Biological materials and uses thereof

[0001] The present invention relates to tenascin-C and its activity in chronic inflammation. Tenascin-C modulators and its biological activity are also provided.

[0002] Inflammation is the complex biological response of tissues to harmful stimuli, such as pathogens, tissue damage or irritants. It is an attempt at tissue protection to remove harmful stimuli as well as to start the tissue healing process. Inflammatory abnormalities include a large group of unrelated disorders that underlie many human diseases (inflammatory disorders). Examples of inflammatory diseases include (but are not limited to) asthma, autoimmune disease, glomerulonephritis, allergy (hypersensitivity), inflammatory bowel disease, reperfusion injury, rheumatoid arthritis and transplant rejection. [0003] In particular, chronic inflammation is a debilitating and serious condition associated with many of the above diseases and characterized by persistent inflammation at the site of infection or injury, or in connection with altered immune responses such as autoimmune disease.

[0004] Rheumatoid arthritis (RA) is a typical example, although by no means the only, chronic inflammation. RA is characterized by synovitis and destruction of articular cartilage and bone by sustained synthesis of proinflammatory cytokines and matrix metalloproteinases (MMPs). Biological compounds that inhibit the synthesis of inflammatory cytokines such as TNFα and IL-6 are short-term effective in the treatment of RA. However, repetition of therapy is required, which makes this therapeutic approach expensive and does not provide long-term remission. Furthermore, in the case of complete systemic inhibition of cytokine function, there are problems associated, for example, with an increased infectious risk. Therefore, despite advances in care, there is an unmet need for an economical treatment regimen for chronic inflammatory diseases that is long-term effective (Smolen (2006) and Williams (2007)).

[0005] The mechanisms underlying chronicity of the disease remain unclear and the factor (s) that drive (s) the prolonged expression of inflammatory and destructive mediators are currently unknown.

[0006] Toll-like receptors (TLRs) play a key role in stimulating the production of inflammatory mediators in RA, and blocking TLR function may have significant clinical advantages (reviewed by Brentano (2005) and O'Neill (2002)). This family of receptors is an integral part of the immune system. TLRs mediate host defense against infection and damage by recognizing both pathogen-related molecular patterns (PAMP) and damage-related molecular patterns (DAMP) (Matzinger (2002)). DAMP are endogenous pro-inflammatory molecules produced after tissue damage and include intracellular molecules released from damaged or necrotic cells, extracellular matrix fragments (ECM) or ECM molecules produced at a higher level after injury (reviewed in Bianchi (2007) and Gordon (2002)).

[0007] After activation, TLRs stimulate both innate and acquired immune responses including stimulation of proinflammatory cytokine and MMP expression (Medzhitov (2002)). TLRs are expressed at a high level in the synovial tissue of RA patients (Radstake (2004), Roelofs (2005), Sacre (2007) and (Sacre, composite manuscript 2008) and mice with targeted deletions or loss-of-type mutations in TLR4 are protected against experimental arthritis (Choe (2003) and Lee (2005). In addition, TLR4 inhibitors may reduce destructive arthritis in mice (AbdollahiRoodsaz (2007)) and the putative TLR4 inhibitor improved symptoms in 15 of 23 patients with moderate to severe RA in a preliminary phase I study (Vanags (2006). However, it is unclear which TLR ligand (s) are involved in the pathogenesis of the disease.

[0008] Tenascin-C is an ECM glycoprotein that is associated with tissue damage and wound repair. Tenascin-C is expressed especially during active tissue remodeling during embryogenesis, with it being observed for the first time during gastrulation and somite formation. In the later stages of development, expression is limited to the morphogenesis sites of the mammary gland and lung branches, in the developing skeleton, cardiovascular system and connective tissue at sites of epithelial-mesenchymal transition. Expression decreases after these processes and before embryogenesis is complete (Jones (2000)).

[0009] Tenascin-C is normally not expressed in healthy adult tissues, but in adults its expression is specifically and transiently increased during acute inflammation and is constantly expressed in chronic inflammation (review Chiquet-Ehrismann (2003)). Immunohistochemical results show that little tenascin-C is expressed in normal human joints, but its levels are significantly elevated in RA synovial fluid, in areas of inflammation and fibrosis, and especially below the synovial lining, in the invasive cusp, and around the blood vessels (Cutolo ( 1992), MacCachren (1992) and Salter (1993)). There was also a significant increase in tenascin-C levels in the synovial fluid of RA patients (Chevalier (1994) and Hasegawa (2007)) and in the cartilage of RA (Salter (1993) and Chevalier (1994)).

[0010] Tenascin-C is a large hexameric protein with a mass of 1.5 million Da. Each chain contains different domains, including splicing domains (TA), EGF-like repeats (EGF-L), fibronectin-like repeat type III (TNIII) and fibrinogen-like globe type (FBG) (review Orend (2005 )). The sequences of tenascin-C and its domains are shown in Figure 13.

[0011] Until now, the role of tenascin-C in inflammation has been uncertain, with evidence showing different effects on different immune cells. For example, tenascin-C has been shown to support the adhesion and turning of human primary peripheral blood lymphocytes and tonsils, suggesting its role in stimulating lymphocyte migration (Clark (1997)). In addition, mice lacking tenascin-C show reduced lymphocyte infiltration and lower levels of IFN, TNF and IL-4 mRNA after concanavalin-induced liver damage in mice (El-Karef (2007)). Thus, evidence suggests that tenascin-C is involved in stimulating the activity of acute inflammatory cells. However, it has also been described that tenascin-C inhibits monocyte chemotaxis in vitro (Loike (2001)) and mice lacking tenascin-C show increased migration of monocytes and macrophages in breast tumor stroma (Talts (1999)). Thus, these data suggest that tenascin-C plays a role in inhibiting inflammatory cells.

[0012] The inventors have shown that tenascin-C is an endogenous TLR4 ligand that is required for the destructive arthritis observed in arthritis.

[0013] Furthermore, it has now been shown that tenascin-C is not associated with causing inflammation (acute inflammatory response), but instead is involved in prolonging the inflammatory response characterizing chronic inflammation. In particular, tenascin-C has been shown to be an endogenous TLR4 activator and has been shown to be required for destructive arthritis.

[0014] The role of tenascin-C in mediating the immune response in the joint has been demonstrated by inducing arthritis after intra-articular injection of tenascin-C FBG domain into mice in vivo. In addition, zymosan-induced acute arthritis was not as prolonged in tenascin-C deficient mice. Wild-type and tenascin-C depleted mice responded to zymosan's acute inflammation in the same way, indicating that tenasacin-C is not involved in initiating the inflammatory process. However, the shorter-lived synovitis in mice lacking tenascin-C indicates a role in maintaining arthritis. The importance of tenascin-C in prolonging arthritis was emphasized by the observation that targeted deletion of tenascin-C protected mice from prolonged erosive arthritis during arthritis induced by mBSA immunization.

[0015] Tenascin-C has been shown to be able to activate cells in the joint and the main active domain of tenascin-C has been mapped to a globe-type fibrinogen-like domain (FBG), 227 amino acid (26.9 kDa), C-terminal globular domain of the molecule (Siri (1991)).

[0016] The addition of FBG to synovial culture from RA patients increased the spontaneous release of proinflammatory cytokines. It also stimulated the synthesis of TNF-α, IL-6 and IL-8 in primary human macrophages and IL-6 in synovial RA fibroblasts by activating TLR4 and MyD88 dependent signaling pathways.

[0017] It has been shown that, as in the case of LPS, expression of TLR4 is necessary for the induction of cytokine synthesis by FBG. However, unlike LPS, neither CD14 nor MD-2 appears to be required for TLR-4 activation. CD14 is unnecessary for TLR4 activation by other ligands. TLR4 is not required to respond to lipid A in a MyD88 dependent manner (Jiang (2005)), EDA fibronectin can activate mast cells even in the absence of CD14 (Gondokaryono (2007)) and hyaluronic acid activation of human monocytic THP-1 cells requires a complex TLR4, CD44 and MD-2, but not CD14 (Taylor (2007)).

[0018] The formation of separate receptor complexes by each TLR4 ligand may facilitate the recruitment of different adapter / signal intracellular molecules. This may be responsible for the different cellular responses observed for FBG and

LPS, for example for the lack of IL-8 induction by FBG in RA synovial fibroblasts. Similarly, activation of the TLR4 and CD44 complex by hyaluronic acid induces a gene expression pattern in mouse alveolar macrophage cell lines that is different than for LPS (Taylor (2007)). The fact that FBG induces IL-8 synthesis in human macrophages suggests that there is cell type specific ligand recognition and / or signaling.

[0019] The tightly regulated expression pattern of tenascin-C makes it an attractive target for the treatment of chronic inflammation. It is mainly absent in healthy adults, however, its expression is specifically induced after tissue damage. Tenascin-C is transiently expressed during acute inflammation: induction often precedes inflammation and both mRNA and protein are absent in the tissue when the inflammation is cured (review Chiquet-Ehrismann (2003)).

[0020] It has now been shown that sustained expression of tenascin-C is associated with chronic inflammation. In addition to RA, increased levels of tenascin-C are observed in other autoimmune diseases, including multiple sclerosis (Gutowski (1999)) and Sjogren's disease (Amin (2001)) and in non-healing wounds and diabetic and varicose ulcers (Loots (1998 )). Tenascin-C synthesis de novo correlates well with the intensity of inflammation in oral mucosa diseases and plasma levels of tenascin-C are a reliable indicator of the activity of inflammatory bowel diseases before and after treatment or surgery (reviewed by Chiquet-Ehrismann (2003)).

[0021] In a first aspect of the invention, there is provided an agent for modulating a chronic inflammatory response, which agent modulates the biological activity of tenascin-C, wherein the agent is an antibody or antigen-binding fragment thereof having specificity for the FBG domain of tenascin-C.

[0022] The agent of the first aspect of the invention may modulate the biological activity of tenascin-C by changing the binding properties of tenascin-C.

[0023] Also described herein are agents that can modulate the biological activity of tenascin-C by changing the transcription and / or translation of tenascin-C.

[0024] Such agents can be identified by methods well known in the art, such as:

(a) determining the effect of the test agent on tenascin-C expression level, for example using Southern blotting or similar hybridization techniques;

(b) determining the effect of the test agent on the level of tenascin-C protein, for example in immunoassays using anti-tenascin-C antibodies;

and (c) determining the effect of the test agent on the functional marker or the result of tenascin-C, for example, by the methods of the examples.

[0025] The agents disclosed herein may reduce the biological activity of tenascin-C.

[0026] The agents disclosed herein may increase the biological activity of tenascin-C. The desire to increase the immunological and inflammatory activity of molecules and cells is important for the development of therapy for patients with immune and inflammatory disorders and for the development of vaccines (see Harandi (2009)).

[0027] The agent described herein may be a tenascin-C transcription inhibitor.

[0028] The agent described herein may be a tenascin-C translation inhibitor.

[0029] The agent of the first aspect of the invention may be an inhibitor of tenascin-C binding properties. For example, the agent may change the conformation of tenascin-C so that it is no longer able to bind to its receptor.

[0030] The agent of the first aspect of the invention may be a competitive inhibitor of tenascin-C binding. It will also be understood by those skilled in the art that the agent may also inhibit the biological activity of tenascin-C by blocking the action of the tenascin-C receptor directly (by acting as a tenascin-C receptor antagonist ) or indirectly (by binding to intermediate or helper molecules).

[0031] The agent of the first aspect of the invention may be a TLR-4 receptor antagonist.

[0032] It will be understood by those skilled in the art that the inhibition of the biological activity of tenascin-C by the agent of the invention may be total or partial. For example, the agent may inhibit the biological activity of tenascin-C by at least

10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably 100% compared to the biological activity of tenascin-C on inflammatory cells that have not been exposed to this agent.

[0033] The agent described herein may be selected from the group consisting of small interfering RNA (siRNA) molecules, short RNA hairpin structure (shRNA) molecules, antisense oligonucleotides, compounds with tenascin-C binding affinity, antibodies (polyclonal or monoclonal) and antigen-binding fragments, small inhibitory compounds, polypeptides and proteins.

[0034] In one example, the agent is siRNA. RNA interference is a two-step process. In the first stage, which is referred to as the initiation stage, the initial dsRNA is digested into 21-23 nucleotide (nt) small interfering RNA (siRNA), probably as a result of the action of the Dicer protein, a member of the RNase III family of dsRNA-specific ribonucleases that it processes (cleaves ) dsRNA (introduced directly or via a transgene or virus) in an ATP-dependent manner. Subsequent cleavage events degrade RNA into 19-21 bp duplexes (siRNAs), each with 230 nucleotide 3 'protruding ends (Hutvagner and Zamore, 2002, Curr. Opin. Genetics and Development 12: 225-232; Bernstein, 2001, Nature 409: 363-366).

[0035] In the effector step, siRNA duplexes bind to the nuclease complex to form RNA-induced silencing complex (RISC). ATP-dependent siRNA duplex development is required for RISC activation. Active RISC is then directed to a homologous transcript through base pairing interactions and cleaves the mRNA into 12-nucleotide fragments from the 3 'end of siRNA (Hutvagner and Zamore, 2002, supra; Hammond et al., 2001, Nat. Rev. Gen. 2: 110-119 (2001); Sharp, 2001, Genes Dev 15: 485-90). Although the cleavage mechanism is still not clear up, studies have shown that each RISC contains one siRNA and RNase (Hutvagner and Zamore, 2002, supra).

[0036] Considering the extraordinary potency of RNAi, an amplification step in the RNAi pathway has been suggested. Amplification can occur by copying the initial dsRNA, which would produce more siRNAs, or by replicating the produced siRNAs. Alternatively or additionally, amplification can occur through many RISC trading events (Hammond et al., 2001, supra; Hutvagner and Zamore, 2002, supra.). Additional information about RNAi can be found in the following review papers, Tuschl, 2001, Chem. Biochem. 2: 239-245, Cullen, 2002, Nat. Immunol. 3: 597-599 and Brantl, 2002, Biochem. Biophys Act. 1575: 15-25.

[0037] The synthesis of RNAi molecules suitable for use in the present invention can be carried out as follows. First, the tenascin-C mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. The occurrence of each AA and 3'19 neighboring nucleotides is recorded as potential siRNA target sites. Preferably, siRNA targets are selected from the open reading frame because the non-translated regions (UTRs) are richer in regulatory protein binding sites. UTR binding proteins and / or translation initiation complexes may interfere with the binding of the siRNA endonuclease complex (Tuschl, ChemBiochem. 2: 239-245). However, it will be appreciated that siRNAs targeting non-translated regions may also be effective.

[0038] Secondly, potential targets are compared to the appropriate genomic database (e.g. human, mouse, rat, etc.) using a sequence-matching program such as BLAST (<a href="http://www.ncbi.nlm.nih.gov/BLAST/">www.ncbi.nlm.nih.gov/BLAST/</a>). Putative destinations that show significant homology to other coding sequences are filtered.

[0039] Eligible target sequences are selected as template for siRNA synthesis. Sequences involving a low G / C content are preferred because they have been shown to be more effective in directing gene silencing compared to those with a G / C content higher than 55%. Preferably, several targets are selected along the length of the target gene for evaluation. For better evaluation of selected siRNAs, a negative control is preferably used together. The negative siRNA control preferably has the same nucleotide composition as siRNA, but no significant homology to the genome. Thus, preferably, the mixed siRNA nucleotide sequence is used, provided that it does not show significant homology to another gene.

[0040] Suitable siRNA molecules can be synthesized as described above so that they are complementary and thus bind to the entire nucleotide sequence of tenascin-C or parts thereof. The nucleotide sequence of tenascin-C is shown in Figure 14. [0041] In one example, the agent may be a short RNA hairpin structure (ShRNA).

[0042] A small or short RNA molecule with a hairpin structure (shRNA) is an RNA sequence that produces a tight hairpin turn that can be used to silence gene expression by RNA interference. shRNA uses a vector (usually adenoviral or lentiviral) introduced into cells and uses the U6 promoter to ensure that shRNA is always always expressed. This vector is usually passed on to daughter cells, allowing inheritance of gene silencing. The shRNA hairpin structure is cleaved by cellular machinery for siRNA, which is then bound to RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that matches the siRNA that has been bound to it (McIntyre (2006) and Paddison (2002)) [0043] The agent of the first aspect of the invention may be a tenascin-C domain or a variant thereof. The FBG domain has been shown to be primarily involved in tenascin-C interaction with its target for persistence of chronic inflammation. Accordingly, the preferred domain is the FBG domain (sequence shown in Figure 13) or variants thereof.

[0044] In an alternative example, the agent is an antisense oligonucleotide.

[0045] Designing antisense molecules that can be used to efficiently reduce tenasacin-C levels / activity requires consideration of two aspects important for an antisense solution. The first aspect is to provide an oligonucleotide to the cytoplasm of cancer cells, while the second aspect is to design an oligonucleotide that specifically binds the designated mRNA within the cells in a way that inhibits its translation.

[0046] The prior art describes many delivery strategies that can be used to efficiently deliver oligonucleotides to many different cell types (for example, see Luft, 1998, J Mol Med 76: 75-6; Kronenwett et al. 1998, Blood

91: 852-62; Rajur et al., 1997, Bioconjug Chem 8: 935-40 ;. Lavigne et al., 1997, Biochem Biophys Res Commun 237: 566-71; Aoki et al., 1997, Biochem Biophys Res Commun 231: 540-5).

[0047] In addition, sequence identification algorithms are available with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that takes into account the energy of structural changes on the target mRNA and oligonucleotide (see, for example, Walton et al. 1999, Biotechnol Bioeng 65: 1- 9).

[0048] Several approaches are also known for designing and predicting the efficacy of specific oligonucleotides using an in vitro system (see, for example

Matveeva et al., 1998, Nature biotechnology 16: 1374-1375).

[0049] Several clinical trials have demonstrated the safety, applicability and activity of antisense oligonucleotides. For example, antisense oligonucleotides useful for treating cancer have been used successfully (Holmlund et al., 1999, Curr Opin Mol Ther 1: 372-85; Gerwitz, 1999, Curr Opin Mol Ther 1: 297-306).

Recently, it has been reported that suppression of human heparanase gene expression mediated by antisense oligonucleotides inhibits pleural dissemination of human tumor cells in a mouse model (Uno et al., 2001, Cancer Res 61: 7855-60).

[0050] Thus, those skilled in the art will be able to easily design and implement antisense approaches suitable for modulating tenascin-C expression.

[0051] Preferably, the antisense oligonucleotide is 15 to 35 bases in length. For example, 20-merc oligonucleotides have been shown to inhibit expression of epidermal growth factor receptor mRNA (Witters et al., Breast Cancer Res Treat 53: 41-50 (1999)) and 25-merc oligonucleotides have been shown to reduce expression of adrenocorticotropic hormone by more than 90% (Frankel et al., J Neurosurg 91: 261-7 (1999)). However, it is understood that it may be desirable to use oligonucleotides of lengths outside this range, for example 10, 11, 12, 13 or 14 bases or 36, 37, 38, 39 or 40 bases.

[0052] In addition, one skilled in the art will recognize that oligonucleotides are broken down and inactivated by endogenous cell nucleases. To counteract this problem, it is possible to use modified oligonucleotides, e.g. with altered internucleotide linkages, in which the naturally occurring phosphodiester linkages have been replaced by another linkage. For example, Agrawal et al. (1988) Proc. Natl. Natl. Acad. Sci. USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-1 using phosphoramidite and phosphorothioate oligonucleotides. Sarin et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451 showed increased inhibition of HIV-1 using methylphosphonate oligonucleotides. Agrawal et al. (1989) Proc. Natl. Natl. Acad. Sci. USA 86, 7790-7794 demonstrated inhibition of HIV-1 replication in both early-stage and chronically infected cell cultures using nucleotide sequence specific phosphorothioate oligonucleotides. Leither et al. (1990) Proc. Natl. Natl. Acad. Sci. USA 87, 3430-3434 describe inhibition in tissue culture of influenza virus replication by phosphorothioate oligonucleotides.

[0053] Oligonucleotides containing artificial bonds have been shown to be resistant to in vivo degradation. For example, Shaw et al. (1991) in Nucleic Acids Res. 19, 747-750, describe that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3'-end by certain protective structures, and that unprotected phosphorothioate oligonucleotides are not degraded in vivo.

[0054] For a detailed description of the H-phosphonate approach for the synthesis of phosphorothioate oligonucleotides, see Agrawal and Tang (1990) Tetrahedron Letters 31, 75417544, the disclosure of which is incorporated herein by reference. The synthesis of methylphosphonates, phosphorodithioates, phosphoramidites, phosphate esters, bridged phosphoramidites and oligonucleoside bridged phosphorothioates are known in the art. See, for example, Agrawal and Goodchild (1987) Tetrahedron Letters 28, 3539; Nielsen et al. (1988) Tetrahedron Letters 29, 2911; Jager et al. (1988) Biochemistry 27, 7237; Uznanski et al. (1987) Tetrahedron Letters 28, 3401; Bannwarth (1988) Helv. Chim. Acta. 71, 1517; Crosstick and Vyle (1989) Tetrahedron Letters 30, 4693; Agrawal et al. (1990) Proc. Natl. Natl. Acad. Sci. USA, 87, 1401-1405, the disclosure of which is incorporated herein by reference. Other synthesis or preparation methods are also possible. In a preferred embodiment, the oligonucleotide is deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences can also be synthesized and used.

[0055] Oligonucleotides useful in the examples described herein are preferably designed to be resistant to degradation by endogenous nucleolytic enzymes. Oligonucleotide degradation in vivo produces oligonucleotide degradation products of reduced length. Such degradation products are more likely to engage in nonspecific hybridization and are less likely to be effective compared to their full length counterparts. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and that are able to reach target cells. Current oligonucleotides can be made more resistant to degradation in vivo by substituting one or more internal artificial internucleotide linkages in place of native phosphodiester linkages, for example by replacing phosphate in sulfur. Examples of bonds that can be used include phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, various phosphoramidite, phosphate esters, bridged phosphorothioate, and bridged phosphorothioate. These examples are provided to illustrate, rather than limit the scope, because other internucleotide linkages are well known in the art. The synthesis of oligonucleotides containing one or more of these bonds substituted in place of phosphodiester internucleotide linkages is well known in the art, including synthetic routes for the production of oligonucleotides containing mixed internucleotide linkages.

[0056] Oligonucleotides can be made resistant to elongation by endogenous enzymes by protecting their ends or introducing similar groups containing 5'- or 3'-end nucleotides. The end seal reagent is commercially available as Amino-Link II ™ from Applied BioSystems Inc, Foster City, CA. Methods of protecting the ends are described, for example, in Shaw et al. (1991) Nucleic Acids Res. 19, 747750 and Agrawal et al. (1991) Proc. Natl. Natl. Acad. Sci. USA 88 (17), 7595-7599.

[0057] A further way of producing oligonucleotides resistant to nuclease attack is that they are "auto-stabilized" as described in Tang et al. (1993) Nucl. Acids Res. 21, 2729-2735. Auto-stabilized oligonucleotides have hairpin loop structures at their 3 'ends and exhibit increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I, and fetal bovine serum. The autostabilized region of the oligonucleotide does not interfere with hybridization with complementary nucleic acids, and pharmacokinetic and stability studies in mice showed increased in vivo persistence of auto-stabilized oligonucleotides relative to their linear counterparts.

[0058] In an example where the agent is a compound having tenascin-C binding affinity, the compound may bind essentially reversibly or substantially irreversibly at the active site of tenascin-C. In a further example, the compound may bind to a portion of tenascin-C that is not an active site such as to interfere with tenascin-C binding to the receptor or ligand. In yet another example, the compound may bind to a portion of tenascin-C to reduce protein activity by allosteric action. This allosteric activity may be an allosteric activity that is involved in the natural regulation of tenascin-C activity, for example in the activation of tenascin C by an "up-activating activator".

[0059] Methods for detecting the interaction between a test compound and tenascin-C are well known in the art. For example, ultrafiltration methods with ion scattering mass spectrometry / HPLC or other physical and analytical methods can be used.

In addition, fluorescence resonance energy transfer (FRET) methods can be used in which the binding of two fluorescently labeled particles can be measured by measuring the interaction of the fluorescent labels when they are close together.

[0060] Alternative methods for detecting binding of a polypeptide to macromolecules, for example DNA, RNA, proteins and phospholipids, include a surface plasmon resonance assay, for example as described in Plant et al. 1995, Analyt, Biochem 226 (2), 342-348. The methods can utilize the use of a polypeptide that is labeled with, for example, a radioactive or fluorescent label.

[0061] A further way of identifying a compound that is capable of binding a polypeptide is one in which the polypeptide is exposed to the compound and each binding of the compound to said polypeptide is detected and / or measured. A binding constant for binding a compound to a polypeptide can be determined. Suitable methods for detecting and / or measuring (quantifying) binding of a compound to a polypeptide are well known to those skilled in the art and can be implemented, for example, using a high throughput method, for example a chip-based method. The new technology, called VLSIPS ™, has made it possible to produce very small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or arrays have probes arranged in arrays, with each probe being assigned to a specific location. Biological chips have been produced in which each place is on a scale of, for example, ten microns. Chips can be used to determine if target molecules interact with any of the probes on the chip. After exposing the matrix to target molecules under selected conditions, the scanning devices can examine each location on the matrix and determine whether the target molecule interacted with the probe at that location.

[0062] Another way to identify compounds with binding affinity for tena-scin-C is a two-hybrid yeast system in which the polypeptides of the invention can be used to "capture" tenascin-C binding proteins. A two-hybrid yeast system is described in Fields and Song, Nature 340: 245-246 (1989).

[0063] In a further example, the agent is a compound that has the ability to bind ligand to tenascin-C.

[0064] For example, the agent may be a soluble fragment of the tenascin-C receptor (such as FPRL1). Alternatively, the agent may be a high affinity molecule that mimics the antibody (so-called "affibody") (for example, see Patent Specification

US 5831012 and <a href="http://www.affibody.se">www.affibody.se</a>). These ligands are small, simple proteins consisting of a bundle of three helices based on a scaffold from one of the IgG Protein A binding domains (Staphylococcus aureus surface protein). This scaffold has excellent affinity ligand properties and can be designed to bind with high affinity to any given target protein.

[0065] The agent of the first aspect of the invention is an antibody or antigen binding fragment thereof. The antigen binding fragment can be selected from the group consisting of Fv fragments (e.g. single chain Fv and Fv with disulfide linkages), Fab-like fragments (e.g. Fab fragments, Fab 'fragments and F (ab) 2 fragments), single variable domains (e.g. VH and VL domains) and domain antibodies (dAb, including single and double formats [i.e. dAb-dAb-linker]].

[0066] The antibody may preferably specifically bind to the FBG domain that it activates

TLR4.

[0067] The advantages of using antibody fragments rather than whole antibodies are various. Smaller fragment sizes can lead to improved pharmacological properties, such as better penetration into solid tissue. In addition, antigen-binding fragments, such as Fab, Fv, scFv fragments, and dAb fragments of the antibody can be expressed and secreted from E. coli, which allows large amounts of said fragments to be easily produced.

[0068] Also included within the scope of the invention are modified versions of the antibodies and antigen-binding fragments thereof, e.g. modified by covalent attachment of polyethylene glycol or another suitable polymer.

[0069] Methods for producing antibodies and antibody fragments are well known in the art. For example, antibodies can be produced by any of several methods that utilize the induction of antibody molecule production in vivo, screening of immunoglobulin libraries (Orlandi et al., 1989. Proc. Natl. Natl. Acad. Sci. USA 86: 383330 3837; Winter et al. , 1991, Nature 349: 293-299) or the production of monoclonal antibody molecules by cell lines in culture. This includes, but is not limited to, hybridoma technique, hybridoma technique using human B lymphocytes, and hybridoma technique using Epstein-Barr virus (EBV) (Kohler et al., 1975 Nature

256: 4,950,497; Kozbor et al., 1985 J. Immunol Methods 81: 31-42; Cote et al., 1983 Proc.

Natl. Acad. Sci. USA 80: 2026-2030; Cole et al., 1984 Mol Cell Biol 62: 109-120).

[0070] Suitable monoclonal antibodies against selected antigens can be produced by known methods, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H. Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications" JGR Hurrell (CRC Press, 1982).

[0071] Antibody fragments can be obtained using methods well known in the art (see, for example, Harlow and Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York). For example, the antibody fragments of the present invention can be produced by proteolytic hydrolysis of the antibody or expression in E. coli or mammalian cells (e.g. in Chinese hamster ovary cell culture or other protein expression systems) DNA encoding this fragment. Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.

[0072] It will be understood by those skilled in the art that humanized antibodies are preferably used in human therapy or diagnosis. Humanized forms of non-human (e.g., murine) antibodies are genetically modified chimeric antibodies or antibody fragments, preferably containing minimal parts derived from non-human antibodies. Humanized antibodies include antibodies in which the complementarity determining regions of the human antibody (recipient antibody) are replaced by residues from non-human complementarity determining regions (donor antibody), such as mouse, rat or rabbit showing the desired function. In some cases, human antibody Fv framework residues are replaced with corresponding non-human residues. Humanized antibodies can also include residues that are not present in either the recipient antibody or the introduced complementarity determining region or framework sequences. Generally, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all complementarity determining regions correspond to those of a non-human antibody, and all or substantially all of the framework regions correspond to those of the corresponding human consensus sequence. Humanized antibodies optimally also contain at least part of the antibody constant region, such as the Fc region, usually derived from a human antibody (see, for example, Jones et al., 1986 Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-329; Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596).

[0073] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody contains one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues, often referred to as introduced residues, are usually taken from the introduced variable domain. Humanization can generally be carried out as described (see, for example, Jones et al., 1988, Nature 321: 522525; Reichmann et al., 1988 Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 15341536I, US 4816567) by replacing human complementarity determining regions with relevant rodent complementarity determining regions. Accordingly, such humanized antibodies are chimeric antibodies in which substantially less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies can usually be human antibodies in which some residues of the complementarity determining region and optionally some framework residues are substituted by residues from analogous sites in rodent antibodies.

[0074] Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom and Winter, 1991, J. Mol Biol 227: 381; Marks et al., 1991, J. Mol Biol 222: 581, Cole et al., 1985, in Monoclonal antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner et al., 1991 J. Immunol 147: 86-95).

[0075] Once the appropriate antibodies have been obtained, they can be tested for activity, for example using ELISA analysis.

[0076] The agent of the first aspect of the invention may be an antibody or antigen-binding fragment thereof having specificity for a Toll-like 4 receptor (TLR4), Toll-like receptor 4 co-receptors (in binding tenascin4, tenascin C or any domain thereof).

[0077] Co-receptors of major receptors, such as TLR4, help to bind the signal receptor for the main receptor to facilitate ligand recognition and binding, and to initiate / maintain the biological process resulting from receptor binding.

[0078] The agent of the first aspect of the invention may be an antibody or antigen-binding fragment thereof with specificity for the FBG domain of tenascin-C.

[0079] Also described herein is a method of identifying an agent that modulates tenascin-C activity, comprising the steps of:

(i) providing one or more funds of the candidates;

(ii) contacting one or more cells with tenascin-C and one or more candidate agents;

(iii) contacting one or more cells with tenascin-C and without the agent agent;

(iv) determining whether said candidate agent modulates the action of tenascin-C on one or more cells in step (ii) compared to the cell (s) of the control step (iii).

[0080] Methods to determine whether a candidate agent modulates the effect of tenasacin-C can be carried out using the methods of the examples.

[0081] The method may lead to an increase in tenascin-C activity.

[0082] The method may lead to a reduction in tenascin-C activity.

[0083] The method may include cells of steps (ii) and (iii) (described above) expressing Toll-like receptor 4 (TLR4).

[0084] This method may include one or more cells selected from the group consisting of inflammatory cells, fibroblasts, fibroblast-like cells (including RA synovial fibroblasts, also known as synoviocytes), mouse embryonic fibroblasts, human embryonic kidney cells.

[0085] Inflammatory cells may be selected from the group consisting of macrophages, dendritic cells, monocytes, lymphocytes, monocyte-like and macrophage-like cells.

[0086] Also described herein is a method of identifying an agent that modulates a chronic inflammatory response by performing a method of identifying an agent that modulates tenascin-C activity.

[0087] In this method, chronic inflammation can be associated with any condition associated with abnormal inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel disease, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, Sjogren's disease, diabetes, lupus erythematosus (including systemic lupus erythematosus), asthma, fibrotic diseases (including cirrhosis), lung fibrosis, UV damage and psoriasis.

[0088] In particular, but not exclusively, chronic inflammation is associated with rheumatoid arthritis (RA).

[0089] Also described herein is an agent identified according to the method of the second and third aspects of the invention. Such an agent can modulate a chronic inflammatory response.

[0090] The agent may reduce a chronic inflammatory response.

[0091] The agent may increase a chronic inflammatory response.

[0092] The agent may be selected from the group consisting of small interfering RNA (siRNA) molecules, short RNA hairpin structure (shRNA) molecules, antisense oligonucleotides, compounds with tenascin-C binding affinity, antibodies (polyclonal or monoclonal) and their antigen binding fragments, small inhibitory compounds, polypeptides and proteins.

[0093] In a first aspect of the invention, chronic inflammation may be associated with any condition associated with abnormal inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel disease, non-healing times, multiple sclerosis, cancer, atherosclerosis, Sjogren's disease, diabetes, lupus erythematosus (including systemic lupus erythematosus), asthma fibrotic diseases (including cirrhosis), lung fibrosis, UV damage and psoriasis.

[0094] In a second aspect of the invention there is provided a composition comprising an agent as defined in the first aspect of the invention and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0095] Those skilled in the art will appreciate that such an effective amount of the agent or its preparation may be administered in the form of a single bolus dose (i.e. acute administration) or, more preferably, in several doses over time (i.e. prolonged administration).

[0096] The agents of the invention may be formulated at various concentrations depending on the efficacy / toxicity of the compound used and the indication for which it is used. Preferably, the formulation comprises the agent of the invention at a concentration between 0.1 μΜ and 1 mM, more preferably between 1 μΜ and 100 μΜ, between 5 μΜ and 50 μΜ, between 10 μΜ and 50 μΜ, between 20 μΜ and 40 μΜ, and most preferably about 30 μΜ. For in vitro applications, the formulations may contain a lower concentration of the compound of the invention, for example between 0.0025 μΜ and 1 μΜ.

[0097] Those skilled in the art will appreciate that the agents of the invention will typically be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected for the intended route of administration and standard pharmaceutical practice (see, for example, The Science and Practice of Pharmacy, 19th edition , 1995, edited by Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA).

[0098] For example, the agents of the invention may be administered orally, buccal or sublingually in the form of tablets, capsules, globules, elixirs, solutions or suspensions which may contain flavoring or coloring agents for immediate, delayed or controlled use. The agents of the invention may also be administered by intra-cavernous injection.

[0099] Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrating agents such as starch (preferably corn starch, potato starch or tapioca starch), sodium starch glycolate , croscarmellose sodium and certain complex silicates and binders for granulation, such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0100] Solid compositions of a similar type may also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. In the case of aqueous suspensions and / or elixirs, the compounds of the invention may be combined with various sweetening or flavoring agents, coloring agents or dyes, with emulsifying and / or suspending agents, and with diluents such as water, ethanol, propylene glycol and glycerin and their combinations.

[0101] The agents of the invention may also be administered parenterally, for example intravenously, intraarticularly, intraarterially, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial, intramuscular or subcutaneous, or may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution that may contain other substances, for example, enough salt or glucose to make the solution isotonic with blood. Aqueous solutions should be properly buffered (preferably to a pH of 3 to 9) if necessary. The preparation of suitable parenteral preparations under sterile conditions is easy to perform by standard pharmaceutical techniques well known to those skilled in the art.

[0102] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, bacteriostatic agents and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations can be in single-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, for example water for injection, immediately before use. Solutions and suspensions prepared immediately prior to use can be prepared from sterile powders, granules and tablets of the type previously described.

[0103] For oral and parenteral administration to humans, the daily dosage level of the agents of the invention will typically be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg / kg), administered once or in divided doses.

[0104] The agents of the invention may be administered intranasally or by inhalation, and conveniently be provided in the form of a dry powder inhaler or aerosol spray administered from a pressurized container, pump, spray or nebulizer using a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas . In the case of a pressurized aerosol, the dosage unit may be determined by a valve enabling delivery of a metered amount. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound, for example using a mixture of ethanol and propellant as a solvent, which may additionally contain a lubricant, for example sorbitan trioleate. Capsules and cartridges (made, for example, of gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

[0105] Aerosol or dry powder formulations are preferably adjusted such that each metered dose or "puff" contains at least 1 mg of a compound of the invention for delivery to a patient. It will be understood that the total daily aerosol dose will vary from patient to patient and may be administered in a single dose or, more typically, in divided doses throughout the day.

[0106] Alternatively, the agents of the invention may be administered in the form of a suppository or pessary, or may be applied topically in the form of a liquid, solution, cream, ointment or dusting powder. The compounds of the invention may also be administered transdermally, for example by applying a patch to the skin. They can also be administered by eye.

[0107] When applied to the eyes, the agents of the invention may be formulated as micronized suspensions in an isotonic, sterile, pH-adjusted saline solution, or preferably as solutions in an isotonic, sterile, pH-adjusted saline solution, optionally in combination with a preservative such like benzylalene chloride. Alternatively, they may be formulated in an ointment, such as yellow petrolatum.

For topical application to the skin, the agents of the invention may be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture of one or more of the following ingredients: mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene compound polyoxypropylene, emulsifying wax and water. Otherwise, they may be formulated in the form of a suitable liquid or cream, suspended or dissolved in, for example, a mixture of one or more of the following components: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, wax from cetyl esters, cetearyl alcohol , 2-octyldodecanol, benzyl alcohol and water.

[0109] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles containing the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouth rinses containing the active ingredient in a suitable liquid carrier. [0110] When the agent is a polypeptide, it may be beneficial to use a sustained release drug delivery system such as microspheres. They are specifically designed to reduce the frequency of injections. An example of such a system is Nutropin

Depot, which has enclosed human recombinant growth hormone (rhGH) in biodegradable microspheres that, when injected, release rhGH slowly over an extended period of time.

[0111] Alternatively, the polypeptide agents of the present invention may be administered in a surgically implanted device that releases the drug directly at the required site.

[0112] Electroporation therapy (EPT) systems can also be used to administer proteins and polypeptides. A device that delivers a pulsed electric field to the cells increases the permeability of cell membranes to the drug, which results in a significant increase in intracellular drug delivery.

[0113] Proteins and polypeptides may also be provided by electroincorporation (EI).

El occurs when small particles up to 30 microns in diameter on the surface of the skin experience electrical impulses identical or similar to those used in electroporation. In EI, these particles are introduced through the stratum corneum and into the deeper layers of the skin. The particles can be loaded or coated with drugs or genes, or they can simply act as "balls" that create pores in the skin through which drugs can pass.

[0114] An alternative method for delivering proteins and polypeptides is thermosensitive injected ReGel. Below body temperature, ReGel is an injectable fluid, while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered at a time when the biopolymers dissolve.

[0115] Protein and polypeptide drugs may also be administered orally. One such system uses the body's natural process of taking vitamin B12 by mouth to co-administer proteins and polypeptides. By releasing the vitamin B12 uptake system, the protein or polypeptide can pass through the intestinal wall. Complexes are formed between vitamin B12 analogues and a drug that retains both significant affinity for the intrinsic factor (IF) in the vitamin B12 part of the complex and the significant biological activity of the drug part of the complex.

[0116] Methods of administering oligonucleotide or polynucleotide agents of the invention are also well known in the art (see Dass, 2002, J Pharm Pharmacol 54 (1): 3-27; Dass, 2001, Drug Deliv. 8 (4): 191-213 ; Lebedeva et al., 2000, Eur J Pharm Blopharm 50 (1): 101-19; Pierce et al., 2005, Mini Rev Med Chem 5 (1): 41-55; Lysik and WuPong, 2003, J Pharm Sci . 2003 2 (8): 1-559-73; Dass, 2004, Biotechnol Appl Biochem. 40 (Pt 2): 113-22; Medina, 2004, Curr Pharm Des. 10 (24): 2981-9.

[0117] The composition of the second aspect of the invention may further comprise at least one other agent.

[0118] Such additional agent may be an anti-inflammatory agent that includes, but is not limited to, a nonsteroidal anti-inflammatory drug (NSAID), disease modifying anti-rheumatic drug (LMPCh), a statin (including HMG-CoA reductase inhibitors such as simvastatin), biological agent (biological agents), steroid, immunosuppressant, salicylate and / or bactericide. Nonsteroidal anti-inflammatory drugs include anti-metabolite agents (e.g., methotrexate) and gold-type anti-inflammatory agents (including sodium thiocyanate gold, aurothioja, or gold salts such as auranofin). Biological agents include, but are not limited to, anti-TNF agents (including adalimumab, etanercept, infliximab, anti-IL-1 agents, anti-IL-6 agents, anti-B cell agents (retoximab), anti-T lymphocyte agents (anti-CD4 antibodies), anti-IL-15 agents, anti-CLTA4 agents, anti-RAGE agents), antibodies, soluble receptors, receptor binding proteins, cytokine binding proteins, mutated proteins with altered or attenuated function, RNAi, polynucleotide aptemers, antisense oligonucleotides or omega-3 fatty acids. Steroids (also known as corticosteroids) include cortisone, prednisolone or dexamethasone. Immunosuppressants include cyclosporin, FK506, rapamycin, and mycophenolic acid. Salicylates include aspirin, sodium salicylate, choline salicylate, and magnesium salicylate. Bactericides include quinine and chloroquine. For example, the agent may be administered in combination with one or more NSAIDs, LMPCh or an immunosuppressant.

[0119] Also described herein is an agent or composition as defined herein for use as a medicament.

[0120] In a third aspect of the invention there is provided an agent or composition as defined in the first or second aspect of the invention for use in the treatment of chronic inflammation, wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), inflammatory bowel disease, atherosclerosis arteries and / or psoriasis.

[0121] In a fourth aspect of the invention there is provided the use of an agent or composition as defined in the first or second aspect of the invention for the manufacture of a medicament for the treatment of chronic inflammation, wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), inflammatory bowel diseases , atherosclerosis and / or psoriasis.

[0122] Also described herein is a method of treating chronic inflammation comprising administering to the subject an effective amount of an agent or composition as defined herein. [0123] The agent, composition, use or method defined herein may refer to the treatment of a chronic inflammation in which the condition is associated with any condition associated with abnormal inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel disease, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, Sjogren's disease, diabetes, lupus erythematosus (including systemic lupus erythematosus) , asthma, fibrotic diseases (including cirrhosis), pulmonary fibrosis, UV damage and psoriasis.

[0124] Also described herein is a set of parts for performing the methods described herein, comprising:

(i) one or more cells (ii) control sample of one or more cells (iii) tenascin-C sample (iv) instructions for use [0125] The kit may further comprise:

(v) agent candidate.

[0126] The kit may optionally further comprise (vi) agents for determining the effect of the candidate agent on any ten-scin-C activity or chronic inflammation.

[0127] In a fifth aspect of the present invention there is provided a kit of parts comprising:

(i) an agent or composition as defined in the first or second aspect of the invention (ii) administration agents (iii) instructions for use [0128] The kit according to the fifth aspect of the invention may further optionally comprise (iv) at least one other agent.

Definitions [0129] The term "inflammation" includes the meaning of local accumulation of fluid, plasma proteins and white blood cells that is caused by tissue damage, infection, or a local immune response.

[0130] By "acute inflammation" is meant the significance of the initial stages (onset) of inflammation and the short-term transient inflammatory response immediately following injury, infection or local immune response. Usually, acute inflammation disappears quickly and lasts from several minutes to no more than a few days.

[0131] By "chronic inflammation" is meant persistent and / or transient inflammation. It is often associated with inadequate destruction of healthy tissue. It can be progressive and can last for weeks or more. Chronic inflammation is usually associated with a persistent infection or disease, including, but not limited to, autoimmune conditions.

[0132] The term "chronic arthritis" includes the importance of persistent inflammation that progresses and continues continuously for weeks to months, causing deformity of the joint in which it occurs, and radiographic evidence of cartilage and bone destruction as seen in human disease (Kelly, Harris, Ruddy and Sledge, Textbook of Rheumatology, 4th edition).

[0133] In experimental mouse models, chronic arthritis is characterized by inflammation that persists and causes inappropriate tissue damage, even over a relatively short period of time. It is characterized (and can be identified) histologically by the prolonged presence of inflammatory cells in the synovium of the joint space, death of chondrocytes and erosion of cartilage and bone.

[0134] The term "agent" includes all chemical compounds, for example, oligonucleotides, polynucleotide, polypeptides, peptidomimetics and small compounds.

[0135] By "fragment" we mean at least 10 nucleotides, for example at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0136] By "variant" is meant that the nucleotide sequence has at least 90% sequence identity with the full-length sequence of interest, for example at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0137] The percentage of sequence identity between two polynucleotide sequences can be determined using appropriate computer programs, for example, the GAP program from the University of Wisconsin Genetic Computing Group, and it should be noted that the percent identity is calculated with respect to polynucleotides whose sequences have been optimally matched.

[0138] Alignment can alternatively be performed using the Clustal program

In (as described in Thompson et al., 1994, Nuc. Acid Res. 22: 4673-4680).

[0139] The parameters used may be the following:

Parameters for quick matching in pairs: size K-tuple (words); 1, window size; 5, penalty for a break; 3, the number of best diagonals; 5. Scoring method: x percent.

Multiple matching parameters: penalty for opening a break; 10, penalty for extending the break; 0.05.

Scoring matrix: BLOSUM.

[0140] Alternatively, the BESTFIT program can be used to determine local sequence matches.

[0141] The term "antibody" includes substantially intact antibody molecules as well as chimeric antibodies, humanized antibodies, human antibodies (at least one amino acid mutated relative to naturally occurring human antibodies), single chain antibodies, bispecific antibodies, heavy chain antibodies, lightweight antibody chains homodimers and heterodiomers of heavy and / or light chains of antibodies, and antigen-binding fragments and derivatives thereof.

[0142] By "antigen binding fragment" is meant a functional antibody fragment that is capable of binding tenascin-C.

[0143] The term "subject" means all animals including humans. Examples of individuals include humans, cows, dogs, cats, goats, sheep and pigs. The term "patient" means an individual with a disorder in need of treatment.

[0144] As used herein, "pharmaceutical formulation" means a therapeutically effective formulation according to the invention.

[0145] "Therapeutically effective amount" or "effective amount" or "therapeutically effective", as used herein, refers to an amount that provides therapeutic effect for a given condition and schedule of administration. It is a predetermined quantity of active substance calculated to produce the desired therapeutic effect in combination with the required additive and diluent, i.e. the vehicle or vehicle for administration. In addition, this is intended to mean an amount sufficient to reduce, and most preferably prevent, a clinically significant deficit in host activity, function and response. Alternatively, a therapeutically effective amount is sufficient to cause a clinically significant improvement in the host. As will be appreciated by those skilled in the art, the amount of compound may vary depending on its specific activity. Appropriate dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods of preparation and use in the composition of the invention, a therapeutically effective amount of the active ingredient is provided. A therapeutically effective amount can be determined by an ordinary qualified healthcare or veterinary practitioner based on the patient's characteristics such as age, weight, sex, health condition, complications, other diseases, etc., as is well known in the art.

[0146] Below, embodiments of aspects of the invention will be described with reference to the following drawings, in which:

Figure 1. Accelerated treatment of acute inflammation in tenascin-C deficient mice.

(a) paw edema in wild-type (+ / +) mice (white bars) and tenascin-C (- / -) (black bars) post-zymosan injection. Data are presented as mean increase in paw diameter compared to paw diameter before +/- SEM injection (n = 24 mice per genotype). ** = p &lt;0.01. (be) representative ankle sections in wild (b, c) and tenascin-C (d, e) mice 4 days after zymosan injection, stained with hemotoxylin and eosin (b, d) and safranin-O (c, e) . Boxes indicate synovial membrane of the joint (s) and cartilage proteoglycan (cp). X10 magnification. Quantification of arthritis (f) and chondrocyte death (g) in the knee joints 4 days after zymosan injection in wild-type (white bars) and tenascin-C (black bars) mice. Data are expressed as means (+/- SD) (n = 24 mice per genotype). * = p &lt;0.05.

Figure 2. Synovitis caused in tenascin-C deficient mice after antigen injection.

(ab, g) Representative knee sections of wild-type mice injected with an inert substance (cf, hi). Representative knee joint sections in wild-type (c, d, h) or tenascin-C (e, f, i) mice 24 h after intra-articular injection of mBSA. Inflammation of inflammatory cells in the capsule, meniscus and joint space of both wild-type and tenascin-C-deficient mice is indicated by (cap), (M) and (J), respectively. (S) indicates normal synovial membrane in mice injected with an inert substance that is no more than 1-3 cells in thickness across the entire bone surface, and (ST) indicates the synovial fluid of wild-type and tenascinC-deficient mice, both of which are clearly thickened. The sections were stained with hemotoxylin and eosin (a, c, e, g, h, i) and safranin-O (b, d, f). Magnification x10 (af) or x40 (gi). (n = 5 mice per genotype).

Figure 3. Synovitis quickly resolves in tenascin-C deficient mice.

Representative cross-sections of the knee joint of wild-type (a, b, f) or tenascin-C (c, d, e) mice 3 days after intra-articular injection of mBSA. (a, c) The line indicates increased bursitis in wild-type mice compared to mice lacking tenascin-C. (b, d) (cp) indicates increased loss of cartilage proteoglycan in wild-type mice compared to mice lacking tenascin-C. (e, f) Significant synovial hypertrophy (line), cell and fibrin deposits in the joint space (arrow) and invasion of the scales (arrowheads) are observed in wild-type mice compared to mice lacking tenascin-C. Sections were stained with hemotoxylin and eosin (a, c, e, f) and safranin-O (b, d) Magnification x10 (ad) or x20 (ef). (n = 5 mice per genotype).

Figure 4. Mice lacking tenascin-C are protected against tissue destruction during antigen-induced arthritis.

(ab) Representative knee sections of wild-type mice 7 days after intra-articular injection of mBSA, stained with hemotoxylin and eosin (a) and safranin-O (b). X10 magnification. (n = 24 mice per genotype). The arrowhead indicates the area of ​​bone erosion. The arrow indicates the invasion of the scales to the articular cartilage. (cd) Representative knee sections of mice lacking tenascin-C 7 days after intra-articular injection of mBSA, stained with hemotoxylin and eosin (c) and safranin-O (d). X10 magnification. (n = 24 mice per genotype). J indicates articular space and AC intact articular cartilage. (e) Histological scoring of knee arthritis 24 h, 3 days and 7 days after mBSA injection in wild-type mice (white bars) and tenascin C-deficient mice (black bars). Data represent mean +/- SD (n = 5 per genotype (24 h, 3 days) or 24 per genotype (7 days)). (f) Quantification of chondrocyte death, cartilage erosion and bone erosion after mBSA injection into the knee joints of wild-type mice (white bars) and tenascin-C-deficient mice (black bars). Death of chondrocytes is presented after 24 h, 3 days and 7 days, and erosion of cartilage surface and bone erosion after 7 days. Data represent mean +/- SD (n = 5 per genotype (24 h, 3 days) or 24 per genotype (7 days)).

Figure 5. Tenascin-C induces the synthesis of TNF-α, IL-6 and IL-8 in primary human macrophages and synovial fibroblasts of RA.

(ab) Primary human macrophages (a) and synovial fibroblasts RA (b) were not stimulated (without addition) or stimulated with LPS (1 ng / ml (a) or 10 ng / ml (b)) or recombinant tenascin-C (1) , 0 μΜ - 1.0 nM) for 24 h. Data shown are means of three replicates (+/- SD) from one of three representative experiments. (c) Primary human macrophages were not stimulated (without addition) or stimulated with LPS (1 ng / ml) or recombinant tenascin-C (1.0 μΜ) for 24 h. (-) Indicates that the cells were pre-incubated with medium alone. (P) Before stimulation, cells were preincubated with 25 μg / ml polymyxin B for 30 min. (H) Cells were incubated with medium without addition or contained LPS or tenascin-C, which were boiled for 15 minutes before being added to the cells. Data shown are means of three replicates (+/- SD) from one of three representative experiments.

Figure 6. The FBG domain of tenascin-C mediates the stimulation of cytokine synthesis in vivo and in vitro.

(a) Primary human macrophages were not stimulated (without addition) or stimulated with LPS (1 ng / ml), recombinant tenascin-C (TNC) or 1.0 μΜ tenascin-C domains (TA, EGF-L, TNIII1-5, TNIII1-3, TNIII3-5, TNIII5-7, TNIII6-8 and

FBG) for 24 h. Data shown are means of three replicates (+/- SD) from one of three representative experiments. (b) RA synovial cells were not stimulated (without addition) or stimulated with LPS (10 ng / ml) or recombinant FBG (1.0 - 0.01 μΜ) for 24 h. Data shown are mean% change from level cytokines in unstimulated cells (+ / SEM) from five different patients. (ch) Representative knee sections of wild-type mice 3 days after intra-articular injection of PBS (ce) or 1 μg FBG (fh). Sections were stained with hemotoxylin and eosin (c, d, f, g) or safranin-O (e, h). Magnification x10 (c, f) or x25 (d, e, g, h) (n = 5 mice per genotype). (i) Quantification of joint inflammation, bone erosion, cartilage surface erosion and chondrocyte death in the knee joints of wild-type mice 3 days after intra-articular injection of PBS (black bars) or 1 μg FBG (white bars). Data represent mean +/- SD (n = 5 per genotype).

Figure 7. FBG-mediated cytokine synthesis is dependent on MyD88.

(a) Human synovial fibroblasts RA were either uninfected, infected with GFP-expressing adenovirus (AdGFP) or infected with adenovirus expressing dominant negative MyD88 (AdMyD88dn). Cells were not stimulated, stimulated with LPS (10 ng / ml) or stimulated with FBG (1 μΜ) for 24 h. Data shown is the average of three independent experiments (+/- SEM). (b) Mouse embryonic fibroblasts isolated from wild-type (+ / +) or MyD88-deficient (- / -) mice were not stimulated (-) or stimulated with ΡΑΜ3 (100 ng / ml), LPS (100 ng / ml), TNFa (100 ng / ml), IL-1 (5 ng / ml) and FBG (1 μΜ) for 24 h. Data shown are the average of three independent experiments (+/- βΕΜ).

Figure 8 FBG-mediated cytokine synthesis is TLR4 dependent, but does not require CD14 or MD-2.

(a) Primary human macrophages were pre-incubated with medium alone or medium containing antibodies blocking TLR2 (10 μg / ml), TLR4 (25 μg / ml) or isotype control antibodies (25 μg / ml) for 30 minutes before stimulation. Cells were not stimulated or stimulated with LPS (1 ng / ml), FBG (1 μΜ) or PAY13 (10 ng / ml) for 24 h. Data shown are the average of three independent experiments (+/- βΕΜ). (b) Mouse embryonic fibroblasts isolated from wild-type, TLR2 (TLR2 - / -) or TLR4 (TLR4 / -) mice were not stimulated or stimulated with PAY13 (100 ng / ml), LPS (100 ng / ml), IL -1 (5 ng / ml) and FBG (1 μΜ) for 24 h. Data shown are the average of three independent experiments (+/- βΕΜ). (c) Bone marrow macrophages isolated from wild-type mice lacking TLR2 (TLR2 - / -) or TLR4 (TLR4 - / -) were not unstimulated or stimulated with PAM3 (100 ng / ml), LPS (100 ng / ml) or FBG (1 μΜ) for 24 h. Data shown is the average of three independent experiments (+/- SEM). (d) Human macrophages were pre-incubated without inhibitor, with 1 μg / ml msbB LPS or 10 μg / ml anti-CD14 antibody for 30 min before stimulation with LPS (1 ng / ml), FBG (1 μΜ) or PAM3 (10 ng / ml ) for 24 hours. The data presented are the average of three independent experiments (+/- SEM).

Figure 9 Swelling of the paws after zymosan injection.

Exemplary images of paw without injection in mice lacking tenascin-C (a, e) (diameter 1.6 mm), mice lacking tenascin-C after 24 h (d, f) (diameter 2.5 mm) and 4d (b, h ) (diameter 1.7 mm) after injection of zymosan and wild-type 4d mice after injection of zymosan (c, g) (diameter 2.1 mm).

Figure 10. Synthesis of recombinant proteins.

(a) Domain structure of tenascin-C monomer comprising various domains, including splicing domain (TA), 14 and half EGF-like repeats (EGFL), 17 fibronectin-like repeats type III (TNIII) (8 constitutively expressed (1-8 ) and 9, which may alternatively be combined, and globe-type fibrinogen-like (FBG). (b) Areas covered by recombinant proteins that have been synthesized, the corresponding amino acid residues, and the molecular weight of each protein.

Figure 11. Analysis of protein purity.

Silver-stained gel representing 1 μg of each recombinant protein analyzed by SDS-PAGE under reducing conditions. Tracks: 1 (TA), 2 (EGF-L), 3 (TNIII1-5), 4 (TNIII5-7), 5 (TNIII6-8), 6 (TNIII1-3), 7 (TNIII3-6) and 8 (FBG).

Figure 12. FBG-mediated arthritis in vivo requires TLR4 expression.

Representative knee sections of mice lacking TLR2 (a) and TLR4 (b) 3 days after intra-articular injection of 1 μg FBG. Sections were stained with hemotoxylin and eosin. X10 magnification (n = 5 mice per genotype). (c) Quantification of arthritis, bone erosion, cartilage surface erosion and chondrocyte death in the knee joints of mice lacking TLR2 (white bars) and TLR4 (black bars) 3 days after intra-articular injection of 1 μg FBG. Data represent mean +/- SD (n = 5 per genotype).

Figure 13. Amino acid sequence of human tenascin C and its domains

Figure 14. Nucleotide sequence of human tenascin C

Figure 15. Synthesis of TNF in response to specific FBG peptides.

TNF synthesis by cultures of RA membranes incubated for 24 h without or with addition

100 μΜ of each of the FBG peptides (P1, P3-P9).

Figure 16 Synthesis of TNF and IL8 in response to different concentrations of specific FBG peptides.

Synthesis of TNF and IL8 by cultures of RA membranes incubated for 24 h without addition or with 25, 100 or 250 μΜ FBG peptide.

Figure 17. Synthesis of IL8 in response to LPS, the entire FBG domain or specific FBG peptides.

IL8 synthesis by macrophages after 24 h incubation without addition, with 1 ng / ml LPS, 1 μΜ whole FBG domain (FBG) or 1 or 20 μΜ FBG peptides (P1, P3-P9).

Figure 18. Synthesis of IL8 and TNF in response to LPS and FBG after pre-incubation with FBG peptides.

Synthesis of TNF and IL8 by macrophages after 24 h incubation without addition, with 1 ng / ml LPS or 1 μΜ whole FBG domain (FBG) with or without pre-incubation with 20 μΜ FBG peptides.

Figure 19. Synthesis of IL8 and TNF in response to siRNA targeted to tenascinC.

Tenascin-C mRNA levels in RA fibroblasts transfected with luciferase-specific siRNA (control) or tenascin-C siRNA: oligo 1 (si 1), oligo-2 (si 2) or oligo 1 +2 (si + 1 2) combination . IL-6 synthesis in RA fibroblasts transfected with luciferase siRNA (control) or oligo 1 + 2 (siRNA) combination directed to tenascin-C in the presence or absence of 10 ng / ml LPS for 24 h.

Example 1 - General methods

Reagents [0147] Zymosan, methylated BSA and Freund's complete adjuvant, anti-FLAG Μ2 antibody (mouse monoclonal antibody), blasticidin and isotype control antibodies (Mouse IgG2a, IgG1) were from Sigma-Aldrich (Dorset, UK). Hypnorm was from VetaPharma Ltd. (Leeds, United Kingdom). The Limulus amaebocyte lysate test was from Associates of Cape Cod (Liverpool, UK). Embryonic wild-type human kidney cells (HEK 293-EBNA) were from Invitrogen (Groningen, The Netherlands). M-CSF and mouse IL-Ιβ were from PeproTech (Neuilly-Sur-Seine,

France). DMEM, RPMI 1640, fetal bovine serum (FBS), penicillin / streptomycin, an antibiotic-antifungal solution in PSA and β-mercaptoethanol were from PAA Laboratories (Yeovil, UK). HEK293 cell lines stably expressing human TLR2 and TLR4 / CD14 / MD-2, polymyxin B, msbB LPS and TLR2 blocking function (Clone: ​​TL2.1 Isotype: Mouse IgG2a) and TLR4 antibodies (Clone:

HTA125 Isotope: Mouse IgG2a) were from Invivogen (Calne, United Kingdom). LPS (rough and smooth) Escherichia coli purified with phenol-chloroform and Pam3CysSer-Lys4 (Pam3C) were from Alexis (Birmingham, United Kingdom). Mouse TNF-α and IL-1 receptor antagonist (IL-1ra-IL-1F3) were from R&amp;D Systems (Abingdon, UK). Function blocking anti-CD14 antibodies (Isotype: Mouse IgG1) were from Abcam (Cambridge, UK). ELISA assays for human and mouse TNFα, IL-6 and IL-8 were from Pharmingen (Oxford, UK).

Purification of full-length tenascin-C [0148] To ensure that cytokine production is not attributed to bacterial contaminants such as LPS and LPS-associated molecules, purified full-length recombinant human tenascin-C from conditioned medium labeled His-transfected HEK293 cell line human tenascin-C in the pCEP-pu vector as described (Lange (2007)). Tenascin-C was purified to homogeneity as described (Lange (2007) and determined to be free of LPS contamination using the Limulus amaebocyte lysate assay according to the manufacturer's instructions.

Synthesis of recombinant proteins [0149] Proteins corresponding to each tenascin-C domain were synthesized (TA, EGF-1, different TNIII and FBG repeats) and purified. See Example 2.

Measurement of LPS contamination in recombinant proteins [0150] To ensure that LPS levels in each recombinant protein were used, the Limulus amaebocyte lysate test was used according to the manufacturer's instructions (sensitivity ~ 0.7 ± 0.5 pg LPS per mg protein). All recombinant proteins used in this study had LPS levels that were lower than 10pg / ml.

Adenoviral vectors and their amplification [0151] Recombinant adenoviral vectors with wild type replication deficit

MyD88 (AdMyD88wt), dominant negative forms of MyD88 (AdMyD88dn) and GFP control (AdGFP) were constructed on site. The synthesis of these viruses is provided in

Andreakos (2004). All viruses used in this study had an E1 / E3 deletion and belonged to the Ad5 serotype. Viruses were propagated in human embryonic kidney cells

293, purified by ultracentrifugation through two cesium chloride gradients, and virus titers were determined in a plaque assay as previously described (Sacre (2007)).

Animals [0152] Tenascin-C homozygous mice from the original strain described by Saga (1992) in inbred 129 / sv mice with white belly and agouti-like background appearance were provided by prof. Charles French-Constant (University of Edinburgh, United Kingdom). Age-matched ingenic 129 / sv wild-type inbred mice were obtained from Charles River (Margate, UK). All mice lacking tenascin-C and wild type 129 / sv were male and between 8-10 weeks of age during the experiment.

[0153] Homozygous mice lacking TLR2 and TLR4 in the background of the C57BL / 6 (inbred black-colored mouse strain) were obtained from B&amp;K Universal (Hull, UK), Hoshi no (1999) and Takeuchi (1999). C57BL / 6 homozygous mice lacking MyD88 in the background were provided by the Sanger Institute (Cambridge, UK). Age-matched wild-type C57B / L6 inbred mice were obtained from Charles River (Margate, UK). For the isolation of mouse embryonic fibroblasts, one female at the age of 8-10 weeks was crossed with two males at the age of 8-10 weeks 20 days. For the isolation of bone marrow macrophages, mice were female and between 10-12 weeks of age during the experiment.

[0154] All animals were fed standard rodent feed and had free access to water and were placed (&lt;6 mice / cage) in sawdust cages in an air-conditioned environment with a 12-hour light / dark cycle.

All procedures were approved by the Institute's ethics committee.

Statistical methods [0155] Average, SD, SEM and statistical tests were calculated using GraphPad version 3 (GraphPad Software Inc., San Diego, CA). The means of many groups were analyzed using one-way analysis of variance followed by a multiple comparison test

Dunnett's where appropriate. The unpaired t-test was used for experiments using only two groups.

Example 2 - Synthesis of recombinant proteins [0156] Proteins corresponding to each tenascin-C domain were synthesized (TA, EGF-L, different TNIII and FBG repeats) and purified. The synthesized recombinant proteins are shown in Figure 9.

Reagents [0157] Pfu Turbo polymerase was from Stratagene (Amsterdam, The Netherlands). Tubes

PCR Easy mix 50 were from Molecular Bioproducts (Lutterworth, UK). RNeasy sets and Ni columns^{2 +}-NTA-agarose originated from Qiagen (Crawley, UK). The pCR Blunt vector, the pCEP4 plasmid vector, human embryonic kidney cells (HEK293-EBNA) and 4-12% Bis-Tris gels were from Invitrogen (Groningen, The Netherlands). The pET32b vector and Rosetta BL21 (DE3) cells were from Novagen (Kent, United

Britain). HiTrap Q columns, HiTrap S columns, Sephacryl S500 HR column and heparin and sepharose columns were from Amersham (Buckinghamshire, UK). [0158] Restriction enzymes were obtained from New England Biolabs (Hitchin, United Kingdom). DMEM, fetal bovine serum (FBS) and penicillin / streptomycin were from PAA laboratories (Yeovil, UK). FuGENE6 transfection reagent was from

Roche Applied Science (Basel, Switzerland).

[0159] Anti-FLAG M2 antibody (mouse monoclonal antibody), anti-FLAG M2 agarose, FLAG peptide were from Sigma-Aldrich (Dorset, UK). The anti-tetra-His (mouse monoclonal antibody) was from Qiagen (Crawley, UK). Alkaline phosphatase-conjugated goat anti-(IgG mouse) IgG antibody and stabilized substrate for Western Blue alkaline phosphatase were from Promega (Southampton, UK). Precision Protein Standards for SDS-PAGE were from BioRad (Hemel Hempstead, UK). Designing of primers [0160] Domain borders were determined using matches published in human tenascin-C sequence (Siri (1991), accession number P24821 (Swiss-Prot)). To clone each domain, PCR primers were designed that were both top and bottom primers containing 18-21 bases corresponding to the 5 'and 3' end sequences of the required coding sequence. The upper primer contained an Nde1 restriction site followed by an N-terminal His tag immediately before the coding sequence. The final 3 bases of the Nde1 site form the initiating methionine codon ATC.

The downstream primer contained the TTA stop codon immediately after the coding sequence, followed by a BamH1 or Kpn1 site to allow unidirectional cloning into pET32b expression vectors.

Table 1

<td>Protein Name</td><td>Upper starter Bottom starter</td>

<td>TA</td><td>FW: ATACA TA 7GCATCATCATCATCATCATGGGGTCCTGA AG AAAGTCATCCGG RV: GCCGGATCCTTAGCCTGCTCCTGCAGTACATTG</td>

<td>EGF-L</td><td>PCR1 FW: ACAGTGG7ACCACCATGGGGGCCATGGGGGCCATGACT CAGCTGTTG RV: CTTGTCATCGTCGTCCTTGTAGTCACCTTCGGTAGCGAG GGCAAG PCR2 FW: GACTAGAAGGACGACGATGACAAGTGCTGTCTCCAGCC TGCCAC RV: G ACAGCGGA 7CCTTAATG ATG ATGATGATG ATGTGAGCA GTCTTCTCCGCTGTAGC</td>

<td>TN1-5</td><td>FW: ATACA7A7GCATCATCATCATCATCATGAGGTGTCTCCTCC CAAAGA RV: GCCGGTACCTTAAGTGGATGCCTTCACACGTGC</td>

<td>TN1-3</td><td>FW: ATACA7A7GCATCATCATCATCATCATGAGGTGTCTCCTC CCAAAGA RV: GCCGGTACCTTATGTTGTGAAGGTCTCTTT GGC</td>

<td>TN3-5</td><td>FW: ATACA7A7GCATCATCATCATCATCATCGCTTGGATGCC CCCAGCCAGAT RV: GCCGGTACCTTAAGTGGATGCCTTCACACGTGC</td>

<td>TN5-7</td><td>FW: ATACA TA TGC ATC ATC ATC ATC ATC AT GAGTTG GAC ACG CCCAAGGAC RV: GCCGGATCCTTATGTTGTGAACTTGGCAGTGATGGTTG</td>

<td>TN6-8</td><td>FW: ATACA TA 7GCATCATCATCATCATCATGCCATGGGCTCCCC AAAGGAA RV: GCCGGA TCCTTATGTGGTGAAGATGGTCTGGATCAT</td>

<td>FBG</td><td>FW: ATACA TA 7GCATCATCATCATCATCATATTGGACTCCTGTAC CCCTTCC RV: GCCGGATCCTTATGCCCGTTTGCGCCTGCCT TCAA</td>

[0161] All of the above primers were recorded from 5 'to 3'. Flag sequences are bolded, His tags (CATCATCATCATCATCAT) are underlined, and restriction enzyme cleavage sites (CATATG = Nde1 site, GGATCC = BamH1, GGTACC = Kpn1 site) are shown in bold italics.

PCR [0162] PCR amplification was performed using 10 pmol / μΐ each primer, μg matrix, 5 μl DMSO and 1.25 units of Pfu Turbo polymerase in a final volume of 25 μ 25. This was added to buffer and dNTP in Easy mix 50 tubes. The template used in all reactions was cDNA prepared from U87MG human glioblastoma cells using RNA isolated using RNeasy kits. The reaction was carried out 40 times in denaturation, attachment and extension cycles at 95 ° C, 55-65 ° C respectively (depending on the melting point (Tt) of the primers) and 72 ° C.

Cloning [0163] PCR products were ligated into pCR Blunt vectors and sequenced to ensure that errors were not introduced by PCR. Clones that had no errors or silent mutations were selected. The inserts were then ligated into pET32b using Nde1 and BamH1 restriction sites introduced into the primers (TN5-7 and TN68). Human tenascin-C has internal BamH1 sites in the TA domain (position 494) and TNIII2 (position 2509). Thus TA and TN1-8 were cloned using the Nde1 site in the FW primer and the Kpn1 site at the pCRBLUNT cloning site. Human tenascin-C does not contain internal Kpn1 sites. TN1-5, TN1-3 and TN3-5 were cloned using Nde1 and Kpn1 sites in primers. FBG contains an internal Nde1 site (position 6439) and was therefore cloned using two-step ligation of Nde1 and BamH1 digestion followed by Nde1 digestion. (Positions refer to sites in the full-length tenascin C nucleotide sequence shown in Figure 14)

Bacterial culture, induction and lysis [0164] Plasmids were transformed into BL21 (DE3) Rosetta cells grown in 3 L Luria-Bertani medium containing 50 μg / ml carbenicillin and induced with 1 mM isopropyl 25-D-thiogalactopyranoside. After 3 hours, cells were harvested by centrifugation at

4000 RPM for 20 minutes, washed twice with ice-cold wash buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl and 1 mM EDTA) and lysed using a French press. Inclusion bodies were collected by centrifugation at 12,000 rpm for 20 minutes at 4 ° C. With the exception of TA and FBG, the proteins were completely in the supernatant. Recombinant TA and FBG proteins were extracted from inclusion bodies with 6M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.0 and 10 mM β-mercaptoethanol at room temperature with continuous stirring for 2 hours.

Purification of bacterial proteins [0165] The solution containing the recombinant protein was applied to a Ni column²⁺-NTAagarose and washed with 50 mM Tris-HCl, pH 8.0 containing 20 mM imidazole. The column was then washed with 50 mM Tris-HCl, pH 8.0 and the protein eluted with 50 mM Tris-HCl, pH 8.0 containing 60 mM imidazole. For TA and FBG, each wash and elution buffer contained 6 M guanidine hydrochloride. After Ni chromatography, TA and FBG did not require further purification. TN1-3 and TN6-8 were further purified by ion exchange chromatography using a HiTrap Q column, TN1-5, TN3-5 and TN5-7 by cation exchange chromatography using a HiTrap S column and TN1-8 using a HiTrap S column, followed by by gel filtration using a column

Sephacryl S500 HR.

Refolding of insoluble proteins [0166] TA and FBG were refolded by dilution to 20 μg / ml in 50 mM Tris-HCl, pH 8.0 containing 6 M guanidine hydrochloride followed by treatment with 20 mM cystamine with stirring for 16 hours in 4 ° C. The solution was then dialyzed twice against 15 volumes of 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl, 10 mM CaCl 2, 5 mM β-mercaptoethanol and 1 mM 2-hydroxyethyl disulfide for 24 h at 4 ° C, twice against 20 mM Tris-HCl, pH 8.0, for 8 hours at 4 ° C, followed by centrifugation at 12,000 rpm for 30 minutes at 4 ° C. Refolding was assessed by size changes using SDS PAGE under reducing and non-reducing conditions. Protein activity was confirmed by TA domain polymerization and FBG binding to heparin and sepharose in columns.

Synthesis of EGF-L domain using mammalian cells [0167] Initial attempts to express and purify the EGF-L repeat region using an E. coli expression system were unsuccessful. This is most likely due to the difficulty of obtaining protein folding due to the total number of 91 cysteines in this region.

Because TNF-like EGF-like domains were expressed using cells

HEK293 cells.

[0168] Two PCR reactions were performed. The first PCR product contained the KpnI restriction enzyme site, the Kozak sequence, followed by the TN-C signal sequence. The second PCR product contained the FLAG peptide, the sequence of the EGF-like domain, the next histidine tag and the sequence for the BamH1 restriction enzyme. [0169] Two PCR products were ligated together as described by Ho (1989). PCR reactions were carried out as described above. The entire construct was cloned into the PCR blunt vector and sequenced. It was then cloned into pCEP4 vector. DNA was transfected into HEK293 cells using Fugene and the cells were selected for hygromycin resistance (200 μg / ml) in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v / v) fetal calf serum, penicillin (100 units / ml) and streptomycin (100 units / ml). 2 liters of conditioned medium (harvested after culturing the cells in the medium) from stably transfected cells was collected and combined. The combined conditioned medium (2 L) was centrifuged at 3000 rpm to separate cell debris from the medium.

[0170] Then the medium was applied to an anti-FLAG column. The material was collected in 5010 ml fractions for the fraction flowing through the column. The column was washed with 10 column volumes of 1 M NaCl solution, 50 mM Tris-HCl, pH 7.5, and then washed with 10 column volumes of 60% isopropanol solution to ensure removal of LPS. The column was washed with 50 mM Tris-HCl buffer, pH 7.5 and finally the protein was eluted using 200 μg / ml FLAG peptide in 50 mM Tris-HCl buffer, pH 7.5.

Protein purity analysis [0171] Each protein was dialyzed against 1000 volumes of 150 mM NaCl and 50 mM Tris, pH 7.5. Protein purity was analyzed by SDS PAGE under reducing conditions. To this end, 1 μg of each purified recombinant protein was separated on a BisTris gel with a 4-12% gradient and the gel was then stained with silver to show a single band (Figure 10). Western blotting was also performed. Proteins separated by SDS-PAGE were electrotransferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA in Tris buffered saline and then incubated with FLAG M2 recognizing primary antibodies (1: 2000 dilution) (EGF-1) or tetra-His (1: 2000) (all other proteins). The membrane was then incubated with an alkaline phosphatase-conjugated secondary antibody and the protein bands were visualized using a stabilized Western Blue substrate, the gels showing a single specific band recognized by each Mw predicted antibody (not shown).

Example 3 - animal models

Zymosan-induced arthritis [0172] Zymosan-induced arthritis (ZIA) was induced in mice lacking tenascin-C and wild-type by injection of zymosan (Saccharomyces cerevisiae) as described in Keystone (1977). Zymosan was prepared by dissolving 15 mg zy38 mosan in 1 ml sterile PBS. The solution was boiled twice and sonicated. Mice were euthanized by intraperitoneal injection of 150 μl Hypnorm diluted 1:10 in sterile water, followed by injection of zymosan (10 μL into the right paw pad (d = 0).

[0173] Control mice received a 10 μl injection of PBS alone or did not receive an injection.

For macroscopic assessment of arthritis, the thickness of each hind paw was measured daily with a microcircle (Kroeplin, Schluchlem, Germany) and the diameter was expressed as the mean for each hind paw with inflammation per mouse.

[0174] At the end of the experiment (day = 4), mice were sacrificed and hind paws fixed in 10% (v / v) buffered formalin, delimited in 10% EDTA and embedded in paraffin.

Antigen-induced arthritis [0175] Antigen-induced arthritis (AIA) was induced in mice lacking tenascin-C and wild type as previously described by Brackertz (1977). Briefly, on day 0, mice were anesthetized by intraperitoneal injection of 150 μl Hypnorm diluted 1:10 in sterile water, followed by immunization with 200 μg methylated BSA. mBSA was emulsified into 0.2 ml of complete Freund's adjuvant and injected intradermally at the base of the tail.

[0176] On day 7, arthritis induced by intra-articular injection of mBSA (100 μg in 10 μl sterile PBS) into the right knee joint using sterile microcannula size 33. Control mice received a 10 L injection of PBS alone or did not receive an injection.

[0177] On day 14, mice were sacrificed, knee joints excised and placed in 10% (volume / volume) buffered formalin, delimited with 10% EDTA and embedded in paraffin.

Injection of FBG [0178] Wild type mice were anesthetized by an intraperitoneal injection of 150 μl Hypnorm diluted 1:10 in sterile water, followed by injection of 100 ng, 1 or 3 μg FBG in 10 μl sterile PBS into the right knee joint using a sterile 33 microcannula. Control mice received an injection of 10 μl PBS or did not receive an injection.

[0179] On days 3 and 7, the mice were sacrificed, knee joints excised and fixed in 10% (volume / volume) buffered formalin, delimited with 10% EDTA and embedded in paraffin.

Histology of the knee joints [0180] Frontal tissue sections (4 μm) were made at 7 depths through the joint; 80 μm apart and stained with hematoxylin and eosin or safranin-O to assess joint pathology. Histopathological changes were assessed using the following parameters as described in Van Lent (2006).

[0181] Inflammation (inflammatory cell infiltration (infiltration) and joint cavity (exudate) was assessed using a conventional scale from 0 (no inflammation) to 3. (severe inflammation). Death of chondrocytes was determined as a percentage of cartilage surface containing empty cavities in relation to the total surface The erosion of the cartilage surface was determined as the amount of cartilage lost in relation to the total surface of cartilage. Bone destruction was assessed at 10 different locations throughout the knee joint. The destruction was scored on a scale of 0 (no damage) to 3 (complete loss of bone structure). Histological analysis was carried out by a researcher who had knowledge of experimental groups. The average score for each animal in the experimental group was calculated by averaging the histopathological results at at least 5 depths of the cross-section.

Results

Zymosan-induced arthritis is not persistent in mice lacking tenascin-C [0182] Injection of zymosan into the paw pad was used to induce acute synovitis in mice. Wild-type mice exhibited rapid paw swelling, reaching maximum paw diameter for 24 h (2.56 mm, 62% increase from base paw diameter). This was maintained for another 24 hours. After 2 days, the paw diameter decreased, but the paws remained swollen for 4 days (2.08 mm, increase by 32%) (Figure 1a). Tenascin-C-depleted mice showed a similar degree of paw edema as wild-type mice 24 h after injection (2.41 mm, increase by 57% relative to baseline paw diameter). However, swelling in mice lacking tenascin-C resolved faster than in wild-type mice; paw diameter was significantly reduced after 2 days and decreased to 1.7 mm (increase by 11%) for 4 days (Figure 1A). On day 4 after injection, the paws of wild-type mice were still clearly swollen and red, while the paws of mice lacking tenascin-C were not visibly swollen or red and resembled paws not injected (Figure 9).

[0183] This difference was histologically reflected after 4 days. The articular gout of wild-type mice was significantly inflamed and showed cell infiltration, and loss of cartilage proteoglycan was observed (Figure 1B, C). In contrast, the synovium of mice lacking tenascin-C did not show synovial inflammation, cellular infiltration, or loss of cartilage proteoglycan (Figure 1d, e), and resembled the joints of mice injected with inert material and non-injected mice (data not shown). Quantitative assessment of arthritis revealed that while there was little exudate (cellular mass in the joint cavity) in wild-type or Tenascin-C deficient mice, the level of infiltration (synovial cell mass) was significantly reduced in Tenascin-C deficient mice (Figure 1f ). No cartilage and bone erosion was found in the mice of each genotype (data not shown), however, low chondrocyte death occurred in wild-type mice that were not seen in tenascin-C-deficient mice (Figure 1g). Thus, tenascin-C expression appears to be conducive to maintaining acute inflammation.

Tenascin-C-deficient mice are protected against persistent inflammation and structural damage during antigen-induced arthritis [0184] To determine if tenascin-C also contributes to the more destructive arthritis disease, erosive arthritis is induced by intra-articular injection of mBSA to the knee after immunization with mBSA. This model covers both cellular and humoral immune responses and causes pathological changes similar to those found in people with RA (Brackertz (1977)). Injection of mBSA elicited a similar inflammatory response in both Tenascin-C and wild-type mice. Cell infiltration and thickening of the synovium was visible for 24 h in mice of both genotypes (Figure 2c-f, h, i) compared to the injection of the inert substance (Figure 2a, b, g) or in the non-injected mice (data not shown ).

[0185] However, this did not persist in mice lacking tenascin-C as in wild-type mice. Three days after injection, wild-type mice showed increased meniscus and bursitis, synovial hyperplasia, cell and fibrin deposits in the joint space, scaling and local loss of cartilage proteoglycan (Figure 3a, b, f). In contrast, after 3 days, in mice lacking tenascin C, the inflammation was restricted to the capsule, synovitis resolved and there were no fibrin / cell aggregates present in the joint space, no scaling or loss of cartilage proteoglycan was found (Figure 3c, d, e ).

[0186] After 7 days, wild-type mice exhibited persistent inflammatory cell infiltration and joint effusion, extensive synovitis and scaling, and cartilage destruction and bone erosion (Figure 4a, b). Injected knees and knees of injected mice were healthy and showed no inflammation or joint damage (data not shown). Mice lacking tenascin C also had healthy joints that showed only mild infiltration of inflammatory cells, without exudate into the joint space, synovitis, scaling, destruction of articular cartilage or bone erosion (Figure 4c, d). Joints from tenascin-C-depleted mice injected with neutral substance or mice that were not injected were also healthy (data not shown).

[0187] These histological data are visible after quantifying the disease of the joints as described in the materials and methods. The level of cell infiltration and exudate observed in both wild-type and tenascin-C 24 h mice after injection did not differ significantly. However, while cell mass continued to increase in wild-type mice over time, this response was suppressed in mice lacking tenascin-C and the number of cells in the joint decreased over time (Figure 4e). Increasing levels of chondrocyte death occurred in the cartilage of wild-type mice over time, but no significant chondrocyte death was observed in mice lacking tenascin-C (Figure 4f). In wild-type mice there was no pronounced erosion of cartilage and bone erosion after 24 hours or 3 days (data not shown), but significant tissue destruction occurred after 7 days. In contrast, mice lacking tenascin-C did not show tissue damage after 24 hours, 3 days (data not shown) and 7 days (Figure 4f). These data indicate that while the onset of arthritis (influx of cells into the synovium and joint space) does not change in mice lacking tenascin-C, unlike wild-type mice, the disease does not lead to tissue destruction and cell death. These results indicate that tenascin-C expression is required for persistent synovitis and joint destruction in this model.

Example 4 - Cell culture

Samples from patients [0188] Human monocytes were isolated from London Blood Bank and macrophages were derived from monocytes after 4 days differentiation from 100 ng / ml M-CSF as previously described (Foxwell (1998)).

[0189] RA cell membranes (representing a mixed population of all types of synovial cells) were isolated from synovial membranes obtained from patients undergoing joint replacement surgery as previously described (Brennan, (1989)). RA synovial fibroblasts were isolated from a mixed population of RA cell membranes as previously described (Brennan (1989)). The study was approved by the local ethics committee Trust (Riverside NHS Research Committee), and waste tissue (synovium after joint replacement surgery) was obtained only after receiving the patient's signed informed consent and tissue anonymization to protect the patient's identity.

[0190] Immediately after isolation, RA cell membranes and macrophages were cultured at 1x10⁵ cells / well in RPMI 1640 containing 10% (v / v) FBS and 100 U / ml (unit / ml) penicillin / streptomycin in 96 well tissue culture plates for 24 h before stimulation. Synovial fibroblasts (used for passage number 2 or 3) were grown at 1x10⁴ cells / well in DMEM containing 10% (v / v) FBS and 100 U / ml (units / ml) penicillin / streptomycin in 96 well tissue culture plates for 24 h before stimulation.

Mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages (BMDM) [0191] MEFs express high levels of mRNA for all 9 mouse TLRs and are specifically and highly sensitive to TLR ligand activation. MEFs from mice with targeted deletions TLR2, TLR4 and MyD88 show deep defects in their IL-6 response to specific ligands (Kurt-Jones (2004)). MEF was isolated from d13 embryos collected from age-matched, pregnant wild type females, TLR2, TLR4 and null mice (as described in Todaro (1963)). Fibroblasts were cultured at 2x10⁴ cells / well in DMEM containing 10% (v / v) FBS and 100 U / ml penicillin / streptomycin in 96 well tissue culture plates for 24 h before stimulation.

[0192] BMDM was obtained by aspiration of wild-type female thighs, TLR2 and TLR4 deficient mice as described in Butler (1999)) and cells were cultured for 7 days in DMEM, 20% (v / v) FBS, 10 ml / l (v / v) solution of the antibiotic PSA, 50 μM β-mercaptoethanol and 10 ng / ml M25 CSF. Macrophages were then grown at 1x10⁵ cells / well in DMEM, 20% (v / v) FBS, 10 ml / l (v / v) solution of the antibiotic PSA, 50 μM β-mercaptoethanol in 96 well tissue culture plates for 24 h before stimulation.

HEK293 cell lines [0193] HEK293 cell lines expressing TLR2 and TLR4 / CD14 / MD-2 were grown at 1x10⁴ cells / well in DMEM containing 10% (v / v) FBS and 10 μg / ml blasticidin in 96 well tissue culture plates for 24 h before stimulation.

Cell stimulation and evaluation of cytokine synthesis [0194] Cells were incubated for 24 h at 37 ° C and at the indicated doses of tenascin-C and recombinant fragments of tenascin-C (1.0 μM - 1.0 nM). Cells were also stimulated where indicated by LPS (1 ng / ml for human macrophages, 10 ng / ml for human fibroblasts, RA and HEK membrane cells, 100 ng / ml for MEF and BMDM, 10 ng / ml for HEK), PAM3 (10 ng / ml for human macrophages, human fibroblasts and HEK, 100 ng / ml for MEF and BMDM), mouse IL-1 (5 ng / ml for MEF) and mouse TNF-α (100 ng / ml for MEF). Unless otherwise specified, raw LPS was used for in vitro studies.

[0195] For adenovirus gene transfer experiments, RZS human synovial fibroblasts were incubated with adenoviral vectors at an infection rate of 100, washed after 2 hours, cultured in complete medium for 24 h and then stimulated for 24 h, after which the supernatants were collected.

[0196] Where found, cells were pre-incubated with 10 μg / ml anti-CD14 antibody, 10 μg / ml IL-1 receptor antagonist, 10 μg / ml anti-TLR2 antibody,

25 μg / ml anti-TLR4 antibody, 10 or 25 Lig / ml isotype control antibody, μg / ml polymyxin B or 1 Lig / ml msbB LPS for 30 minutes at 37 ° C before stimulation. Where found, recombinant tenascin-C and FBG and LPS were boiled for 15 minutes before adding to the cells [0197] In all cases, cell viability was not significantly altered throughout the experiment period when tested in MTT cell viability assay (Sigma, Poole, Great Britain).

[0198] Supernatants were then analyzed for the presence of TNF-α, IL-6 and IL-8 cytokines using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems

Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent program (Thermo Labsystems, Altrincham, UK).

Results

Tenascin-C induces the synthesis of TNF-α, IL-6 and IL-8 in primary human synovial RA cells and macrophages [0199] It was then examined whether tenascin-C could activate an innate immune response. Tenascin-C was used to stimulate primary human macrophage and synovial fibroblasts RA and the production of TNF-α, IL-6 and IL-8 proinflammatory cytokines was studied. The cell wall component of LPS bacteria was used as a positive control. Tenascin-C induced a cell type specific cytokine profile that was significantly different from LPS. It stimulated in a dose-dependent manner the production of TNF-α, IL-6 and IL-8 in human macrophages (Figure 5a). However, tenascin-C only induced IL-6 synthesis in synovial fibroblasts, while LPS induced both IL-6 and IL-8 (Figure 5b). Neither LPS nor tenascin-C induced TNF-α synthesis in fibroblasts (data not shown). Tenascin-C stimulation of IL-6 (Figure 5c), IL-8 and TNF-α in human macrophages and IL-6 in synovial fibroblasts (data not shown) was temperature sensitive and was not affected by LPS inhibitor polymyxin B. these results provide strong evidence that the induction of cytokines by tenascin-C is not due to LPS contamination.

The globe-type fibrinogen-like domain (FBG) mediates the activation of tenascin-C cells.

[0200] Tenascin C is a large hexameric molecule whose each domain binds to various cell surface receptors (reviewed in Orend (2005)). Understanding the mechanism of action of tenascin-C requires identifying which domain (s) is critical to triggering cytokine production. Recombinant proteins containing different domains of the molecule were synthesized (Figure 10). Each domain was generated in E. coli, purified (Figure 11) and was found to contain &lt;10 pg / ml LPS by performing a purified protein test on Limulus amaebocyte lysate. Only one tenascin-C domain was active. The globe-type fibrinogen-like domain (FBG) stimulated TNF-α synthesis in human macrophages (Figure 6a), IL-6 and IL-8 synthesis in human macrophages (data not shown) and IL-6 in RA synovial fibroblasts (data not shown) equally compared to full-length tenascin-C. Like tenascin-C, full-length FBG did not induce IL-8 synthesis in RA synovial fibroblasts, for which it was stimulated by LPS (data not shown). FBG-induced cytokine synthesis was also temperature sensitive and resistant to polymyxin B (data not shown).

Tenascin-C FBG domain induces cytokine production in human synovium RA and arthritis in mice.

[0201] It was investigated whether FBG could stimulate inflammatory cytokine expression in synovial membranes from RA patients. This RA model (containing a mixed population of all synovial cell types) spontaneously produces high levels of IL-6, IL-8 and TNF-α (Brennan (1989)) (Figure 6b). FBG further enhanced the synthesis of all these cytokines (Figure 6b). To determine whether FBG can cause inflammation in vivo, wild-type mice were injected intra-articularly with FBG. Transient and dose-dependent stimulation of joint inflammation was observed. No inflammation or loss of proteoglycan was observed in the uninjected mice or in the mice injected with PBS (Figure 6c-e) or 100 ng FBG (data not shown). Inflammatory cell infiltration (Figure 6f), mild synovitis, plaque formation (6g) and loss of proteoglycan (Figure 6h) were observed in mice injected with 1 μg FBG. A similar response was seen in mice injected with 3 μg FBG (data not shown). After histological quantification, a high level of cellular infiltrate, and exudation and death of chondrocytes were observed in mice injected with FBG, including a small amount of cartilage surface erosion and bone damage (Figure 6i). FBG-mediated cytokine synthesis is dependent on MyD88 [0202] Many DAMPs, including fibrinogen (Smiley (2001)), have been shown to stimulate an innate immune response as a result of TLR activation. Thus, we examined whether TLRs can also mediate tenokcin-C-induced cytokine production. Myeloid differentiation factor 88 (MyD88) is required for signaling by all TLRs except TLR3 (O'Neill (2008)). Infection of synovial fibroblasts with adenovirus expressing dominant negative MyD88 but not the GFP control virus abolished IL-6 induction by FBG (Figure 7a). These data suggest that FBG-induced inflammation is dependent on functional MyD88. FBG does not appear to mediate IL-1 because the addition of IL-1 receptor antagonist did not inhibit cytokine induction (data not shown). To confirm that FBG is dependent on MyD88, FBG has not been shown to stimulate cytokine synthesis in embryonic fibroblasts isolated from mice with targeted deletions in the MyD88 gene. TLR2 PAM3 ligand, TLR4 LPS ligand and IL-1 all signal through MyD88. Their stimulation was also abolished in MEF from mice by their deficiency. However, this did not affect TNF-α, which does not signal through MyD88 (Figure 7b). Re-transfection of wild-type MyD88 restored the response of these cells to FBG, PAM3, LPS and IL-1 (data not shown).

FBG signals via TLR4 [0203] TLR has specificity for endogenous ligands; proteins recognized by one or both TLR2 and 4 (review by O'Neill (2008)). Neutralizing anti-TLR4 antibodies inhibited both FBG and LPS-induced IL-6, IL-8 and TNF-α synthesis in human macrophages and IL-6 synthesis in RA synovial fibroblasts, but had no effect on the TLR2 ligand, PAM3. Anti-TLR2 antibodies inhibited PAM3-mediated cytokine synthesis, but had no effect on LPS or FBG-induced cytokine synthesis. Isotype-matched controls did not affect the cytokine synthesis induced by each of the ligands (TNF-α synthesis by human macrophages is shown in Figure 8a). To confirm that FBG activity is dependent on TLR4, we have shown that FBG does not stimulate cytokine synthesis in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR4 gene. FBG-mediated cytokine synthesis was not altered in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR2 gene. Cells isolated from mice lacking TLR2 did not respond to PAM3, but responded to LPS and IL-1. Cells isolated from mice lacking TLR4 did not respond to LPS but responded to PAM3 and IL-1 (Figure 8b, c). In addition, TLR4 expression was required for arthritis-inducing FGB in vivo; FBG was able to induce arthritis in TLR2-deficient mice, but not in TLR4-deficient mice (Figure 12).

Different co-receptor requirements for FBG and LPS [0204] LR signal transduction by TLR4 is mediated by a receptor complex comprising soluble MD-2 protein and surface-mounted or soluble CD14 linked to GPI (review Fitzgerald (2004)). It was then examined whether CD14 and MD-2 are required for activation of TLR4 by FBG. As a positive control, the activity of smooth glycosylated LPS was tested, which requires both MD-2 and CD14 (Jiang (2005)). LPS-mediated IL-6, IL-8 and TNF-α synthesis by human macrophages and IL-6 synthesis by synovial fibroblasts RA was inhibited by anti-CD14 antibodies and antagonist LPS derived from the E. coli msbB mutant that competes with LPS for MD-2 binding (Coats (2007)). On the other hand, both PAM3, which does not require these co-receptors for TLR2 activation, and FBG-mediated cytokine synthesis were not altered by anti-CD14 or LPS antibodies from the msbB mutant (Figure 8d shows the synthesis of TNF-α by human macrophages ). These data suggest that neither CD14 nor MD-2 are required for FBG-mediated cytokine synthesis. Thus, while LPS and FBG both signal by activation of TLR4, they may have different co-receptor requirements.

Example 5 - Inhibition of tenascin-C activity and synthesis in human tissue [0205] In this example the effect of (1) preventing the pro-inflammatory effect of tenascin-C and (2) inhibiting tenascin-C expression in the human synovium of RA is examined.

methods

Peptide synthesis [0206] Nine overlapping peptides spanning the entire FBG domain (Table 2) were synthesized in Biogenes, Germany. Peptides were cleaved at room temperature (cleavage mixture 90% trifluoroacetate, 5% thioanisole, 3% ethanedithiol, 2% anisole), purified by reverse phase high performance liquid chromatography and characterized by MALDI TOF mass spectrum analysis. Peptide purity was&gt; 85%, as determined by high performance liquid chromatography.

[0207] The unit was unable to synthesize peptide 7, probably due to the formation of a secondary structure that prevented peptide chain extension (as previously described (LaFleur (1997)).

Table 2. Overlapping peptides that encompass the entire FBG domain of human tenascin-C

<td>Peptide #</td><td>Amino Acid Sequence</td>

<td> 1</td><td>TIGLLYPFPKDCSQAMLNGDTTSGLYTIYL</td>

<td> 2</td><td>YTIYLNGDKAEALEVFCDMTSDGGGWIVFL</td>

<td> 3</td><td>WIVFLRRKNGRENFYQNWKAYAAGFGDRRE</td>

<td> 4</td><td>GDRREEFWLGLDNLNKITAQGQYELRVD</td>

<td> 5</td><td>ELRVDLRDHGETAFAVYDKFSVGDAKTRYK</td>

<td> 6</td><td>KTRYKLKVEGYSGTAGDSMAYHNGRSFST</td>

<td> 7</td><td>RSFSTFDKDTDSAITNCALSYKGAFWYRN</td>

<td> 8</td><td>WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG</td>

<td> 9</td><td>FHWKGHEHSIQFAEMKLRPSNFRNLEGRRKRA</td>

Samples from patients and cell culture [0208] RA cell membranes (representing a mixed population of all synovial cell types) were isolated from synovial membranes obtained from patients undergoing joint replacement surgery (Brennan (1989)). The synovium was digested in ΚΓΜΙ 1640 medium (GIBCO) containing 5% fetal calf serum (FCS) (Gibco), 5 mg / ml type IV collagenase (Sigma) and 0 15 mg / ml type I DNAse (Sigma) and incubated at 37 ° C for 2 hours.

[0209] After incubation, the tissue was pipetted through a nylon mesh into a sterile beaker. Cells were then washed three times with complete medium (ΚΓΜΙ 1640 supplemented with 10% FCS). RA synovial fibroblasts were isolated from a mixed population of RA cell membranes by selection in DMEM (Bio-Whittaker) supplemented with 10% FBS, 1 μΜ glutamine, 100 U / ml penicillin and streptomycin. Human monocytes were isolated from peripheral blood (London

Blood Bank), and macrophages originated from monocytes after differentiation for 4 days with 100 ng / ml M-CSF.

[0210] The study was approved by the local ethics committee and waste tissue (synovium after joint replacement surgery) was obtained only after receiving informed patient consent and tissue anonymization to protect the patient's identity.

Cell stimulation and cytokine synthesis assessment [0211] Immediately after isolation, RA cells were cultured at 1x10⁵ cells / well in RPMI 1640 containing 10% (v / v) FBS and 100 U / ml penicillin / streptomycin in 96 well tissue culture plates. Cells were incubated for 24 h at 37 ° C without additions, in control buffer solution (PBS buffer, 1% BSA, 0.01% NaN3) or with 25 μ 100, 100 μΜ and 250 μΜ of each of the FBG-containing peptides.

[0212] Synovial fibroblasts (used for passage number 2 or 3) were plated at a concentration of 5x10⁴ cells / well in a 3.5-ml plate. siRNA was transfected at a final concentration of 10 nM using Lipofectamine 2000 (Invitrogen) for 4 hours in serum free OptiMEM I. Two different siRNAs were used against human tenascin-C (s7069 and s229491) (Applied Biosystems).

[0213] The siRNA sequences of s7069 are: (5 'CGCGAGAACUUCUACCAAAtt 3' sense, 5 'UUUGGUAGAAGUUCUCGCGtc 3' antisense) and s229491 are (5 'GGAAUAUGAAUAAAGAAGAtt 3', 5 'UAUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUCAUAUT Luciferase siRNA (Dharmacon) was transfected as non-targeted control.

[0214] Four hours after transfection, the medium was changed to pre-equilibrated Dulbecco's modified Eagle's medium containing 10% FBS (v / v) and the cells were incubated for a further 48 h and 72 h. The cells were then stimulated with 10 ng / ml LPS for 24 h at 37 ° C. Tenascin-C mRNA and protein levels were quantified using PCR and Western blot, respectively. Total RNA was extracted from the cells using the QIAamp RNA Blood mini kit (Qiagen, Germany). cDNA was synthesized from appropriate amounts of total RNA using SuperScript® III Reverse Transcriptase (Invitrogen) and 18-mer oligo dT (Eurofins MWG Operon).

[0215] Gene expression was analyzed using the delta-delta ct method based on real-time quantitative PCR with a set of TaqMan primers for human tenascin-C (Hs01115663-m1) and control of endogenous human ribosomal protein (RPLPO) (4310879E) (Applied Biosystems) on a Corbett Rotor-gen 6000 (Corbett Research Ltd). Tenascin-C protein was detected in cell supernatants and cell lysates using SDS PAGE and Western blotting using body anti-body MAB1908 (Millipore).

[0216] Macrophages were grown at 1x10⁵ cells / well in RPMI 1640 containing

5% (v / v) FBS, 100 U / ml penicillin / streptomycin in 96-well tissue culture plates for 24 h before stimulation. Cells were incubated for 24 h at 37 ° C without addition, with 1.0 μM FBG, 1 ng / ml LPS or 1 or 20 μM FBG peptide. Where indicated, cells were pre-incubated with 20 PM FBG peptides for 15 minutes.

[0217] Cell viability was not significantly changed over the entire experiment period when tested in the MTT cell viability assay (Sigma, Poole, UK). Supernatants were tested for the presence of TNF-α, IL-6 and IL-8 cytokines by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&amp;D Systems). Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using Ascent software (Thermo Labsystems, Altrincham, UK).

Statistical methods [0218] Mean, SD, SEM using GraphPad (GraphPad Software Inc., San Diego,

CA)

Results

Blockade of cytokine synthesis in RZS membrane cultures by specific FBG peptides [0219] The peptide inhibition method was successfully used to identify the integrin binding site of αvβ3 in the FBG domain of tenascin-C and to prevent cell adhesion in response to this tenascin-C domain (Lafleur (1997) and Yokoyama (2000)). [0220] A series of 8 overlapping ~ 30 amino acid peptides covering the entire FBG sequence were synthesized (Table 2). Peptides were tested for their ability to block spontaneous cytokine synthesis in RA synovial culture. TNF synthesis and

IL8 was inhibited by peptides 3 and 8, but not by any other peptide (TNF is shown in Figure 15). Peptides 3 and 8 inhibited cytokine synthesis in a dose-dependent manner, with 95% and 56% inhibition respectively at the highest concentrations (Figure 16). While peptide 5 had no effect on TNF synthesis, it blocked IL8 synthesis in a dose dependent manner in RA cells with maximal inhibition of 81% (Fi30gura 16).

[0221] To map the active domain in FBG, which is responsible for inducing cytokine production, primary human macrophages were stimulated with each of the FBG peptides. Peptides 1, 5 and 6 all induced cytokine synthesis in a dose-dependent manner. (Figure 17). [0222] To determine if any peptide can block induced cytokine synthesis

FBG in human macrophages, cells were preincubated with each of the FBG peptides before stimulation with all FBG or LPS. Peptide 5 specifically blocked FBG-mediated cytokine synthesis, while peptide 8 blocked cytokine synthesis in response to both LPS and FBG (Figure 18).

[0223] Thus, peptide 8 non-specifically blocks cytokine production induced by any stimulus. This domain is an FBG integrin binding domain that mediates cell adhesion and can therefore act to prevent cell adhesion on tissue culture plates. Peptide 5 specifically blocks FBG-induced cytokine synthesis, suggesting that targeting this domain may be useful in preventing tenascin-C-induced inflammation.

Silencing tenascin-C gene expression inhibits cytokine synthesis in RA synovial fibroblasts [0224] A study on the inhibition of tenascin-C expression in human synovial RA has identified synovial fibroblasts as a major source of tenascin-C in RA (Figure 1

C) (in Goh 2010).

[0225] siRNA-mediated silencing of tenascin-C expression in these cells showed a maximum yield between 94-96% (Figure 19). In siRNA transfected cells for tenascin-C, both basal cytokine levels and cytokine synthesis and LPS-induced cytokine production were inhibited by 38% and 44% compared to control cells (Figure 19) [0226] These data show that silencing tenascin-C synovial fibroblasts RA reduces the synthesis of pro-inflammatory cytokines and suggests that the removal of tenascin-C expression is a possible strategy to suppress inflammation in the synovium.

[0227] The present work showed that blocking tenascin-C (peptide) activity and tenascin-C (siRNA) expression reduces the synthesis of inflammatory cytokines in human synovial membrane RA. These data show that tenascin-C blockade is a potential clinical benefit in the treatment of RA and other inflammatory diseases.

Literature items [0228]

1. Smolen, JS and Maini, RN Interleukin-6: a new therapeutic target. Arthritis Res Ther

Suppl 2, S5 (2006).

2. Williams, RO, Paleolog, E. and Feldmann, M. Cytokine inhibitors in rheumatoid arthritis and other autoimmune diseases, Curr Opin Pharmacol 7, 412-417 (2007).

3. Brentano, F., Kyburz, D., Schorr, O., Gay, R. and Gay, S. The role of Toll-like receptor signalling in the pathogenesis of arthritis. Cell Immunol 233, 90-96 (2005).

4. O'Neill, LA Primer: Toll-like receptor signaling pathways-what do rheumatologists need to know? Nat Clin Pract Rheumatol (2008).

5. Matzinger, P. The danger model: a renewed sense of self. Science 296, 301-305 (2002).

6. Bianchi, ME DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol 81, 1-5 (2007).

7. Gordon, S. Pattern recognition receptors: doubling up for the innate immune response. Cell 111, 927-930 (2002).

8. Medzhitov, R. and Janeway, CA, Jr. Decoding the patterns of self and nonself by the innate immune system. Science 296, 298-300 (2002).

9. Radstake, TR et al. Expression of toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-gamma. Arthritis Rheum 50, 3856-3865 (2004).

10. Roelofs, MF et al. The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells. Arthritis Rheum 52, 2313-2322 (2005).

11. Sacre, SM et al. The Toll-like receptor adapter proteins MyD88 and MaI / TIRAP contribute to the inflammatory and destructive processes in a human model of rheumatoid arthritis. Am J Pathol 170, 518-525 (2007).

12. Choe, JY, Crain, B., Wu, SR and Corr, M. Interleukin 1 receptor dependence of serum transferred arthritis can be circumvented by toll-like receptor 4 signaling. J Exp Med 197, 537-542 (2003).

13. Lee, EK, Kang, SM, Paik, DJ, Kim, JM and Youn, J. Essential roles of Toll-like receptor-4 signaling in arthritis induced by type II collagen antibody and LPS. Int Immunol 17, 325-333 (2005).

14. Abdollahi-Roodsaz, S. et al. Inhibition of Toll-like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis. Arthritis Rheum 56, 2957-2967 (2007).

15. Vanags, D. et al. Therapeutic efficacy and safety of chaperonin 10 in patients with rheumatoid arthritis: a double-blind randomized trial. Lancet 368, 855-863 (2006).

16. Chiquet-Ehrismann, R. and Chiquet, M. Tenascins: regulation and putative functions during pathological stress. J Pathol 200, 488-499 (2003).

17. Cutolo, M., Picasso, M., Ponassi, M., Sun, MZ and Balza, E. Tenascin and fibronectin distribution in human normal and pathological synovium. J Rheumatol 19, 1439-1447 (1992).

18. McCachren, SS and Lightner, VA Expression of human tenascin in synovitis and its regulation by interleukin-1. Arthritis Rheum 35, 1185-1196 (1992).

19. Salter, DM Tenascin is increased in cartilage and synovium from arthritic knees. Br J Rheumatol 32, 780-786 (1993).

twenty. Chevalier, X., Groult, N., Larget-Piet, B., Zardi, L. and Hornebeck, W. Tenascin distribution in articular cartilage from normal subjects and from patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum 37, 1013-1022 (1994).

21. Hasegawa, M. et al. Expression of large tenascin-C splice variants in synovial fluid of patients with rheumatoid arthritis. J Orthop Res 25, 563-568 (2007).

22. Orend, G. Potential oncogenic action of tenascin-C in tumorigenesis. Int J Biochem Cell Biol 37, 1066-1083 (2005).

23. Brackertz, D., Mitchell, GF and Mackay, IR Antigen-induced arthritis in mice. I. Induction of arthritis in various strains of mice. Arthritis Rheum 20, 841-850 (1977).

24. Brennan, FM, Chantry, D., Jackson, A., Maini, R. and Feldmann, M. Inhibitors effect of TNF alpha antibodies on synovial cell interleukin-1 production in rheumatoid arthritis. Lancet 2, 244-247 (1989).

25. Smiley, ST, King, JA and Hancock, WW Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4. J Immunol 167, 2887-2894 (2001).

26. Fitzgerald, KA, Rowe, DC and Golenbock, DT Endotoxin recognition and signal transduction by the TLR4 / MD2-complex. Microbes Infect 6, 1361-1367 (2004).

27. Jiang, Z. et al. CD14 is required for MyD88-independent LPS signaling. Nat Immunol 6, 565-570 (2005).

28. Coats, SR, Do, CT, Karimi-Naser, LM, Braham, PH and Darveau, RP Antagonistic lipopolysaccharides block E. coli lipopolysaccharide function at human TLR4 via interaction with the human MD-2 lipopolysaccharide binding site. Cell Microbiol 9, 11911202 (2007).

29. Siri, A. et al. Human tenascin: primary structure, pre-mRNA splicing patterns and localization of the epitopes recognized by two monoclonal antibodies. Nucleic Acids Res 19, 525-531 (1991).

thirty. Gondokaryono, SP et al. The extra domain A of fibronectin stimulates murine mast cells via toll-like receptor 4. J Leukoc Biol 82, 657-665 (2007).

31. Taylor, KR et al. Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2. J Biol

Chem 282, 18265-18275 (2007).

32. Kim, HM et al. Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130, 906-917 (2007).

33. Schaefer, L. et al. The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages. J Clin Invest 115, 2223-2233 (2005).

34. Foell, D., Wittkowski, H. and Roth, J. Mechanisms of disease: a 'DAMP' view of inflammatory arthritis. Nat Clin Pract Rheumatol 3, 382-390 (2007).

35. Taniguchi, N. et al. High mobility group box chromosomal protein 1 plays a role in the pathogenesis of rheumatoid arthritis as a novel cytokine. Arthritis Rheum 48, 971981 (2003).

36. Pullerits, R. et al. High mobility group box chromosomal protein 1, a DNA binding cytokine, induces arthritis. Arthritis Rheum 48, 1693-1700 (2003).

37. Kokkola, R. et al. Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity. Arthritis Rheum 48, 2052-2058 (2003).

38. Gutowski, NJ, Newcombe, J. and Cuzner, ML Tenascin-R and C in multiple sclerosis lesions: relevance to extracellular matrix remodelling. Neuropathol Appl Neurobiol 25, 207-214 (1999).

39. Amin, K. et al. Inflammation and structural changes in the airways of patients with primary Sjogren's syndrome. Respir Med 95, 904-910 (2001).

40. Loots, MA et al. Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds. J Invest Dermatol 111, 850-857 (1998).

41. Lange, K. et al. Endothelin receptor type B counteracts tenascin-C-induced endothelin receptor type A-dependent focal adhesion and actin stress fiber disorganization. Cancer Res 67, 6163-6173 (2007).

42. Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. and Aizawa, S. Mice develop normally without tenascin. Genes Dev 6, 1821-1831 (1992).

43. Hoshino, K. et al. Cutting edge: Toll-like receptor 4 (TLR4) -deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 162, 3749-3752 (1999).

44. Takeuchi, O. et al. Differential roles of TLR2 and TLR4 in recognition of gramnegative and gram-positive bacterial cell wall components. Immunity 11, 443-451 (1999).

45. Keystone, EC, Schorlemmer, HU, Pope, C. and Allison, AC Zymosan-induced arthritis: a model of chronic proliferative arthritis following activation of the alternative pathway of complement. Arthritis Rheum 20, 1396-1401 (1977).

46. van Lent, PL et al. Fcgamma receptors directly mediate cartilage, but not bone, destruction in murine antigen-induced arthritis: uncoupling of cartilage damage from bone erosion and joint inflammation. Arthritis Rheum 54, 3868-3877 (2006).

47. Foxwell, B. et al. Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent. Proc Natl Acad Sci USA 95, 8211-8215 (1998).

48. Kurt-Jones, EA et al. Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity. J Endotoxin Res 10, 419-424 (2004).

49. Todaro, GJ and Green, H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17, 299-313 (1963).

50. Butler, DM, Malfait, AM, Maini, RN, Brennan, FM and Feldmann, M. Anti-IL-12 and anti-TNF antibodies synergistically suppress the progression of murine collageninduced arthritis. Eur J Immunol 29, 2205-2212 (1999).

51. Ho, SN, Hunt, HD, Horton, RM, Pullen, JK and Pease, LR Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59 (1989).

52. Clark, RA, Erickson, HP and Springer, TA Tenascin supports lymphocyte rolling. J Cell Biol 137, 755-765 (1997).

53. El-Karef, A. et al. Deficiency of tenascin-C attenuates liver fibrosis in immunemediated chronic hepatitis in mice. J Pathol 211, 86-94 (2007).

54. Loike, JD, Cao, L., Budhu, S., Hoffman, S. and Silverstein, SC Blockade of alpha 5 beta 1 integrins reverses the inhibitors effect of tenascin on chemotaxis of human monocytes and polymorphonuclear leukocytes through three-dimensional gels of extracellular matrix proteins. J Immunol 166, 7534-7542 (2001).

55. Talts, JF, Wirl, G., Dictor, M., Muller, WJ and Fassler, R. tenascin-C modulates tumor stroma and monocyte / macrophage recruitment but not tumor growth or metastasis in a mouse strain with spontaneous mammary cancer. J Cell Sci 112 (Pt 12), 1855-1864 (1999).

56. Jones (2000) Matrix Biol., 19, 581-96

57. Harandl (2009) Expert Review of Vaccines, 8, 293-298

58. McIntyre (2006) BMC Biotechnol. 6: 1

59. Paddison (2002) Genes Dev. 16 (8): 948-58

60. Andreakos (2004) Blood, 103, 2229-37

61. Goh, FG, Piccinini, AM, Krausgruber, T., Udalova, IA and Midwood, KS Transcriptional regulation of the endogenous danger signal tenascin-C: a novel autocrine loop in inflammation. J Immunol 184, 2655-2662 (2010).

62. Midwood, K. et al. Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease. Nat Med 15, 774-780 (2009).

63. LaFleur, DW et al. Aortic smooth muscle cells interact with tenascin-C through its fibrinogen-like domain. J Biol Chem 272, 32798-32803 (1997).

64. Taylor, PC and Feldmann, M. Anti-TNF biologic agents: still the therapy of choice for rheumatoid arthritis. Nat Rev Rheumatol 5, 578-582 (2009).

65. Yokoyama, K., Erickson, HP, Ikeda, Y. and Takada, Y. Identification of amino acid sequences in fibrinogen gamma-chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3. J Biol Chem 275, 16891 -16898 (2000).