endogenous proteinases, cartilage was treated with APMA to activate matrix metalloproteinases (Ganu et al., Arthritis Rheum. , 1998, 41:21.
43-51).

Among the molecules released into the medium in buffer, chondroadherin showed little unique extraction. Zone velocity centrifugation of the released material in a glycerol gradient shows that collagen and chondroadherin co-fractionate. This is unexpected given their vastly different masses. Therefore, it seems that the two molecules occurred within the same complex, which
Represents an interaction. To further identify and define the binding site (s), negative staining and electron microscopy was performed on material from tissue extracts and from in vitro recombinant collagen and chondroadherin. A pooled analysis showed that chondroadherin and collagen actually interact and that the binding sites are identical in the material released from the tissue and in the complex formed by the pure constituents. . In both systems, one major and one minor binding site were identified at 185 nm and 267 nm, respectively, from the C-terminus of collagen. To study the quality of this interaction, pure components were analyzed using surface plasmon resonance within the BIAcore. The interaction K _D was calculated for both interactions when chondroadherin was immobilized and when collagen was immobilized. The K _{D in the} nanomolar range is somewhat different depending on which component in the interacting pair it was immobilized on. The tighter binding of collagen to immobilized chondroadherin enhances the binding strength compared to chondroadherin that interacts with the immobilized collagen, of two binding sites for chondroadherin on collagen. Can result. This hypothesis is based on the model K _D, which allows one and two binding sites on the analyte, respectively.
: Further strengthened by the difference of ₅ . It is also possible that the microenvironment on the chip will depend in part on which ligand it is immobilized on. The complex released from cartilage and characterized by electron microscopy consists of collagen type II with one or two chondroadherin molecules attached. Neither collagen nor chondroadherin appear to be degraded, indicating that its release is dependent on its anchoring division within the tissue. The complex is composed of intact chondroadherin and intact collagen, which further reinforces the hypothesis that these complexes occur in vivo. Collagen in the complex is a monomer but no peptide in its mature form. This is of particular interest in view of the very low yield of non-aggregated collagen molecules which have a very high degree of interaction. Since collagen is not in its pro form, it appears to have been processed in tissue. It is not clear whether this is the result of induced proteolysis. However, all collagen was processed at the same time as fragmentation of other protein-like chondroadherins and COMP was restricted, indicating that the processing was prior to APMA treatment.

In artificial cartilage, chondroadherin is periluminal, while most other LRR proteins also known to interact with collagen, such as decorin and fibromodulin, are Within the periluminal space with structural function. It is rational to speculate that proteins closer to the cell play a regulatory role in communicating between the cell and the 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 multiple different integrins on their surface, including α ₁ β ₁ and α ₂ β ₁ integrins (Durr et al., Exp. Ce.
ll Res. 1993, 207; 235-44, Woods et al., Arthritis Rheum, 1
994, 37; 537-44). The α ₂ β ₁ integrin has been shown to be a receptor for chondroadherin (Camper et al., J. Cell. Biol. 1
997, 138; 1159-67), but both of these integrins are known to bind to collagen type II. Only collagen can induce the spreading of cells grown on collagen and chondroadherin, respectively, even though they partially interact with the same integrin (Sommarin et al., E
xp. Cell Res. 1989, 184: 181-92). By partially sharing the same receptors on chondrocytes and interacting with each other, a complex system is created. This potentially involves bridging a certain number of receptors. Future research work should elucidate the consequences of the interaction of the individual components with respect to, for example, subsequent intracellular signaling and cell function as compared to that of the complex.

The apparent function of collagen type II within artificial cartilage is to form highly organized myofibrils that are of fundamental importance to the mechanical properties of the tissue. Studies have shown that the assembly of collagen fibers is influenced by numerous other molecules such as collagen type IX and substrate proteins within the LRR protein family such as decorin, fibromodulin and lumican. They all bind to myofibrillar collagen type I and type 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). As a result of this interaction,
They will be able to modulate myofibril formation in vitro and influence myofibril size (Vogel et al., Biochem. J. 1984, 223; 5).
87-97). Numerous connective tissue disorders have been linked to altered collagen assembly and thus function. This is believed to be due to either defects in the collagen itself or defects in the protein modifications involved in the assembly of collagen myofibrils. LR
Genes for some members of the R protein family have been functionally deleted in the mouse. Lumican (Chakravarti et al., J. Cell. Biol. 19
98, 141; 1277-86), decorin (Danielson et al. J. Cell. Biol.
1997, 136; 729-43), and fibromodulin (Svensson et al.
al., J. Biol. Chem. 1999, 274; 9636-47).
One major phenotype is the dysregulation of collagen myofibril formation. The result is irregular thick or thin collagen myofibrils. These studies are LRR
We confirm that the in vitro observation of the interaction between protein and collagen is of biological importance. Chondroadherin is also a member of this LRR protein family and has now been shown to interact with collagen. This protein also has a high probability of affecting the function of aggregates and thus myofibrillar collagen. Chondroadherin is abundant in the periluminal matrix and may therefore play a particular role in early events in collagen assembly.
Indeed, the finding of monomeric soluble collagen with bound chondroadherin represents one role in regulating collagen assembly. This role is particularly involved in preventing premature association of collagen molecules within myofibrils.

The molecular complexes released from cartilage at the time of APMA treatment separated into two chondroadherin-containing classes show a major difference in ionic charge. One fraction is now shown to represent the monomeric collagen chondroadherin complex.
The other fraction was much more anionic. Chondroadherin appears to be bound to the anionic "carrier" protein as it does not bind to the DEAE cellulose used for separation at neutral pH. Given the presence of biglycan in this fraction, there may be a complex containing biglycan and chondroadherin and optionally other molecules. Thus, this fraction may represent yet another interaction with the potentially beneficial chondroadherin.

Therefore, the interaction between chondroadherin and collagen II should be utilized for medical purposes such as to treat and / or alleviate symptoms of arthritic diseases such as rheumatoid and rheumatoid arthritis. You can One symptom of such a disease is that both chondroadherin and collagen II are released into synovial fluid and serum in association with the degradation of matrix components. As already mentioned above,
Cytokines are believed to cause this degradation by inducing the production of proteinases.

The present invention relates to a) a chondroadherin fragment capable of specifically binding to collagen II and b) a cytokine, a cytokine antagonist and / or
Alternatively, a conjugate comprising an arthritic drug substance such as a protease inhibitor is provided. Examples of suitable cytokine antagonists include interleukins such as IL-1, tumor necrosis factor alpha (TNFα), α ₁ AT, PR3 and MPO. As mentioned above, the symptoms of arthritic arthritis, such as rheumatoid and rheumatoid arthritis, are characterized by the degradation of matrix components. This degradation is caused by proteases, especially metalloproteinases. Therefore, tissue inhibitors of metalloproteinases (TIMP-1 to TIMP-4) [Brew et al.,
Biochim. Biophys. Acta 1477; 267-83 (2000)] is particularly preferred as the arthritic drug substance. alpha ₁ - can be used as a thrombin III and other protease inhibitors such likewise, arthritis acting pharmaceutical substances - antitrypsin, alpha ₁ - anti Chemo antitrypsin, inter α- trypsin inhibitor, alpha ₂ - macroglobulin and anti is there.

A conjugate according to the invention containing chondroadherin linked to a protease such as TIMP-1, TIMP-2, TIMP-3 or TIMP-4 is used to protect cartilage tissue from proteolysis. be able to. The chondroadherin portion of the zygote binds to collagen II, thus placing the zygote within cartilage tissue. Thus, the protease moiety has the ability to directly inhibit the protease within cartilage tissue.

The conjugate according to the invention can be produced by chemically cross-linking a) chondroadherin, a collagen II binding fragment thereof, and b) an arthritic pharmaceutical substance. This can be done by reactions involving common and well-known chemical crosslinkers such as glutaraldehyde.

The conjugates of the present invention can, of course, be produced using recombinant DNA technology. a) a nucleic acid sequence encoding chondroadherin or a collagen II-binding fragment thereof, and b) an arthritic drug is functionally efficient.
) Chondroadherin or collagen II binding fragment thereof, and b) ligated together for the purpose of obtaining a fused nucleic acid encoding a fusion protein comprising a functionally efficient arthritic pharmaceutical substance. Suitable techniques that can be used to obtain such fusion proteins can be found in Sambrook; Molecular Cloning, Laboratory Manual, Second Edition.

The chondroadherin portion of the conjugate according to the invention may be the complete chondroadherin as disclosed in FIG. 6 or, alternatively, at least one.
It may be a truncated derivative containing 0 amino acids. However, such truncated chondroadherin derivative must be able to specifically bind to collagen type II.

The invention also provides a pharmaceutical composition comprising a 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 the tissue suffering from an articular disease such as the affected joint. In such cases, the composition contains a therapeutically effective amount of the conjugate in sterile saline.

Experimental Section : Materials and Methods: Cloning and Sequencing of Human Chondroadherin cDNA: λZA made from human chondrocytes (provided by Dr. Michael Bayliss).
The PII cDNA library was screened with a 450 bp bovine chondroadherin cDNA probe. The 1444 bp clone was isolated and purified with the plasmid Midi kit (Qiagen). Complete sequencing was performed on both strands by standard double-stranded dideoxy terminated sequencing using T3, T7 and synthetic endospecific primers.

Expression of Recombinant Chondroadherin and Generation of Antibodies to Human Chondroadherin One from the C-terminal region of the protein (GQHIRDDTDAFRS, SEQ ID NO: 2) linked to keyhole limpet hemocyanin using glutaraldehyde Immunizing rabbits with the peptide generated polyclonal antisera against human chondroadherin (Collawn et al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons. Inc. 1989, 11.15). Production of anti-peptide antibody p2-3). This antiserum has been shown to react only with chondroadherin in crude tissue extracts.

The 1.4 kbp human chondroadherin cDNA Not I-fragment was transformed into the Epstein-Barr virus-based eukaryotic expression vector pCEP4 (Invitrogen).
e, California, USA). This vector is maintained extrachromosomally in eukaryotic cells and expresses high levels of recombinant protein (Young et al., Gene 198).
8, 62, 171-85). Transcription is driven by the cytomegavirus promoter contained within the pCEP4 vector. The endogenous signal peptide of human chondroadherin was used. The construct in pCE04 was labeled with lipofectamine (
Gibco, BRL) was used to transfect human EBNA cells. Following transfection, cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Gibw, BRL) containing 10% fetal bovine serum. 48
After some time, hygromycin (Calbiochem) (160μ)
g / ml) was added. When the cells were expanded and left to confluence, the medium was changed to fetal calf serum-free DMEM. The cells were left for 48 hours, after which the medium was harvested and centrifuged for 5 minutes at 3000 xg. Phenylmethylsulfonyl fluoride (1 mM, final concentration) was added as a protease inhibitor. One liter of collected medium was diluted 4-fold to a final concentration of 20 mM Tris pH 7.3 and applied to a CM52 column pre-equilibrated in 20 mM Tris pH 7. The column was washed with 20 mM Tris, 0-0 in 20 mM Tris, pH 7.
Eluted with a gradient of 0.5 M NaCl.

Protein purity and identification was confirmed by Western blotting and SDS-PAGE using an antibody raised against the C-terminal region of human chondroadherin.

Separation of Chondroadherin Complex from Cartilage Fresh calf articular cartilage was dissected into 2-3 mm pieces, washed twice in PBS, followed by Hepes buffered saline supplemented with CaCl _2. Washed twice (HBSCa
) (50 mM Hepes, 0.15 M NaCl, 1 mM CaCl ₂ , pH 7.4). 1
Overnight overnight in 15 volumes (weight / volume) of HBSCa supplemented with mM APMA (mercuric p-amino-phenylacetate, Sigma) and 10 μM cycloheximide.
The cartilage was incubated at 7 ° C. Supernatants were collected by brief centrifugation to remove coarser pieces of cartilage, followed by centrifugation at 17700 xg for 30 minutes. The proteinase activated by APMA was inhibited by adding EDTA to a final concentration of 9.8 mM. 43000 rpm for 1 hour at 4 ° C (
The supernatant was finally rendered particle-free by centrifugation in a Beckman Ti50.2 rotor at 100,000 xg).

Ion Exchange Chromatography 200 ml of supernatant from cartilage incubation was added to 9.8 mM EDTA, pH 7.4.
Was applied to a 32 ml column of DE52 (Whatman) equilibrated in Hepes buffered saline containing The flow through was saved and analyzed further. Sodium acetate (0
Elution was performed with a gradient of ~ 1.5M, pH 7.4).

Zone Velocity Centrifuge A gradient (4 ml) of 10-30% glycerol in Hepes buffered saline with EDTA was prepared. Specimens in the same buffer (200 μl) were applied on top, followed by centrifugation at 180 ° C. for 15 hours in a Beckman Sw60.1 rocking bucket rotor at 50,000 rpm (˜257,000 × g). 200 μl
Gradient was drawn from the bottom in the fraction.

SDS-PAGE and Western Blotting: Specimens of the fractions from zonal speed centrifugation were prepared for SDS-PAGE by precipitation with 9 volumes of 95% ethanol containing 50 mM sodium acetate (Paulsson. et al., Biochem. J. 1983, 212; 659-67). The specimen
Separated on a 4-16% polyacrylamide gel. Proteins can be stained directly or PVD using a tank blot system (Mini Trans-Blot BioRad)
The film was transferred to an F film (fluorotrans transfer film, Pal Filtron). The blotted membranes were blocked against additional protein binding to 3% (W / v) milk powder in 10 mM Tris, 0.15 M NaCl, 0.2% Tween 20, pH 7.4.
A primary antibody raised against bovine chondroadherin was applied in the same buffer solution. Bound antibody was detected after incubation with secondary antibody conjugated to horseradish peroxidase by chemiluminescence using the ECL system (Amersham Life Science).

Separation and Purification of Collagen II: Collagen II was prepared as described previously (Miller, Biochemistry 1972).
, 11, 4903-9; Vogel et al., Biochem. J. 1984, 223, 587.
-97) Collagen II was purified from pepsin digested bovine articular cartilage. The purity of collagen II was confirmed by SDS-PAGE (Fig. 1).

Electron Microscopy Chondroadherin- and collagen-containing specimens from the DE52 column flow-through were examined for electron microscopy either directly or with affinity-purified serum labeled with gold against bovine chondroadherin. Prepared either after incubation. Recombinant chondroadherin and collagen type II were preincubated and processed in the same manner as specimens separated from tissue extracts. The sample was adsorbed on a 400-mesh carbon-coated copper grid that was previously rendered hydrophilic by low-pressure glow discharge in the atmosphere. Blotting the grid immediately,
It was washed with 2 drops of water and stained for 15 seconds with 0.75% uranyl formate. At the point of use, as previously described (Baschong et al., J. Electron. Microsc. Tech. 19
90, 14; 313-23) The antibody was labeled with 3 nm size colloidal gold.

Jeol 1200EX operated at an acceleration voltage of 60 kV and a magnification of 75,000 times
The specimen was observed in a transmission electron microscope. Images were recorded on Kodak ESTAR Thick Base 4489 plates without pre-irradiation, typically at a dose of 2000 electrons / nm ² . Data from electron micrographs were evaluated (Engel et al., Meth.
ods Enzymol. 1987, 145: 3-78). An antibody against the C-terminal region of collagen type II (donated by Dr. Rikard Holmdahl) was used to determine the polarity of the type II collagen molecule.

Interactions Studied by Surface Plasmon Resonance To further characterize the interaction and determine the binding properties between chondroadherin and type II collagen, the BoAcore ^™ 20 system (BIAcoreAB, Sweden).
)It was used.

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 surface of the carboxymethylated dextran on the chip (CM5 sensor chip, BIAcore AB, Uppsala Sweden) was subjected to 35 μl of 50 nM N-hydroxysuccinimide and 35 μl 200 mM N-ethyl-N ′-(dimethyl) at a flow rate of 5 μl / ml at 25 ° C. Activation with aminopropyl) carbodiimide. Chondroadherin (35 μl at 100 μg / ml) was immobilized at 25 ° C. with a flow rate of 5 μl / min. One surface was used as a negative control and contained no conjugated protein. The remaining activated groups were blocked with 40 μl of 1 M ethanolamine, pH 8.5. Immobilization of recombinant chondroadherin
This resulted in 4000 resonance units (RU) (1000 RU = 1 ng / mm ² ).

10 mM Hepes pH 7.4, 0.15M NaCl applied at 40 μl / min
Binding assays were performed at 25 ° C. by using pepsin-treated collagen II at different concentrations within 0.09-6 μg / ml in 0.05% Tween. 0
0.5M acetic acid, 2M NaCl followed by 0.1M NaHCO ₃ , 2M Na
The surface was regenerated by using Cl, pH 9.2.

In another set of experiments, pepsin-treated collagen type II (2.1 mg / ml in 0.1 M acetic acid) was added to 100 μg / ml in 10 mM sodium citrate, pH 3.2.
Dilute to ml and immediately use the same conditions as above (running buffer: 10m
Immobilization under M Hepes, 0.15 M NaCl, 0.05% Tween, pH 7.4). Immobilization of collagen type II resulted in 3000 RU.

10 mM Hepes pH 7.4, 0.15 M NaCl applied at 40 μl / min
Binding assays were performed at 25 ° C. by using recombinant chondroadherin at different concentrations within the range 0.03-4 μg / ml in 0.05% Tween. 10 mM
Hepes, 2M NaCl, pH 7.4 followed by 0.1M NaHCO ₃ , 2M
The surface was regenerated by using NaCI, pH 9.2.

BIA evaluation 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: As a result of screening a human cDNA library, a clone of 1444 bp was isolated. The deduced amino acid sequence was published (Grover et al., Genomics
1997, 45; 379-85). However, there were some differences. Leucine replaces valine at position 114 in the sequence, 1
At position 66, alanine was found instead of proline. Our sequence at these positions has been found in the mouse (Landgren et al., Genomics 1998, 47; 84-9.
1), rat (Shen et al., Biochem, J. 1998, 330; 549-57) and cow (Neame et al. J. Biol. Chem. 1994, 269; 21547-5).
It was found to match the sequence from 4).

Expression of Native Recombinant Protein: 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 1-2 μg per ml of medium. The protein was SDS-PAGE (Fig. 1
) And appears to be pure as shown by Western blotting with antibodies raised against the C-terminal region of the protein. Chondroadherin exists in two forms that differ by 9 amino acids at the C-terminus. Both forms are expressed by EBNA cells and upon purification on the CM52 ion exchanger, they elute differently in the partially 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 analyzed further. Gel filtration of the supernatant representing released protein on Superose & showed that chondroadherin eluted in the early fractions, indicating that it occurred within a very large size complex. Was shown (data not shown). Ultracentrifugation at 100,000 g for 1 hour showed that chondroadherin remained in the supernatant and was therefore not present within the particles. The supernatant was subjected to anion exchange chromatography for further study of the complex. At this time, the chondroadherin pool was separated into two fractions, that is, a fraction passing through the column and a fraction retained on the column. The latter was eluted with several proteins, including biglycan as the major component, at a sodium acetate concentration of 0.7-0.8M. DE
The pool through the column was replenished with collagen IIα1-chain (as deduced from migration on SDS-PAGE under non-reducing and reducing conditions) and chondroadherin and other (as detected by Western blot). It contained several components. Among these other components, the C-terminal propeptide of collagen II was identified as a prominent band by Western blot (data not shown). Pools containing collagen and chondroadherin as well as other components were further fractionated by zone velocity centrifugation and analyzed by electron microscopy.

Zone Velocity Centrifuge: Flow-through specimens from DE52 columns containing multiple different molecular units were further fractionated by zone centrifugation in a glycerol gradient. The composition of the various fractions was studied by SDS-PAGE electrophoresis and Western blotting of non-reducing specimens. The upper part of the gradient with the slowest settling (Fig. 2
Collagen type II was detected in parentheses. The same fraction from the same gradient also contained chondroadherin as demonstrated by Western blotting (Fig. 2).

Electron Microscopy: Electron microscopy showed filaments of intact monomeric collagen molecules with attached globules with a canonical trilobal appearance of LRR protein at completely different locations (FIG. 3A). . To confirm its properties, the complexes were incubated with affinity-purified antibodies labeled with gold against chondroadherin before electron microscopy. The image then showed the canonical IgG structure of three globules associated with the putative chondroadherin globules bound to collagen (FIG. 3B). Thus, the isolated complex is representative of collagen with bound chondroadherin. No other molecule appeared to bind at these sites on collagen. Similar complexes could be formed with recombinant chondroadherin and isolated pepsin-treated collagen II.
An equimolar amount of collagen II was defined by an antibody that recognizes an epitope near its C-terminus (donated by Dr. Rikard Holmdahl, Lund). Collagen C
The binding site was defined by measuring the distance from the end to the chondroadherin binding site. The relative frequencies of chondroadherin binding at different sites on collagen are shown in FIG. These data show that there are two different binding sites at 185 nm and 267 nm from the C-terminus of collagen, and that both of these sites occupy similar regions in vivo (Figure 4A) and in vitro (Figure 4B). Demonstrate to show status.

In vitro interaction between chondroadherin and collagen II; chondroadherin and collagen II immobilized in the first set of experiments
The type interactions were studied with different concentrations of collagen, resulting in FIG.
The association and dissociation curves shown in Figure 3 were obtained. Evaluation using a Langmuir model predicting a 1: 1 interaction revealed a dissociation constant of 0.6 nM. Using a model that tolerates a bivalent analyte (collagen II), a significantly lower K _D was obtained for the stronger of the two putative binding sites.

In another set of experiments, collagen type II was immobilized and the interaction with chondroadherin was studied (FIG. 5B). Evaluation of these data revealed that the dissociation constant for the interaction in this system was 40 mM.

[Brief description of drawings]

FIG. 1 discloses SDS-PAGE of collagen II and recombinant chondroadherin used in the surface plasmon resonance assay.

FIG. 2 shows zone velocity sedimentation of chondroadherin-containing complexes on a glycerol gradient. Above is the SDS- fraction of the flow-through glycerol gradient centrifugation from ion exchange chromatography of bovine cartilage treated with APMA.
PAGE and Coomassie staining are shown. The lower fraction is on the left and the upper fraction is on the right. Collagen type II is mainly present in the upper fraction. The lower panel shows a Western blot analysis of chondroadherin within the same fractions as above. Chondroadherin is mainly present in the upper fraction.

FIG. 3 relates to electron microscopy of collagen II-chondroadherin complex. Figure 3
A shows electron microscopy of collagen type II-chondroadherin complex in flow-through from ion exchange chromatography of medium from APMA-treated bovine cartilage. The complex is indicated by an arrow. The polarity of collagen is defined by the antibody F4 indicated by the arrow head. FIG. 3B shows the same as FIG. 3A, but
Here, affinity-purified antibodies labeled with gold generated against bovine chondroadherin were added, indicating that the protein that interacts with collagen type II is chondroadherin. . The complex is indicated by an arrow. The polarity of collagen is defined by the antibody F4 indicated by the arrow head. FIG. 3C depicts electron microscopy of a reconstituted complex of recombinant chondroadherin and collagen type II. The complex is indicated by a single arrow. The polarity of collagen is defined by the antibody F4 indicated by the arrow head.

FIG. 4: Distribution of bound chondroadherin along monomeric collagen II. Plate (A) above shows collagen I found in the complex separated from cartilage.
1 shows the distribution of chondroadherin along type I. Relative frequency represents the fraction of chondroadherin molecules bound to a given site on Collagen II. C
The ends are at 0 nm as determined by using an antibody against the C-terminus of collagen type II. Plate (B) below shows the binding site of the in vitro reconstituted collagen II-chondroadherin complex.

FIG. 5 shows the characteristics of chondroadherin collagen II interaction. Surface plasmon resonance assay (BIAcore) showing interaction between collagen type II and chondroadherin
). The figure shows the association step (0-60 seconds) and the dissociation step (0-60 seconds). Plate A shows the interaction of collagen type II on immobilized chondroadherin. Plate B shows the interaction of chondroadherin on immobilized collagen type II.

FIG. 6 discloses the cDNA sequence for human chondroadherin and its putative amino acid sequence. Signal sequences are shown in bold. Stop codons are indicated by dots.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 29/00 101 C07K 14/52 43/00 111 14/78 ZNA C07K 14/52 C12N 15/00 A 14 / 78 ZNA A61K 37/02 C12N 15/09 37/04 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM , KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, A , AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA , MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF F terms (reference) 4B024 AA01 CA04 4C084 AA01 AA07 AA14 AA19 BA01 BA08 BA18 BA23 BA41 BA44 CA17 CA20 CA34 DA01 DA12 DA25 DA58 DC32 DC34 DC35 MA02 NA14 4H960 ZB2ZA0 AA30 BA41 CA40 DA01 DA56 EA22 EA28

Claims (10)

[Claims] 1. A conjugate comprising a) chondroadherin or collagen II binding fragment thereof and b) an arthritic drug substance. 2. The conjugate according to claim 1, characterized in that the arthritic pharmaceutical substance is selected from the group consisting of cytokines, cytokine antagonists and protease inhibitors. 3. A substance which affects arthritis is a tissue inhibitor of metalloproteinase (TIMP-1, TIMP-2, TIMP-3, TIMP-4), α _1.
Conjugate according to claim 2, characterized in that it is selected from the group consisting of: anti-trypsin, α ₁ -antichymotrypsin, inter-α-trypsin inhibitor, α ₂ -macroglobulin and anti-thrombin III. . 4. The conjugate according to any one of claims 1 to 3, wherein the chondroadherin moiety and the arthritic drug are joined using chemical crosslinking. 5. The conjugate according to any one of claims 1 to 3, which is a fusion protein containing a) chondroadherin or a collagen II-binding fragment thereof, and b) an arthritic drug substance. 6. A nucleic acid sequence encoding the fusion protein of claim 5. 7. The chondroadherin moiety comprises at least 10 consecutive amino acid residues from the amino acid sequence disclosed in FIG.
The joined body according to any one of 5 above. 8. The joined body according to any one of claims 1 to 5 or 7 for medical use. 9. Use of the conjugate according to claim 8 for preparing a pharmaceutical composition for treating and / or alleviating the symptoms of arthritis, for example rheumatoid and rheumatoid arthritis. 10. A pharmaceutical composition comprising a conjugate according to any one of claims 1-5 or 7 in combination with a pharmaceutically acceptable carrier.