Biological materials and uses thereof

DESCRIPTION

BIOLOGICAL MATERIALS AND USES THEREOF

The present invention relates to tenascin-C and its activity in chronic inflammation. Modulators of tenascin-C and its biological activity are also provided.

Inflammation is the complex biological response of tissues to harmful stimuli, such as pathogens, tissue damage or irritants. It is an attempt to protect the tissue from removing the harmful stimuli as well as starting the healing process for the tissue. Anomalies associated with inflammation comprise a large, unrelated group of disorders that underlie a variety of human diseases (inflammatory disorders). Examples of diseases with an inflammatory appearance include (but are not limited to) asthma, autoimmune disease, glomerulonephritis, allergy (hypersensitivity), inflammatory bowel disease, reperfusion injury, rheumatoid arthritis, and transplant rejection.

In particular, chronic inflammation is a debilitating and severe condition associated with many of the aforementioned diseases, and is characterized by persistent inflammation at the site of infection or injury, or in relation to altered immune responses, such as autoimmune disease.

Rheumatoid arthritis (RA) is a typical example of, although by no means the only, chronic inflammatory condition. RA is characterized by synovial inflammation and destruction of joint and bone cartilage, mediated by persistent synthesis of

402406280Β1 proinflammatory cytokines and matrix metalloproteinases (MMP). Biological compounds that suppress the synthesis of inflammatory cytokines, such as TNFα and IL-6, are successful in short-term treatment of RA. However, repeated treatments are required, which makes this therapeutic approach expensive, and does not provide long-term remission. Furthermore, total systemic suppression of cytokine function is not without its inherent problems, such as increased infectious risk. Thus, despite advances in care, there remains an unmet need for a cost-effective, long-term cost-effective mode of treating chronic inflammation (Smolen (2006) and Williams (2007)).

The mechanisms that support the chronic state of the disease are not yet clear, and the factor (s) driving the prolonged expression of inflammatory and destructive mediators is currently unknown.

Toll-like receptors (TLR) play a key role in driving the production of inflammatory mediators in RA, and blockade of TLR function may be of significant clinical benefit (reviewed in Brentano (2005) and O'Neill (2002). ). This family of receptors forms an integral part of the immune system. TLRs mediate host defense against infection and injury by recognizing both pathogen-associated molecular patterns (PAMP) and injury-associated molecular patterns (DAMP) (Matzinger (2002)). DAMPs are endogenous proinflammatory molecules generated after tissue damage, and include intracellular molecules released from damaged or necrotic cells, fragments of extracellular matrix (ECM) molecules, or over-regulated ECM molecules (revised in Bianchi ( 2007)

22406280-1 and Gordon (2002)).

Following activation, TLRs promote both innate and adaptive immune responses, including stimulation of proinflammatory cytokine and MMP expression (Medzhitov (2002)). TLRs are expressed at high levels in the synovial tissue of RA patients (Radstake (2004), Roelofs (2005), Sacre (2007), and (Sacre, manuscript submitted in 2008), and mice with targeted deletions or loss mutations. of function in TLR4 are protected from experimental arthritis (Choe (2003) and Lee (2005)). In addition, TLR4 inhibitors may reduce destructive arthritis in mice (Abdollahi-Roodsaz (2007)), and a putative TLR4 inhibitor improved symptoms in 15 of 23 patients with moderate to severe RA in a preliminary phase I trial. (Vanags (2006). However, it is not clear which TLR ligand (s) are involved in the pathogenesis of the disease.

Tenascin-C is an ECM glycoprotein that is associated with tissue injury and wound repair. Tenascin-C is specifically expressed during active tissue remodeling during embryogenesis and is first observed during gastrulation and sommits. In later stages of expression is restricted to the morphogenesis sites of branching of the mammary gland and lung, in the developing squirrel, cardiovascular system and connective tissues at sites of epithelial to mesenchymal transformation. Expression is down-regulated once these processes cease and before embryogenesis ends (Jones (2000)).

development training

Tenascin-C is not a healthy adult, but normally expressed in adult tissue, is transiently overregulated specifically during inflammation.

Aguda2406280Β1 acute and persistently expressed in chronic inflammation (reviewed in Chiguet-Ehrismann (2003)). Immunohistochemical studies show that tenascin-C is poorly expressed in normal human joints, but levels increase considerably in RA synovium, areas of inflammation and fibrosis, specifically under the synovial lining, invasive pannus, and around vessels. (Cutolo (1992), MacCachren (1992) and Salter (1993)). There is also a significant increase in tenascin-C levels in synovial fluid in RA patients (Chevalier (1994) and Hasegawa (2007)) and in RA cartilage (Salter (1993) and Chevalier (1994)).

Tenascin-C is a large 1.5 million Da hexameric protein. Each chain is composed of different domains, including a mounting domain (TA), EGF-like repeats (EGF-L), fibronectin-like repeats. III (TNIII) and a fibrinogen-like globe (FBG) (reviewed in Orend (2005)). The tenascin-C sequences and their domains are shown in figure 13.

Previously, the role of tenascin-C in inflammation was uncertain, with evidence showing varying effects on different immune cells. For example, tenascin-C has been shown to support adhesion and lamination of human primary peripheral blood lymphocytes and tonsils, thus suggesting a role in stimulating lymphocyte migration (Clark (1997)). In addition, tenascin-C null mice show reduced lymphocyte infiltration and lower levels of IFN, TNF and IL-4 mRNA following concanavalin-A-induced liver damage in mice (El-Karef (2007 )). Thus, evidence suggests that tenascin-C is involved in promoting the activity of acute inflammatory cells. However, the

ΕΡ2406280Β1 tenascin-C has also been described as an inhibitor of in vitro monocyte chemotaxis (Loike (2001)), and null mice in tenascin-C exhibit increased monocyte and macrophage migration in mammary tumor stroma (Talts (1999)). ). Therefore, this evidence suggests

<td>that tenascin-C inflammatory.</td><td>has a role in</td><td>inhibition</td><td>in</td><td>cells</td>

<td>The inventors</td><td colspan="2">showed that tenascin-C is</td><td>one</td><td>calling</td>

<td>TLR4 endogenous</td><td>What is necessary</td><td>to the</td><td colspan="2">inflammation</td>

<td colspan="2">destruction of the joint observed in</td><td>arthritis.</td><td></td><td></td>

In addition, it is now shown that tenascin-C is not involved in inducing inflammation (acute inflammatory response), but rather is involved in prolonging the inflammatory response that characterizes chronic inflammatory disease. In particular, tenascin-C has now been shown to be an endogenous activator of TLR4 and it has been shown that this molecule is required for destructive joint inflammation.

A role for tenascin-C has been demonstrated in mediating an immune response in the joint by inducing joint inflammation by intra-articular injection of the tenascin-C FBG domain in mice in vivo. In addition, zimosan-induced acute joint inflammation was not so prolonged in tenascin-C deficient mice. Both wild-type and tenascin-C mice responded to zimosan inflammation, also demonstrating that tenascin-C does not appear to be involved in the initiation of inflammation. However, the less persistent synovitis exhibited by null mice in tenascin-C indicates a role in maintaining joint inflammation. The importance of tenascin-C in prolonging joint inflammation was acute-induced null mice.

ΕΡ2406280Β1 is underlined by the observation that the targeted deletion of tenascin-C protected mice from prolonged erosive joint inflammation during mBSA immunization-induced arthritis.

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

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

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

Formation of distinct receptor complexes by each TLR4 ligand may facilitate the recruitment of different adapter / signaling intracellular molecules. This may contribute to the answers

ΕΡ2406280Β1 differential cells that we observed with FBG and LPS, for example, the lack of FBG-induced IL-8 in RA synovial fibroblasts. Similarly, activation of the TLR4 and CD44 complex hyaluronic acid induces a pattern of gene expression in mouse alveolar macrophage cell lines, which is different from LPS (Taylor (2007)). The fact that FBG induces IL-8 synthesis in human macrophages suggests that recognition and / or signaling of the cell type-specific ligand occurs.

Tenascin-C's tightly regulated expression pattern makes it an attractive target for the treatment of absent and induced induction. During chronic inflammation, healthy adults,

Predominantly however, expression specifically after tissue injury, acute inflammation to tenascin-C is transiently expressed: induction often precedes inflammation, and both mRNA and protein are absent from tissue when inflammation is resolved (reviewed in Chiquet-Ehrismann (2003)).

Persistent expression of tenascin-C has now been shown to be associated with chronic inflammation. In addition to RA, increased tenascin-C levels are seen in other autoimmune diseases, including multiple sclerosis (Gutowski (1999)) and Sjogren's disease (Amin (2001)), and in non-healing wounds, and in diabetic and venous ulcers (Loots (1998)). De novo synthesis of tenascin-C correlates well with the intensity of inflammation in oral mucosal diseases, and tenascin-C plasma levels are a reliable indicator for the activity of inflammatory bowel disease before and after medication. or surgery (reviewed in Chiquet-Ehrismann (2003)).

402406280Β1

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

The first aspect agent of the invention may modulate the biological activity of tenascin-C by altering the binding properties of tenascin-C.

Also described are agents which may modulate the biological activity of tenascin-C by altering the transcription and / or translation of tenascin-C.

Such agents may be identified using methods well known in the art, such as:

(a) by determining the effect of a test agent on tenascin-C expression levels, for example by Southern blotting or related hybridization techniques;

(b) by determining the effect of a test agent on tenascin-C protein levels, for example, by immunoassays using antitenascin-C antibodies; and (c) by determining the effect of a test agent on a functional marker or result of tenascin-C activity, for example by the methods of the examples.

The disclosed agents may downregulate the biological activity of tenascin-C.

The disclosed agents may over-regulate the

ΕΡ2406280Β1 biological activity of tenascin-C. The desirability of overregulating the activity of immune and inflammatory molecules and cells is relevant for the production of therapies for immune and inflammatory compromised patients, and for the development of vaccines (see Harandi (2009)).

The above described agent may be transcription of tenascin-C.

be an inhibitor of

The agent described below is translation of tenascin-C.

may be an inhibitor of

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

The agent of the first aspect of the invention may be a competitive tenascin-C binding inhibitor. It will be appreciated by persons skilled in the art that the agent may also inhibit the biological activity of tenascin-C by blogging the function of the tenascin-C receptor, either directly (by acting as a tenascin-C receptor antagonist) or indirectly (by bind to intermediate or auxiliary molecules).

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

It will be appreciated by persons skilled in the art that inhibition of the biological activity of tenascin-C by an agent of the invention may be wholly or partly. For example, the agent may inhibit the biological activity of

Enas2406280Β1 tenascin-C by at least 10%, preferably by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably by 100% compared to activity of Tenascin-C in inflammatory cells that were not exposed to the agent.

The above-described agent can be selected from the group consisting of small interfering RNA (siRNA) molecules, small stapled RNA (shRNA) molecules, antisense oligonucleotides, tenascin-C binding affinity compounds, (polyclonal) antibodies or monoclonal) and antigen binding fragments thereof, small inhibitory compounds, polypeptides and proteins.

In one example, the agent is an siRNA. RNA interference is a two-step process. The first step, which is referred to as the priming step, the incoming dsRNA is digested into small 21 - 23 nucleotide (nt) interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of specific dsRNA ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or virus) in an ATP-dependent manner. Successive cleavage events degrade RNA in 19-21 bp duplexes (siRNAs), each with 2 nucleotide overhangs at the 3 'terminus (Hutvagner &amp; Zamore, 2002, Curr. Opin. Genetics and Development 12: 225-232; Bernstein, 2001, Nature 409:

<td colspan="6"> 363-366).</td>

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<td>complex of</td><td>nuclease</td><td>in</td><td>to form the</td><td>complex</td><td>in</td>

<td>silencing</td><td>induced</td><td colspan="2">by RNA (RISC). IS</td><td>required</td><td>one</td>

<td colspan="2">dependent unfolding</td><td>in</td><td>ATP duplex</td><td>siARN to</td><td>The</td>

<td>activation of</td><td>RISK 0</td><td colspan="2">Active RISC drives</td><td>-if then</td><td>to</td>

ΕΡ2406280Β1 homologous transcript, through base pairing interactions, and cuts mRNA into siRNA 3 'terminal 12 nucleotide fragments (Hutvagner &amp; Zamore, 2002, supra; Hammond et al., 2001, Nat. Rev. Gen. 2: 110119 (2001); Sharp, 2001, Genes, Dev. 15: 485-90). Although the cleavage mechanism remains to be elucidated, research indicates that each RISC contains a single siRNA and one RNase (Hutvagner &amp; Zamore, 2002, supra.).

Given the remarkable potency of siRNA, an amplification step within the siRNA pathway has been suggested. Amplification may occur by copying the input dsRNAs that would generate more siRNAs, or by replicating the formed siRNAs. Alternatively, or additionally, amplification may be effected by various RISC turnover events (Hammond et al., 2001, supra; Hutvagner &amp; Zamore, 2002, supra). Additional information about RNAi can be found in the following reviews, Tuschl, 2001, Chem. Biochem. 2: 239-245, Cullen 2002, Nat. Immunol. 3: 597-599 and Brantl, 2002, Biochem. Biophys Ata.

1575: 15-25.

Synthesis of appropriate RNAi molecules for use with the present invention may be performed as follows. First, the tenascin-C mRNA sequence is screened downstream of the AUG initiation codon for AA dinucleotide sequences. Occurrence of each AA and adjacent nucleotides in the 3 'direction is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, since untranslated regions (RTUs) are rich in regulatory protein binding sites. RTU-binding proteins and / or translation initiation complexes may interfere with the binding of siRNA endonuclease complex (Tuschl,

402406280Β1

ChemBiochem. 2: 239-245). However, it will be appreciated that siRNAs targeting untranslated regions may also be effective.

Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat, etc.) using sequence alignment software such as BLAST (www.ncbi.nlm.nih. gov / BLAST /). Putative target sites that exhibit significant homology to other coding sequences are filtered.

Qualified target sequences are selected as template for siRNA synthesis. Preferred sequences are those which include low G / C content, as these have proven to be more effective in mediating gene silencing compared to those with G / C content greater than 55%. Preferably various target sites are selected for evaluation along the length of the target gene. For a better evaluation of the selected siRNAs a negative control is preferably used together. Negative siRNA control preferably includes the same nucleotide composition as siRNAs, but lacks significant homology to the genome. Thus, a siRNA encoded nucleotide sequence is preferably used, provided that it shows no significant homology to any other gene.

Suitable siRNA molecules may be synthesized as described above so that they are complementary and therefore bind to the entire tenascin-C nucleotide sequence or portions thereof. The nucleotide sequence of tenascin-C is shown in Figure 14.

402406280Β1

In one example, the agent may be a small staple RNA (shRNA).

A small stapled RNA or a short stapled RNA (shRNA) is an RNA sequence that makes a tight staple curve that can be used to silence gene expression through interfering RNA. The shRNA utilizes a vector (usually adenovirus or lentivirus) introduced into the cells, and uses the U6 promoter to ensure that the shRNA is always expressed. This vector is normally passed to daughter cells, allowing gene silencing to be inherited. The shRNA staple structure is cleaved by the cellular maguaria to siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds and cleaves the mRNAs that match the siRNA that is attached to it (McNtyre (2006) and Paddison (2002)).

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 predominantly involved in the interaction of tenascin-C with its target in relation to persistence of chronic inflammation. Therefore, the preferred domain is the FBG domain (sequence shown in Figure 13) or variants thereof.

In an alternative example, the agent is an antisense oligonucleotide.

The design of antisense molecules that can be used to efficiently decrease tenascin-C levels / activity leads to consideration of two important aspects of the antisense approach. The first aspect is the delivery of the oligonucleotide to the cancer cell cytoplasm, while the second aspect is the design of an oligonucleotide that binds.

ΕΡ2406280Β1 specifically to the designated mRNA within the cells, thereby inhibiting its translation.

The prior art discloses a number of delivery strategies which can be used to efficiently deliver oligonucleotides to a wide variety of cell types (e.g., 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

<td rowspan="2">al., al.,</td><td rowspan="2"> 1997, 1997,</td><td colspan="3">Biochem Biophys Res Commun 237: 566-71;</td><td rowspan="2">Aoki et</td>

<td>Biochem Biophys Res</td><td>Commun</td><td> 231: 540-5).</td>

<td></td><td>For</td><td>In addition, they are</td><td colspan="3">algorithms available for</td>

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maximum predicted target mRNA based on a thermodynamic cycle that takes into account the energy of structural alternations in both the target mRNA and the oligonucleotide (for example, see Walton et al., 1999, Biotechnol Bioeng 65: 1- 9).

Various approaches to designing and predicting the efficiency of specific oligonucleotides using an in vitro system are also known (for example, see Matveeva et al., 1998, Nature Biotechnology 16: 1374-1375).

Several clinical trials have demonstrated the safety, viability and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for cancer treatment have been successfully used (Holmlund et al., 1999, Curr Opin Mol Ther 1: 372-85; Gerwitz, 1999, Curr Opin Mol Ther 1: 297-306). More recently, suppression of antisense-mediated human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model (Uno et al., 2001, Cancer Res

402406280Β1

61: 7855-60) .

Thus, persons skilled in the art are readily able to devise and implement appropriate antisense approaches to modulating tenascin-C expression.

Advantageously, the antisense oligonucleotide is from 15 to 35 bases in length. For example, 20-mer oligonucleotides have been shown to inhibit epidermal growth factor receptor mRNA expression (Witters et al., Breast Cancer Res Treat 53: 41-50 (1999)), and 25-mer oligonucleotides have been shown to decrease adrenocorticotropic hormone expression by more than 90% (Frankel et al., J Neurosurg 91: 261-7 (1999)). However, it is appreciated 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.

It will be further appreciated by those skilled in the art that oligonucleotides are subject to being degraded or inactivated by endogenous cellular nucleases. To counteract this problem, modified oligonucleotides may be used, for example having altered internucleotide bonds, whereupon naturally occurring phosphodiester bonds have been replaced with another one. For example, Agrawal et al. (1988) Proc.

Natl. Acad. Know. USA 85, 7079-7083, have shown increased inhibition in HIV-1 tissue culture using phosphoramidate and phosphorothioate oligonucleotides. Sarin et al. (1988) Proc. Natl. Acad. Know. USA 85, 7448-7451, demonstrated increased inhibition of HIV-1 using methylphosphonate oligonucleotides. Agrawal et al. , (1989)

Proc. Natl. Acad. Know. USA 86, 7790-7794, have shown inhibition of HIV-1 replication in both cultures of

402406280Β1 early infected and chronically infected cells using nucleotide sequence-specific phosphorothioate oligonucleotides. Leither et al. , (1990) Proc. Natl. Acad. Know. USA 87, 3430-3434, report an inhibition of influenza virus replication tissue culture by phosphorothioate oligonucleotides.

Artificially linked oligonucleotides have been shown to be resistant to degradation in vivo. For example, Shaw et al. (1991) in Nucleic Acids Res. 19, 747750, report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3 'end by certain encapsulation structures, and that unencapsulated phosphorothioate oligonucleotides are not degraded in vivo.

A detailed description of the H-phosphonate approach for phosphorothioate oligonucleotide synthesis is provided in Agrawal and Tang (1990) Tetrahedron Letters 31, 7541-7544, the teachings of which are incorporated herein by reference. Syntheses of methylphosphonate oligonucleosides, phosphorodithioates, phosphoramidates, phosphate esters, bridged phosphoramidates and 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. Ata. 71, 1517; Crosstick and Vyle (1989) Tetrahedron Letters 30, 4693; Agrawal et al. (1990) Proc. Natl. Acad. Know. USA 87, 1401-1405, the teachings of which are incorporated herein by reference. Other methods for synthesis or production are also possible. In a preferred embodiment, the oligonucleotide is a deoxyribonucleic acid (DNA), although it may also be synthesized and applied.

402406280Β1 ribonucleic acid (RNA) sequences.

Oligonucleotides useful in the examples described herein are preferably designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces reduced length oligonucleotide decomposition products. Such decomposition products are more likely to engage in non-specific hybridization, and are less likely to be effective relative to their full length equivalents. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and capable of reaching the target cells. Current oligonucleotides may become more resistant to degradation in vivo by replacing one or more internal artificial internucleotide bonds with native phosphodiester bonds, for example by substituting phosphate for sulfur in the bond. Examples of bonds that may be used include phosphorothioates, methylphosphonates, sulfone, sulfate, cetyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phosphorothioates and bridged phosphoramidates. Such examples are illustrative, not limiting, as other internucleotide linkages are well known in the art. Synthesis of oligonucleotides having one or more of these bonds substituted by phosphodiester internucleotide bonds, including synthetic pathways for the production of oligonucleotides with mixed internucleotide bonds, is well known in the art.

Oligonucleotides may be made resistant to extension by endogenous enzymes by encapsulation, or by incorporation of similar groups into the 5 'or 3' terminal nucleotides. A packaging reagent such as Amino-Link II ™ of

402406280Β1

Applied BioSystems Inc, Foster City, CA. Encapsulation methods are described, for example, by Shaw et al. , (1991) Nucleic Acids Res. 19, 747-750 and Agrawal et al. , (1991) Proc. Natl. Acad. Know. USA 88 (17), 7595-7599.

Another method of preparing nuclease-resistant oligonucleotides is that they are self-stabilizing as described by Tang et al. (1993) Nuc. Acids Res. 21, 2729-2735. Self-stabilizing oligonucleotides have staple loop structures at their 3 'ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I, and fetal bovine serum. The self-stabilized region of the oligonucleotide does not interfere with hybridization to complementary nucleic acids, and mouse pharmacokinetic and stability studies showed an in vivo increase in the persistence of self-stabilized oligonucleotides relative to their linear counterparts.

In one example where the agent is a tenascin-C binding affinity compound, the compound may bind substantially reversibly or substantially irreversibly to an active tenascin-C site. In another example, the compound may bind to a portion of tenascin-C, which is not the active site, so as to interfere with the binding of tenascin-C to a ligand or receptor. In yet another example, the compound may bind to a portion of tenascin-C in order to decrease protein activity by an allosteric effect. This allosteric effect may be an allosteric effect which is involved in the natural regulation of tenascin-C activity, for example the activation of tenascin-C by an upstream activator.

Methods for the

ΕΡ2406280Β1 detection of interactions between a test compound and tenascin-C. Ultrafiltration with ion spray mass spectroscopy / HPLC methods or other physical and analytical methods may be used for example. In addition, resonance energy fluorescence transfer (FRET) methods may be used, whereby the binding of two fluorescently labeled entities can be measured by measuring the interaction of the fluorescent markers while being in close proximity to each other.

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

Another method of identifying a compound which is capable of binding to the polypeptide is one in which the polypeptide is exposed to the compound, and any binding of the compound to said polypeptide is detected and / or measured. The binding constant for binding of the compound to the polypeptide can be determined. Appropriate methods for detecting and / or measuring (guantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be carried out, for example, using a method capable of high throughput, e.g. chip based method. The new technology, called VLSIPS ™, has enabled the production of extremely poor chips that contain hundreds of thousands or more of different molecular probes. These biological chips, or arrays, have probes arranged in arrays, each probe has been assigned a specific location. Biological chips have been

402406280Β1 produced so that each location has a dimension of, for example, ten microns. Chips can be used to determine if target molecules interact with either probe on the chip. After exposing the matrix to target molecules under selected test conditions, screening devices can examine each location in the matrix and determine if a target molecule interacted with the probe at that location.

Another method of identifying tenascin-C-binding affinity compounds is the yeast two-hybrid system, wherein the polypeptides of the invention can be used to capture tenascin-C-binding proteins. The two yeast hybrid system is described in Fields &amp; Song, Nature 340: 245-246 (1989).

In another example, the agent is a compound having tenascin-C ligand binding capacity.

For example, the agent may be a soluble fragment of a tenascin-C receptor (such as FPRL1). Alternatively, the agent may be a high affinity molecule that mimics an antibody (one called affibody) (for example, see US 5,831,012 and www.affibody.se). These ligands are small, simple proteins composed of a three-helix beam based on the backbone of one of the Protein A IgG binding domains (a surface protein from Staphylococcus aureus bacteria). This backbone has excellent characteristics as an affinity ligand, and can be designed to bind with high affinity to any given target protein.

An agent of the first aspect of the invention is an antibody or antigen binding fragment thereof. The antigen binding fragment may be selected from

402406280Β1 from the group consisting of Fv fragments (e.g. single chain Fv and disulfide linked Fv), Fab type fragments (e.g. Fab fragments, Fab 'fragments and F (ab) 2 fragments), one domain (s). single variable (for example, V domains_H and V_L) and domain antibodies (dAb, including single and double formats [i.e. dAb - binding agent - dAb]).

The antibody may preferably specifically bind to the active FBG domain TLR4.

There are several advantages to using antibody fragments instead of whole antibodies. Smaller fragment size may lead to improved pharmacological properties, such as better penetration of solid tissue. In addition, antigen binding fragments, such as Fab, Fv, ScFv and dAb antibody fragments, can be expressed in, and secreted from, E. coli, thus allowing easy production of large amounts of said fragments.

Also included within the scope of the invention are modified versions of antibodies and antigen binding fragments thereof, for example, modified by covalent bonding of polyethylene glycol or other suitable polymer.

Methods for producing antibodies and antibody fragments are well known in the art. For example, antibodies may be generated by any of several methods employing in vivo induction of antibody molecule production, screening for immunoglobulin libraries (Orlandi et al., 1989. Proc. Natl. Acad. Sci. USA 86: 3833-3837; Winter et al., 1991, Nature 349: 293-299) or the production of

402406280 linhas1 monoclonal antibodies by cultured cell lines. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus hybridoma (EBV) technique (Kohler et al., 1975. Nature 256: 4950497; 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. Biol 62: 109-120).

Suitable monoclonal antibodies for antigen selection can be prepared by known techniques, for example those disclosed in Monoclonal antibodies: A manual of techniques, H Zola (CRC Press, 1988) and Monoclonal hybridoma antibodies: techniques and applications JGR Hurrell (CRC Press , 1982).

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

It will be appreciated by those skilled in the art that humanized antibodies are preferably used for the therapy or diagnosis of humans. Humanized forms of non-human (e.g. murine) antibodies are genetically chimeric antibodies.

Modified ΕΡ2406280Β1, or antibody fragments preferably having minimal portions derived from non-human antibodies. Humanized antibodies include antibodies in which the complementarity determining regions of a human antibody (receptor antibody) are replaced by residues of a region of a nonhuman species (donor antibody), such as mouse, rat or rabbit, having the desired functionality. In some cases, Fv framework residues of the human antibody are replaced by the corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the receptor antibody nor in the imported complementarity-determining region or structure sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all complementarity determining regions correspond to those of a non-human antibody, and all or substantially all regions of structure correspond to those of a relevant human consensus track. Humanized antibodies also optimally include at least a portion of an antibody constant region, such as an 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: 593596).

Methods for the humanization of nonhuman antibodies are well known in the art. Generally, the humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. THE

Humanization can be performed essentially as described (see, for example, Jones et al., 1988, Nature 321: 522-525; Reichmann et al., 1988, Nature 332: 323-327;

Verhoeyen et al. , 1988, Science 239: 1534-15361; US 4,816

567), replacing complementarity determining human regions with corresponding complementarity determining rodent regions. Thus, such humanized antibodies are polymeric antibodies in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies may typically be human antibodies wherein some complementarity-determining region residues, and possibly some framework residues, are replaced by analogs in rodent antibodies.

of waste sites from

Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom &amp; 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, page 77; Boerner et al., 1991, J. Immunol. -95).

When suitable antibodies are obtained they can be tested for activity, for example by ELISA.

An agent of the first aspect of the invention may be an antibody, or antigen-binding fragment thereof, which has specificity for Toll-type receptor 4 (TLR4), Toll-type receptor 4 correctors (in connection with tenascin-4, tenascin-C or a domain of any of these.

402406280Β1

Correctors for primary receptors, such as TLR4, assist with the binding of a signaling molecule to the primary receptor in order to facilitate ligand recognition and binding, and to initiate / maintain the biological process resulting from receptor binding.

An agent of the first aspect of the invention may be an antibody, or antigen-binding fragment thereof, which has specificity for the tenascin-C FBG domain.

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

(i) provide one or more candidate agents;

(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 no candidate agents;

(iv) determine if said candidate agent modulates

<td>the effect of</td><td>tenascin-C in</td><td>an</td><td>or more cells</td><td>at</td>

<td>step (ii)</td><td>in comparison</td><td>with</td><td>the cells)</td><td>in</td>

<td>control of</td><td>step (iii).</td><td></td><td></td><td></td>

<td>The methods</td><td>to determine</td><td>if</td><td colspan="2">the candidate agent</td>

modulating the effect of tenascin-C can be accomplished using the methods of the examples.

This method may result in tenascin-C activity being overregulated.

This method may result in tenascin-C activity being downregulated.

The method may include the cells of steps (ii) and (iii) (described above), which express the Toll-type receptor 4 (TLR4).

The method may have 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.

Inflammatory cells can be selected from the group consisting of macrophages, dendritic cells, monocytes, lymphocytes, monocyte cells and macrophage cells.

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

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

Of particular but not exclusive interest, chronic inflammation is associated with rheumatoid arthritis (RA).

402406280Β1

Also described herein is an agent identified according to the method of the second and third aspects of the invention. Such an agent may modulate the chronic inflammatory response.

agent may under-regulate the chronic inflammatory response.

agent may overregulate the chronic inflammatory response.

The agent can be selected from the group consisting of small interfering RNA (siRNA) molecules, small staple RNA (shRNA) molecules, antisense oligonucleotides, tenascin-C binding affinity compounds, antibodies (polyclonal or monoclonal) and antigen binding fragments thereof, composed of small inhibitors, polypeptides and proteins.

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

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.

402406280Β1

It will be appreciated by those skilled in the art that such effective amount of the agent, or formulation thereof, may be delivered as a single dose (ie acute administration) or more preferably as a series of doses over time ( ie chronic administration).

The agents of the invention may be formulated at various concentrations, depending upon the efficacy / toxicity of the compound to be used, and the indication for which one is being used. Preferably, the formulation comprises the agent of the invention at a concentration of 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 more preferably about 30 μΜ. For in vitro applications, formulations may include a lower concentration of a compound of the invention, for example between 0.0025 μ and 1 μΜ.

It will be appreciated by those skilled in the art that the agents of the invention will generally be administered in admixture with a suitable pharmaceutical diluent, excipient or carrier selected with respect to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19^The 1995 edition, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania; USA).

For example, the agents of the invention may be administered orally, buccally or sublingually in the form of tablets, capsules, ova, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate, delayed or controlled release applications. The agents of the invention may also be administered via intracavernous injection.

402406280Β1

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

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

The agents of the invention may also be administered parenterally, for example intravenously, intraarticularly, intraarterially, intraperitoneally, intrathecally, 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 which may contain other substances, for example, sufficient salts or glucose to make the solution isotonic with blood. Aqueous solutions, if

If necessary, they should be adequately buffered (preferably at a pH of from 3 to 9). Preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the recipient's blood; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit dose or multidose containers, for example ampoules and sealed vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

For oral and parenteral administration to human patients, the daily dosage level of the agents of the invention will normally be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg / kg) administered in single or divided doses.

The agents of the invention may also be administered intranasally or by inhalation, and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer using a suitable propellant, for example the

ΕΡ2406280Β1 dichlorodifluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane heptafluoropropane other appropriate gas, trichlorofluoromethane or hydrofluoroalkane such as (HFA 134A3 or 1,1,1,2,3,3,3 (HFA 227EA3) In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a measured amount. 0 Pressurized container, pump, sprayer or nebulizer may contain a solution or suspension of the active compound, for example using a mixture of ethanol and the propellant as solvent, which may additionally contain a lubricant, for example sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator, may be formulated to contain a powder mixture of a compound of the invention and a suitable base powder such as lactose or starch. .

Aerosol or dry powder formulations are preferably arranged such that each metered or puffed dose contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or more usually in divided doses throughout the day.

Alternatively, the agents of the invention may be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The compounds according to the invention may also be administered transdermally, for example by the use of a skin patch. They can also be administered via the eye.

For ophthalmic use, the agents of the invention may be

402406280Β1 formulated as micronized suspensions in sterile pH adjusted isotonic saline or, preferably, as sterile pH adjusted isotonic saline solutions, optionally in combination with a preservative such as benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petroleum jelly.

Vaseline mixture emulsifying compound

For topical application to the skin, the agents of the invention may be formulated as an appropriate ointment which contains the active compound suspended or dissolved in, for example, one or more of the following: mineral oil, liquid, white vaseline, propylene glycol, polyoxyethylene polyoxypropylene, wax 5 water. Alternatively, they may be formulated as an appropriate lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, ester wax cetyls, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

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 comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

When the agent is a polypeptide, it may be preferable to use a controlled release drug delivery system such as microspheres. These are specifically designed to reduce the frequency of injections. An example of such a system is Nutropin Depot, which encapsulates recombinant human growth hormone (rhGH) in

ΕΡ2406280Β1 Biodegradable microspheres which, once injected, slowly release rhGH over an extended period.

Alternatively, the polypeptide agents of the present invention may be administered by a surgically implanted device, which delivers the drug directly to the returned site.

Electroporation therapy (EPT) systems may also be used for the administration of proteins and polypeptides. A device that provides a pulsed electric field to cells increases the permeability of cell membranes to the drug, resulting in a significant increase in intracellular drug delivery.

Proteins and polypeptides may also be delivered by electroincorporation (EI). IE occurs when small particles up to 30 microns in diameter on the skin surface experience electrical impulses identical or similar to those used in electroporation. In IS, these particles are carried through the stratum corneum and into the deepest layers of the skin. The particles may be charged or coated with drugs or genes, or may simply act as bullets that generate pores in the skin, through which drugs may enter.

An alternative method of protein and polypeptide delivery is the injectable, thermosensitive ReGel. Below body temperature, ReGel is an injectable liquid, while at body temperature it immediately forms a slowly corroding gel reservoir and dissolves in known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.

402406280Β1

Protein and polypeptide drugs may also be delivered orally. Such a system employs a natural process for the oral absorption of vitamin B12 in the body to co-administer proteins and polypeptides. When assembling the vitamin B12 absorption system, the protein or polypeptide may move through the intestinal wall. Complexes are produced between the vitamin B12 analogs and the drug, which retain both significant affinity for intrinsic factor (IF) in the vitamin B12 portion of the complex and significant bioactivity of the drug portion of the complex.

Methods for 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, Drugs Deliv. 8 ( 4): 191-213; Lebedeva et al., 2000, Eur J Pharm Biopharm 50 (1): 101-19; Pierce et al., 2005, Mini Rev Med Chem. 5 (1): 41-55; Lysik &amp; Wu-Pong, 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.

The composition of the second aspect of the invention may comprise at least one further agent.

Such an additional agent may be an anti-inflammatory agent which includes, but is not limited to, a non-steroidal anti-inflammatory agent (NSAID), an antirheumatic disease modifying drug (DMARD), a statin (including HMG-reductase inhibitors). CoA, such as simvastatin), a biological (biological) agent, a steroid, an immunosuppressive agent, a salicylate and / or a microbicidal agent. Non-steroidal agents include antimetabolite agents (such as methotrexate) and gold antiinflammatory agents (including

402406280Β1 sodium, aurothiomalate or gold salts such as auranofin). Biologicals include anti-TNF agents (including adalimumab, etanercept, infliximab, anti-IL1 reagents, anti-IL6 reagents, anti-B-cell reagents (retoximab), anti-T-cell reagents (anti-CD4 antibodies), anti-ILl5 reagents. -CLTA4, antiRAGE reagents), antibodies, soluble receptors, receptor binding proteins, cytokine binding proteins, mutant proteins with altered or attenuated functions, RNAi, polynucleotide aptamers, antisense oligonucleotides or omega 3 fatty acids. Steroids (also known as corticosteroids) include cortisone, prednisone or dexamethasone. Immunosuppressive agents include cyclosporine, FK506, rapamycin, mycophenolic acid. Salicylates include aspirin, sodium salicylate, choline salicylate and magnesium salicylate. Microbicidal agents include guinine and chloroguine. For example, the agent may be administered in combination with one or more of an NSAID, DMARD, or immunosuppressant.

Also as defined hereinbefore described is an agent or composition such as for use as a medicament.

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

In one aspect of the invention there is provided the use of an agent or composition as defined in the first or second aspects of the invention in the manufacture of

ΕΡ2406280Β1 is a medicament for the treatment of a chronic inflammatory condition wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), inflammatory bowel disease, atherosclerosis and / or psoriasis.

Also described herein is a method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an agent or composition as defined herein.

The agent, composition, use or method as defined herein may be related to the treatment of a chronic inflammatory condition, wherein the condition is associated with any condition associated with inadequate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, 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.

Also described herein is a kit of parts for carrying out the methods described herein, comprising:

(i) one or more cells (ii) a control sample of one or more cells (iii) a tenascin-C sample (iv) the instructions for their kit use may optionally comprise:

(v) a candidate agent.

The kit may optionally further comprise (vi) means for determining the effect of a candidate agent on either tenascin-C activity or chronic inflammation.

In a fifth aspect of the invention there is provided a kit of parts comprising:

(i) an agent or composition as defined in the first or second aspects of the invention (ii) the administration means (iii) the instructions for its use of the fifth aspect of the invention may optionally further comprise (iv) at least another agent.

Definitions

By inflammation we include the meaning of local fluid accumulation, plasma proteins, and white blood cells, which is initiated by injury, infection or a local immune response in the tissues.

By acute inflammation we include the meaning of the early stages (initiation) of inflammation and short-term transient inflammatory response immediately following injury, infection or local immune response. Acute inflammation usually resolves quickly, lasting from a matter of minutes to no more than a few days.

By chronic inflammation we include the meaning of persistent and / or unresolved inflammation. It is often

402406280Β1 associated with inadequate destruction of healthy tissue. This can be progressive, and last over a period of weeks or more. Chronic inflammation is typically associated with persistent infection or disease, including, but not limited to, autoimmune conditions.

By chronic joint inflammation we include the meaning of persistent inflammation, which is progressive and constant over a period of weeks to months, resulting in distortion of the affected joint, and radiographic evidence of cartilage and bone destruction as seen in the disease. human (Kelly, Harris, Ruddy and Sledge, Textbook of Rheumatology, 4^The edition).

In experimental murine models, chronic joint inflammation is characterized by non-ceasing inflammation and improper tissue destruction, 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 synovial membrane and joint space, chondrocyte death, and cartilage and bone erosion.

By one agent we include all chemical entities, for example oligonucleotides, polynucleotides, polypeptides, peptide mimetics and small compounds.

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.

By variant we mean that the nucleotide sequence shares at least 90% identity of

Com2406280Β1 sequence with the full length sequence of interest, e.g. at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

The percent sequence identity between two polynucleotides may be determined using suitable computer programs, for example the University of Wisconsin Genetic Computing Group GAP program, and it will be appreciated that the percent identity is calculated relative to the polynucleotides whose sequences have been aligned. optimized way.

Alignment may alternatively be performed using the Clustal W program (as described in Thompson et al., 1994, Nuc. Acid Res. 22: 4673-4680).

The parameters used can be as follows:

Fast pair alignment parameters: Ktuple size (word); 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: open gap penalty; 10, gap extension penalty; 0.05.

Score matrix: BLOSUM.

Alternatively, the BESTFIT program can be used to determine local sequence alignments.

By antibody we include molecules of substantially chimeric, intact, as well as humanized antibodies, humans (wherein at least one amino acid is antibody antibodies mutated in

Ocorrência2406280Β1 to naturally occurring human antibodies), single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, antibody heavy and / or light chain homodimers and heterodimers, and fragments of antigen binding, and derivatives thereof.

By antigen-binding fragment we can say a functional fragment of an antibody that is capable of binding to tenascin-C.

subject term means all animals, including humans. Examples of subjects include humans, cows, dogs, cats, goats, sheep and pigs. The term patient means a subject with a disorder in need of treatment.

As used herein, pharmaceutical formulation means a therapeutically effective formulation according to the invention.

Effective therapeutic amount, or effective amount, or effective therapy, as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined amount of active material calculated to produce a desired therapeutic effect in association with the additive and diluent, i.e. a carrier or delivery vehicle. In addition, it is intended to be of sufficient amount to reduce, and more preferably prevent, a clinically significant deficit in host activity, function and response. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a condition.

ΕΡ2406280Β1 clinically significant in a host. As appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined amount of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for the manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount may be determined by the medical professional or veterinarian based on patient characteristics such as age, weight, gender, condition, complications, other diseases, etc., as is well known in the art.

Examples will now be described which embody an aspect of the invention with reference to the following figures, wherein:

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

(a) Paw swelling in wild type (+ / +) mice (white bars) and tenascin-C (- / -) null mice (black bars) over time after zimosan injection. Data are presented as the mean increase in paw diameter versus paw diameter before injection +/- SEM (n = 24 mice per genotype). ** = p &lt;0.01. (be) Representative sections of ankle joint of wild-type (b, c) and null mice in tenascin-C (d, e), 4 days after zymosan injection, dyed with hemotoxylin and eosin (b, d ) and safranin-0 (c, e). The boxes highlight the joint synovial membrane (s) and the proteoglycans of the

402406280Β1 cartilage (cp). X10 magnification. Quantification of joint inflammation (f) and chondrocyte death (g) in the knee joint 4 days after zimosan injection of wild-type mice (white bars) and null mice in tenascin-C (black bars). Data are expressed as the mean (+/- SD) (n = 24 mice per genotype). * = p &lt;0.05.

Figure 2. Synovial inflammation is induced in tenascin-C deficient mice following antigen injection.

(ab, g) Representative knee joint sections of wild-type mice with a fake injection. (cf, hi) Representative sections of the knee joint of wild type (c, d, h), or tenascin-C (e, f, i) null mice 24 hours after intra-articular injection of mBSA . Infiltration of inflammatory cells into the capsule, meniscus and joint space of both wild type and null mice in tenascinin is highlighted by (cap), (M) and (J), respectively.

(S) highlights healthy synovial membrane of farce-injected mice, which are no longer than 1-3 cells thick along the surface of the entire bone, and (ST) highlights synovial of wild type mice and mice nulls in tenascin-C which are both significantly thicker. The sections are stained with hemotoxylin and eosin (a, c, e, g, h, i), and safranin-0 (b, d, f). Magnification x10 (af) or x40 (gi). (n = 5 mice per genotype).

Figure 3. Synovial inflammation rapidly decreases in tenascin-C deficient mice.

Representative sections of the knee joint of

ΕΡ2406280Β1 wild type mice (a, b, f) or tenascin-C null mice (c, d, e) 3 days after intraarticular injection of mBSA. (a, c) The line highlights increased capsule inflammation in wild type mice compared to null mice in tenascin-C. (b, d) (cp) highlights the increased loss of cartilage proteoglycans in wild type mice compared to null mice in tenascin-C. (e, f) Significant synovial hyperplasia (line), joint and fibrin deposits in the joint space (arrow), and pannus invasion (arrowheads) are observed in wild type mice compared to tenascin-C null mice . The sections are stained with hemotoxylin and eosin (a, c, e, f) and safranin-0 (b, d). Magnification x10 (ad) or x20 (ef). (n = mice per genotype).

Figure 4. Tenascin-C deficient mice are protected from tissue destruction during antigen-induced arthritis.

(ab) Representative knee joint sections of wild-type mice 7 days after intra-articular injection of mBSA, stained with hemotoxylin and eosin (a) and safranin-0 (b). X10 magnification. (n = 24 mice per genotype). The arrowhead highlights the erosion area of ​​the bone. The arrow highlights the invasion of pannus in the cartilage of the joint. (cd) Representative sections of knee joint of null mice in tenascin-C 7 days after intraarticular injection of mBSA, stained with hemotoxylin and eosin (c) and safranin-0 (d). X10 magnification. (n = 24 mice per genotype). J highlights the joint space and AC the intact joint cartilage. (e) Histological score of knee joint inflammation 24 hours, 3 days, and 7 days after mBSA injection of

ΕΡ2406280Β1 wild type mice (white bars) and tenascin-C null mice (black bars). Data represent the mean +/- SD (n = 5 per genotype (24 h, 3 d) or 24 per genotype (7 d)). (f)

Quantification of chondrocyte death, cartilage surface erosion and bone erosion following mBSA injection into knee joints of wild-type mice (white bars) and tenascin-C null mice (black bars). Chondrocyte death is shown at 24 hours, 3 days and 7 days, and cartilage surface erosion and bone erosion at 7 d. Data represent the mean +/- SD (n = 5 per genotype (24 h, 3 d) or 24 per genotype (7 d)).

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

(ab) Human primary macrophages (a) and RA synovial fibroblasts (b) were not stimulated (without addition) or were 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 the mean of triplicate (+/- SD) values ​​from one of three representative experiments. (c) Human primary macrophages were not stimulated (without addition) or were stimulated with LPS (1 ng / ml) or recombinant tenascin-C (1.0 μΜ) for 24 h. (-) indicates that cells were preincubated with medium alone. (P) Cells were preincubated with 25 pg / ml polymyxin B for 30 min before stimulation. (H) Cells were incubated with medium without addition or without containing LPS or tenascin-C which was boiled for 15 minutes prior to addition to cells. The data presented are the average of the values ​​in

Triplicate 622406280Β1 (+/- SD) from one of three representative experiments.

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

(a) Human primary macrophages were not stimulated (without addition) or stimulated with LPS (1 ng / ml), recombinant tenascin-C (TNC) or 1.0 μΜ of tenascin-C domains (TA, EGF -L, TNIII-15, TNIII-3, TNIII3-5, TNIII5-7, TNIII6-8 and FBG) for 24 h. Data shown are the mean of triplicate (+/- SD) values ​​from one of three representative experiments. (b) RA synovial membrane cells were not stimulated (without addition) or were stimulated with LPS (10 ng / ml) or recombinant FBG (1.0 - 0.01 μΜ) for 24 hours. Data presented are mean% change in cytokine levels compared to unstimulated cells (+ / - SEM) from five different patients, (ch) Representative knee joint sections of wild type mice 3 days post injection PBS (ce) or 1 pg FBG (fh). The sections are stained with hemotoxylin and eosin (c, d, f, g) or safranin-0 (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 knee joints of wild type mice 3 days after intraarticular injection of PBS (black bars) or 1 pg of FBG (white bars). Data represent the mean +/- SD (n = 5 per genotype).

402406280Β1

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

(a) Human RA synovial fibroblasts were either uninfected, infected with GFP alone expressing adenovirus (AdGFP) or infected with MyD88 negative dominant adenovirus (AdMyD88dn). Cells were not stimulated, were stimulated with LPS (10 ng / ml) or were stimulated with FBG (1 μΜ) for 24 hours. Data shown are 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 PAM3 (100 ng / ml), LPS ( 100 ng / ml), TNFα (100 ng / ml), IL-1 (5 ng / ml) and FBG (1 μΜ) for 24 hours. Data shown are the average of three independent experiments (+/- SEM).

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

(a) Human primary macrophages were only preincubated with medium or medium containing TLR2 (10 pg / ml) function blocking antibodies, TLR4 (25 pg / ml) or isotype control antibodies (25 pg / ml) for 30 minutes before stimulation. Cells were not stimulated, or were stimulated with LPS (1 ng / ml), FBG (1 μΜ) or PAM3 (10 ng / ml) for 24 h. Data shown are the average of three independent experiments (+/- SEM). (b) Mouse embryonic fibroblasts isolated from wild-type, TLR2 (TLR2 - / -) or TLR4 (TLR4 - / -) deficient mice were not stimulated or were stimulated with PAM3 (100 ng / ml),

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LPS (100 ng / ml), IL-1 (5 ng / ml) and FBG (1 μΜ) for 24 hours. Data shown are the average of three independent experiments (+/- SEM). (c) Bone marrow derived macrophages isolated from wild-type, TLR2 (TLR2 - / -) or TLR4 (TLR4 - / -) deficient mice were not stimulated or were stimulated with PAM3 (100 ng / ml), LPS (100 ng / ml) or FBG (1 μΜ) for 24 hours. Data shown are the average of three independent experiments (+/- SEM). (d) Human macrophages were preincubated without inhibitor with 1 pg / ml msbB LPS or 10 pg / ml anti-CDl4 antibody for 30 minutes prior to stimulation with LPS (1 ng / ml), FBG (1 μΜ) or PAM3 (10 ng / ml) for 24 h. Data shown are the average of three independent experiments (+/- SEM).

Figure 9. Paw swelling over time after zimosan injection.

Representative images of paws of uninjected tenascin-C null mice (a, e) (1.6 mm diameter), 24 h (d, f) tenascin-C null mice (2.5 mm diameter) and 4 d (b, h) (diameter 1.7 mm) after zimosan injection, and wild-type mice 4 d after zimosan injection (c, g) (2.1 mm diameter).

Figure 10. Synthesis of recombinant proteins.

(a) Tenascin-C monomer domain structure comprising different domains, including the mounting domain (TA), 14 and a half EGF-like repeats (EGF-L), 17 fibronectin-like repeats

III (TNIII) (8 constitutively expressed (1-8) and 9 g which may alternatively be excised, and one globe

ΕΡ2406280Β1 fibrinogenic type (FBG). (b) The regions covered by the recombinant proteins which were synthesized, the corresponding amino acid residues and the molecular weight of each protein.

Figure 11. Analysis of protein purity.

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

Figure 12. FBG-mediated inflammation of the joints in vivo requires TLR4 expression.

Representative knee joint sections of null mice in TLR2 (a) and TLR4 (b) 3 days after intra-articular injection of 1 pg FBG. The sections are stained with hemotoxylin and eosin. Magnification x10 (n = 5 mice per genotype). (c) Quantification of joint inflammation, bone erosion, cartilage surface erosion and chondrocyte death in knee joints of null mice in TLR2 (white bars) and TLR4 (black bars) 3 days after 1 pg intra-articular injection of FBG. Data represent the mean +/- SD (n = 5 per genotype).

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

Figure 14. Human tenascin-C nucleotide tracking.

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

TNF synthesis by AR membrane cultures incubated for 24 hours without or with the addition of 100 μΜ of each FBG peptide (Pl, P3-P9).

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

TNF and IL8 synthesis by AR membrane cultures incubated for 24 hours without addition or with the addition of 25, 100 or 250 μΜ FBG peptide.

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

Macrophage IL8 synthesis after 24-hour incubation without addition or with addition of 1 ng / ml LPS, 1 μΜ of the entire FBG domain (FBG) or 1 or 20pM FBG peptides (Pl, P3-P9).

Figure 18. Synthesis of IL8 and TNF in response to LPS and FBG following preincubation with FBG peptides.

TNF and IL8 synthesis by macrophages after 24-hour incubation without addition or with addition of 1 ng / ml LPS or 1 μΜ of the entire FBG domain (FBG), war with or without preincubation with 20pM FBG peptides .

Figure 19. Synthesis of IL8 and TNF in response to tenascin-C target siRNA.

Tenascin-C mRNA levels in luciferase specific siRNA (control) or siRNA transfected fibroblasts

ΕΡ2406280Β1 C-tenascin target: oligo 1 (si 1), oligo 2 (si 2) or a combination of oligos 1 + 2 (si 1 + 2). IL-6 synthesis in luciferase siRNA transfected (control) or a combination of tenascin-C 1 + 2 target oligos (siRNA) in the presence or absence of 10 ng / ml LPS for 24 hours .

Example 1 - General Methods

Reagents

Zimosan, methylated BSA and Freund's complete adjuvant, anti-FLAG M2 antibody (mouse monoclonal antibody), blasticidine, and isotype control antibodies (IgG2a, mouse IgGl) from Sigma-Aldrich (Dorset, UK). Hypnorm was from VetaPharma Ltd. (Leeds, UK). Limulis amaebocyte lysate assay was from Associates of Cape Cod (Liverpool, UK). Wild-type human embryonic kidney cells (HEK293-EBNA) were from Invitrogen (Groningen, the Netherlands). Murine M-CSF and IL-β were from PeproTech (Neuilly-Sur-Seine, France). DMEM medium, RPMI 1640, fetal bovine serum (FBS), penicillin / streptomycin, antimicotic antibiotic PSA solution 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 the TLR2 blogging function (Clone:

TL2.1 Isotype: Mouse IgG2a) TLR4 antibodies (Clone: ​​HTA125 Isotype: Mouse IgG2a) were from Invivogen

Purified Escherichia coli LPS and Pam3Cys-Ser-Lys4 UK). (Calne, UK) with phenol - chloroform (smooth and rough) (Pam3C) were from Alexis (Birmingham TNF-α receptor antagonist and murine IL-1 (IL-lra-IL1 F3) were from R &amp; D Systems (Abingdon, UK) Function blogging anti-CDl4 antibodies (isotype: IgG1 of

ΕΡ2406280Β1 mouse) were from Abcam (Cambridge, UK). Human and murine TNF-α, IL-6 and IL-8 ELISAs were from Pharmingen (Oxford, UK).

Purification of full-length tenascin-C

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

Recombinant protein synthesis

Proteins corresponding to each tenascin-C domain (TA, EGF-L, various TNIII and FBG repeats) were synthesized and purified. See example 2.

LPS contamination measurement in recombinant proteins

To determine the LPS levels in each recombinant protein the Limulus amaebocyte lysate assay was used according to the manufacturer's instructions (sensitivity of ~ 0.7 ± 0.5 pg LPS per mg protein). All recombinant proteins used in this study had LPS levels which were lower than 10 pg / ml.

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Adenovirus vectors and their spread

We constructed the replication-deficient recombinant adenoviral vectors encoding wild type MyD88 (AdMyD88wt), the dominant negative forms of MyD88 (AdMyD88dn) and the GFP control (AdGFP). A description of the synthesis of these viruses is in Andreakos (2004). All viruses used in the present study are deleted E1 / E3, belong to serotype Ad5. Viruses were propagated in human embryonic kidney 293 cells, purified by ultracentrifugation through two cesium chloride gradients, and viral titers were determined by plaque assay as described above (Sacre (2007)).

Animals

Tenascin-C deficient homozygous mice from the original stock described by Saga (1992) at 129 / sv, a pure strain of mice with an agouti and white bellied-looking background were provided by Professor Charles French-Constant (University of Edinburgh , UK). Wild type congenital inbred 129 / sv mice of the same age were obtained from Charles River (Margate, UK). All wild-type tenascin-C and 129 / sv deficient mice were male and were between 8—10 weeks of age at the time of experimentation.

TLR2 and TLR4 deficient homozygous mice on a background of C57BL / 6 (a inbred strain of black-haired mice) were obtained from B&amp;K Universal (Hull, UK) Hoshino (1999) and Takeuchi (1999). MyD88 deficient homozygous mice in a C57BL / 6 background were provided by the Sanger Institute

402406280Β1 (Cambridge, UK). Inbred congenic wild type C57B / L6 mice of the same age were obtained from Charles River (Margate, UK). For isolation of mouse embryo fibroblasts an 8-10 week old female was mated with two 8-10 week old males. For isolation of bone marrow derived macrophages the mice used were females 10-12 weeks old at the time of experimentation.

All animals were fed standard rodent chow and water ad libitum, and were housed (&lt;6 mice / cage) in sawdust-lined cages in an air-conditioned environment with 12-hour light / dark cycles. All animal procedures were approved by the institutional ethics committee.

Statistical methods

Mean, SD, SEM, and statistical tests were calculated using GraphPad version 3 (GraphPad Software Inc., San Diego, CA). The means of multiple groups were analyzed by unilateral variance analysis, followed by Dunnett's multiple comparison test, which was always appropriate. The unpaired t-test was used for experiments involving only two groups.

Example 2 - Synthesis of Recombinant Proteins

Proteins corresponding to each tenascin-C domain (TA, EGF-L, various TNIII and FBG repeats) were synthesized and purified. The synthesized recombinant proteins are shown in Figure 9.

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Reagents

Pfu Turbo polymerase was from Stratagene (Amsterdam, the Netherlands). The easy mix 50 PCR tubes were from Molecular Bioproducts (Lutterworth, UK). RNeasy Kits and Ni Speakers²⁺-NTA-agarose were from Qiagen (Crawley, UK). The pCR blunt vector, the pCEP4 plasmid vector, the human embryonic kidney cells (HEK293-EBNA) and 4-12% Bis-Tris gradient gels were from Invitrogen (Groningen, The Netherlands). The pET32b vector and Rosetta BL21 (DE3) cells were from Novagen (Kent, UK). HiTrap Q columns, HiTrap S columns, Sephacryl S500 HR column and sepharose heparin columns were from Amersham (Buckinghamshire, United Kingdom).

Restriction enzymes were obtained from New England Biolabs (Hitchin, United Kingdom). DMEM medium, fetal bovine serum (FBS) and penicillin / streptomycin were from PAA laboratories (Yeovil, UK). The transfection reagent FuGENE6 was from Roche Applied Science (Basel, Switzerland).

Anti-Flag M2 antibody (mouse monoclonal antibody), anti-FLAG M2-agarose, FLAG peptide were from Sigma-Aldrich (Dorset, United Kingdom). The antitetrahis antibody (mouse monoclonal antibody) was from Qiagen (Crawley, United Kingdom). Alkaline phosphatase-conjugated goat anti-mouse IgG and Western substrate

Blue stabilized for alkaline phosphatase were from Promega (Southampton, UK). Protein accuracy standards for SDS-PAGE were from BioRad (Hemel Hempstead,

UK).

Primer oligonucleotide design

Domain boundaries were determined using

402406280Β1 alignments published following human tenascin-C sequence (Siri (1991), accession number P24821 (Swiss-Prot)). To clone each domain we designed PCR primer oligonucleotides where both forward and reverse primer oligonucleotides contained 18 - 21 bases corresponding to the 5 'and 3' terminal sequences of the required coding sequence. 0 The forward primer oligonucleotide contained an Ndel restriction site, followed by an N-terminal his tag just before the coding sequence. The final three bases of the Ndel site form the ATC methionine initiation code. The reverse primer oligonucleotide included a TTA stop codon immediately after the coding sequence, followed by a BamH1 or Kpn1 site to allow unidirectional cloning into pET32b expression vectors.

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<td></td><td>Table 1</td>

<td>Name of</td><td>Sense primer oligonucleotide</td>

<td>protein</td><td>Antisense oligonucleotide primer</td>

<td>OK</td><td>FW; ATACA WrGCATCATCATCATCATCATGGGGTCCTCAAÔ AAAGTCATCCGG RV: GCCGGATCCTTAGCCTGGTCGTGGAGTACATTG</td>

<td>EGF-L</td><td>PCR1 FW: ACAGTGGTACCACCATGGGGGCGATGGGGGCCATGACT CAGCTGTTG RV: CTTGTCATÇGTCGTCCTTGTAGTCACCTTCGGTAGCGAG GGGAAG PCR2 FW; GACTAGAAGGACGACGATGACAAGTGCTGTCTCCAGCC TGCCAC RV: GAGAGCGGATCCTTAATGATGATGATGATGATGTGAGCA GTCTTCTCCGCTGTAGC</td>

<td>TN1-5</td><td>FW: ATACATATGCATCATCATCATCATCATGAGGTGTCTCCTCC CÁAAGA RV: GCCGG MCCTTAAGTGGATGCCTTCACACGTGC</td>

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

<td>TN3-5</td><td>FW: ATTACK rGCÁTCAT.CATGATCATGATCÔCTTGGATGCC CCCAGCCAGAT RV: GCCGGTACCTTAAGTGGATGCCTTCACACGTGC</td>

<td>TN5-7</td><td>FW: ATACATATGCATCATCATCATCATCArGAGTTGGACACG CCCAAGGAC RV: GCCGGATCCTTATGTTGTGAACTTGGCAGTGATGGTTG</td>

<td>TN6-8</td><td>FW: ATACATA TGCATCATCATCATCATCATGCCATGGCCTCCCC AAAGGAA RV: GCCGGA TCCTTATGTGGTGAAGATGGTCTGGATCAT</td>

<td>FBG</td><td>FW: ATTACK TA TCCATCATCATCATCATCATATTGÔACTCCTGTAC CCCTTCC RV: GCCGGA TCCTTATGCCCGTTTGCGCCTGCCT TCAA</td>

All of the above primer oligonucleotides are written from 5 'to 3'. Flag strings are bolded, his (CATCATCATCATCATCAT) markers are underlined, and restriction enzyme cleavage sites (CATATG = Ndel site, GGATCC = BamHl site, GGTACC = Kpnl site) are bold and italic.

PCR

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PCR amplification was performed using 10 pmol / μΐ of each primer oligonucleotide, 1 pg template, 5 μΐ DMSO and 1.25 Pfu Turbo polymerase units, in a final volume of 25 μΐ. This was added to the buffer and dNTP in easy mix 50 tubes. The template used for all reactions was cDNA prepared from U87MG human glioma cells using RNA isolated with RNeasy kits. The reaction was repeated 40 times with denaturation, annealing and elongation temperatures of 95 ° C, 55 - ° C (depending on the fusion temperature (Tm) of the primer oligonucleotides) and 72 ° C, respectively.

Cloning

PCR products were ligated into pCR Blunt vectors and sequenced to ensure no errors were introduced by PCR. Clones that had no errors or silent mutations were selected. The inserts were then ligated into pET32b using Ndel and BamH1 restriction sites designed on primer oligonucleotides (TN5-7 and TN6-8). Human tenascin-C has BamH1 internal sites within the TA (position 494) and TNIII2 (position 2509) domain. TA and TNl-8 were therefore cloned using the Ndel site at the forward primer and the Kpnl site at the pCR Blunt cloning site. Human tenascin-C does not contain internal KpnI sites. TNl-5, TNl-3 using Ndel sites

Kpnl initiators. FBG contains one site and TN3-5 were cloned into Ndel internal oligonucleotides (position

6439) and therefore was cloned using a two-step ligation of Ndel and BamH1 digestion followed by Ndel digestion. (Positions refer to the locations within the full length nucleotide sequence of tenascin-C, shown in Figure 14)

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Bacterial growth, induction and lysis

The plasmids were transformed into Rosetta BL21 (DE3) cells, cultured in 3 Luria-Bertani medium containing 50 pg / ml carbenicillin, and induced with 1 mM isopropyl-β-D-thiogalactopyranoside. After 3 hours, cells were harvested by centrifugation at 4000 rpm for 20 min, washed twice with ice cold wash buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM EDTA), and Used with a french press. Inclusion bodies were obtained by centrifugation at 12,000 rpm for 20 min at 4 ° C. Except for TA and FBG, the proteins were entirely localized in the supernatant. Recombinant TA and FBG proteins were extracted from inclusion bodies with 6 M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.0, and 10 mM β-mercaptoethanol at room temperature with constant stirring for 2 hours. .

Bacterial Protein Purification

The solution containing the recombinant protein was applied to a Ni column.²⁺-NTA-agarose and washed with 50 mM Tris-HCl, pH 8.0, containing 20 mM imidazole. The column was subsequently washed with 50 mM Tris-HCl pH 8.0 and the protein was 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, TA and FBG chromatography, no further purification was required. TNl-3 and TN6-8 were further purified by anion exchange chromatography using a HiTrap Q column, TNl-5, TN3-5 and TN5-7 by cation exchange chromatography using a HiTrap S column, and TNl-8 using a column. HiTrap S followed by gel filtration using a column

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Sephacryl S500 HR.

Refolding of insoluble proteins

RT and FBG were refolded by diluting to 20 pg / ml with 50 mM Tris-HCl, pH 8.0 containing 6 M guanidine hydrochloride and then treating with 20 mM cystamine with stirring for 16 hours at 4 ° C. ° C. The solution was then

<td>dialyzed twice</td><td>against</td><td> 15</td><td>50 mM volumes</td><td>in</td><td>Tris-</td>

<td>HCl, pH 8.0 containing</td><td>150 mM</td><td>in</td><td colspan="2">NaCl, 10 mM CaCR</td><td>0.5 mM</td>

<td>β-mercaptoethanol</td><td>and 1</td><td>mM</td><td colspan="2">disulfide</td><td>of 2-</td>

<td>hydroxyethyl for</td><td colspan="2">24 hours at</td><td>4 ° C, twice</td><td colspan="2">against 20</td>

<td>mM Tris-HCl, pH 8</td><td colspan="2">, 0 during</td><td>8 hours at 4 ° C,</td><td>and</td><td>after</td>

<td colspan="2">centrifuged at 12,000 rpm</td><td colspan="2">for 30 min at</td><td> 4</td><td>° C. THE</td>

Refolding was assessed by size changes using SDSPAGE under reducing and non-reducing conditions. Protein activity was confirmed by TA domain polymerization and FBG binding to heparin sepharose columns.

EGF-L domain synthesis using mammalian cells

Initial attempts to express and purify the EGF-L repeat region using an E. coli expression system were unsuccessful. Most likely this is attributable to the difficulty in obtaining protein folding due to a total of 91 cysteine ​​residues in this region. Therefore, TN-C EGF-like domains were expressed using HEK293 cells.

Two PCR reactions were performed. The first PCR product consisted of a KpnI restriction enzyme site, a Kozak sequence followed by the TN-C signal sequence. The second PCR product consisted of a FLAG peptide, the EGF-like domain followed by a

402406280Β1 histidine marker and a restriction enzyme sequence BamHl.

The two PCR products were ligated together as described by Ho (1989). PCR reactions were performed as described above. The entire construct was cloned into the blunt PCR vector and sequenced. It was then subcloned into the pCEP4 vector. 0 DNA was transfected into HEK293 cells using Fugene, and cells were selected for hygromycin resistance (200 pg / 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 (collected after cells were cultured in medium) were collected and combined from stably transfected cells. Pooled conditioned medium (2 liters) was centrifuged at 3000 rpm to separate cellular debris from the medium.

The medium was then applied to an anti-FLAG column. The material was collected in 50 ml fractions for flow. The column was washed with 10 column volumes of 1 M NaCl, 50 mM Tris-HCl, pH 7.5 and then washed with 10 column volumes of 60% isopropanol to ensure LPS removal. The column was then washed with a 50 mM Tris-HCl buffer, pH 7.5, and finally the protein was eluted using 200 pg / ml FLAG peptide in 50 mM Tris-HCl buffer, pH 7.5.

Protein Purity Analysis

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 do this 1 pg of each recombinant protein was run

402406280Β1 purified on a 4-12% Bis-Tris gradient gel, and the gel was subsequently silver stained to demonstrate a single band (Figure 10). Western blotting analyzes were also performed. Proteins separated by SDS-PAGE were electrotransferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA in Tris-buffered saline and then incubated with primary antibodies recognizing FLAG M2 (1: 2000 dilution) (EGF-L) or tetra-his antibodies (1: 2000) ( all other proteins). The membrane was then incubated with alkaline phosphatase-conjugated secondary antibody, and protein bands were visualized using Western Blue stabilized substrate where the gels show a specific single band recognized by each of the antibodies at the expected PM (not shown).

Example 3 - Animal Models

Zimosan-induced arthritis

Zymosan-induced arthritis (ZIA) was induced in tenascin-C-deficient and wild-type mice by zymosan injection (Saccharomyces cerevisiae) as described in Keystone (1977). Zimosan was prepared by dissolving 15 mg zimosan in 1 ml sterile PBS. The solution was boiled twice and sonicated. Mice were anesthetized by intraperitoneal injection of 150 μΐ Hypnorm diluted 1:10 in sterile water, then injected with zimosan (10 μΐ) into the right paw pad (d = 0).

Control mice received a 10 μΐ injection of PBS alone or were not injected. For macroscopic evaluation of arthritis, the thickness of each hind paw was measured daily with microcalipers (Kroeplin,

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Schluchlem, Germany), and the diameter was expressed as an average for each rat-inflamed hind paw.

Upon completion of the experiment (day = 4), mice were sacrificed and their hind paws fixed on 10% (v / v) buffered formalin, decalcified with 10% EDTA and processed for paraffin.

Antigen-induced Arthritis

Antigen-induced arthritis (AIA) was induced in wild-type and tenascin-C deficient mice as previously described by Brackertz (1977). Briefly, on day 0 mice were anesthetized by intraperitoneal injection of 150 μΐ of Hypnorm diluted 1:10 in sterile water, then immunized with 200 pg of methylated BSA. The mBSA was emulsified in 0.2 ml Freund's complete adjuvant and injected intradermally at the base of the tail.

On day 7, arthritis was induced by intraarticular injection of mBSA (100 µg in 10 µl sterile PBS) into the right knee joint using a 33 gauge sterile microcannula. Control mice received either a 10 µl injection of PBS alone or have not been injected.

On day 14, the mice were sacrificed, the knee joints were excised and fixed in 10% (volume / volume) buffered formalin, decalcified with 10% EDTA, and processed for paraffin.

FBG Injection

Wild type mice were anesthetized by intraperitoneal injection of 150 µl of 1:10 diluted Hypnorm

402406280Β1 in sterile water, and then injected with 100 ng, 1 or 3 pg FBG in 10 pi sterile PBS, into the right knee joint using a 33 gauge sterile microcannula. Control mice received a 10 pi injection of PBS. alone or not injected.

On days 3 and 7, mice were sacrificed, knee joints were excised and fixed in 10% (volume / volume) buffered formalin, decalcified with 10% EDTA and processed for paraffin.

Knee Joints Histology

Coronal tissue sections (4 pm) were cut at 7 depths along the joint; 80 pm apart and stained with hematoxylin and eosin or safranin0 to assess joint pathology. Histopathological changes were recorded with the following parameters as described in Van Lent (2006).

Inflammation (the influx of inflammatory cells in the synovial membrane (infiltrate) and joint cavities (exudate) was rated according to an arbitrary scale from 0 (no inflammation) to 3 (severe inflammation). Chondrocyte death was determined as the percentage of cartilage area that contains empty gaps in relation to the total area. Erosion of the cartilage surface was determined as the amount of cartilage lost relative to the total cartilage area. Bone destruction was determined in 10 different areas of the total knee joint section. Destruction was graded on a scale from 0 (no damage) to 3 (complete loss of bone structure). Histological analysis was performed by an investigator who was unaware of the experimental groups. The average score for each animal in a group

Experimental ΕΡ2406280Β1 was calculated by averaging histopathological scores in at least 5 section depths per joint.

Results

Zymosan-induced joint inflammation is not sustained in tenascin-C deficient mice

Zimosan injection into the paw pad was used to induce acute synovitis in mice. Wild-type mice exhibited rapid paw swelling reaching a maximum paw diameter by 24 hours (2.56 mm, a 62% increase in initial paw diameter). This was maintained for a further 24 hours. After 2 days the paw diameter decreased, but the paws remained swollen for 4 days (2.08 mm, a 32% increase) (figure 1a). Tenascin-C-deficient mice exhibited a similar degree of paw swelling as tyrosine mice. wild type 24 hours after injection (2.41 mm, a 57% increase in paw initial diameter). However, swelling in tenascin-C null mice decreases faster than in wild-type mice; paw diameter was significantly reduced at 2 days and fell to 1.7 mm (an increase of only 11%) at 4 days (figure 1a). On day 4 post-injection the wild-type mouse paws were still visibly swollen and red, while the tenascin-C null mouse paws were not visibly swollen or red and resembled uninjected paws (Figure 9). .

This difference was reflected histologically at 4 days. The synovium of wild-type mice was significantly inflamed and exhibited cellular infiltration and cartilage proteoglycan loss was observed.

402406280Β1 (figure lb, c). In contrast, the synovial membrane of tenascin-C-deficient mice exhibited no synovitis, cellular infiltration or loss of cartilage proteoglycan (Figure 1d, e), and resembled the joints of sham-injected or uninjected mice. shown). Guantification of joint inflammation revealed that while there was little exudate (cell mass in the joint cavity) in wild-type or null mice in tenascin-C, infiltrate levels (synovial layer cell mass) were significantly reduced in null mice in tenascin-C (Figure 1f). No cartilage or bone erosion occurred in mice of both genotypes (not shown), however, a low level of chondrocyte death occurred in wild type mice, which was not observed in null mice in tenascin-C (Figure 1g). ). Thus, tenascin-C expression seems to favor the maintenance of acute inflammation.

Tenascin-C null mice are protected against persistent inflammation and structural damage during antigen-induced arthritis.

To determine if tenascin-C also contributes to the most destructive inflammatory joint disease, erosive arthritis was induced by intraarticular injection of mBSA into the knee joint following immunization with mBSA. This model involves both humoral and cellular immune responses and induces pathological changes similar to human RA (Brackertz (1977)). Injection of mBSA induced a similar inflammatory response in both tenascin-C and wild type null mice. Cell infiltration and synovial thickening is apparent at 24 hours in mice of both genotypes (Figure 2c-f, h, i) compared to sham-injected (Figure 2a, b, g) or non-injected mice (Figure 2a-b, g). not shown).

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However, this does not persist in tenascin-C null mice as it does in wild type mice. At 3 days after injection, wild-type mice exhibit increased inflammation of the meniscus and capsule, synovial hyperplasia, articular cell and fibrin deposits, pannus formation, and localized loss of cartilage proteoglycans (Figure 3a, b, f ). In contrast, at 3 days in tenascin-C null mice inflammation is limited to the capsule, synovial inflammation has decreased and there are no fibrin aggregates / cells present in the joint space, no pannus formation and no loss of proteoglycan. cartilage (Figure 3c, d, e).

At 7 days, wild-type mice exhibited persistent inflammatory cell infiltration and joint space exudate, extensive synovitis and pannus formation, and joint cartilage destruction and bone erosion (Figure 4a, b). The farce-injected knees and the knees of mice that had undergone any injections were healthy and exhibited no joint inflammation or destruction (not shown). Tenascin-C-deficient mice also had healthy joints which had only mild inflammatory cell infiltration, no joint space exudate, synovitis, pannus formation, joint cartilage destruction, or bone erosion (Figure 4c, d). The joints of tenascin-C-deficient mice that had undergone farce injection or had no injections were also healthy (not shown).

These histological data are reflected after joint disease scoring as described in the materials and methods. The levels of cellular infiltrate and exudate observed in both wild type and

ΕΡ2406280Β1 in tenascin-C null mice 24 hours after injection were not significantly different. However, although cell mass continued to increase in wild-type mice over time, this response was attenuated in tenascin-C-null mice and cell numbers in the joint decreased over time (Figure 4e). Higher levels of chondrocyte death occurred in cartilage of wild-type mice over time, but no significant death was observed in null mice in tenascin-C (Figure 4f). Cartilage surface erosion and bone erosion were not evident in 24-hour or 3-day wild type mice (not shown), but significant tissue destruction had occurred by 7 days. In contrast, tenascin-C null mice exhibited no tissue destruction at 24 hours, 3 days (not shown) or 7 days (Figure 4f). These data indicate that although the onset of joint inflammation (influx of cells to the synovium and joint space) is unaffected in null mice in tenascinina, unlike wild-type mice the disease does not progress to tissue destruction and cell death. These results demonstrate that tenascin-C expression is required for persistent synovial inflammation and joint destruction in this model.

Example 4 - Cell Culture

Patient Samples

Human monocytes were isolated from peripheral blood (Blood Bank of London) and macrophages were obtained from monocytes after differentiation for 4 days with 100 ng / ml M-CSF as previously described (Foxwell (1998). )).

402406280Β1

RA membrane cells (representing a mixed population of all synovial cell types) were isolated from synovial membranes obtained from patients undergoing joint replacement surgery as previously described (Brennan (1989)). . RA synovial fibroblasts were isolated from the mixed population of RA membrane cells as described previously (Brennan (1989)). 0 The study was approved by the local fund ethics committee (Riverside NHS Research Committee), and waste tissue (synovium after joint replacement surgery) was obtained only after receiving informed consent from the patient and providing anonymity to the tissue to protect it. the identity of the patient.

Immediately after isolation, AR membrane cells and macrophages were cultured at 1x10⁵cells / well in RPMI 1640 medium containing 10% (v / v) FBS and 100 U / ml (units / ml) penicillin / streptomycin in 96 well tissue culture plates for 24 hours prior to stimulation. Synovial fibroblasts (used only in each passage number 2 or 3) were grown at 1x10⁴ cells / well in DMEM medium containing 10% (v / v) FBS and 100 U / ml penicillin / streptomycin in 96-well tissue culture plates for 24 hours prior to challenge.

Mouse embryonic fibroblasts (MEF) and bone marrow derived macrophages (BMDM)

MEFs express high levels of mRNA from all 9 murine TLRs and are specifically and highly responsive to TLR ligand activation. MEF from TLR2, TLR4 and MyD88 target deletion mice demonstrate profound defects in their IL-6 response to specific ligands (Kurt-Jones

(240408080) (2004)). FEMs were isolated from dl3 embryos collected from wild-age, matched, pregnant female TLR2, TLR4 and null mice (as described in Todaro (1963)). Fibroblasts were grown at 2x10⁴cells / well in DMEM medium containing 10% (v / v) FBS and

100 U / ml penicillin / streptomycin in 96-well tissue culture plates for 24 hours prior to stimulation.

BMDM were derived by aspiration of femurs from wild-age matched females, TLR2 and TLR4 null mice as described in Butler (1999)), and cell culture for 7 days in DMEM medium, 20% (v / v) FBS, 10 ml / 1 (v / v) antibiotic - antimycotic PSA solution, 50 μΜ β-mercaptoethanol and 10 ng / ml MCSF. The macrophages were then grown at 1x10⁵cells / well in DMEM medium, 20% (v / v) FBS, 10 ml / 1 (v / v) antibiotic - antimycotic PSA solution, 50 μΜ βmercaptoethanol in 96 well tissue culture dishes for 24 hours before the stimulus.

HEK293 Cell Lines

HEK293 cell lines expressing TLR2 and TLR4 / CD14 / MD-2 were grown at 1x10⁴ cells / well in DMEM medium containing 10% (v / v) FBS and 10 pg / ml blasticidine in 96-well tissue culture plates for 24 hours prior to stimulation.

Cell stimulation and cytokine synthesis evaluation

Cells were incubated for 24 hours at 37 ° C with the indicated doses of tenascin-C and recombinant tenascin-C fragments (1.0 μΜ - 1.0 nM). Cells were also stimulated where indicated with LPS (1 ng / ml

ΕΡ2406280Β1 for human macrophages, 10 ng / ml for human fibroblasts, AR and HEK membrane cells, 100 ng / ml for MEFS and BMDM and 10 ng / ml for HEKS), PAM3 (10 ng / ml for human macrophages, human fibroblasts and HEK, 100 ng / ml for MEF and BMDM), murine IL-1 (5 ng / ml for MEFS) and murine TNFα (100 ng / ml for MEFS). Unless specifically stated otherwise rough LPS was used for in vitro studies.

For adenoviral gene transfer experiments, human synovial RA fibroblasts were incubated with adenoviral vectors, at a multiplicity of infection, washed after 2 hours, cultured in complete culture medium for 24 hours, then stimulated for 24 hours. hours after this time the supernatants were collected.

Where indicated, cells were preincubated with 10 pg / ml anti-CD14 antibody, 10 pg / ml IL-1 receptor antagonist, 10 pg / ml anti-TLR2 antibody, 25 pg / ml anti-CDL4 antibody. -TLR4, 10 or 25 pg / ml isotype control antibody, 25 pg / ml polymyxin B, or 1 pg / ml msbB LPS, for 30 minutes at 37 ° C prior to stimulation. Where indicated, recombinant tenascin-C and FBG, and LPS were boiled for 15 minutes prior to addition of cells.

In all cases, cell viability was not significantly affected during the experimental time period when examined by the MTT cell viability assay (Sigma, Poole, UK).

Supernatants were further analyzed for the presence of TNF-α, IL-6 and IL-8 cytokines by enzyme linked enzyme linked immunosorbent assay (ELISA) according to

ΕΡ2406280Β1 manufacturer's instructions. Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, United Kingdom).

Results

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

We then investigated whether tenascin-C could activate the innate immune response. Tenascin-C was used to stimulate primary human macrophages and RA synovial fibroblasts, and the production of proinflammatory cytokines TNF-α, IL-6 and IL-8 was examined. The bacterial cell wall LPS component was used as a positive control. Tenascin-C induced a cell type-specific cytokine profile, which was significantly different from LPS. Dose-dependent stimulation of TNF-α, IL-6 and IL-8 production in human macrophages (Figure 5a). However, tenascin-C only induced IL-6 synthesis in synovial fibroblasts, whereas LPS induced both IL-6 and IL-8 (Figure 5b). Neither LPS nor tenascin-C induced synthesis of TNF-α in fibroblasts (data not shown). The stimulation of IL-6 by tenascin-C (Figure 5c), IL-8 and TNF-α by human macrophages and IL-6 by synovial fibroblasts (not shown) was heat sensitive and unaffected by the LPS inhibitor polymyxin. B. Taken together, these results provide strong evidence that tenascin-C cytokine induction is not due to LPS contamination.

fibrinogen-like globe (FBG) mediates cell activation by tenascin-C.

402406280Β1

Tenascin-C is a large hexameric molecule, each domain binding to different cell surface receptors (reviewed in Orend (2005)). Understanding the mechanism of action of tenascin-C will require the identification of which domain (s) are critical for promoting cytokine production. We synthesized recombinant proteins comprising different domains of the molecule (Figure 10). Each domain was performed on E. coli, purified (Figure 11), and found to contain &lt;10 pg / ml LPS by subjecting the pure protein to the Limulus amaebocyte lysate assay. Only one domain of tenascin-C was active. Fibrinogen-like globe (FBG) stimulated TNF-α synthesis in human macrophages (Figure 6a), IL-6 and IL-8 synthesis in human macrophages (not shown) and IL-6 in RA synovial fibroblasts (not shown) to an extent equal to full-length tenascin-C. Like full-length tenascin-C, FBG did not induce IL-8 synthesis in RA synovial fibroblasts where LPS did (data not shown). FBG-induced cytokine synthesis was also heat sensitive and unaffected by polymyxin B (data not shown).

The FBG domain of tenascin-C induces cytokine production in human RA synovium and joint inflammation in mice.

We investigated whether FBG could promote the expression of inflammatory cytokines in the synovial membranes of RA patients. This AR tissue model (which comprises a mixed population of all synovial cell types) spontaneously produces elevated 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 if FBG could induce inflammation in vivo, wild type mice were injected intra72.

402406280Β1 jointly with FBG. Transient and dose-dependent stimulation of joint inflammation was observed. No inflammation or loss of proteoglycan occurred in uninjected mice or mice injected with PBS (Figure 6c-e) or 100 ng FBG (data not shown). In mice injected with 1 pg FBG inflammatory cell infiltration (Figure 6f), mild synovitis, pannus formation (Figure 6g) proteoglycans (Figure 6h) were observed. Loss and similar response was observed in mice injected with 3 pg FBG (data not shown) observed exudate and

Following histological quantification levels, cellular infiltrate and chondrocyte death were increased in FBG-injected mice, along with a modest amount of cartilage surface erosion and bone damage (Figure 6i).

FBG-mediated cytokine synthesis is MyD88 dependent

Many DAMP, including fibrinogen (Smiley (2001)), have been shown to stimulate the innate immune response by TLR activation. Therefore, we investigated whether TLRs could also mediate tenascin-C-induced cytokine production. Myeloid differentiation factor 88 (MyD88) is required for signaling all TLRs except TLR3 (O'Neill (2008)). Infection of synovial fibroblasts with adenovirus expressing dominant negative MyD88, but not GFP virus control, abolished FBG-induced IL-6 (Figure 7a). These data suggest that FBG-induced inflammation is dependent on functional MyD88. This effect of FBG did not appear to be mediated by IL-1 since the addition of IL-1 receptor antagonist did not inhibit cytokine induction (data not shown). To confirm that FBG action is dependent on MyD88 we demonstrated that FBG does not stimulate cytokine synthesis in fibroblasts.

Embryonic ΕΡ2406280Β1 isolated from mice with targeted deletions in the MyD88 gene. TLR2 ligand, PAM3, TLR4 ligand, LPS, and IL-1 all signal via MyD88. Stimulation with these was also abolished in MEF from deficient mice. However, TNF-α, which does not signal via MyD88, was not affected (Figure 7b). A new 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

TLRs exhibit specificity for endogenous ligands; proteins are recognized by one or both of TLR2 and 4 (reviewed in O'Neill (2008)). Neutralizing antibodies to TLR 4 inhibited 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 function of ligand TLR2, PAM3. Antibodies to TLR2 inhibited PAM3-mediated cytokine synthesis, but had no effect on LPS or FBG-induced cytokine synthesis. Corresponding isotype controls had no effect on ligand-induced cytokine synthesis (synthesis of TNF-α by human macrophages is shown in Figure 8a). To confirm that FBG action is TLR4 dependent we have demonstrated that FBG does not stimulate cytokine synthesis in embryonic fibroblasts or macrophages isolated from mice with target deletions in the TLR4 gene. FBG-mediated cytokine synthesis was not affected in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR2 gene. Cells isolated from TLR2-deficient mice were insensitive to PAM3, but responsive to LPS and IL-1. Cells isolated from TLR4 deficient mice were insensitive to LPS, but responded to PAM3 and IL-1 (Figure 8b, c). Furthermore,

402406280Β1 TLR4 expression was required for arthritogenic action of FBG in vivo; FBG was able to induce joint inflammation in TLR2 null mice, but not in TLR4 null mice (Figure 12).

Different Broker Requirements for FBG and LPS

LPS signaling via TLR4 is mediated by a receptor complex including soluble GPI-bound or soluble cell surface protein MD-2 and CD14 (reviewed in Fitzgerald (2004)). Next, we examined whether CD14 and MD-2 are required for FBG activation of TLR4. As an acute positive control we examined the activity of glycosylated smooth LPS that regenerate both MD-2 and CD14 (Jiang (2005)). LPS mediated the synthesis of IL-6, IL-8 and TNF-α by human macrophages, and IL-6 synthesis by RA synovial fibroblasts was inhibited by anti-CD14 antibodies and an E mutant msbB LPS-derived antagonist. coli, which competes for linking LPS to MD-2 (Coats (2007)). On the other hand, both PAM3, which does not require these correctors for TLR2 activation, and FBG-mediated cytokine synthesis was affected by mutant anti-CDl4 or LPS msbB antibodies (Figure 8d shows macrophage synthesis of TNF-α humans) . These data suggest that neither CD14 nor MD-2 is required for FBG-mediated cytokine synthesis. Therefore, while LPS and FBG both signal by activating TLR4, they may have different corrector requirements.

Example 5 - Inhibition of tenascin-C action and synthesis in human tissues

This example studies the effect of (1) preventing the proinflammatory action of tenascin-C and (2) inhibiting expression of tenascin-C in the human RA synovial membrane.

402406280Β1

Methods

Peptide Synthesis

Nine overlapping peptides comprising the entire FBG domain (Table 2) were synthesized by Biogenes, Germany. The peptides were cleaved at room temperature (cleavage mixture: 90% trifluoroacetate, 5% thioanisol, 3% ethanedithiol, 2% anisole), were purified by reverse phase high performance liquid chromatography and were characterized. by MALDI TOF mass spectrum analysis. Purity of the peptides was&gt; 85% as determined by high performance liquid chromatography.

The facility was unable to synthesize peptide 7, possibly due to the formation of secondary structures that prevented elongation of the peptide chain (as previously reported (LaFleur (1997)).

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

<td>Peptide no.</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>

402406280Β1

Patient samples and cell culture

RA membrane cells (representing a mixed population of all synovial cell types) were isolated from synovial membranes obtained from patients undergoing joint replacement surgery (Brennan (1989)). Synovial membrane tissue was digested in RPMI 1640 medium (GIBCO) containing 5% fetal calf serum (FCS) (GIBCO), 5 mg / ml type IV collagenase (Sigma) and 0 15 mg / ml DNAse from type I (Sigma), and was incubated at 37 ° C for 2 h.

After incubation, the tissue was pipetted through a nylon mesh into a sterile beaker. The cells were then washed three times in complete medium (RPMI 1640 supplemented with 10% FCS). Synovial RA fibroblasts were isolated from the mixed AR membrane cell population by selection in DMEM (BioWhittaker) medium 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 were obtained from monocytes after differentiation for 4 days with 100 ng / ml M-CSF.

The study was approved by the local fund ethics committee, and waste tissue (synovium after joint replacement surgery) was obtained only after receiving informed consent signed by the patient and providing tissue anonymity to protect the patient's identity.

Cell stimulation and cytokine synthesis evaluation

Immediately after isolation, the cells of the

ΕΡ2406280Β1 AR membrane were cultured at lx10⁵ cells / well in RPMI 1640 medium 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 addition of control buffer (PBS, 1% BSA, 0.01% NaN₃), or at 25 pm, 100 μΜ or 250 μΜ of each peptide comprising FBG.

Synovial fibroblasts (used only in each passage number 2 or 3) were plated at a concentration of 5x10⁴ cells in 3.5 cm plates. SiRNA was transfected to a final concentration of 10 nM using

Lipofectamine 2000 OptiMEM I (Invitrogen) free for 4 hours in serum medium. Two different siRNAs were used against human tenascin-C (s7069 and s229491) (Applied Biosystems).

CGCGAGAACUUCUACCAAAtt's Followings

UUUGGUAGAAGUUCUCGCGtc GGAAUAUGAAUAAAGAAGA11 UCUUCUUUAUUCAUAUUCCgg siARN's

3',

') and

3',

3 '). SiARN vs. s7069 are: (s229491 anti-sense 5 is anti-sense '

(5' '

luciferase (Dharmacon) was transfected as a non-target control.

Four hours after transfection, the medium was changed with Dulbecco's pre-equilibrated and cells were incubated h

The cells were then ° C. The were Eagle's modified medium containing 10% FBS (v / v) for an additional 48 h and 72 stimulated with 10 ng / ml LPS for 24 hours at 37 tenascin-C mRNA levels and PCR-guarantied protein and Western blotting, respectively. Total RNA was extracted from cells using a QiaAmp RNA Blood mini kit (Qiagen, Germany). CDNA was synthesized from eguivalent amounts of

Total RNA using SuperScript® III reverse transcriptase

402406280Β1 (Invitrogen) and 18-mer oligo dT (Eurofins MWG Operon).

Gene expression was analyzed by ct delta-delta methods based on real-time guantitative PCR with the human tenascin-C TagMan primer oligonucleotide set (Hs01115663-ml) and endogenous human ribosomal protein (RPLPO) control (4310879E) (Applied Biosystems) on a Corbett Rotor-gene 6000 machine (Corbett Research Ltd). Tenascin-C protein was detected in cell supernatants and cellular used by SDS-PAGE and Western blotting using MAB1908 antibodies (Millipore).

Macrophages were grown at 1x10⁵ cells / well in RPMI 1640 medium containing 5% (v / v) FBS and 100 U / ml penicillin / streptomycin in 96-well tissue culture plates for 24 hours prior to stimulation. Cells were incubated for 24 hours at 37 ° C without addition, 1.0 μΜ FBG, 1 ng / ml LPS, or 1 or 20 μΜ FBG peptide. Where indicated, cells were preincubated with 20 μΜ FBG peptides for 15 minutes.

Cell viability was not significantly affected during the experimental time period as analyzed by the MTT cell viability assay (Sigma, Poole, UK). Supernatants were analyzed for the presence of TNF-α, IL-6 and IL-8 cytokines by enzyme linked enzyme linked immunosorbent assay (ELISA) according to manufacturer's instructions (R &amp; D systems). Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, United Kingdom).

402406280Β1

Statistical methods

Average, SD and SEM were calculated using GraphPad (GraphPad Software Inc., San Diego, CA).

Results

Blockade of cytokine synthesis in AR membrane cultures by specific FBG peptides

The peptide inhibition approach has been successfully used to identify the ανβ3 integrin binding site in the tenascin-C FBG domain, and to prevent cell adhesion in response to this tenascin-C domain (Lafleur (1997) and Yokoyama ( 2000)).

We synthesized a series of 8 overlapping -30 amino acid peptides that span the entire FBG sequence (Table 2). The peptides were tested for their ability to blog spontaneous cytokine synthesis in RA synovial membrane cultures. The synthesis of TNF and IL8 was inhibited by peptides 3 and 8, but not by any other peptide (TNF shown in Figure 15). Peptides 3 and 8 inhibited dose-dependent cytokine synthesis, with the highest concentrations reaching 95% and 56% inhibition, respectively (Figure 16). Although peptide 5 had no effect on TNF synthesis, it dose-dependently blogged IL8 synthesis in AR membrane cells, with a maximum inhibition of 81% (Figure 16).

To map the active domain within FBG responsible for inducing cytokine production we stimulated human primary macrophages with each FBG peptide. Peptides 1, 5 and 6 all induced cytokine synthesis.

ΕΡ2406280Β1 in a dose dependent form (Figure 17).

To determine if any peptide could block FBG-induced cytokine synthesis in human macrophages, cells were preincubated with each FBG peptide prior to stimulation with either full 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).

Therefore, peptide 8 non-specifically blocks cytokine production induced by any stimuli. This domain is the integrin-binding FBG domain that mediates cell adhesion and therefore may be acting to prevent cell attachment in the tissue of the 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 of tenascin-C gene expression inhibits cytokine synthesis in RA synovial fibroblasts

Analysis of the effect of inhibiting tenascin-C expression on the human RA synovial membrane identified synovial fibroblasts as the major source of tenascin-C in RA (Figure 1 C) (in Goh 2010).

SiRNA-mediated blockade of tenascin-C expression in these cells has been demonstrated with a maximum efficiency of 94 - 96% (Figure 19). In cells transfected with tenascin-C siRNA, both the basal level of cytokine synthesis and LPS-induced cytokine production were inhibited by 38% and 44% respectively,

ΕΡ2406280Β1 compared to control cells (Figure 19).

These data reveal that tenascin-C silencing in RA synovial fibroblasts reduces the synthesis of proinflammatory cytokines, and suggests that ablation of tenascin-C expression is a viable strategy to inhibit inflammation in the synovial membrane.

This work established that blogging tenascin-C activity (with peptides) and tenascin-C expression (with siRNA) reduces the synthesis of inflammatory cytokines in human RA synovium. These data show that tenascin-C blogging is of potential clinical benefit in the treatment of RA and other inflammatory diseases.

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62. Midwood, K. et al., Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for

Inflammation2406280Β1 maintaining inflammation in arthritic joint disease. Nat Med 15, 774-780 (2009).

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