EP3356388A2

EP3356388A2 - Compounds binding to the fbg domain of tenascin and uses thereof

Info

Publication number: EP3356388A2
Application number: EP16784408.3A
Authority: EP
Inventors: Kim Suzanne Midwood; Lorena ZULIANI-ALVAREZ; Anna Maria Piccinini
Original assignee: Imperial Innovations Ltd
Current assignee: Ip2ipo Innovations Ltd
Priority date: 2015-09-29
Filing date: 2016-09-29
Publication date: 2018-08-08
Also published as: WO2017055521A3; GB201517184D0; WO2017055521A2

Abstract

There is provided agents for modulation of a chronic inflammatory response wherein the agent modulates the biological activity of tenascin, wherein the agents have binding specificity for and/or modulate the biological activity associated with one or more portions of the FBG domain of tenascin. There is also provided methods of identifying agents such agents and uses of such agents. In particular, the tenascin is tenascin-C, tenascin-R or tenascin-W.

Description

BIOLOGICAL MATERIALS SPECIFIC FOR THE TENASCIN FBG DOMAIN AND USES THEREOF

The disclosure of priority application GB1517184.6 is incorporated herein by reference.

The present invention relates to particular portions of the FBG domain of tenascin and their importance in chronic inflammation. There is also provided modulators of those particular portions of the FBG domain of tenascin and their biological activity and further uses of those particular portions of the FBG domain of tenascin in the identification of agents that up-regulate or down-regulate chronic inflammation. In particular, the tenascin is tenascin-C, tenascin-R or tenascin-W.

Inflammation is the complex biological response of tissues to harmful stimuli, such as pathogens, tissue damage, or irritants. It is a protective attempt by the tissue to remove the injurious stimuli as well as initiate the healing process for the tissue. Abnormalities associated with inflammation comprise a large, unrelated group of disorders which underlie a variety of human diseases (inflammatory disorders).

Chronic inflammation is a debilitating and serious condition associated with many o diseases and is characterised by persistent inflammation at a site of infection or injury, or in relation to altered immune responses such as in autoimmune disease.

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

Toll-like receptors (TLRs) 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 receptor family forms an integral part of the immune system. TLRs mediate host defence against infection and injury by recognising both pathogen- associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (Matzinger (2002)). DAMPs are endogenous pro-inflammatory molecules generated upon tissue injury and include intracellular molecules released from damaged or necrotic cells, fragments of extracellular matrix (ECM) molecules or ECM molecules up regulated upon injury (reviewed in Bianchi (2007) and Gordon (2002)).

Upon activation, TLRs promote both innate and adaptive immune responses including stimulation of expression of pro-inflammatory cytokines and MMPs (Medzhitov (2002)). TLRs are expressed at high levels in synovial tissue from RA patients (Radstake (2004), Roelofs (2005), Sacre (2007), and (Sacre, manuscript submitted 2008) and mice with targeted deletions or loss of function mutations in TLR4 are protected from experimental arthritis (Choe (2003) and Lee (2005). Furthermore, inhibitors of TLR4 can reduce destructive arthritis in mice (Abdollahi- Roodsaz (2007)) and a putative TLR4 inhibitor improved symptoms in 15 out of 23 patients with moderate to severe RA in a preliminary phase I trial (Vanags (2006). However, it is unclear which TLR ligand(s) are involved in disease pathogenesis.

The inventors have shown previously that tenascin-C is an endogenous TLR4 ligand that is required for destructive joint inflammation observed in arthritis and is involved in the prolonging of the inflammatory response characterising the chronic inflammatory condition. In particular, tenascin-C has been shown to be an endogenous activator of TLR4 and demonstrated that this molecule is required for destructive joint inflammation (WO 2010/103289).

In WO 2010/103289, a role for tenascin-C in mediating an immune response in the joint was demonstrated by induction of joint inflammation upon intra-articular injection of the FBG domain of tenascin-C in mice in vivo. Moreover, acute joint inflammation induced by zymosan was not as prolonged in tenascin-C deficient mice. Both the wild type and tenascin-C null mice responded to acute inflammation induction by zymosan equally, demonstrating that tenascin-C does not appear to be involved in the initiation of inflammation. However, the less persistent synovitis exhibited by tenascin-C null mice indicated a role in the maintenance of joint inflammation. The importance of tenascin-C in prolonging joint inflammation was underscored by the observation that targeted deletion of tenascin-C protected mice from sustained erosive joint inflammation during arthritis induced by immunization with mBSA.

Tenascin-C was shown to be capable of activating 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 C terminal of the molecule (Siri (1991)).

Addition of FBG to synovial membrane cultures from RA patients enhanced the spontaneous release of pro-inflammatory cytokines. It also stimulated synthesis of TNF-a, IL-6 and IL-8 in primary human macrophages and IL-6 in RA synovial fibroblasts via activation of TLR4 and MyD88 dependent signalling pathways.

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

Formation of distinct receptor complexes by each TLR4 ligand may facilitate recruitment of different intracellular adapter/signalling molecules. This may account for the differential cellular responses we observe with FBG and LPS, for example lack of IL-8 induction by FBG in RA synovial fibroblasts. Similarly, hyaluronic acid activation of the TLR4 and CD44 complex induces a pattern of gene expression in mouse alveolar macrophage cell lines that is different to LPS (Taylor (2007)). That FBG induces IL-8 synthesis in human macrophages, suggests cell type specific ligand recognition and/or signalling occurs.

The tightly regulated pattern of expression of tenascin-C makes it an attractive target for treating chronic inflammation. It is predominantly absent from healthy adults, however expression is specifically induced upon tissue injury. During acute inflammation tenascin-C is transiently expressed: induction often precedes inflammation and both mRNA and protein are absent from the tissue by the time 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 observed in other autoimmune diseases including multiple sclerosis (Gutowski (1999)) and Sjogrens disease (Amin (2001)), and in non-healing wounds and diabetic and venous ulcers (Loots (1998)). De novo synthesis of tenascin-C correlates well with the intensity of inflammation in diseases of the oral mucosa and plasma levels of tenascin-C are a reliable indicator for the activity of inflammatory bowel diseases before and after medication or surgery (reviewed in Chiquet-Ehrismann (2003)).

In summary, the inventors have found previously that tenascin-C induces the expression of pro-inflammatory cytokines in primary human macrophages and synovial fibroblasts, as well as in mixed population of cells from synovial membrane of individuals with RA. In addition, it was demonstrated using a model of erosive arthritis, that mice lacking tenascin-C are protected from persistent joint inflammation and destruction compared to wild-type mice, suggesting that tenascin-C is essential for the maintenance of inflammation in the arthritic joint (Midwood et al. 2009). Tenascin-C is a large (320 kDa for a monomer) multi-domain extracellular matrix protein and the inventors previously identified the 27 kDa C-terminus domain (fibrinogen-like globe or FBG) as the region of tenascin-C that induces cytokine synthesis in primary human cells and in vivo. The FBG domain of tenascin-C (FBG-C) exerts this action by activating the pattern recognition receptor toll-like receptor 4 (TLR4) (Midwood et al. 2009). TLR4 has been extensively studied in the context of pathogenic activation by the membrane bacterial lipopolysaccharide LPS, however little is known about how endogenous stimuli can activate this receptor.

In addition to tenascin-C, the tenascin family also includes tenascin-R, -W and -X (See Table 3 in Example 6 for properties). Each has a distinct pattern of expression in adults: tenascin-R is mainly expressed in the brain and the central nervous system; tenascin-W is expressed in smooth muscle and bone; and tenascin-X is expressed in loose connective tissue. Only tenascin-C is expressed at universally sites of inflammation. The tenascin family have a similar domain organisation and all contain an FBG domain, which is highly homologous amongst all members (Table 4 in Example 6). However, it was unknown if the FBG domain of the other tenascins could induce an immune or inflammatory response.

WO 2010/103289 described agents for modulation of a chronic inflammatory response wherein the agent modulates the biological activity of tenascin-C and their use in treating conditions associated with chronic inflammation. However, there remains an ongoing need for new and improved treatments for such conditions.

WO 2015/104564 further showed that tenascin-C can be citrullinated in vitro and that citrullinated tenascin-C is preferentially found in patients with a chronic inflammatory condition.

The inventors have now identified, surprisingly, how particular portions of the FBG domain of tenascin activate TLR4 and consequently provide specific agents for modulation of a chronic inflammatory response, wherein those agents modulate the biological activity associated with and/or have binding specificity for one or more of those particular portions of the FBG domain. The inventors equally surprisingly identified that, as well as tenascin-C, tenascin-R and W can induce TLR4-mediated inflammation. SUMMARY OF THE DISCLOSURE

In one independent aspect there is provided a binding domain comprising two variable domains wherein each variable domain comprises 3 CDRs and a framework wherein the binding domain is specific to an FBG domain of a Tenascin, and binds to at least residues 123 and 127, and at least one of the residues 125 or 129 of SEQ ID NO: 1, 2 or 3.

In one embodiment the domain binds residues 123, 125 and 127 and optionally 129, for example residues 123, 125, 127 are lysine, arginine and lysine respectively (and for example residue 129 is lysine).

In one embodiment the domain binds residue 123, 127 and 129, for example residues 123, 127 and 129 are arginine, lysine and arginine respectively.

In one embodiment the domain binds a further, 1, 2, 3, 5, 6, 7, 8, 9 or more residues in range 116 to 132 of said sequences.

In one independent aspect there is provided a binding domain comprising two variable domains wherein each variable domain comprises 3 CDRs and a framework wherein the binding domain is specific to an FBG domain of a Tenascin, and binds to at least residues 155 and 157, and at least one of the residues 159 or 153 of SEQ ID NO: 1, 2 or 3.

In one embodiment the domain binds residues 153, 155 and 157 and optionally 159, for example residues 153, 155, 157 are aspartic acid, aspartic acid and aspartic acid respectively (and for example residue 159 is aspartic acid or alanine).

In one independent aspect there is provided a binding domain comprising two variable domains wherein each variable domain comprises 3 CDRs and a framework wherein the binding domain is specific to an FBG domain of a Tenascin, and binds to at least residues 225, 226 and 227, and optionally residue 228 of SEQ ID NO: 1, 2 or 3.

In one embodiment the binding domain binds SEQ ID NO: 17 and/or 18, in particular a binding domain which binds specifically to one or both of said sequences.

In one embodiment the binding domain binds SEQ ID NO: 27 and/or 28, in particular a binding domain which binds specifically to one or both of said sequences.

In one embodiment the binding domain binds SEQ ID NO: 37 and/or 38, in particular a binding domain which binds specifically one or both of said sequences.

In one embodiment the domain binds a further, 1, 2, 3, 5, 6, 7, 8, 9 or more residues in range 199 to 240 of said sequences.

In one embodiment there is a binding domain according to the present disclosure which is human.

In one embodiment there is provided a binding domain according to claim 1 or 2, which is humanised.

In one embodiment there is provided a binding domain with affinity in the range of 5pM to

500nM.

In one embodiment there is provided a binding domain according to the present disclosure, which is specific to loop 5 of Tenascin-C. In one embodiment there is provide an antibody or binding fragment thereof comprising a binding domain according to the present disclosure, for example wherein the antibody is a full length antibody or a binding fragment such as a Fab, Fab', modified Fab', F(ab') 2, Fv, single domain antibodies (e.g. VH or VL or VHH), or a scFv,

In one embodiment the antibody binding or binding fragment is neutralising.

In one embodiment there is provided a chimeric antigen receptor comprising a binding domain according to the present disclosure.

In one embodiment there is provided a polynucleotide squence encoding a binding domain or an antibody or a chimeric antigen receptor according to the present disclosure.

In one embodiment there is provide a vector comprising a polynucleotide according to the present disclosure

Also provided is a cell comprising a polynucleotide or a vector according to the present disclosure.

In a further aspec the present disclosure provides a pharmaceutical formulation comprising an antibody disclosed herein..

The present disclosure aslo extends to a method of treating a patient compring administering a therapeutically effective amout of an antibody or binding fragment a chimeric antigen receptor or a pharmaceutical formulation as disclosed herein.

The present disclosure extends to an antibody or binding, a chimeric antigen receptor or a pharmaceutical formulation as disclosed herein for use in treatment, in particular for use in the treatment of a chronic inflammatory response.

In a further aspect there is provided use of an antibody or binding fragment, a chimeric antigen receptor or a pharmaceutical formulation for the manufacture of a medicament for the treatment of a chronic inflammatory response.

In an independent aspect there is provided an epitope of no more than 18 amino acids from the P domain of a Tenacin comprising at least residues 129 and 127, and at least one of the residues 125 or 123 of SEQ ID NO; 1, 2 or 3, for example further comprising 1, 2, 3, 5, 6, 7, 8, 9 or more residues in range 116 to 132 of said sequences.

In one embodiment the epitope is SEQ ID NO: 17 or 18. In one embodiment the epitope is SEQ ID NO: 27 or 28. In one embodiment the epitope is SEQ ID NO: 37 or 38.

In one embodiment the epitope according to the present disclosure is conjugated to a carrier.

Also provided is use of an epitope according to the present disclosure for immunizing a host animal or to interrogate a library.

In an independent aspect there is provided an agent, for example as disclosed herein, such as an antibody or binding fragment thereof specific to the FBG domain, in particular the P subdomain or tenascin-R. In one embodiment the agent is a modulator of the activity of the FBG domain, in particular an inhibitor thereof.

In an independent aspect there is provided an agent, for example as disclosed herein, such as an antibody or binding fragment thereof specific to the FBG domain, in particular the P subdomain or tenascin-W. In one embodiment the agent is a modulator of the activity of the FBG domain, in particular an inhibitor thereof.

DETAILED DISCLOSURE

In a first aspect of the invention there is provided an agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with the P subdomain within the FBG domain of tenascin-C.

The full amino acid sequence of the FBG domain of tenascin-C is shown in SEQ ID NO: 1. The P subdomain of the FBG domain of tenascin-C comprises the sequence shown in SEQ ID NO: 10, corresponds to residues 121-206 in the full sequence of the FBG domain of tenascin-C (SEQ ID NO: 1).

For example, by "biological activity associated with the P subdomain" we mean that the P subdomain is required to effect the biological activity which is modulated. It may be that the full P domain is required to effect that biological activity, or only one or more regions/sequences within the P subdomain.

The P subdomain within FBG can be broken down into various portions, as shown in Figure

21. The specific sequences of those portions which are preferred with respect to tenascin-C are as follows:

SEQ ID NO: 11 : KFS VGD AKT RYK LKV, corresponding to residues 116-130 in SEQ ID NO: 1 (designated "loop 5" in Figure 21). SEQ ID NO: 12 : D AKT RYK LKV, corresponding to residues 121-130 in SEQ ID NO: 1 (designated "loop 5" in Figure 21, omitting residues falling outside of the P domain). SEQ ID NO: 13: SFS TFD KDT DSA IT, corresponding to residues 148-161 in SEQ ID NO: 1 (designated "loop 7" in Figure 21). SEQ ID NO: 14: KGA FWY RNC HRV, corresponding to residues 168-179 in SEQ ID NO: 1 (designated "loop 8" in Figure 21). SEQ ID NO: 15: NCALSY, corresponding to residues 162-167 in SEQ ID NO: 1 (designated "alpha helix" in Figure 21).

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 11, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 11 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 12, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 12 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 13, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 13 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function. In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 14, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 14 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 15, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 15 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In reference to all aspects of the invention, by "modulation of a chronic inflammatory response", we include both up-regulation and down-regulation of the chronic inflammatory response. In one set of embodiments, the chronic inflammatory response is down-regulated.

In reference to all aspects of the invention, the use of "modulates", "modulated" and "modulation" in relation to "biological activity", e.g. biological activity associated with specific portions of the FBG domain of tenascin-C, includes both up-regulation and down-regulation of that biological activity. In one set of embodiments, the biological activity is down-regulated.

In one embodiment of the first aspect of the invention, the agent modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 12 ("loop 5"); SEQ ID NO: 13 ("loop 7"); SEQ ID NO: 14 ("loop 8"); and SEQ ID NO: 15 ("alpha helix").

In a preferred embodiment of the first aspect of the invention, the agent modulates the biological activity associated with SEQ ID NO: 12 ("loop 5").

In an second aspect of the invention there is provided an agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with one or more portions of the FBG domain of tenascin-C, wherein the one or more portions are selected from SEQ ID NO: 11 ("loop 5"); SEQ ID NO: 13 ("loop 7"); SEQ ID NO: 14 ("loop 8"); and SEQ ID NO: 15 ("alpha helix").

For example, by "associated with one or more portions" we mean that any one, or any group of, those portions of the FBG domain may be required to effect the biological activity which is modulated. It may be that the full specified portion(s) or sequence thereof is required to effect that biological activity, or only one or more regions/sequences within the specified portion(s).

In one embodiment of the second aspect, the agent modulates the biological activity associated with SEQ ID NO: 11 ("loop 5").

For example, by "modulated the biological activity associated with SEQ ID NO: 11" we mean that this portion of the FBG domain is required to effect the biological activity which is modulated. It may be that the full sequence of SEQ ID NO: 11 is required to effect that biological activity, or only a sub region within that sequence/loop structure.

In embodiments of the first and second aspects of the invention, wherein the agent modulates the biological activity associated with SEQ ID NO: 11 or SEQ ID NO: 12, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 13, 14, 15, 16 ("loop 10", see below) and 19 (see below).

By "additionally modulates the biological activity associated with one or more of SEQ ID NOs: 13, 14, 15, 16 and 19" we include that the agent modulates the biological activity which requires each of SEQ ID NOs: 11 (or 12) and 13, 14, 15, 16 and 19 together, or the agent modulates the biological activity which requires SEQ ID NOs 11 (or 12) and any one of, or a subset of, SEQ ID NOs: 13, 14, 15, 16 and 19.

An additional preferred portion of the FBG domain of tenascin is designated "Loop 10" and the sequences shown in Figure 29. The specific amino acid sequence of this domain in tenascin-C is as follows: SEQ ID NO: 16: NLE GRR KRA, residues 220-228 in SEQ ID NO: 1.

A preferred region within this sequence is made up of the positively charged residues RRKR (SEQ ID NO: 40) plus the following alanine, i.e. the sequence: RRKRA (SEQ ID NO: 19).

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 16, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 16 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 19, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 19 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In certain embodiments of the first and second aspects of the invention, the agent modulates the biological activity associated with one or more of SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15. Optionally, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 11, 12, 16 and 19.

The agents of the first and second aspects may modulate the biological activity associated with one or more particular regions within the portions defined in the embodiments above.

Preferred regions within the portion designated as "loop 5" in Figure 21 are as follows for tenascin-C: KTRYKLK (SEQ ID NO: 17). SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11.

KTRYK (SEQ ID NO: 18). SEQ ID NO: 18 corresponds to residues 8-12 of SEQ ID NO: 11.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 17, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 17 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 18, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 18 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

Therefore, in certain embodiments of the first and second aspects, the agent may modulate the biological activity associated with only a portion of the one or more sequences described in the embodiments above (i.e. only a portion of the designated loop or helix structures). In other words, the agent modulates the biological activity associated with only a sub portion of the specified sequence(s)/loop(s)/helix. Alternatively, the agent may modulate the biological activity associated with the whole of the specified sequence(s)/loop(s) /helix. Where the agent modulates the biological activity associated with more than one specified sequence(s)/loop(s)/helix, the agent may modulate the biological activity associated with only a portion of one or more of the specified sequence(s)/loop(s)/helix, and to the whole of one or more of those other specified sequence(s) /loop(s) /helix.

In one embodiment, the agent of the first and second aspects modulates the biological activity associated with three or more of the positively charged amino acids from the sequence KTRYKLK (SEQ ID NO: 17) of "loop 5" of the FBG domain of tenascin-C. SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11. For example, the agent may modulate the biological activity associated with three or more of the positively charged amino acids K, R, K and K from the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. In other words, the agent modulates the biological activity associated with three or more of the residues 1, 3, 5 and 7 of SEQ ID NO: 17, which are equivalent to residues 8, 10, 12 and 14 in SEQ ID NO: 11 above.

By "modulates the biological activity associated with three or more of the positively charged amino acids" we mean that the three (or more) positively charged amino acids are required to effect the biological activity which is modulated.

Optionally, the agent modulates the biological activity associated with the positively charged amino acids K, R, K and K from the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. In other words, the agent modulates the biological activity associated with residues 1, 3, 5 and 7 of SEQ ID NO: 17, which are equivalent to residues 8, 10, 12 and 14 in SEQ ID NO: 11 above.

By "modulates the biological activity associated with the positively charged amino acids" we mean that those positively charged amino acids are all required to effect the biological activity which is modulated.

In a further embodiment, the agent of the first and second aspect modulates the biological activity associated with the sequence KTRYK (SEQ ID NO: 18) of loop 5 of the FBG domain of tenascin-C. SEQ ID NO: 18 corresponds to residues 8-12 of SEQ ID NO: 11.

In an alternative embodiment, the agent of the first and second aspect modulates the biological activity associated with the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11.

In a further embodiment, the agent of the first and second aspects modulates the biological activity associated with one or more of residues 152, 157, 160 and 162 (as defined in SEQ ID NO: 1) of the FBG domain of tenascin-C. These residues are located in regions designated "loop 7" or the "alpha helix" and are considered to be particularly preferred.

Optionally, the agent of the first and second aspects modulates the biological activity associated with one or more of the hydrophobic and/or positively charged amino acids from the sequence defined by residues 148-179 in SEQ ID NO: 1. In particular, the agent may modulate the biological activity associated with one or more of the hydrophobic amino acids A159, 1160, T161 and F152 (residue numbers correspond to SEQ ID NO: 1). The biological activity of these one or more amino acids may be modulated in addition to or independently of residues 157 and/or 162 (of SEQ ID NO: 1).

The agent of the first or second aspect of the invention may modulate the biological activity associated with the specified domain or portion of tenascin-C by altering the transcription, translation and/or binding properties of tenascin-C or the specified domains or portions therein.

The agent of the first or second aspect preferably prevents the one or more particular regions of the FBG domain described above in relation to modulating their biological activity from binding to or activating TLR4. This may include an agent that binds near the particular region(s) in FBG and prevents binding to or activation of TLR4 by steric hindrance, an agent that binds to FBG in a completely different region but changes the conformation so as to prevent TLR4 binding or activation, an agent that binds to TLR4 in the region where these residues of FBG bind or activate, or an agent that binds to TLR4 in a completely different region but changes the conformation so as to prevent FBG binding or activation.

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

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

(b) by determining the effect of a test agent on levels of tenascin-C protein, for example by immunoassays using anti- tenascin-C antibodies; and

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

The agent of the first or second aspect of the invention may down-regulate the biological activity of tenascin-C.

The agent of the first or second aspect of the invention may up-regulate the biological activity of tenascin-C. The desirability of up-regulating activity of immune and inflammatory molecules and cells is relevant to the production of therapies for compromised immune and inflammatory patients and in the development of vaccines, (see Harandi (2009)).

The agent of the first or second aspect of the invention may be an inhibitor of transcription of tenascin-C.

The agent of the first or second aspect of the invention may be an inhibitor of translation of tenascin-C.

The agent of the first or second aspect of the invention may be an inhibitor of the binding properties associated with the specified domain or portion(s) of the FBG domain of tenascin-C. For example, the agent may alter the conformation of tenascin-C or the specified domain or portion(s) of the FBG domain of tenascin-C such that it is no longer able to bind to its receptor. In preferred embodiments, the binding refers to binding to TLR4.

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 in whole or in part. For example, the agent may inhibit the biological activity of tenascin-C by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably by 100% compared to the biological activity of tenascin-C on inflammatory cells which have not been exposed to the agent.

In one set of embodiments of the first and second aspects, the agent binds to or within tenascin-C, preferably to the FBG domain of tenascin-C.

By "binds to or within", we mean that the agent binds specifically to or within that protein / domain / portion / region/sequence.

By reference to binding to or within a particular amino acid sequence, we also include binding to or within the nucleotide sequence encoding that amino acid sequence.

In one embodiment the agent binds to or within the P domain within the FBG domain of tenascin-C. By "binds to or within the P domain" we include an agent which binds to the whole P domain or to one or more regions/sequences anywhere within the P domain.

In one embodiment the agent binds to or within one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

In an alternative embodiment, the agent binds to or within one or more portions of the FBG domain of tenascin-C, wherein the one or more portions are selected from SEQ ID NO: 11; SEQ ID

NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

By "binds to or within one or more portions" we include that the agent may bind to or within any one, or any group of, those portions of the FBG domain. It may be that the agent binds to the full specified portion(s), or only to one or more regions/sequences within the specified portion(s).

The agent may preferably bind to or within SEQ ID NO: 11 or SEQ ID NO: 12. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 13, 14, 15, 16 and 19.

In one embodiment, the agent binds to or within one or more of SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 11, 12, 16 and 19.

In one embodiment, the agent of the first and second aspect binds to three or more of the positively charged amino acids from the sequence KTRYKLK (SEQ ID NO: 17) of "loop 5" of the FBG domain of tenascin-C. SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11. For example, the agent binds to three or more of the positively charged amino acids K, R, K and K from the sequence KTRYKLK (SEQ ID NO: 17) of "loop 5" of the FBG domain of tenascin-C. In other words, the agent binds to three or more of the residues 1, 3, 5 and 7 of SEQ ID NO: 17, which are equivalent to residues 8, 10, 12 and 14 in SEQ ID NO: 11 above.

By "binds to three or more of the positively charged amino acids" we mean that the agent binds to all three (or more) of those positively charged amino acids. Optionally, the agent binds to the positively charged amino acids K, R, K and K from the sequence KTRYKLK (SEQ ID NO: 17) of "loop 5" of the FBG domain of tenascin-C. In other words, the agent binds to residues 1, 3, 5 and 7 of SEQ ID NO: 17, which are equivalent to residues 8, 10, 12 and 14 in SEQ ID NO: 11 above.

By "binds to the positively charged amino acids" we mean that the agent binds to all of those positively charged amino acids.

In a further embodiment, the agent binds to or within the sequence KTRYK (SEQ ID NO: 18) of "loop 5" of the FBG domain of tenascin-C. SEQ ID NO: 18 corresponds to residues 8-12 of SEQ ID NO: 11.

In an alternative embodiment, the agent binds to or within the sequence KTRYKLK (SEQ ID

NO: 17) of "loop 5" of the FBG domain of tenascin-C. SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11.

The agent may bind to one or more of residues 152, 157, 160 and 162 (as defined in SEQ ID NO: 1) of the FBG domain of tenascin-C.

Optionally, the agent may bind to one or more of the hydrophobic and/or positively charged amino acids from the sequence defined by residues 148-179 in SEQ ID NO: 1. In particular, the agent may bind to one or more of the hydrophobic amino acids A159, 1160, T161 and F152 (residue numbers correspond to SEQ ID NO: 1). The agent may bind to these one or more amino acids in addition to or independently of residues 157 and/or 162 (as defined in SEQ ID NO: 1).

The agent of the first and second aspect of the invention may be selected from the group consisting of antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, aptamers, small inhibitor compounds, polypeptides and proteins, compounds with binding affinity for tenascin-C, and short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides.

In one embodiment of the invention the agent is an siRNA. RNA interference is a two-step process. The first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the Rnase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA) each with 2-nucleotide 3' overhangs (Hutvagner & Zamore, 2002, Curr. Opin. Genetics and Development 12:225-232; Bernstein, 2001, Nature 409:363-366).

In the effector step, the siRNA duplexes bind to a nuclease complex to form the RNA- induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA (Hutvagner & Zamore, 2002, supra.; Hammond et al., 2001, Nat. Rev. Gen. 2:110-119 (2001); Sharp, 2001, Genes. Dev. 15:485-90). Although the mechanism of cleavage is still to be elucidated, research indicates that each RISC contains a single siRNA and an RNase (Hutvagner & Zamore, 2002, supra.). In view of the remarkable potency of RNAi, an amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively, or additionally, amplification could be effected by multiple turnover events of the RISC (Hammond et al., 2001, supra.; Hutvagner & Zamore, 2002, supra.). Additional information on 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 Act. 1575:15-25.

Synthesis of RNAi molecules suitable for use with the present invention can be effected as follows. First, the tenascin-C mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex (Tuschl, ChemBiochem. 2:239-245). It will be appreciated, however, that siRNAs directed at 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 the BLAST (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.

Suitable siRNA molecules can be synthesised as described above such that they are complementary and therefore bind to the nucleotide sequence encoding the required portion(s) of the FBG domain of tenascin. The nucleotide sequence of tenascin-C is found in figure 14.

In one embodiment the agent may be a short hairpin RNA (shRNA).

A small hairpin RNA or short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector (typically adenovirus or lentivirus) introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that it is bound to it. (Mclntyre (2006) and Paddison (2002)) The agent of the first and second aspect of the invention may be a domain of tenascin-C or variant thereof. The FBG domain has been shown to be predominantly involved in the interaction of tenascin-C with its target in relation to the persistence of chronic inflammation. Accordingly the preferred domain is the FBG domain (sequence shown in figure 13) or subdomain(s), portion(s) or variants thereof. Preferred subdomains include the P subdomain of the FBG domain of tenascin-C, "loops" 5, 7 and/or 8, and optionally also "loop 10", as well as the "alpha helix" separating loops 7 and 8. Particularly preferred subdomains include the sequence KTRYKLK of loop 5, specifically 3 or more (preferably 4) of the positively charged amino acids of that subdomain, such as 3 or more (preferably all 4) selected from K, R, K and K, the sequence KTRYK (SEQ ID NO: 18) of "loop 5", and one or more of residues 152, 157, 160 and 162 (as defined in SEQ ID NO: 1) of "loop 7"/"alpha helix" of the FBG domain of tenascin-C.

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

The design of antisense molecules which can be used to decrease efficiently tenascin-C (or a domain, subdomain, or amino acid sequence thereof) levels/activity requires consideration of two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the cancer cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.

The prior art teaches a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types (for example, see Luft, 1998, / Mol Med 76:75-6; Kronenwett et al., 1998, Blood 91:852-62; Rajur et al., 1997, Bioconjug Chem 8:935-40; Lavigne et al, 1997, Biochem Biophys Res Commun 237:566-71; Aoki et al, 1997, Biochem Biophys Res Commun 231:540-5).

In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetics of structural alternations in both the target mRNA and the oligonucleotide are available (for example, see Walton et al., 1999, Biotechnol Bioeng 65:1-9).

Several approaches for designing and predicting 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 trails have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer 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, antisense-mediated suppression of 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 61:7855-60).

Thus, persons skilled in the art are readily able to design and implement antisense approaches suitable for modulating expression of tenascin (or a domain, subdomain, or amino acid sequence thereof).

Advantageously, the antisense oligonucleotide is 15 to 35 bases in length. For example, 20- mer oligonucleotides have been shown to inhibit the expression of the epidermal growth factor receptor mRNA (Witters et al, Breast Cancer Res Treat 53 :41-50 (1999)) and 25-mer oligonucleotides have been shown to decrease the expression of adrenocorticotropic hormone by greater than 90% (Frankel et al, J Neurosurg 91:261-7 (1999)). However, it is appreciated that it may be desirable to use oligonucleotides with 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 person skilled in the art that oligonucleotides are subject to being degraded or inactivated by cellular endogenous nucleases. To counter this problem, it is possible to use modified oligonucleotides, e.g. having altered internucleotide linkages, in which the naturally occurring phosphodiester linkages have been replaced with another linkage. For example, Agrawal et al (1988) Proc. Natl. Acad. Sci. USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-1 using oligonucleotide phosphoramidates and phosphorothioates. Sarin et al (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451 demonstrated increased inhibition of HIV-1 using oligonucleotide methylphosphonates. Agrawal et al (1989) Proc. Natl. Acad. Sci. USA 86, 7790-7794 showed inhibition of HIV-1 replication in both early-infected and chronically infected cell cultures, using nucleotide sequence-specific oligonucleotide phosphorothioates. Leither et al (1990) Proc. Natl. Acad. Sci. USA 87, 3430-3434 report inhibition in tissue culture of influenza virus replication by oligonucleotide phosphorothioates.

Oligonucleotides having artificial linkages have been shown to be resistant to degradation in vivo. For example, Shaw et al (1991) in Nucleic Acids Res. 19, 747-750, report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3 ' end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.

A detailed description of the H-phosphonate approach to synthesising oligonucleoside phosphorothioates is provided in Agrawal and Tang (1990) Tetrahedron Letters 31, 7541-7544, the teachings of which are hereby incorporated herein by reference. Syntheses of oligonucleoside methylphosphonates, phosphorodithioates, phosphoramidates, phosphate esters, bridged phosphoramidates and bridge phosphorothioates are known in the art. See, for example, Agrawal and Goodchild (1987) Tetrahedron Letters 28, 3539; Nielsen et al (1988) Tetrahedron Letters 29, 2911; Jager et al (1988) Biochemistry 27, 7237; Uznanski et al (1987) Tetrahedron Letters 28, 3401; Bannwarth (1988) Helv. Chim. Acta. 71, 1517; Crosstick and Vyle (1989) Tetrahedron Letters 30, 4693; Agrawal et al (1990) Proc. Natl. Acad. Sci. USA 87, 1401-1405, the teachings of which are incorporated herein by reference. Other methods for synthesis or production also are possible. In a preferred embodiment the oligonucleotide is a deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences may also be synthesised and applied.

The oligonucleotides useful in the invention preferably are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are more likely to engage in nonspecific hybridisation and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and which are able to reach the targeted cells. The present oligonucleotides can be rendered more resistant to degradation in vivo by substituting one or more internal artificial internucleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulphur in the linkage. Examples of linkages that may be used include phosphorothioates, methylphosphonates, sulphone, sulphate, ketyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phosphorothioates and bridged phosphoramidates. Such examples are illustrative, rather than limiting, since other internucleotide linkages are well known in the art. The synthesis of oligonucleotides having one or more of these linkages substituted for the phosphodiester internucleotide linkages is well known in the art, including synthetic pathways for producing oligonucleotides having mixed internucleotide linkages.

Oligonucleotides can be made resistant to extension by endogenous enzymes by "capping" or incorporating similar groups on the 5' or 3' terminal nucleotides. A reagent for capping is commercially available as Amino-Link II™ from Applied BioSystems Inc, Foster City, CA. Methods for capping are described, for example, by Shaw et al (1991) Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991) Proc. Natl. Acad. Sci. USA 88 (17), 7595-7599.

A further method of making oligonucleotides resistant to nuclease attack is for them to be

"self-stabilised" as described by Tang et al (1993) Nucl. Acids Res. 21, 2729-2735. Self-stabilised oligonucleotides have hairpin loop structures at their 3' ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I and foetal bovine serum. The self-stabilised region of the oligonucleotide does not interfere in hybridisation with complementary nucleic acids, and pharmacokinetic and stability studies in mice have shown increased in vivo persistence of self-stabilised oligonucleotides with respect to their linear counterparts.

In an embodiment where the agent is a compound with binding affinity for tenascin-C (or a domain, subdomain, or amino acid sequence thereof), the compound may bind substantially reversibly or substantially irreversibly to an active site of tenascin-C. In a further example, the compound may bind to a portion of tenascin-C that is not the active site so as to interfere with the binding of the tenascin-C to a ligand or receptor. In a still further example, the compound may bind to a portion of tenascin-C so as to decrease the proteins activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the activity of tenascin-C, for example in the activation of the tenascin-C by an "upstream activator".

Methods for detecting interactions between a test compound and tenascin-C (or a domain, subdomain, or amino acid sequence thereof) are well known in the art. For example ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods may be used. In addition, Fluorescence Energy Resonance Transfer (FRET) methods may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when 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 plasmon resonance assay, for example as described in Plant et al, 1995, Analyt Biochem 226(2), 342-348. Methods may make use of a polypeptide that is labelled, for example with a radioactive or fluorescent label. A further method of identifying a compound that is capable of binding to the polypeptide is one where the polypeptide is exposed to the compound and any binding of the compound to the said polypeptide is detected and/or measured. The binding constant for the binding of the compound to the polypeptide may be determined. Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example, using a method capable of high throughput operation, for example a chip-based method. New technology, called VLSIPS™, has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or arrays have probes arranged in arrays, each probe assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns. The chips can be used to determine whether target molecules interact with any of the probes on the chip. After exposing the array to target molecules under selected test conditions, scanning devices can examine each location in the array and determine whether a target molecule has interacted with the probe at that location.

Another method of identifying compounds with binding affinity for tenascin (or a domain, subdomain, or amino acid sequence thereof) is the yeast two-hybrid system, where the polypeptides of the invention can be used to "capture" proteins that bind tenascin-C. The yeast two-hybrid system is described in Fields & Song, Nature 340:245-246 (1989).

In a further embodiment of the invention, the agent is a compound which has ligand- binding capacity for tenascin-C (or for a domain, subdomain, or amino acid sequence thereof).

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 (a so-called 'affibody') (for example, see US 5,831,012 and www.affibody.se). These ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A (a surface protein from the bacterium Staphylococcus aureus). This scaffold has excellent features as an affinity ligand and can be designed to bind with high affinity to any given target protein. The agent may also be an aptamer. Libraries based on aptamers are discussed in Kenan et al, 1999, Methods Mol Biol 118, 217-31.

The agent of the first and second aspect of the invention may be an antibody or antigen- binding fragment thereof. The antigen-binding fragment may be selected from the group consisting of Fv fragments [e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab) 2 fragments), single variable domains [e.g. VH and VL domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments. Also included within the scope of the invention are modified versions of antibodies and an antigen-binding fragments thereof, e.g. modified by the covalent attachment of polyethylene glycol or other suitable polymer.

Methods of generating antibodies and antibody fragments are well known in the art. For example, antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al, 1991, Nature 349:293-299) or generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV) -hybridoma technique (Kohler et al, 1975. Nature 256:4950497; Kozbor et al, 1985. Immunol. Methods 81:31-42; Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984. Mol. Cell. Biol. 62 : 109-120).

Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982).

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

It will be appreciated by persons skilled in the art that for human therapy or diagnostics, humanised antibodies are preferably used. Humanised forms of non-human [e.g. murine) antibodies are genetically engineered chimaeric antibodies or antibody fragments having preferably minimal-portions derived from non-human antibodies. Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non-human species (donor antibody) such as mouse, rat of rabbit having the desired functionality. In some instances, Fv framework residues of the human antibody are replaced by corresponding non-human residues. Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences. In general, the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence. Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988, Nature 332 :323-329; Presta, 1992, Curr. Op. Struct. Biol. 2 :593-596). Methods for humanising non-human antibodies are well known in the art. Generally, the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et al, 1986, Nature 321:522-525; Reichmann et al, 1988. Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-15361; US 4,816,567) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions. Accordingly, such humanised antibodies are chimaeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.

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

Once suitable antibodies are obtained, they may be tested for activity, for example by ELISA.

The agent of the first or second aspect of the invention may be an antibody or antigen- binding fragment thereof which has specificity for Toll Like Receptor 4 (TLR4) or co-receptors of Toll Like Receptor 4.

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

The agent of the first and second aspects of the invention may be an antibody or antigen- binding fragment.

In a third aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-C comprising the steps of:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with Tenascin-C or FBG-C or one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 and the one or more candidate agents;

(iii) contacting one or more cells with Tenascin-C or FBG-C or one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 and no candidate agent;

(iv) determining whether said candidate agent modulates the effect of Tenascin-C or FBG-C or one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

In a preferred embodiment of the third aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-C comprising the steps of:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 and the one or more candidate agents; (iii) contacting one or more cells with one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 and no candidate agent;

(iv) determining whether said candidate agent modulates the effect of the one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

In a fourth aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-C comprising:

(i) providing one or more candidate agents;

(ii) contacting TLR4 with Tenascin-C or FBG-C or one or more of SEQ ID NOs: 10-15 and 17-18 and the one or more candidate agents;

(iii) contacting TLR4 with Tenascin-C or FBG-C or one or more of SEQ ID NOs: 10-15 and 17-18 and no candidate agent;

(iv) determining whether said candidate agent modulates the binding of TLR4 to the Tenascin-C or FBG-C or one or more of SEQ ID NOs: 10-15 and 17-18 in step (ii) in comparison to the binding of TLR4 to Tenascin-C or FBG-C or one or more of SEQ ID NOs: 10-15 and 17-18 in step (iii).

In a preferred embodiment of the fourth aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-C comprising:

(i) providing one or more candidate agents;

(ii) contacting TLR4 with one or more of SEQ ID NOs: 10-15 and 17-18 and the one or more candidate agents;

(iii) contacting TLR4 with one or more of SEQ ID NOs: 10-15 and 17-18 and no candidate agent;

(iv) determining whether said candidate agent modulates the binding of TLR4 to the one or more of SEQ ID NOs: 10-15 and 17-18 in step (ii) in comparison to the binding of TLR4 to one or more of SEQ ID NOs: 10-15 and 17-18 in step (iii).

In one embodiment of the methods of the third and fourth aspects, the one or more cells or TLR4 contacted in steps (ii) and (iii) is contacted with the P domain (SEQ ID NO: 10).

In another embodiment of the methods of the third and fourth aspects, the one or more cells or TLR4 contacted in steps (ii) and (iii) is contacted with SEQ ID NO: 11. The one or more cells or TLR4 may be additionally contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 13, 14, 15, 16 and 19.

In one embodiment of the method of the fourth aspect, TLR4 is contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 13, 14 and 15. TLR4 may be additionally contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 11, 16 and 19.

In certain embodiments of the methods of the third and fourth aspects, the one or more cells or TLR4 are contacted in steps (ii) and (iii) with three or more of the positively charged amino acids from the sequence KTRYKLK (SEQ ID NO: 17) of the FBG domain of tenascin-C. The one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the positively charged amino acids K, R, K and K from the sequence KTRYKLK (residues 1, 3, 5 and 7 of SEQ ID NO: 17) of the FBG domain of tenascin-C.

The one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the sequence KTRYK (SEQ ID NO: 18) of the FBG domain of tenascin-C. Alternatively, the one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the sequence KTRYKLK (SEQ ID NO: 17) of the FBG domain of tenascin-C.

In some embodiments of the methods of the third and fourth aspects, the one or more cells or TLR4 may be contacted in steps (ii) and (iii) with one or more of residues 152, 157, 159, 160, 161 and 162 (as defined in SEQ ID NO: 1) of the FBG domain of tenascin-C.

In certain embodiments of the methods of the third and fourth aspects, the activity of tenascin-C is up-regulated. Alternatively, the activity of tenascin-C is down-regulated.

In one set of embodiments of the methods of the third and fourth aspects, the agent identified by the method binds to or within tenascin-C, preferably to or within the FBG domain of tenascin-C.

By "binds to or within", we mean that the agent binds specifically to that whole protein/domain/portion/region/sequence, or to a particular sub-region within that protein/domain/portion/region/sequence.

In one embodiment the agent identified by the method binds to or within the P subdomain within the FBG domain of tenascin-C. By "binds to or within the P subdomain" we include an agent which binds to the whole P domain or to one or more portions/regions/sequences anywhere within the P domain.

In one embodiment the agent identified by the method binds to or within one or more portions within the P subdomain, wherein the one or more portions are selected from SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

In an alternative embodiment, the agent identified by the method binds to or within one or more portions of the FBG domain of tenascin-C, wherein the one or more portions are selected from SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

The agent identified by the method may preferably bind to or within SEQ ID NO: 11 or SEQ ID NO: 12. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 13, 14, 15, 16 and 19.

In one embodiment, the agent identified by the method binds to or within one or more of SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 11, 12, 16 and 19.

In one embodiment, the agent identified by the method binds to three or more of the positively charged amino acids from the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11. For example, the agent binds three or more of the positively charged amino acids K, R, K and K from the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. In other words, the agent binds three or more of the residues 1, 3, 5 and 7 of SEQ ID NO: 17, which are equivalent to residues 8, 10, 12 and 14 in SEQ ID NO: 11 above.

By "binds to three or more of the positively charged amino acids" we mean that the agent binds to all three or more of those positively charged amino acids.

Optionally, the agent identified by the method binds to the positively charged amino acids K, R, K and K from the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin- C. In other words, the agent binds to residues 1, 3, 5 and 7 of SEQ ID NO: 17, which are equivalent to residues 8, 10, 12 and 14 in SEQ ID NO: 11 above.

In a further embodiment, the agent identified by the method binds to or within the sequence KTRYK (SEQ ID NO: 18) of loop 5 of the FBG domain of tenascin-C. SEQ ID NO: 18 corresponds to residues 8-12 of SEQ ID NO: 11.

In an alternative embodiment, the agent identified by the method binds to or within the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11.

The agent identified by the method may bind to one or more of residues 152, 157, 159, 160,

161 and 162 (as defined in SEQ ID NO: 1) of the FBG domain of tenascin-C.

The agent identified by the methods of the third and fourth aspects may modulate the biological activity associated specifically with the P domain within the FBG domain of tenascin-C.

In one embodiment, the agent identified by the method modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 12 ("loop 5"); SEQ ID NO: 13 ("loop 7"); SEQ ID NO: 14 ("loop 8"); and SEQ ID NO: 15 ("alpha helix").

In a preferred embodiment, the agent identified by the method modulates the biological activity associated with SEQ ID NO: 12 ("loop 5").

The agent identified by the method of the third or fourth aspects may modulate the biological activity associated with one or more portions of the FBG domain of tenascin-C, wherein the one or more portions are selected from SEQ ID NO: 11 ("loop 5"); SEQ ID NO: 13 ("loop 7"); SEQ ID NO: 14 ("loop 8"); and SEQ ID NO: 15 ("alpha helix").

In one embodiment, the agent identified by the method modulates the biological activity associated with SEQ ID NO: 11 ("loop 5"). For example, by "modulates the biological activity associated with SEQ ID NO: 11" we mean that this portions of the FBG domain is required to effect the biological activity which is modulated. It may be that the full sequence of SEQ ID NO: 11 is required to effect that biological activity, or only a sub region within that sequence.

In certain embodiments, wherein the agent identified by the method modulates the biological activity associated with SEQ ID NO: 11 or SEQ ID NO: 12, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 13, 14, 15, 16 and 19.

By "additionally modulates the biological activity associated with one or more of SEQ ID NOs: 13, 14, 15, 16 and 19" we include that the agent modulates the biological activity which requires each of SEQ ID NOs: 11 or 12, and 13, 14, 15, 16 and 19 together, or the agent modulates the biological activity which requires SEQ ID NOs 11 or 12 and any one of, or a subset of, SEQ ID NOs: 13, 14, 15, 16 and 19.

In certain embodiments of the third and fourth aspects of the invention, the agent identified by the method modulates the biological activity associated with one or more of SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15. Optionally, the agent may additionally modulates the biological activity associated with one or more of SEQ ID NOs: 11, 12, 16 and 19.

The agent identified by the method may modulate the biological activity associated with one or more particular regions within the portions defined in the embodiments above.

In certain embodiments, the agent identified by the method may modulate the biological activity associated with only a portion of the one or more sequences described in the embodiments above (i.e. only a portion of the designated loop or helix structures). In other words, the agent modulates the biological activity associated with only a sub portion of the specified sequence(s)/loop(s)/helix. Alternatively, the agent may modulate the biological activity associated with the whole of the specified sequence (s) /loop (s) /helix. Where the agent modulates the biological activity associated with more than one specified sequence(s)/loop(s)/helix, the agent may modulate the biological activity associated with only a portion of one or more of the specified sequence(s)/loop(s)/helix, and to the whole of one or more of those other specified sequence(s) /loop(s) /helix.

In one embodiment, the agent identified by the method modulates the biological activity associated with three or more of the positively charged amino acids from the sequence KTRYKLK (SEQ ID NO: 17) of "loop 5" of the FBG domain of tenascin-C. SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11. For example, the agent may modulate the biological activity associated with three or more of the positively charged amino acids K, R, K and K from the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. In other words, the agent identified by the method modulates the biological activity associated with three or more of the residues 1, 3, 5 and 7 of SEQ ID NO: 17, which are equivalent to residues 8, 10, 12 and 14 in SEQ ID NO: 11 above.

By "modulates the biological activity associated with three or more of the positively charged amino acids" we mean that the three or more positively charged amino acids are required to effect the biological activity which is modulated. Optionally, the agent identified by the method modulates the biological activity associated with the positively charged amino acids K, R, K and K from the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. In other words, the agent modulates the biological activity associated with residues 1, 3, 5 and 7 of SEQ ID NO: 17, which are equivalent to residues 8, 10, 12 and 14 in SEQ ID NO: 11 above.

By "modulates the biological activity associated with the positively charged amino acids" we mean that those positively charged amino acids are required to effect the biological activity which is modulated.

In a further embodiment, the agent identified by the method of the third and fourth aspect modulates the biological activity associated with the sequence KTRYK (SEQ ID NO: 18) of loop 5 of the FBG domain of tenascin-C. SEQ ID NO: 18 corresponds to residues 8-12 of SEQ ID NO: 11.

In an alternative embodiment, the agent identified by the method of the third and fourth aspect modulates the biological activity associated with the sequence KTRYKLK (SEQ ID NO: 17) of loop 5 of the FBG domain of tenascin-C. SEQ ID NO: 17 corresponds to residues 8-14 of SEQ ID NO: 11.

In a further embodiment, the agent identified by the method modulates the biological activity associated with one or more of residues 152, 157, 159, 160, 161 and 162 (as defined in SEQ ID NO: 1) of the FBG domain of tenascin-C. These residues are located in regions designated "loop 7" or the "alpha helix" and are considered particularly preferred.

The agent identified by the method of the third and fourth aspects may modulate the biological activity associated with the specified domain or portion of tenascin-C by altering the transcription, translation and/or binding properties of tenascin-C or the specified domains or portions therein.

Methods of determining whether the candidate agent modulate the effect of tenascin-C can be carried out using the methods of the examples.

The method of the third and fourth aspect of the invention may result in the activity of tenascin-C or the specified domain or portion(s) being upregulated.

The method of the third and fourth aspect of the invention may result in the activity of tenascin-C or the specified domain or portion(s) being downregulated.

The method of the third and fourth aspect of the invention may include the cells of steps (ii) and (iii) (described above) expressing Toll-like receptor 4 (TLR4).

The method of the third and fourth aspect of the invention may have the 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, THP1 cell lines.

The inflammatory cells may be selected from the group consisting of macrophages, dendritic cells, monocytes, lymphocytes, monocyte like cells and macrophage like cells.

In a fifth aspect of the invention there is provided a method of identification of an agent that modulates a chronic inflammatory response by conducting the method of the third or fourth aspects of the invention. In this method the chronic inflammation may be associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, alzheimer's disease and parkinson's disease.

Of particular, but non-exclusive interest, the chronic inflammation is associated with rheumatoid arthritis (RA).

In a sixth aspect of the invention there is provided an agent identified according to the method of the third and fourth aspects of the invention. Such an agent may modulate a chronic inflammatory response.

The agent of the sixth aspect may down-regulate the chronic inflammatory response.

The agent of the sixth aspect may up-regulate the chronic inflammatory response.

The agent of the sixth aspect may be selected from the group consisting of short interfering

RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides, compounds with binding affinity for tenascin-C, antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, aptamers, small inhibitor compounds, polypeptides and proteins.

The agent of the sixth aspect may itself have any of the properties set out above for the agent of the first and second aspects of the invention.

In the first, second or sixth aspects of the invention the chronic inflammation may be associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, alzheimer's disease and parkinson's disease.

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

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

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

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

For example, the agents of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The agents of invention may also be administered via intracavernosal injection.

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 glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (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 flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The agents of the invention can also be administered parenterally, for example, intravenously, intra-articularly, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they 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, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The 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 injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For oral and parenteral administration to human patients, the daily dosage level of the agents of the invention will usually 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 can 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 pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or 'puff 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 can 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 of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered by the ocular route.

For ophthalmic use, the agents of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the agents of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable 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, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Where the agent is a polypeptide, it may be preferable to use a sustained-release drug delivery system, such as a microspheres. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

Alternatively, polypeptide agents of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site.

Electroporation therapy (EPT) systems can also be employed for the administration of proteins and polypeptides. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.

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

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

Protein and polypeptide pharmaceuticals can also be delivered orally. One such system employs a natural process for oral uptake of vitamin B12 in the body to co-deliver proteins and polypeptides. By riding the vitamin B12 uptake system, the protein or polypeptide can move through the intestinal wall. Complexes are produced between vitamin B12 analogues and the drug that 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 know in the art (see Dass, 2002, ] Pharm Pharmacol. 54(l):3-27; Dass, 2001, Drug Deliv. 8(4) :191-213; Lebedeva et ah, 2000, Eur] Pharm Biopharm. 50(1):101-19; Pierce et al, 2005, Mini Rev Med Chem. 5(l):41-55; Lysik & Wu-Pong, 2003, / Pharm Sci. 2003 2(8) :1559-73; Dass, 2004, Biotechnol Appl Biochem. 40(Pt 2):113-22; edina, 2004, Curr Pharm Des. 10(24) :2981-9.

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

Such a further agent may be an anti-inflammatory agent which includes but is not limited to non-steroidal anti-inflammatory agent (NSAID), a disease modifying anti-rheumatic drug (DMARD), a statin (including HMG-CoA reductase inhibitors such as simvastatin), a biological agent (biologicals), a steroid, an immunosuppressive agent, a salicylate and/or a microbicidal agent. Non-steroidal anti-inflammatory agents include anti-metabolite agents (such as methotrexate) and anti-inflammatory gold agents (including gold sodium thiomalate, aurothiomalate or gold salts, such as auranofin). Biologicals include anti-TNF agents (including adalimumab, etanercept, infliximab, anti-IL-1 reagents, anti-IL-6 reagents, anti-B cell reagents (retoximab), anti-T cell reagents (anti-CD4 antibodies), anti-IL-15 reagents, anti-CLTA4 reagents, anti-RAGE reagents), antibodies, soluble receptors, receptor binding proteins, cytokine binding proteins, mutant proteins with altered or attenuated functions, RNAi, polynucleotide aptemers, antisense oligonucleotides or omega 3 fatty acids. Steroids (also know as corticosteroids) include cortisone, prednisolone or dexamethasone. Immunosuppressive agents include cylcosporin, FK506, rapamycin, mycophenolic acid. Salicylates include aspirin, sodium salicylate, choline salicylate and magnesium salicylate. Microbicidal agents include quinine and chloroquine. For example, the agent may be administered in combination with one or more of an NSAID, DMARD, or immunosuppressant

In an eighth aspect of the invention there is provided an agent or composition as defined in the first, second, sixth and seventh aspects of the invention for use as a medicament.

In a ninth aspect of the invention there is provided an agent or composition as defined in the first, second, sixth and seventh aspects of the invention for use in the treatment of a chronic inflammatory condition.

In a tenth aspect of the invention there is provided the use of an agent or composition as defined in as defined in the first, second, sixth and seventh aspects of the invention in the manufacture of a medicament for the treatment of a chronic inflammatory condition.

In an eleventh aspect of the invention there is provided a method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an agent or composition as defined in the first, second, sixth and seventh aspects of the invention.

The agent, composition, use or method as defined in the eighth, ninth, tenth or eleventh aspects of the invention may relate to treatment of a chronic inflammatory condition wherein the condition is associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, alzheimer's disease and parkinson's disease.

In a twelfth aspect of the invention there is provided a kit of parts for performing the method of the third and fourth aspect of the invention comprising:

(i) one or more cells

(ii) a control sample of one or more cells

(iii) a sample of tenascin-C or FBG-C or one or more of SEQ ID NOs: 10, 11, 12, 17 and 18

(iv) instructions for their use In a preferred embodiment of the twelfth aspect of the invention there is provided a kit of parts for performing the method of the third and fourth aspect of the invention comprising:

(i) one or more cells

(ii) a control sample of one or more cells

(iii) a sample of one or more of SEQ ID NOs: 10, 11, 12, 17 and 18

(iv) instructions for their use

In certain embodiments of the kit, the sample of one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 of the FBG domain of tenascin-C may refer to one or more of the specific portions of those particular sequences as defined in the earlier aspects of the invention. Optionally, a sample of SEQ ID NO: 16 or 19 of the FBG domain of tenascin-C is additionally included.

In a thirteenth aspect of the invention there is provided a kit of parts for performing the method of the third and fourth aspect of the invention comprising:

(i) TLR4;

(ii) a control sample of TLR4;

(iii) a sample of tenascin-C or FBG-c or one or more of SEQ ID NOs: 11-15 and 17-18; and

(iv) instructions for their use

In certain embodiments of the kit, the sample of one or more of SEQ ID NOs: 11-15 and 17- 18 of the FBG domain of tenascin-C may refer to one or more of the specific portions of those particular sequences as defined in the earlier aspects of the invention.

In a preferred embodiment of the thirteenth aspect of the invention there is provided a kit of parts for performing the method of the third and fourth aspect of the invention comprising:

(i) TLR4;

(ii) a control sample of TLR4;

(iii) a sample of one or more of SEQ ID NOs: 11-15 and 17-18; and

(iv) instructions for their use

In certain embodiments of the kit, the sample of one or more of SEQ ID NOs: 11-15 and 17-18 of the FBG domain of tenascin-C may refer to one or more of the specific portions of those particular sequences as defined in the earlier aspects of the invention.

The kit of the twelfth and thirteenth aspect of the invention may optionally comprise:

(v) a candidate agent.

The kit of the twelfth and thirteenth aspect of the invention may further optionally comprise

(vi) means of determining the effect of a candidate agent on either tenascin-C activity or chronic inflammation.

The kit of parts of the twelfth and thirteenth aspects may optionally have (i) and (ii) provided as one and split before use.

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

(i) an agent or composition as defined in the first, second, sixth or seventh aspects of the invention (ii) administration means

(iii) instructions for their use.

The kit of the fourteenth aspect of the invention may further optionally comprise

(iv) at least one other agent.

Optionally, the tenascin-C referred to any of the above aspects of the invention is citrullinated tenascin-C. For example, the citrullinated tenascin-C may be citrullinated at the FBG domain. The citrullinated tenascin-C may be citrullinated at only the FBG domain.

"Citrullinated tenascin-C" is intended to include tenascin-C which has been modified to any extent at any position by the post-translational process of citrullination, that is, the conversion of arginine residues to citrulline. "Citrullinated tenascin-C" also includes one or more fragments of citrullinated tenascin-C. It is therefore intended that the agents of the invention may modulate the biological activity of one or more fragments of citrullinated tenascin-C. "Citrullinated tenascin-C" may include tenascin-C which has been citrullinated at one or more specific residue(s), for example, wherein the specific residue(s) may be selected from any of the group comprising residues 50, 51, 55, 72, 120, 169, 173, 209, 214, 219, 220, 222; or combinations thereof (residue numbers as determined from SEQ ID NO: 7). Alternatively, the tenascin-C may be citrullinated at one or more specific residue(s) wherein the specific residue(s) may be selected from any of the group comprising residues 55, 72, 120, 169, 173, 209, 214, 219, and 220; or combinations thereof (residue numbers as determined from SEQ ID NO: 7). The specific citrullinated residues may comprise CIT55, CIT209, CIT214, CIT219, and/or CIT220. The specific citrullinated residue may comprise CIT50. The specific citrullinated residue may comprise CIT51. The specific citrullinated residue may comprise CIT55. The specific citrullinated residue may comprise CIT209. The specific citrullinated residue may comprise CIT214. The specific citrullinated residue may comprise CIT219. The specific citrullinated residue may comprise CIT220. In another embodiment, the specific citrullinated residues may comprise CIT209 and/or CIT214. Alternatively, the specific citrullinated residues may comprise CIT219 and/or CIT220. The specific citrullinated residue may comprise CIT222.

A specific residue that may be citrullinated in "loop 5" of the FBG domain of tenascin-C is residue 120 (from SEQ ID NO: 7).

Specific residues that may be citrullinated in "loop 8" of the FBG domain of tenascin-C are residues 169 and/or 173 (from SEQ ID NO: 7).

Specific residues that may be citrullinated in "loop 10" of the FBG domain of tenascin-C are residues 214, 219 and/or 220 (from SEQ ID NO: 7).

SEQ ID NO: 7:

PFPKDCSQAM LN GDTTSGLYTI YLN GDKAEALE VFCDMTSDGGG Wl VFLRRKN GRE N FYQNWKAYAAGFGDRREEFWLGLDN LN KITAQGQYELRVDLRDHG ETAFAVYD KF SVGDAKTRYKLKVEGYSGTAGDSM AYH NGRSFSTFD DTDSAITN CALSYKGAF WYR N C H R V NLMGRYGDNNHSQGVNWFHWKGHEHSIQFAEMK LR P S N F R N LEG R_R K R

Further details regarding citrullinated tenascin-C and its importance in modulation of a chronic inflammatory response can be found in WO 2015/104564. In a fifteenth aspect of the invention there is provided an agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with tenascin-R, preferably the biological activity is associated with the FBG domain of tenascin-R.

In a preferred embodiment, there is provided an agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with the P domain within the FBG domain of tenascin-R.

The full amino acid sequence of the FBG domain of tenascin-R is shown in SEQ ID NO: 2. The P subdomain of the FBG domain of tenascin-R comprises the sequence shown SEQ ID NO: 20, which corresponds to residues 121-206 in the full sequence of the FBG domain of tenascin-R (SEQ ID NO: 2).

For example, by "biological activity associated with the P domain" we mean that the P domain is required to effect the biological activity which is modulated. It may be that the full P domain is required to effect that biological activity, or only one or more regions/sequences within the P domain.

21. The specific sequences of those portions which are preferred with respect to tenascin-R are as follows:

SEQ ID NO: 21: RFS VED SRN LYK LRI, corresponding to residues 116-130 in SEQ ID NO: 2 (designated "loop 5" in Figure 21). SEQ ID NO: 22 : D SRN LYK LRI, corresponding to residues 121- 130 in SEQ ID NO: 2 (designated "loop 5" in Figure 21, omitting residues falling outside of the P domain). SEQ ID NO: 23: PFS TED RDN DVA VT, corresponding to residues 148-161 in SEQ ID NO: 2 (designated "loop 7" in Figure 21). SEQ ID NO: 24: KGA WWY KNC HRT, corresponding to residues 168-179 in SEQ ID NO: 2 (designated "loop 8" in Figure 21). SEQ ID NO: 25: NCAMSY, corresponding to residues 162-167 in SEQ ID NO: 2 (designated "alpha helix" in Figure 21).

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 21, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 21 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 22, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 22 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 23, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 23 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function. In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 24, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 24 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 25, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 25 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment of the fifteenth aspect of the invention, the agent modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 22 ("loop 5"); SEQ ID NO: 23 ("loop 7"); SEQ ID NO: 24 ("loop 8"); and SEQ ID NO: 25 ("alpha helix").

In a preferred embodiment of the fifteenth aspect of the invention, the agent modulates the biological activity associated with SEQ ID NO: 22 ("loop 5").

In an sixteenth aspect of the invention there is provided an agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with one or more portions of the FBG domain of tenascin-R, wherein the one or more portions are selected from SEQ ID NO: 21 ("loop 5"); SEQ ID NO: 23 ("loop 7"); SEQ ID NO: 24 ("loop 8"); and SEQ ID NO: 25 ("alpha helix").

In one embodiment of the sixteenth aspect, the agent modulates the biological activity associated with SEQ ID NO: 21 ("loop 5").

For example, by "modulated the biological activity associated with SEQ ID NO: 21" we mean that this portion of the FBG domain is required to effect the biological activity which is modulated. It may be that the full sequence of SEQ ID NO: 21 is required to effect that biological activity, or only a sub region within that sequence/loop structure.

In embodiments of the fifteenth and sixteenth aspects of the invention, wherein the agent modulates the biological activity associated with SEQ ID NO: 21 or SEQ ID NO: 22, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 23, 24, 25, 26 ("loop 10", see below) and 29 (see below).

By "additionally modulates the biological activity associated with one or more of SEQ ID NOs: 23, 24, 25, 26 and 29" we include that the agent modulates the biological activity which requires each of SEQ ID NOs: 21 (or 22) and 23, 24, 25, 26 and 29 together, or the agent modulates the biological activity which requires SEQ ID NOs 21 (or 22) and any one of, or a subset of, SEQ ID NOs: 23, 24, 25, 26 and 29. An additional preferred portion of the FBG domain of tenascin is designated "Loop 10" and the sequences shown in Figure 29. The specific amino acid sequence of this domain in tenascin-R is as follows: SEQ ID NO: 26: LMAGRKRQSLQF, residues 220-231 in SEQ ID NO: 2.

A preferred region within this sequence is made up of the positively charged residues RKR (SEQ ID NO: 29).

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 26, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 26 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 29, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 29 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In certain embodiments of the fifteenth and sixteenth aspects of the invention, the agent modulates the biological activity associated with one or more of SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25. Optionally, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 21, 22, 26 and 29.

The agents of the fifteenth and sixteenth aspects may modulate the biological activity associated with one or more particular regions within the portions defined in the embodiments above.

Preferred regions within the portion designated as "loop 5" in Figure 21 are as follows for tenascin-R: RNLYKLR (SEQ ID NO: 27). SEQ ID NO: 27 corresponds to residues 8-14 of SEQ ID NO: 21. RNLYK (SEQ ID NO: 28). SEQ ID NO: 28 corresponds to residues 8-12 of SEQ ID NO: 21.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 27, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 27 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 28, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 28 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

Therefore, in certain embodiments of the fifteenth and sixteenth aspects, the agent may modulate the biological activity associated with only a portion of the one or more sequences described in the embodiments above (i.e. only a portion of the designated loop or helix structures). In other words, the agent modulates the biological activity associated with only a sub portion of the specified sequence(s)/loop(s)/helix. Alternatively, the agent may modulate the biological activity associated with the whole of the specified sequence(s)/loop(s) /helix. Where the agent modulates the biological activity associated with more than one specified sequence(s)/loop(s)/helix, the agent may modulate the biological activity associated with only a portion of one or more of the specified sequence(s)/loop(s)/helix, and to the whole of one or more of those other specified sequence(s) /loop(s) /helix.

In one embodiment, the agent of the fifteenth and sixteenth aspects modulates the biological activity associated with three or more of the positively charged amino acids from the sequence SEQ ID NO: 27 of the FBG domain of tenascin-R. SEQ ID NO: 27 corresponds to residues 8-14 of SEQ ID NO: 21. For example, the agent may modulate the biological activity associated with the positively charged amino acids R, K and R from the sequence RNLYKLR (SEQ ID NO: 27) of loop 5 of the FBG domain of tenascin-R.

By "modulate the biological activity associated with the positively charged amino acids" we mean that those positively charged amino acids are all required to effect the biological activity which is modulated.

In a further embodiment, the agent of the fifteenth and sixteenth aspect modulates the biological activity associated with the sequence SEQ ID NO: 28 of loop 5 of the FBG domain of tenascin-R. SEQ ID NO: 28 corresponds to residues 8-12 of SEQ ID NO: 21.

In an alternative embodiment, the agent of the fifteenth and sixteenth aspects modulates the biological activity associated with the sequence SEQ ID NO: 27 of loop 5 of the FBG domain of tenascin-R. SEQ ID NO: 27 corresponds to residues 8-14 of SEQ ID NO: 21.

In a further embodiment, the agent of the fifteenth and sixteenth aspects modulates the biological activity associated with one or more of residues 152, 157, 160 and 162 (as defined in SEQ ID NO: 2) of the FBG domain of tenascin-R. These residues are located in regions designated "loop 7" or the "alpha helix" and are considered to be particularly preferred.

Optionally, the agent of the fifteenth and sixteenth aspects modulates the biological activity associated with one or more of the hydrophobic and/or positively charged amino acids from the sequence defined by residues 148-179 in SEQ ID NO: 2. In particular, the agent may modulate the biological activity associated with one or more of the hydrophobic amino acids V158, A159, V160 and T161 (residue numbers correspond to SEQ ID NO: 2). The biological activity of these one or more amino acids may be modulated in addition to or independently of residues 152, 157 and/or 162 (as defined in SEQ ID NO: 2).

The agent of the fifteenth and sixteenth aspect of the invention may modulate the biological activity associated with the specified domain or portion of tenascin-R by altering the transcription, translation and/or binding properties of tenascin-R or the specified domains or portions therein.

The agent of the fifteenth and sixteenth aspect preferably modulates the biological activity of the one or more particular regions of the FBG domain described above in relation to modulating their biological activity from binding to or activating TLR4. This may include an agent that binds near the particular region(s) in FBG and prevents binding to or activation of TLR4 by steric hindrance, an agent that binds to FBG in a completely different region but changes the conformation so as to prevent TLR4 binding or activation, an agent that binds to TLR4 in the region where these residues of FBG bind or activate, or an agent that binds to TLR4 in a completely different region but changes the conformation so as to prevent FBG binding or activation.

Such agents may be identified using methods well known in the art and as described above.

The agent of the fifteenth and sixteenth aspect of the invention may down-regulate the biological activity of tenascin-R.

The agent of the fifteenth or sixteenth aspect of the invention may up-regulate the biological activity of tenascin-R. The desirability of up-regulating activity of immune and inflammatory molecules and cells is relevant to the production of therapies for compromised immune and inflammatory patients and in the development of vaccines, (see Harandi (2009)).

The agent of the fifteenth or sixteenth aspect of the invention may be an inhibitor of transcription of tenascin-R.

The agent of the fifteenth or sixteenth aspect of the invention may be an inhibitor of translation of tenascin-R.

The agent of the fifteenth or sixteenth aspect of the invention may be an inhibitor of the binding properties associated with the specified domain or portion(s) of the FBG domain of tenascin-R. For example, the agent may alter the conformation of tenascin-R or the specified domain or portion(s) of the FBG domain of tenascin-R such that it is no longer able to bind to its receptor. In preferred embodiments, the binding refers to binding to TLR4.

It will be appreciated by persons skilled in the art that inhibition of the biological activity of tenascin-R by an agent of the invention may be in whole or in part. For example, the agent may inhibit the biological activity of tenascin-R by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably by 100% compared to the biological activity of tenascin-R on inflammatory cells which have not been exposed to the agent.

In one set of embodiments of the fifteenth or sixteenth aspects, the agent binds to or within tenascin-R, preferably to the FBG domain of tenascin-R.

By "binds to or within", we mean that the agent binds specifically to or within that protein / domain /portion/region/sequence.

In one embodiment the agent binds to or within the P domain within the FBG domain of tenascin-R. By "binds to or within the P domain" we include an agent which binds to the whole P domain or to one or more regions/sequences anywhere within the P domain.

In one embodiment the agent binds to or within one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

In an alternative embodiment, the agent binds to or within one or more portions of the FBG domain of tenascin-R, wherein the one or more portions are selected from SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

The agent may preferably bind to or within SEQ ID NO: 21 or SEQ ID NO: 22. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 23, 24, 25, 26 and 29.

In one embodiment, the agent binds to or within one or more of SEQ ID NO: 23; SEQ ID NO:

24; and SEQ ID NO: 25. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 21, 22, 26 and 29.

In one embodiment, the agent of the fifteenth or sixteenth aspect binds to the positively charged amino acids from the sequence SEQ ID NO: 27 of the FBG domain of tenascin-R. SEQ ID NO: 27 corresponds to residues 8-14 of SEQ ID NO: 21. For example, the agent has binding specificity for the positively charged amino acids R, K and R from the sequence SEQ ID NO: 27 of "loop 5" of the FBG domain of tenascin-R.

In a further embodiment, the agent binds to or within the sequence SEQ ID NO: 28 of "loop

5" of the FBG domain of tenascin-R. SEQ ID NO: 28 corresponds to residues 8-12 of SEQ ID NO: 21.

In an alternative embodiment, the agent binds to or within the sequence SEQ ID NO: 27 of "loop 5" of the FBG domain of tenascin-R. SEQ ID NO: 27 corresponds to residues 8-14 of SEQ ID NO: 21.

The agent may bind to one or more of residues 152, 157, 160 and 162 (as defined in SEQ ID

NO: 2) of the FBG domain of tenascin-R.

Optionally, the agent may bind to one or more of the hydrophobic and/or positively charged amino acids from the sequence defined by residues 148-179 in SEQ ID NO: 2. In particular, the agent may bind to one or more of the hydrophobic amino acids V158, A159, V160 and T161 (residue numbers correspond to SEQ ID NO: 2). The agent may bind to these one or more amino acids in addition to or independently of residues 152, 157 and/or 162 (as defined in SEQ ID NO: 2).

The agent of the fifteenth or sixteenth aspect of the invention may be selected from the group consisting of antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, aptamers, small inhibitor compounds, polypeptides and proteins, compounds with binding affinity for tenascin-R, and short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides.

In one embodiment of the invention the agent is an siRNA as discussed in detail in relation to the first and second aspects.

In one embodiment the agent may be a short hairpin RNA (shRNA), as discussed in detail above in relation to the first and second aspects.

The agent of the fifteenth and sixteenth aspect of the invention may be a domain of tenascin-R or variant thereof. The FBG domain has been shown to be predominantly involved in the interaction of tenascin-R with its target in relation to the persistence of chronic inflammation. Accordingly the preferred domain is the FBG domain (sequence shown in figure 2) or subdomain(s), portion(s) or variants thereof. Preferred subdomains include the P subdomain of the FBG domain of tenascin-R, "loops" 5, 7 and/or 8, and optionally also "loop 10", as well as the "alpha helix" separating loops 7 and 8. Particularly preferred subdomains include the sequence RNLYKLR of loop 5, specifically 3 or more of the positively charged amino acids of that subdomain, such as R, K and R, the sequence RNLYK of "loop 5", and one or more of residues 152, 157, 158, 159, 160, 161 and 162 (as defined in SEQ ID NO: 2) of "loop 7"/"alpha helix" of the FBG domain of tenascin-R.

In an alternative embodiment, the agent is an antisense oligonucleotide, as discussed in detail in relation to the first and second aspects.

In an embodiment where the agent is a compound with binding affinity for tenascin-R (or a domain, subdomain, or amino acid sequence thereof), the compound may bind substantially reversibly or substantially irreversibly to an active site of tenascin-R. In a further example, the compound may bind to a portion of tenascin-R that is not the active site so as to interfere with the binding of the tenascin-R to a ligand or receptor. In a still further example, the compound may bind to a portion of tenascin-R so as to decrease the proteins activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the activity of tenascin-R, for example in the activation of the tenascin-R by an "upstream activator".

Methods for detecting interactions between a test compound and tenascin-R (or a domain, subdomain, or amino acid sequence thereof) are well known in the art and described above in relation to the first and second aspects. Alternative methods of detecting binding of a polypeptide to macromolecules, or of identifying a compound that is capable of binding to the polypeptide are described in relation to the first and second aspects.

In a further embodiment of the invention, the agent is a compound which has ligand- binding capacity for tenascin-R (or for a domain, subdomain, or amino acid sequence thereof).

The agent of the fifteenth and sixteenth aspects of the invention may be an antibody or antigen-binding fragment thereof, as defined in relation to the first and second aspects.

The agent of the fifteenth and sixteenth aspect of the invention may be an antibody or antigen-binding fragment thereof which has specificity for Toll Like Receptor 4 (TLR4) or co- receptors of Toll Like Receptor 4.

The agent of the fifteenth and sixteenth aspects of the invention may be an antibody or antigen-binding fragment.

In a seventeenth aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-R comprising the steps of:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with Tenascin-R or FBG-R or one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 and the one or more candidate agents; (iii) contacting one or more cells with Tenascin-R or FBG-R or one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 and no candidate agent;

(iv) determining whether said candidate agent modulates the effect of Tenascin-R or FBG-R or one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

In a preferred embodiment of the seventeenth aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-R comprising the steps of:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 and the one or more candidate agents;

(iii) contacting one or more cells with one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 and no candidate agent;

(iv) determining whether said candidate agent modulates the effect of the one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

In an eighteenth aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-R comprising:

(i) providing one or more candidate agents;

(ii) contacting TLR4 with Tenascin-R or FBG-R or one or more of SEQ ID NOs: 20- 25 and 27-28 and the one or more candidate agents;

(iii) contacting TLR4 with Tenascin-R or FBG-R or one or more of SEQ ID NOs: 20- 25 and 27-28 and no candidate agent;

(iv) determining whether said candidate agent modulates the binding of TLR4 to the Tenascin-R or FBG-R or one or more of SEQ ID NOs: 20-25 and 27-28 in step (ii) in comparison to the binding of TLR4 to Tenascin-R or FBG-R or one or more of SEQ ID NOs: 20-25 and 27-28 in step (iii).

In a preferred embodiment of the eighteenth aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-R comprising:

(i) providing one or more candidate agents;

(ii) contacting TLR4 with one or more of SEQ ID NOs: 20-25 and 27-28 and the one or more candidate agents;

(iii) contacting TLR4 with one or more of SEQ ID NOs: 20-25 and 27-28 and no candidate agent;

(iv) determining whether said candidate agent modulates the binding of TLR4 to the one or more of SEQ ID NOs: 20-25 and 27-28 in step (ii) in comparison to the binding of TLR4 to one or more of SEQ ID NOs: 20-25 and 27-28 in step (iii).

In one embodiment of the methods of the seventeenth and eighteenth aspects, the one or more cells or TLR4 contacted in steps (ii) and (iii) is contacted with the P domain (SEQ ID NO: 20).

In another embodiment of the methods of the seventeenth and eighteenth aspects, the one or more cells or TLR4 contacted in steps (ii) and (iii) is contacted with SEQ ID NO: 21. The one or more cells or TLR4 may be additionally contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 23, 24, 25, 26 and 29.

In one embodiment of the method of the eighteenth aspect, TLR4 is contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 23, 24 and 25. TLR4 may be additionally contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 21, 26 and 29.

In certain embodiments of the methods of the seventeenth and eighteenth aspects, the one or more cells or TLR4 are contacted in steps (ii) and (iii) with three or more of the positively charged amino acids from the sequence SEQ ID NO: 27.

The one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the positively charged amino acids R, K and R from the sequence SEQ ID NO: 27 of the FBG domain of tenascin-R.

The one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the sequence SEQ ID NO: 28 of the FBG domain of tenascin-R. Alternatively, the one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the sequence SEQ ID NO: 27 of the FBG domain of tenascin- R.

In some embodiments of the methods of the seventeenth and eighteenth aspects, the one or more cells or TLR4 may be contacted in steps (ii) and (iii) with one or more of residues 152, 157, 158, 159, 160, 161 and 162 (as defined in SEQ ID NO: 2) of the FBG domain of tenascin-R.

In certain embodiments of the methods of the seventeenth and eighteenth aspects, the activity of tenascin-R is up-regulated. Alternatively, the activity of tenascin-R is down-regulated.

In one set of embodiments of the methods of the seventeenth and eighteenth aspects, the agent identified by the method binds to or within tenascin-R, preferably to or within the FBG domain of tenascin-R.

By "binds to or within", we mean that the agent binds specifically to that whole protein/domain/portion/region/sequence, or to a particular sub-region within that protein / domain /portion/region/sequence.

In one embodiment the agent identified by the method binds to or within the P domain within the FBG domain of tenascin-R. By "binds to or within the P domain" we include an agent which binds to the whole P domain or to one or more portions/regions/sequences anywhere within the P domain.

In one embodiment the agent identified by the method binds to or within one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

In an alternative embodiment, the agent identified by the method binds to or within one or more portions of the FBG domain of tenascin-R, wherein the one or more portions are selected from SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

By "binds to or within one or more portions" we include that the agent may bind to or within any one, or any group of, those portions of the FBG domain. It may be that the agent binds to the full specified portion(s), or only to one or more regions/sequences within the specified portion(s). The agent identified by the method may preferably bind to or within SEQ ID NO: 21 or SEQ ID NO: 22. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 23, 24, 25, 26 and 29.

In one embodiment, the agent identified by the method binds to or within one or more of SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 21, 22, 26 and 29.

In one embodiment, the agent identified by the method binds to three or more of the positively charged amino acids from the sequence SEQ ID NO: 27. For example, the agent binds to the positively charged amino acids R, K and R from the sequence SEQ ID NO: 27.

In a further embodiment, the agent identified by the method binds to or within the sequence SEQ ID NO: 28. In an alternative embodiment, the agent identified by the method binds to or within the sequence SEQ ID NO: 27.

The agent identified by the method may bind to one or more of residues 152, 157, 158, 159,

160, 161 and 162 (as defined in SEQ ID NO: 2) of the FBG domain of tenascin-R.

The agent identified by the methods of the seventeenth and eighteenth aspects may modulate the biological activity associated specifically with the P domain within the FBG domain of tenascin-R.

In one embodiment, the agent identified by the method modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 22 ("loop 5"); SEQ ID NO: 23 ("loop 7"); SEQ ID NO: 24 ("loop 8"); and SEQ ID NO: 25 ("alpha helix").

In a preferred embodiment, the agent identified by the method modulates the biological activity associated with SEQ ID NO: 22 ("loop 5").

The agent identified by the method of the 17th or 18th aspects may modulate the biological activity associated with one or more portions of the FBG domain of tenascin-R, wherein the one or more portions are selected from SEQ ID NO: 21 ("loop 5"); SEQ ID NO: 23 ("loop 7"); SEQ ID NO: 24 ("loop 8"); and SEQ ID NO: 25 ("alpha helix").

In one embodiment, the agent identified by the method modulates the biological activity associated with SEQ ID NO: 21 ("loop 5").

For example, by "modulates the biological activity associated with SEQ ID NO: 21" we mean that this portions of the FBG domain is required to effect the biological activity which is modulated. It may be that the full sequence of SEQ ID NO: 21 is required to effect that biological activity, or only a sub region within that sequence. In certain embodiments, wherein the agent identified by the method modulates the biological activity associated with SEQ ID NO: 21 or SEQ ID NO: 22, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 23, 24, 25, 26 and 29.

By "additionally modulates the biological activity associated with one or more of SEQ ID NOs: 23, 24, 25, 26 and 29" we include that the agent modulates the biological activity which requires each of SEQ ID NOs: 21 or 22 and 23, 24, 25, 26 and 29 together, or the agent modulates the biological activity which requires SEQ ID NOs 21 or 22 and any one of, or a subset of, SEQ ID NOs: 23, 24, 25, 26 and 29.

In certain embodiments, the agent identified by the method modulates the biological activity associated with one or more of SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25. Optionally, the agent may additionally modulates the biological activity associated with one or more of SEQ ID NOs: 21, 22, 26 and 29.

In one embodiment, the agent identified by the method modulates the biological activity associated with three or more of the positively charged amino acids from the sequence SEQ ID NO: 27. For example, the agent may modulate the biological activity associated with the positively charged amino acids R, K and R from the sequence SEQ ID NO: 27.

In a further embodiment, the agent identified by the method modulates the biological activity associated with the sequence SEQ ID NO: 28. In an alternative embodiment, the agent identified by the method modulates the biological activity associated with the sequence SEQ ID NO: 27.

In a further embodiment, the agent identified by the method modulates the biological activity associated with one or more of residues 152, 157, 158, 159, 160, 161 and 162 (as defined in SEQ ID NO: 2) of the FBG domain of tenascin-R. These residues are located in regions designated "loop 7" or the "alpha helix". The agent identified by the method may modulate the biological activity associated with the specified domain or portion of tenascin-R by altering the transcription, translation and/or binding properties of tenascin-R or the specified domains or portions therein.

Methods of determining whether the candidate agent modulate the effect of tenascin-R can be carried out using the methods of the examples.

The method of the seventeenth or eighteenth aspect of the invention may result in the activity of tenascin-R or the specified domain or portion(s) being upregulated.

The method of the seventeenth or eighteenth aspect of the invention may result in the activity of tenascin-R or the specified domain or portion(s) being downregulated.

The method of the seventeenth or eighteenth aspect of the invention may include the cells of steps (ii) and (iii) (described above) expressing Toll-like receptor 4 (TLR4).

The method of the seventeenth or eighteenth aspect of the invention may have the 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, THP1 cell lines.

In a nineteenth aspect of the invention there is provided a method of identification of an agent that modulates a chronic inflammatory response by conducting the method of the seventeenth or eighteenth aspects of the invention.

In this method the chronic inflammation may be associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, and diseases associated with neurological inflammation such as alzheimer's disease and parkinson's disease.

Of particular, but non-exclusive interest, the chronic inflammation is associated with rheumatoid arthritis (RA), alzheimer's disease and/or parkinson's disease

In a twentieth aspect of the invention there is provided an agent identified according to the method of the seventeenth or eighteenth aspects of the invention. Such an agent may modulate a chronic inflammatory response.

The agent of the twentieth aspect may down-regulate the chronic inflammatory response. The agent of the twentieth aspect may up-regulate the chronic inflammatory response.

The agent of the twentieth aspect may be selected from the group consisting of short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides, compounds with binding affinity for tenascin-R, antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, aptamers, small inhibitor compounds, polypeptides and proteins. The agent of the twentieth aspect may itself have any of the properties set out above for the agent of the 15^th and 16^th aspects of the invention.

In the 15^th, 16^th or 20^th aspects of the invention the chronic inflammation may be associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, , rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, and diseases associated with neurological inflammation such as alzheimer's disease and parkinson's disease.

In a 21st aspect of the invention there is provided a composition comprising an agent as defined in the 15^th, 16^th or 20^th aspects of the invention and a pharmaceutically acceptable carrier, excipient and/or diluent. Further details relating to possible compositions are as discussed above in relation to the seventh aspect.

The composition of the 21st aspect of the invention may further comprise at least one other agent as defined above in relation to the seventh aspect.

In a 22nd aspect of the invention there is provided an agent or composition as defined in the 15^th, 16^th, 20^th and 21^st aspects of the invention for use as a medicament.

In a 23rd aspect of the invention there is provided an agent or composition as defined in the 15^th, 16^th, 20^th and 21^st aspects of the invention for use in the treatment of a chronic inflammatory condition.

In a 24th aspect of the invention there is provided the use of an agent or composition as defined in as defined in the 15^th, 16^th, 20^th and 21^st aspects of the invention in the manufacture of a medicament for the treatment of a chronic inflammatory condition.

In an 25th aspect of the invention there is provided a method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an agent or composition as defined in the 15^th, 16^th, 20^th and 21^st aspects of the invention.

The agent, composition, use or method as defined in the 22^nd to the 25^th aspects of the invention may relate to treatment of a chronic inflammatory condition wherein the condition is associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, , rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, and diseases associated with neurological inflammation such as alzheimer's disease and parkinson's disease.

In a 26th aspect of the invention there is provided a kit of parts for performing the method of the 17^th and 18th aspect of the invention comprising:

(i) one or more cells

(ii) a control sample of one or more cells (iii) a sample of tenascin-R or FBG-R or one or more of SEQ ID NOs: 20, 21, 22, 27 and 28

(iv) instructions for their use

In a preferred embodiment of the 26th aspect of the invention there is provided a kit of parts for performing the method of the 17^th and 18^th aspect of the invention comprising:

(i) one or more cells

(ii) a control sample of one or more cells

(iii) a sample of one or more of SEQ ID NOs: 20, 21, 22, 27 and 28

(iv) instructions for their use

In certain embodiments of the kit, the sample of one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 of the FBG domain of tenascin-R may refer to one or more of the specific portions of those particular sequences as defined in the earlier aspects of the invention. Optionally, a sample of SEQ ID NO: 26 or 29 of the FBG domain of tenascin-R is additionally included.

In a 27th aspect of the invention there is provided a kit of parts for performing the method of the 17^th and 18^th aspect of the invention comprising:

(i) TLR4;

(ii) a control sample of TLR4;

(iii) a sample of tenascin-R or FBG-R or one or more of SEQ ID NOs: 21-25 and 27- 28; and

(iv) instructions for their use

In certain embodiments of the kit, the sample of one or more of SEQ ID NOs: 21-25 and 27-28 of the FBG domain of tenascin-R may refer to one or more of the specific portions of those particular sequences as defined in the earlier aspects of the invention.

In a preferred embodiment of the 27^th aspect of the invention there is provided a kit of parts for performing the method of the 17^th and 18^th aspect of the invention comprising:

(i) TLR4;

(ii) a control sample of TLR4;

(iii) a sample of one or more of SEQ ID NOs: 21-25 and 27-28; and

(iv) instructions for their use

In certain embodiments of the kit, the sample of one or more of SEQ ID NOs: 21-25 and 27- 28 of the FBG domain of tenascin-R may refer to one or more of the specific portions of those particular sequences as defined in the earlier aspects of the invention.

The kit of the 26th and 27th aspect of the invention may optionally comprise:

(v) a candidate agent.

The kit of the 26th and 27th aspect of the invention may further optionally comprise

(vi) means of determining the effect of a candidate agent on either tenascin-R activity or chronic inflammation.

The kit of parts of the 26th and 27th aspects may optionally have (i) and (ii) provided as one and split before use. In a 28th aspect of the invention there is provided a kit of parts comprising:

(i) an agent or composition as defined in the 15^th, 16^th, 20^th and 21^st aspects of the invention

(ii) administration means

(iii) instructions for their use

The kit of the 28th aspect of the invention may further optionally comprise

(iv) at least one other agent.

Optionally, the tenascin-R referred to any of the above aspects of the invention is citrullinated tenascin-R. For example, the citrullinated tenascin-R may be citrullinated at the FBG domain. The citrullinated tenascin-R may be citrullinated at only the FBG domain.

"Citrullinated tenascin-R" is intended to include tenascin-R which has been modified to any extent at any position by the post-translational process of citrullination, that is, the conversion of arginine residues to citrulline. "Citrullinated tenascin-R" also includes one or more fragments of citrullinated tenascin-R. It is therefore intended that the agents of the invention may modulate the biological activity of one or more fragments of citrullinated tenascin-R. "Citrullinated tenascin-R" may include tenascin-R which has been citrullinated at one or more specific residue(s).

Further details regarding citrullinated of tenascin and its importance in modulation of a chronic inflammatory response can be found in WO 2015/104564.

In a 29th aspect of the invention there is provided an agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with tenascin-W, preferably the biological activity is associated with the FBG domain of tenascin-W.

In a preferred embodiment, there is provided an agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with the P domain within the FBG domain of tenascin-W.

The full amino acid sequence of the FBG domain of tenascin-W is shown in SEQ ID NO: 3

The P subdomain of the FBG domain of tenascin-W comprises the following sequence (SEQ ID NO: 30) : SSKERYKL TVGKYRGTAG DALTYHNGWK FTTFDRDNDI ALSNCALTHH GGWWYKNCHL ANPNGRYGET KHSEGVNWEP WKGHEFSI

This P domain sequence corresponds to residues 123-208 in the full sequence of the FBG domain of tenascin-W (SEQ ID NO: 3).

The P domain within FBG can be broken down into various portions, as shown in Figure 21.

The specific sequences of those portions which are preferred with respect to tenascin-W are as follow: SEQ ID NO: 31: FFQ VAS SKE RYK LTV, corresponding to residues 118-132 in SEQ ID NO: 3 (designated "loop 5" in Figure 21). SEQ ID NO: 32 : S SKE RYK LTV, corresponding to residues 123-132 in SEQ ID NO: 3 (designated "loop 5" in Figure 21, omitting residues falling outside of the P domain). SEQ ID NO: 33: KFT TFD RDN DIA LS, corresponding to residues 150-163 in SEQ ID NO: 3 (designated "loop 7" in Figure 21). SEQ ID NO: 34: HGG WWY KNC HLA, corresponding to residues 170-181 in SEQ ID NO: 3 (designated "loop 8" in Figure 21). SEQ ID NO: 35: NCALTH, corresponding to residues 164-169 in SEQ ID NO: 3 (designated "alpha helix" in Figure 21).

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 31, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 31 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 32, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 32 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 33, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 33 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 34, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 34 wherein one or more, preferably one or two, amino acids are replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 35, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 35 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment of the 29th aspect of the invention, the agent modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 32 ("loop 5"); SEQ ID NO: 33 ("loop 7"); SEQ ID NO: 34

("loop 8"); and SEQ ID NO: 35 ("alpha helix").

In a preferred embodiment of the 29th aspect of the invention, the agent modulates the biological activity associated with SEQ ID NO: 32 ("loop 5").

In an 30th aspect of the invention there is provided an agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with one or more portions of the FBG domain of tenascin-W, wherein the one or more portions are selected from SEQ ID NO: 31 ("loop 5"); SEQ ID NO: 33 ("loop 7"); SEQ ID NO: 34 ("loop 8"); and SEQ ID NO:

35 ("alpha helix"). For example, by "associated with one or more portions" we mean that any one, or any group of, those portions of the FBG domain may be required to effect the biological activity which is modulated. It may be that the full specified portion(s) or sequence thereof is required to effect that biological activity, or only one or more regions/sequences within the specified portion(s).

In one embodiment of the 30th aspect, the agent modulates the biological activity associated with SEQ ID NO: 31 ("loop 5").

For example, by "modulated the biological activity associated with SEQ ID NO: 31" we mean that this portion of the FBG domain is required to effect the biological activity which is modulated. It may be that the full sequence of SEQ ID NO: 31 is required to effect that biological activity, or only a sub region within that sequence/loop structure.

In embodiments of the 29^th and 30^th aspects of the invention, wherein the agent modulates the biological activity associated with SEQ ID NO: 31 or SEQ ID NO: 32, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 33, 34, 35, 36 ("loop 10", see below) and 39 (see below).

By "additionally modulates the biological activity associated with one or more of SEQ ID

NOs: 33, 34, 35, 36 and 39" we include that the agent modulates the biological activity which requires each of SEQ ID NOs: 31 (or 32) and 33, 34, 35, 36 and 39 together, or the agent modulates the biological activity which requires SEQ ID NOs 31 (or 32) and any one of, or a subset of, SEQ ID NOs: 33, 34, 35, 36 and 39.

An additional preferred portion of the FBG domain of tenascin is designated "Loop 10" and the sequences shown in Figure 29. The specific amino acid sequence of this domain in tenascin-W is as follows: SEQ ID NO: 36: PVL GRK KRT LRG RLR TF, residues 224-240 in SEQ ID NO: 3.

A preferred region within this sequence is made up of the residues RKKR (SEQ ID NO: 39).

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 36, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 36 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 39, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 39 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In certain embodiments of the 29th and 30th aspects of the invention, the agent modulates the biological activity associated with one or more of SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35. Optionally, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 31, 32, 36 and 39.

The agents of the 29th and 30th aspects may modulate the biological activity associated with one or more particular regions within the portions defined in the embodiments above. Preferred regions within the portion designated as "loop 5" in Figure 21 are as follows for tenascin-W:

KERYKLT (SEQ ID NO: 37). SEQ ID NO: 37 corresponds to residues 8-14 of SEQ ID NO: 31.

KERYK (SEQ ID NO: 38). SEQ ID NO: 38 corresponds to residues 8-12 of SEQ ID NO: 31. In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 37, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 37 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

In one embodiment there is provided a peptide/epitope shown in SEQ ID NO: 38, for example consisting of said sequence. In one embodiment there is provided a derivative of SEQ ID NO: 38 wherein one or more, preferably one or two, amino acids is replaced or deleted, in particular wherein the derivative has reduced deleterious biological function or no deleterious biological function.

Therefore, in certain embodiments of the 29th and 30th aspects, the agent may modulate the biological activity associated with only a portion of the one or more sequences described in the embodiments above (i.e. only a portion of the designated loop or helix structures). In other words, the agent modulates the biological activity associated with only a sub portion of the specified sequence(s) /loop (s) /helix. Alternatively, the agent may modulate the biological activity associated with the whole of the specified sequence (s) /loop (s) /helix. Where the agent modulates the biological activity associated with more than one specified sequence(s)/loop(s)/helix, the agent may modulate the biological activity associated with only a portion of one or more of the specified sequence(s)/loop(s)/helix, and to the whole of one or more of those other specified sequence(s) /loop(s) /helix.

In one embodiment, the agent of the 29th and 30th aspects modulates the biological activity associated with three or more of the positively charged amino acids from the sequence SEQ ID NO: 37 of the FBG domain of tenascin-W. SEQ ID NO: 37 corresponds to residues 8-14 of SEQ ID NO: 31. For example, the agent may modulate the biological activity associated with the positively charged amino acids K, R and K from the sequence KERYKLT (SEQ ID NO: 37) of loop 5 of the FBG domain of tenascin-W.

In a further embodiment, the agent of the 29th and 30th aspect modulates the biological activity associated with the sequence SEQ ID NO: 38 of loop 5 of the FBG domain of tenascin-W. SEQ ID NO: 38 corresponds to residues 8-12 of SEQ ID NO: 31.

In an alternative embodiment, the agent of the 29th and 30th aspects modulates the biological activity associated with the sequence SEQ ID NO: 37 of loop 5 of the FBG domain of tenascin-W. SEQ ID NO: 37 corresponds to residues 8-14 of SEQ ID NO: 31. In a further embodiment, the agent of the 29th and 30th aspects modulates the biological activity associated with one or more of residues 154, 159, 162 and 164 (as defined in SEQ ID NO: 3) of the FBG domain of tenascin-W. These residues are located in regions designated "loop 7" or the "alpha helix" and are considered to be particularly preferred.

Optionally, the agent of the 29th and 30th aspects modulates the biological activity associated with one or more of the hydrophobic and/or positively charged amino acids from the sequence defined by residues 150-181 in SEQ ID NO: 3. In particular, the agent may modulate the biological activity associated with one or more of the hydrophobic amino acids 1160, A161, L162 and F154 (residue numbers correspond to SEQ ID NO: 3). The biological activity of these one or more amino acids may be modulated in addition to or independently of residues 159 and/or 164 (as defined in SEQ ID NO: 3).

The agent of the 29th and 30th aspect of the invention may modulate the biological activity associated with the specified domain or portion of tenascin-W by altering the transcription, translation and/or binding properties of tenascin-W or the specified domains or portions therein.

The agent of the 29th and 30th aspect preferably modulates the biological activity of the one or more particular regions of the FBG domain described above in relation to modulating their biological activity from binding to or activating TLR4. This may include an agent that binds near the particular region(s) in FBG and prevents binding to or activation of TLR4 by steric hindrance, an agent that binds to FBG in a completely different region but changes the conformation so as to prevent TLR4 binding or activation, an agent that binds to TLR4 in the region where these residues of FBG bind or activate, or an agent that binds to TLR4 in a completely different region but changes the conformation so as to prevent FBG binding or activation.

Such agents may be identified using methods well known in the art and as described above. The agent of the 29th and 30th aspect of the invention may down-regulate the biological activity of tenascin-W.

The agent of the 29th or 30th aspect of the invention may up-regulate the biological activity of tenascin-W. The desirability of up-regulating activity of immune and inflammatory molecules and cells is relevant to the production of therapies for compromised immune and inflammatory patients and in the development of vaccines, (see Harandi (2009)).

The agent of the 29th or 30th aspect of the invention may be an inhibitor of transcription of tenascin-W.

The agent of the 29th or 30th aspect of the invention may be an inhibitor of translation of tenascin-W.

The agent of the 29th or 30th aspect of the invention may be an inhibitor of the binding properties associated with the specified domain or portion(s) of the FBG domain of tenascin-W. For example, the agent may alter the conformation of tenascin-W or the specified domain or portion(s) of the FBG domain of tenascin-W such that it is no longer able to bind to its receptor. In preferred embodiments, the binding refers to binding to TLR4.

It will be appreciated by persons skilled in the art that inhibition of the biological activity of tenascin-W by an agent of the invention may be in whole or in part. For example, the agent may inhibit the biological activity of tenascin-W by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably by 100% compared to the biological activity of tenascin-W on inflammatory cells which have not been exposed to the agent.

In one set of embodiments of the 29th or 30th aspects, the agent binds to or within tenascin-W, preferably to the FBG domain of tenascin-W.

In one embodiment the agent binds to or within the P domain within the FBG domain of tenascin-W. By "binds to or within the P domain" we include an agent which binds to the whole P domain or to one or more regions/sequences anywhere within the P domain.

In one embodiment the agent binds to or within one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

In an alternative embodiment, the agent binds to or within one or more portions of the FBG domain of tenascin-W, wherein the one or more portions are selected from SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

The agent may preferably bind to or within SEQ ID NO: 31 or SEQ ID NO: 32. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 33, 34, 35, 36 and 39.

In one embodiment, the agent binds to or within one or more of SEQ ID NO: 33; SEQ ID NO:

34; and SEQ ID NO: 35. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 31, 32, 36 and 39.

In one embodiment, the agent of the 29th or 30th aspect binds to the positively charged amino acids from the sequence SEQ ID NO: 37 of the FBG domain of tenascin-W. SEQ ID NO: 37 corresponds to residues 8-14 of SEQ ID NO: 31. For example, the agent has binding specificity for the positively charged amino acids K, R and K from the sequence SEQ ID NO: 37 of "loop 5" of the FBG domain of tenascin-W.

In a further embodiment, the agent binds to or within the sequence SEQ ID NO: 38 of "loop

5" of the FBG domain of tenascin-W. SEQ ID NO: 38 corresponds to residues 8-12 of SEQ ID NO: 31.

In an alternative embodiment, the agent binds to or within the sequence SEQ ID NO: 37 of "loop 5" of the FBG domain of tenascin-W. SEQ ID NO: 37 corresponds to residues 8-14 of SEQ ID NO: 31. The agent may bind to one or more of residues 154, 159, 162 and 164 (as defined in SEQ ID NO: 3) of the FBG domain of tenascin-W.

Optionally, the agent may bind to one or more of the hydrophobic and/or positively charged amino acids from the sequence defined by residues 150-181 in SEQ ID NO: 3. In particular, the agent may bind to one or more of the hydrophobic amino acids 1160, A161, L162 and F154 (residue numbers correspond to SEQ ID NO: 3). The agent may bind to these one or more amino acids in addition to or independently of residues 159 and/or 164 (as defined in SEQ ID NO: 3).

The agent of the 29th or 30th aspect of the invention may be selected from the group consisting of antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, aptamers, small inhibitor compounds, polypeptides and proteins, compounds with binding affinity for tenascin-W, and short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides.

In one embodiment the agent may be a short hairpin RNA (shRNA), as discussed above in relation to the first and second aspects.

The agent of the 29th and 30th aspect of the invention may be a domain of tenascin-W or variant thereof. The FBG domain has been shown to be predominantly involved in the interaction of tenascin-W with its target in relation to the persistence of chronic inflammation. Accordingly the preferred domain is the FBG domain (sequence shown in figure 2) or subdomain(s), portion(s) or variants thereof. Preferred subdomains include the P subdomain of the FBG domain of tenascin- W, "loops" 5, 7 and/or 8, and optionally also "loop 10", as well as the "alpha helix" separating loops 7 and 8. Particularly preferred subdomains include the sequence KERYKLT (SEQ ID NO: 37) of loop 5, specifically 3 or more of the positively charged amino acids of that subdomain, such as K, R and K, the sequence KERYK (SEQ ID NO: 38) of "loop 5", and one or more of residues 154, 159, 160, 161, 162 and 164 (as defined in SEQ ID NO: 3) of "loop 7"/"alpha helix" of the FBG domain of tenascin-W.

In an embodiment where the agent is a compound with binding affinity for tenascin-W (or a domain, subdomain, or amino acid sequence thereof), the compound may bind substantially reversibly or substantially irreversibly to an active site of tenascin-W. In a further example, the compound may bind to a portion of tenascin-W that is not the active site so as to interfere with the binding of the tenascin-W to a ligand or receptor. In a still further example, the compound may bind to a portion of tenascin-W so as to decrease the proteins activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the activity of tenascin-W, for example in the activation of the tenascin-W by an "upstream activator".

Methods for detecting interactions between a test compound and tenascin-W (or a domain, subdomain, or amino acid sequence thereof) are well known in the art and described above in relation to the first and second aspects. Alternative methods of detecting binding of a polypeptide to macromolecules, or of identifying a compound that is capable of binding to the polypeptide are described in relation to the first and second aspects.

In a further embodiment of the invention, the agent is a compound which has ligand- binding capacity for tenascin-W (or for a domain, subdomain, or amino acid sequence thereof).

The agent of the 29th and 30th aspects of the invention may be an antibody or antigen- binding fragment thereof, as defined in relation to the first and second aspects.

The agent of the 29th and 30th aspect of the invention may be an antibody or antigen- binding fragment thereof which has specificity for Toll Like Receptor 4 (TLR4) or co-receptors of Toll Like Receptor 4.

The agent of the 29th and 30th aspects of the invention may be an antibody or antigen- binding fragment.

In a 31st aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-W comprising the steps of:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with Tenascin-W or FBG-W or one or more of SEQ ID

NOs: 30, 31, 32, 37 and 38 and the one or more candidate agents;

(iii) contacting one or more cells with Tenascin-W or FBG-W or one or more of SEQ ID

NOs: 30, 31, 32, 37 and 38 and no candidate agent;

(iv) determining whether said candidate agent modulates the effect of Tenascin-W or

FBG-W or one or more of SEQ ID NOs: 30, 31, 32, 37 and 38 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

In a preferred embodiment of the 31st aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-W comprising the steps of:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with one or more of SEQ ID NOs: 30, 31, 32, 37 and

38 and the one or more candidate agents;

(iii) contacting one or more cells with one or more of SEQ ID NOs: 30, 31, 32, 37 and

38 and no candidate agent;

(iv) determining whether said candidate agent modulates the effect of the one or more of SEQ ID NOs: 30, 31, 32, 37 and 38 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

In an 32nd aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-W comprising:

(i) providing one or more candidate agents;

(ii) contacting TLR4 with Tenascin-W or FBG-W or one or more of SEQ ID NOs: 30-35 and 37-38 and the one or more candidate agents; (iii) contacting TLR4 with Tenascin-W or FBG-W or one or more of SEQ ID NOs: 30-35 and 37-38 and no candidate agent;

(iv) determining whether said candidate agent modulates the binding of TLR4 to the Tenascin-W or FBG-W or one or more of SEQ ID NOs: 30-35 and 37-38 in step (ii) in comparison to the binding of TLR4 to Tenascin-W or FBG-W or one or more of SEQ

ID NOs: 30-35 and 37-38 in step (iii).

In a preferred embodiment of the 32nd aspect of the invention there is provided a method of identifying an agent that modulates the activity of tenascin-W comprising:

(v) providing one or more candidate agents;

(vi) contacting TLR4 with one or more of SEQ ID NOs: 30-35 and 37-38 and the one or more candidate agents;

(vii) contacting TLR4 with one or more of SEQ ID NOs: 30-35 and 37-38 and no candidate agent;

(viii) determining whether said candidate agent modulates the binding of TLR4 to the one or more of SEQ ID NOs: 30-35 and 37-38 in step (ii) in comparison to the binding of TLR4 to one or more of SEQ ID NOs: 30-35 and 37-38 in step (iii).

In one embodiment of the methods of the 31st and 32nd aspects, the one or more cells or TLR4 contacted in steps (ii) and (iii) is contacted with the P domain (SEQ ID NO: 30).

In another embodiment of the methods of the 31st and 32nd aspects, the one or more cells or TLR4 contacted in steps (ii) and (iii) is contacted with SEQ ID NO: 31. The one or more cells or TLR4 may be additionally contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 33, 34, 35, 36 and 39.

In one embodiment of the method of the 32nd aspect, TLR4 is contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 33, 34 and 35. TLR4 may be additionally contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 31, 36 and 39.

In certain embodiments of the methods of the 31st and 32nd aspects, the one or more cells or TLR4 are contacted in steps (ii) and (iii) with three or more of the positively charged amino acids from the sequence SEQ ID NO: 37.

The one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the positively charged amino acids K, R and K from the sequence SEQ ID NO: 37 of the FBG domain of tenascin-W.

The one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the sequence SEQ ID NO: 38 of the FBG domain of tenascin-W. Alternatively, the one or more cells or TLR4 may be contacted in steps (ii) and (iii) with the sequence SEQ ID NO: 37 of the FBG domain of tenascin- W.

In some embodiments of the methods of the 31st and 32nd aspects, the one or more cells or TLR4 may be contacted in steps (ii) and (iii) with one or more of residues 154, 159, 160, 161, 162 and 164 (as defined in SEQ ID NO: 3) of the FBG domain of tenascin-W.

In certain embodiments of the methods of the 31st and 32nd aspects, the activity of tenascin-W is up-regulated. Alternatively, the activity of tenascin-W is down-regulated. In one set of embodiments of the methods of the 31st and 32nd aspects, the agent identified by the method binds to or within tenascin-W, preferably to or within the FBG domain of tenascin-W.

In one embodiment the agent identified by the method binds to or within the P domain within the FBG domain of tenascin-W. By "binds to or within the P domain" we include an agent which binds to the whole P domain or to one or more portions/regions/sequences anywhere within the P domain.

In one embodiment the agent identified by the method binds to or within one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

In an alternative embodiment, the agent identified by the method binds to or within one or more portions of the FBG domain of tenascin-W, wherein the one or more portions are selected from SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

The agent identified by the method may preferably bind to or within SEQ ID NO: 31 or SEQ ID NO: 32. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 33, 34, 35, 36 and 39.

In one embodiment, the agent identified by the method binds to or within one or more of SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35. Optionally, the agent may additionally bind to or within one or more of SEQ ID NOs: 31, 32, 36 and 39.

In one embodiment, the agent identified by the method binds to three or more of the positively charged amino acids from the sequence SEQ ID NO: 37. For example, the agent binds to the positively charged amino acids K, R and K from the sequence SEQ ID NO: 37.

In a further embodiment, the agent identified by the method binds to or within the sequence SEQ ID NO: 38. In an alternative embodiment, the agent identified by the method binds to or within the sequence SEQ ID NO: 37.

The agent identified by the method may bind to one or more of residues 154, 159, 161, 161,

162 and 164 (as defined in SEQ ID NO: 3) of the FBG domain of tenascin-W.

The agent identified by the methods of the 31st and 32nd aspects may modulate the biological activity associated specifically with the P domain within the FBG domain of tenascin-W.

In one embodiment, the agent identified by the method modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 32 ("loop 5"); SEQ ID NO: 33 ("loop 7"); SEQ ID NO: 34 ("loop 8"); and SEQ ID NO: 35 ("alpha helix").

In a preferred embodiment, the agent identified by the method modulates the biological activity associated with SEQ ID NO: 32 ("loop 5").

The agent identified by the method of the 31^st or 32^nd aspects may modulate the biological activity associated with one or more portions of the FBG domain of tenascin-W, wherein the one or more portions are selected from SEQ ID NO: 31 ("loop 5"); SEQ ID NO: 33 ("loop 7"); SEQ ID NO: 34 ("loop 8"); and SEQ ID NO: 35 ("alpha helix").

In one embodiment, the agent identified by the method modulates the biological activity associated with SEQ ID NO: 31 ("loop 5").

For example, by "modulates the biological activity associated with SEQ ID NO: 31" we mean that this portions of the FBG domain is required to effect the biological activity which is modulated. It may be that the full sequence of SEQ ID NO: 31 is required to effect that biological activity, or only a sub region within that sequence.

In certain embodiments, wherein the agent identified by the method modulates the biological activity associated with SEQ ID NO: 31 or SEQ ID NO: 32, the agent may additionally modulate the biological activity associated with one or more of SEQ ID NOs: 33, 34, 35, 36 and 39.

By "additionally modulates the biological activity associated with one or more of SEQ ID NOs: 33, 34, 35, 36 and 39" we include that the agent modulates the biological activity which requires each of SEQ ID NOs: 31 or 32 and 33, 34, 35, 36 and 39 together, or the agent modulates the biological activity which requires SEQ ID NOs 31 or 32 and any one of, or a subset of, SEQ ID NOs: 33, 34, 35, 36 and 39.

In certain embodiments, the agent identified by the method modulates the biological activity associated with one or more of SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35. Optionally, the agent may additionally modulates the biological activity associated with one or more of SEQ ID NOs: 31, 32, 36 and 39.

In one embodiment, the agent identified by the method modulates the biological activity associated with three or more of the positively charged amino acids from the sequence SEQ ID NO: 37. For example, the agent may modulate the biological activity associated with the positively charged amino acids K, R and K from the sequence SEQ ID NO: 37.

In a further embodiment, the agent identified by the method modulates the biological activity associated with the sequence SEQ ID NO: 38. In an alternative embodiment, the agent identified by the method modulates the biological activity associated with the sequence SEQ ID NO: 37.

In a further embodiment, the agent identified by the method modulates the biological activity associated with one or more of residues 154, 159, 160, 161, 162 and 164 (as defined in SEQ ID NO: 3) of the FBG domain of tenascin-W. These residues are located in regions designated "loop 7" or the "alpha helix".

The agent identified by the method may modulate the biological activity associated with the specified domain or portion of tenascin-W by altering the transcription, translation and/or binding properties of tenascin-W or the specified domains or portions therein.

Methods of determining whether the candidate agent modulate the effect of tenascin-W can be carried out using the methods of the examples.

The method of the 31st or 32nd aspect of the invention may result in the activity of tenascin-W or the specified domain or portion(s) being upregulated.

The method of the 31st or 32nd aspect of the invention may result in the activity of tenascin-W or the specified domain or portion(s) being downregulated.

The method of the 31st or 32nd aspect of the invention may include the cells of steps (ii) and (iii) (described above) expressing Toll-like receptor 4 (TLR4).

The method of the 31st or 32nd aspect of the invention may have the 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, THP1 cell lines.

In a 33rd aspect of the invention there is provided a method of identification of an agent that modulates a chronic inflammatory response by conducting the method of the 31st or 32nd aspects of the invention.

In this method the chronic inflammation may be associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, alzheimer's disease and parkinson's disease.

Of particular, but non-exclusive interest, the chronic inflammation is associated with rheumatoid arthritis (RA) or tumours.

In a 34th aspect of the invention there is provided an agent identified according to the method of the 31st or 32nd aspects of the invention. Such an agent may modulate a chronic inflammatory response.

The agent of the 34th aspect may down-regulate the chronic inflammatory response.

The agent of the 34th aspect may up-regulate the chronic inflammatory response.

The agent of the 34th aspect may be selected from the group consisting of short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides, compounds with binding affinity for tenascin-W, antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, aptamers, small inhibitor compounds, polypeptides and proteins.

The agent of the 34th aspect may itself have any of the properties set out above for the agent of the 29th and 30th aspects of the invention.

In the 29th, 30th or 34th aspects of the invention the chronic inflammation may be associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, alzheimer's disease and parkinson's disease.

In a 35th aspect of the invention there is provided a composition comprising an agent as defined in the 29th, 30th or 34th aspects of the invention and a pharmaceutically acceptable carrier, excipient and/or diluent. Further details relating to possible compositions are as discussed above in relation to the seventh aspect.

The composition of the 35th aspect of the invention may further comprise at least one other agent as defined above in relation to the seventh aspect.

In a 36th aspect of the invention there is provided an agent or composition as defined in the 29th, 30th, 34th and 35th aspects of the invention for use as a medicament.

In a 37th aspect of the invention there is provided an agent or composition as defined in the 29th, 30th, 34th and 35th aspects of the invention for use in the treatment of a chronic inflammatory condition.

In a 38th aspect of the invention there is provided the use of an agent or composition as defined in as defined in the 29th, 30th, 34th and 35th aspects of the invention in the manufacture of a medicament for the treatment of a chronic inflammatory condition. In an 39th aspect of the invention there is provided a method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an agent or composition as defined in the 29th, 30th, 34th and 35th aspects of the invention.

The agent, composition, use or method as defined in the 36th to the 39th aspects of the invention may relate to treatment of a chronic inflammatory condition wherein the condition is associated with any condition associated with inappropriate inflammation. Such conditions include, but are not limited to, rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, alzheimer's disease and parkinson's disease.

In a 40th aspect of the invention there is provided a kit of parts for performing the method of the 31st and 32nd aspect of the invention comprising:

(ix) one or more cells

(x) a control sample of one or more cells

(xi) a sample of tenascin-W or FBG-W or one or more of SEQ ID NOs: 30, 31, 32, 37 & 38

(xii) instructions for their use

In a preferred embodiment of the 40th aspect of the invention there is provided a kit of parts for performing the method of the 31st and 32nd aspect of the invention comprising:

(i) one or more cells

(ii) a control sample of one or more cells

(iii) a sample of one or more of SEQ ID NOs: 30, 31, 32, 37 and 38

(iv) instructions for their use.

In certain embodiments of the kit, the sample of one or more of SEQ ID NOs: 30, 31, 32, 37 and 38 of the FBG domain of tenascin-W may refer to one or more of the specific portions of those particular sequences as defined in the earlier aspects of the invention. Optionally, a sample of SEQ ID NO: 36 or 39 of the FBG domain of tenascin-W is additionally included.

In a 41st aspect of the invention there is provided a kit of parts for performing the method of the 31st and 32nd aspect of the invention comprising:

(v) TLR4;

(vi) a control sample of TLR4;

(vii) a sample of tenascin-W or FBG-W or one or more of SEQ ID NOs: 31-35 & 37-38; and

(viii) instructions for their use.

In certain embodiments of the kit, the sample of one or more of SEQ ID NOs: 31-35 and 37-38 of the FBG domain of tenascin-W may refer to one or more of the specific portions of those particular sequences as defined in the earlier aspects of the invention.

In a preferred embodiment of the 41st aspect of the invention there is provided a kit of parts for performing the method of the 31st and 32nd aspect of the invention comprising:

(ix) TLR4; (x) a control sample of TLR4;

(xi) a sample of one or more of SEQ ID NOs: 31-35 and 37-38; and

(xii) instructions for their use.

The kit of the 40th and 41st aspect of the invention may optionally comprise:

(v) a candidate agent.

The kit of the 40th and 41st aspect of the invention may further optionally comprise

(vi) means of determining the effect of a candidate agent on either tenascin-W activity or chronic inflammation.

The kit of parts of the 40th and 41st aspects may optionally have (i) and (ii) provided as one and split before use.

In a 42nd aspect of the invention there is provided a kit of parts comprising:

(i) an agent or composition as defined in the 29th, 30th, 34th and 35th aspects of the invention

(ii) administration means

(iii) instructions for their use

The kit of the 42nd aspect of the invention may further optionally comprise

(iv) at least one other agent.

Optionally, the tenascin-W referred to any of the above aspects of the invention is citrullinated tenascin-W. For example, the citrullinated tenascin-W may be citrullinated at the FBG domain. The citrullinated tenascin-W may be citrullinated at only the FBG domain.

"Citrullinated tenascin-W" is intended to include tenascin-W which has been modified to any extent at any position by the post-translational process of citrullination, that is, the conversion of arginine residues to citrulline. "Citrullinated tenascin-W" also includes one or more fragments of citrullinated tenascin-W. It is therefore intended that the agents of the invention may modulate the biological activity of one or more fragments of citrullinated tenascin-W. "Citrullinated tenascin-W" may include tenascin-W which has been citrullinated at one or more specific residue(s).

In one independent aspect the present disclosure provides an antibody or antigen-binding fragment specific to a novel peptide or epitope sequence disclosed herein.

In one embodiment there is provided an antibody or an antigen-binding fragment specific to the P domain of a tenascin protein, for example tenascin C, R or W. In one embodiment the tenascin protein is not tenascin X.

"Specific to the P domain of a tenascin protein" as employed herein refers to an antibody or antigen-binding fragment comprising a binding domain that: only recognises the antigen to which it is specific (i.e. the P domain including a epitope or fragment thereof); or has significantly higher binding affinity to the antigen to which it is specific (i.e. the P domain including a epitope or fragment thereof) compared to binding to antigens to which it is non-specific, for example at least 5, 6, 7, 8, 9, 10 times higher binding affinity. The antibody or antigen-binding fragment specific to the P domain will generally bind, recognise, identify and/or modulate the biological activity of the sequence even when the P domain is present as part of a bigger sequence or protein, such as endogenous tenascin, in particular endogenous tenascin C, R or W.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 or a combination thereof, such as specific to two or more of said sequences.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 21,

22, 23, 24, 25, 26, 27, 28, 29 or a combination thereof, such as specific to two or more of said sequences.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39 or a combination thereof, such as specific to two or more of said sequences.

"Specific to a SEQ ID NO" as employed herein refers to an antibody or antigen-binding fragment comprising a binding domain that: only recognises the antigen to which it is specific (i.e. the SEQ ID NO referred to); or has significantly higher binding affinity to the antigen to which it is specific (i.e. the SEQ ID NO referred to) compared to binding to antigens to which it is non-specific, for example at least 5, 6, 7, 8, 9, 10 times higher binding affinity. The antibody or antigen-binding fragment specific to the SEQ ID NO will generally bind, recognise, identify and/or modulate the biological activity of the sequence even when the SEQ ID NO is present as part of a bigger sequence or protein, such as endogenous tenascin, in particular endogenous tenascin C, R or W.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 11 or 12, in particular sequence 12.

Some residues of SEQ ID NO: 11 fall outside the P domain. In one independent aspect there is provide an antibody or antigen-binding fragment specific to SEQ ID NO: 11 which bind partly or wholly outside the P domain and modulates the biological activity of that sequence.

SEQ ID NO: 12 are the residues derived from SEQ ID NO: 11, which fall within the P domain. In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 13.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 14.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 15.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 16.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 17. In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 18.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 19.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 11 or 12, in particular sequence 12. Some residues of SEQ ID NO: 21 fall outside the P domain. In one independent aspect there is provide an antibody or antigen-binding fragment specific to SEQ ID NO: 21 which bind partly or wholly outside the P domain and modulates the biological activity of that sequence.

SEQ ID NO: 22 are the residues derived from SEQ ID NO: 21, which fall within the P domain. In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 23.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 24.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 25.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 26.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 27. In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 28.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 29.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 31 or 32, in particular sequence 32.

Some residues of SEQ ID NO: 31 fall outside the P domain. In one independent aspect there is provide an antibody or antigen-binding fragment specific to SEQ ID NO: 31 which bind partly or wholly outside the P domain and modulates the biological activity of that sequence.

SEQ ID NO: 32 are the residues derived from SEQ ID NO: 31, which fall within the P domain.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 33.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 34. In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 35.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 36.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 37.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 38.

In one embodiment the antigen or antigen-binding fragment is specific to SEQ ID NO: 39. In one embodiment an antibody or antigen-binding fragment according to the present disclosure is human or humanised.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure is chimeric.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure is monoclonal.

In one embodiment there is provided an antibody or antigen-binding fragment which cross-blocks an antibody or antigen-binding fragment according to the present disclosure which is specific to the P domain or tenascin, for example tenascin-C, in particular a sequence selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 or combinations thereof.

In one embodiment there is provided an antibody or antigen-binding fragment which binds the same epitope as an antibody or antigen-binding fragment according to the present disclosure which is specific to the P domain or tenascin, for example tenascin-C, in particular a sequence selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 or combinations thereof. In one embodiment an antibody or antigen-binding fragment according to the present disclosure is an inhibitor, for example an inhibitor of one or more biological activities of the P domain of a tenascin protein, for example tenascin-C. In one embodiment the biological activity or activities is one described herein.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure neutralises one or more deleterious biological effects associated with the P domain of tenascin, for example the P domain of tenascin-C, such as a fragment disclosed in any one of the sequences shown in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 or a combination thereof.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure binds one or more tenascin proteins, for example tenascin-C, R and/or W.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure binds only tenascin-C i.e. specific to tenascin-C.

In one embodiment there is provided an antibody or antigen-binding fragment which cross-blocks an antibody or antigen-binding fragment according to the present disclosure which is specific to the P domain or tenascin, for example tenascin-R, in particular a sequence selected from the group consisting of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29 or combinations thereof.

In one embodiment there is provided an antibody or antigen-binding fragment which binds the same epitope as an antibody or antigen-binding fragment according to the present disclosure which is specific to the P domain or tenascin, for example tenascin-R, in particular a sequence selected from the group consisting of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29 or combinations thereof.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure is an inhibitor, for example an inhibitor of one or more biological activities of the P domain of a tenascin protein, for example tenascin-R. In one embodiment the biological activity or activities is one described herein.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure neutralises one or more deleterious biological effects associated with the P domain of tenascin, for example the P domain of tenascin-R, such as a fragment disclosed in any one of the sequences shown in SEQ ID NO: 21, 22, 22, 23, 24, 25, 26, 27, 28, 29 or a combination thereof.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure binds only tenascin-R i.e. specific to tenascin-R.

In one embodiment there is provided an antibody or antigen-binding fragment which cross-blocks an antibody or antigen-binding fragment according to the present disclosure which is specific to the P domain or tenascin, for example tenascin-W, in particular a sequence selected from the group consisting of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39 or combinations thereof.

In one embodiment there is provided an antibody or antigen-binding fragment which binds the same epitope as an antibody or antigen-binding fragment according to the present disclosure which is specific to the P domain or tenascin, for example tenascin-W, in particular a sequence selected from the group consisting of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39 or combinations thereof. In one embodiment an antibody or antigen-binding fragment according to the present disclosure is an inhibitor, for example an inhibitor of one or more biological activities of the P domain of a tenascin protein, for example tenascin-W. In one embodiment the biological activity or activities is one described herein.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure neutralises one or more deleterious biological effects associated with the P domain of tenascin, for example the P domain of tenascin-W, such as a fragment disclosed in any one of the sequences shown in SEQ ID NO: 31, 32, 32, 33, 34, 35, 36, 37, 38, 39 or a combination thereof.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure binds only tenascin-W i.e. specific to tenascin-W.

In one embodiment an antibody or antigen-binding fragment according to the present disclosure has high binding affinity to the P domain of a tenascin protein, for example tenascin-C protein. High binding affinity includes 500nM or higher affinity, such as 250nM, 200nM, 150nm, ΙΟΟηΜ, 50nM, 500pM, 400pM, 300pM, 200pM, ΙΟΟρΜ, 50pM, ΙΟρΜ or less.

In one embodiment there is provided an antibody or antigen-binding fragment according to the present disclosure or a composition comprising the same for use in treatment, for example for use in the treatment of inflammation, such as chronic inflammation. In one embodiment there is provided an antibody or antigen-binding fragment according to the present disclosure or a composition comprising the same for use in treatment of a condition disclosed herein.

Thus there is provided a method of treating a patient comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment according to the present disclosure or a composition comprising the same, in particular a patient with condition described herein, for example inflammation, such as chronic inflammation.

In one embodiment there is provided use of an antibody or antigen-binding fragment according to the present disclosure for the manufacture of a medicament for the treatment of a condition disclosed herein, for example inflammation, such as chronic inflammation.

Definitions

Tenascin-C is a large hexameric protein of 1.5 million Da. Each chain comprises different domains, including an assembly domain (TA), EGF-like repeats (EGF-L), fibronectin type Ill-like repeats (TNIII) and a fibrinogen-like globe (FBG) (reviewed in Orend (2005)). The sequences of tenascin-C and its domains are shown in Figure 13.

Tenascin-C is an ECM glycoprotein that is associated with tissue injury and wound repair.

Tenascin-C is expressed specifically at during active tissue remodelling during embryogenesis, being first observed during gastrulation and somite formation. In later stages of development expression is restricted to sites of branching morphogenesis of mammary gland and the lung, in the developing skeleton, cardiovascular system and in connective tissues at sites of epithelial to mesenchymal transformation. Expression is down regulated once these processes cease and before embryogenesis is complete (Jones (2000)). Tenascin-C is not normally expressed in healthy adult tissue but, in adults, is specifically and transiently up-regulated during acute inflammation and persistently expressed in chronic inflammation (reviewed in Chiquet-Ehrismann (2003)). Immunohistochemical studies show that little tenascin-C is expressed in normal human joints but levels are greatly increased in RA synovia, in areas of inflammation and fibrosis, specifically below the synovial lining, in the invading pannus and around blood vessels (Cutolo (1992), MacCachren (1992) and Salter (1993)). There is also a significant increase in tenascin-C levels in synovial fluid from RA patients (Chevalier (1994) and Hasegawa (2007)) and in RA cartilage (Salter (1993) and Chevalier (1994)).

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

By "acute inflammation" we include the meaning of the initial stages (initiation) of inflammation and the short-term transient inflammatory response immediately after injury, infection or local immune response. Typically, acute inflammation is rapidly resolved, lasting from a matter of minutes to no longer that a few days.

By "chronic inflammation" we include the meaning of persistent and/or non-resolved inflammation. It is often associated with inappropriate destruction of healthy tissue. This may be progressive and last over a period of weeks or longer. Chronic inflammation is typically associated with persistent infection or disease including, but not limited to, automimmune conditions.

By "chronic joint inflammation" we include the meaning of persistent inflammation that is progressive and unremitting over a period of weeks to months, resulting in distortion of the affected joint and radiographic evidence of cartilage and bone destruction as observed in human disease (Kelly, Harris, Ruddy and Sledge, Textbook of Rheumatology 4th Edition).

In experimental murine models, chronic joint inflammation is characterised by inflammation that does not subside and causes inappropriate tissue destruction, even over a relatively short period of time. This is characterized (and can be identified) histologically by the prolonged presence of inflammatory cells in the synovium and joint space, chondrocyte death, and cartilage and bone erosion.

By an "agent" we include all chemical entities, for example oligonucleotides, polynucleotide, polypeptides, peptidomimetics 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% sequence identity with the full length sequence of interest, for example at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

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

The alignment may alternatively be carried out using the Clustal W program (as described in Thompson et ah, 1994, Nuc. Acid Res. 22:4673-4680). The parameters used may be as follows:

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

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

Scoring matrix: BLOSUM.

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

By "antibody" we include substantially intact antibody molecules, as well as chimaeric antibodies, humanised antibodies, human antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen binding fragments and derivatives of the same.

The antibody molecules of the present invention may comprise a complete antibody molecule having full length heavy and light chains or a binding fragment thereof and may be, but are not limited to Fab, modified Fab, Fab', modified Fab', F(ab') 2, Fv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9) :1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et ah, 1998, Journal of Immunological Methods, 216:165-181). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International patent applications WO05/003169, WO05/003170 and WO05/003171. Multi-valent antibodies may comprise multiple specificities e.g. bispecific or may be monospecific (see for example W092/22853, WO05/113605, WO2009/040562 and

WO2010/035012).

By "antigen-binding fragment" we mean a functional fragment of an antibody that is capable of binding to tenascin.

"Antibody binding fragment" and "antigen-binding fragment" are used interchangeably herein unless the context indicated otherwise.

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

Chimeric antigen receptors are generally made up of three parts, namely:

• the binding domain in the form of an antibody fragment, such as a scFv containing a variable heavy domain (VH) and a variable light domain (VL), which together define the specificity of the binding domain,

• a transmembrane domain which anchors the scFv on the surface of the cell, and

• an intracellular domain (intracytoplasmic domain) responsible for relaying a signal to the cell when the binding domain is engaged appropriately. A detailed description off chimeric antigen receptor is provided in Dotti et al 2009 (Human Gene Therapy 20: 1229-1239 (November 2009). Next generation CARs may comprise CD28 and/or 41BB and/or Ox40 signalling sections. Depending on the chimeric antigen receptor (CAR) construct employed the CAR may have potent signaling capabilities

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

A 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is 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 quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.

Paragraphs:

1. An agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with the P domain within the FBG domain of tenascin-C.

2. An agent as defined in paragraph 1 wherein the agent modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

3. An agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with one or more portions of the FBG domain of tenascin-C, wherein the one or more portions are selected from SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

4. An agent as defined in paragraph 3 wherein the agent modulates the biological activity associated with SEQ ID NO: 11, or an agent as defined in claims 1 or 2 wherein the agent modulates the biological activity associated with SEQ ID NO: 12.

5. An agent as defined in paragraph 4 wherein the agent additionally modulates the biological activity associated with one or more of SEQ ID NOs: 13, 14, 15, 16 and 19.

6. An agent as defined in any of paragraph 1-3 wherein the agent modulates the biological activity associated with one or more of SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

7. An agent as defined in paragraph 6 wherein the agent additionally modulates the biological activity associated with one or more of SEQ ID NOs: 11, 12, 16 and 19. 8. An agent as defined in any previous claim wherein the agent modulates the biological activity associated with three or more of the positively charged amino acids from the sequence KTRYKLK (SEQ ID NO: 17) of the FBG domain of tenascin-C.

9. An agent as defined in paragraph 8 wherein the agent modulates the biological activity associated with the positively charged amino acids K, R, K and K from the sequence KTRYKLK

(residues 1, 3, 5 and 7 of SEQ ID NO: 17) of the FBG domain of tenascin-C.

10. An agent as defined in any previous claim wherein the agent modulates the biological activity associated with the sequence KTRYK (SEQ ID NO: 18) of the FBG domain of tenascin-C.

11. An agent as defined in any previous paragraph wherein the agent modulates the biological activity associated with the sequence KTRYKLK (SEQ ID NO: 17) of the FBG domain of tenascin-C.

12. An agent as defined in any previous paragraph wherein the agent modulates the biological activity associated with one or more of residues 152, 157, 159, 160, 161 and 162 (as defined in SEQ ID NO: 1) of the FBG domain of tenascin-C.

13. An agent as described in any previous paragraph wherein the agent modulates the biological activity associated with the specified domain or portion(s) of the FBG domain of tenascin-C by altering the transcription, translation and/or binding properties of the specified domain or portion(s) of the FBG domain of tenascin-C.

14. An agent as defined in any previous paragraph wherein the agent down-regulates the biological activity associated with the specified domain or portion(s) of the FBG domain of tenascin-C.

15. An agent as defined in any previous paragraph wherein the agent up-regulates the biological activity associated with the specified domain or portion(s) of the FBG domain of tenascin-C.

16. An agent as defined in any previous paragraph wherein the agent is an inhibitor of the binding properties associated with the specified domain or portion(s) of the FBG domain of tenascin-C, preferably the binding properties relate to binding to TLR4.

17. An agent according to any of the preceding paragraph wherein the agent is selected from the group consisting of antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, small inhibitor compounds, a domain of tenascin-C or variant thereof, polypeptides and proteins, compounds with binding affinity for tenascin-C, short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides; optionally wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fv fragments, scFv fragments, Fab, single variable domains and domain antibodies; optionally wherein the antibody or antigen-binding fragment thereof is human or humanised.

18. A method of identifying an agent that modulates the activity of tenascin-C comprising:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 and the one or more candidate agents;

(iii) contacting one or more cells with one or more of SEQ ID NOs: 10, 11, 12, 17 and 18 and no candidate agent; (iv) determining whether said candidate agent modulates the effect of SEQ ID NOs: 10, 11, 12, 17 and 18 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

19. A method of identifying an agent that modulates the activity of tenascin-C comprising:

(i) providing one or more candidate agents;

20. The method of paragraphs 18 or 19 wherein the one or more cells or TLR4 contacted in steps (ii) and (iii) is contacted with SEQ ID NO: 11.

21. The method of paragraph 20 wherein the one or more cells or TLR4 are additionally contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 13, 14, 15, 16 and 19.

22. The method of paragraph 19 wherein TLR4 is contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 13, 14 and 15.

23. The method of paragraph 22 wherein TLR4 is additionally contacted in steps (ii) and (iii) with one or more of SEQ ID NOs: 11, 16 and 19.

24. The method of any of paragraph 18-23 wherein the one or more cells or TLR4 are contacted in steps (ii) and (iii) with three or more of the positively charged amino acids from the sequence KTRYKLK (SEQ ID NO: 17) of the FBG domain of tenascin-C.

25. The method of any of paragraph 18-23 wherein the one or more cells or TLR4 are contacted in steps (ii) and (iii) are contacted with the positively charged amino acids K, R, K and K from the sequence KTRYKLK (residues 1, 3, 5 and 7 of SEQ ID NO: 17) of the FBG domain of tenascin-C.

26. The method of any of paragraph 18-23 wherein the one or more cells or TLR4 are contacted in steps (ii) and (iii) with the sequence KTRYK (SEQ ID NO: 18) of the FBG domain of tenascin-C.

27. The method of any of paragraph 18-23 wherein the one or more cells or TLR4 are contacted in steps (ii) and (iii) with the sequence KTRYKLK (SEQ ID NO: 17) of the FBG domain of tenascin-C.

28. The method of any of paragraph 19-27 wherein the one or more cells or TLR4 are contacted in steps (ii) and (iii) with one or more of residues 152, 157, 159, 160, 161 and 162 (as defined in SEQ ID NO: 1) of the FBG domain of tenascin-C.

29. The method as defined in any of paragraphs 18-28 wherein the activity of tenascin-C is up- regulated.

30. The method as defined in any of paragraphs 18-28 where the activity of tenascin-C is down-regulated. 31. The agent or method as defined in any previous wherein the agent binds to or within tenascin-C, preferably to the FBG domain of tenascin-C.

32. The agent or method as defined in paragraph 31 wherein the agent binds to or within the P domain within the FBG domain of tenascin-C.

33. The agent or method as defined in paragraph 32 wherein the agent binds to or within one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

34. The agent or method as defined in paragraph 31 wherein the agent binds to or within one or more portions of the FBG domain of tenascin-C, wherein the one or more portions are selected from SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

35. The agent or method as defined in any of paragraph 31-34 wherein the agent binds to or within SEQ ID NO: 11 or SEQ ID NO: 12.

36. The agent or method as defined in paragraph 35 wherein the agent additionally binds to or within one or more of SEQ ID NOs: 13, 14, 15, 16 and 19.

37. The agent or method as defined in any of paragraph 31-34 wherein the agent binds to or within one or more of SEQ ID NO: 13; SEQ ID NO: 14; and SEQ ID NO: 15.

38. The agent or method as defined in paragraph 37 wherein the agent additionally binds to or within one or more of SEQ ID NOs: 11, 12, 16 and 19.

39. The agent or method as defined in any previous paragraph wherein the agent binds to three or more of the positively charged amino acids from the sequence KTRYKLK (SEQ ID NO: 17) of the FBG domain of tenascin-C.

40. The agent or method as defined in paragraph 39 wherein the agent binds to the positively charged amino acids K, R, K and K from the sequence KTRYKLK (residues 1, 3, 5 and 7 of SEQ ID NO: 17) of the FBG domain of tenascin-C.

41. The agent or method as defined in any previous paragraph wherein the agent binds to the sequence KTRYK (SEQ ID NO: 18) of the FBG domain of tenascin-C.

42. The agent or method as defined in any previous paragraph wherein the agent binds to the sequence KTRYKLK (SEQ ID NO: 17) of the FBG domain of tenascin-C.

43. The agent or method as defined in any previous paragrapg wherein the agent binds to one or more of residues 152, 157, 159, 160, 161 and 162 (as defined in SEQ ID NO: 1) of the FBG domain of tenascin-C.

44. The method as defined in any of paragraphs 18-43 wherein the activity of tenascin-C which is modulated is the activity associated with one or more of the domains or portion(s) of the FBG domain of tenascin-C as defined for the agents of claims 1-12.

45. The method as described in any of paragraphs 18 and 20-44 wherein the cells of steps (ii) and (iii) express Toll-like receptor 4 (TLR4); and/or wherein the one or more cells are 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; optionally wherein the inflammatory cells are selected from the group consisting of macrophages, dendritic cells, monocytes, lymphocytes, monocyte like cells and macrophage like cells.

46. A method of identifying an agent that modulates a chronic inflammatory response by conducting the method of paragraphs 18-45.

47. A method as described in paragraph 46 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation.

48. A method as described in paragraph 46 or 47 wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis and cardiovascular disease.

49. A method as described in paragraph 48 wherein the chronic inflammation is associated with rheumatoid arthritis (RA).

50. An agent identified according to the method as described in any of paragraph 18-49.

51. An agent as described in paragraph 50 wherein the agent identified modulates a chronic inflammatory response

52. An agent as described in paragraph 51 wherein the agent down-regulates the chronic inflammatory response.

53. An agent as described in paragraph 52 wherein the agent up-regulates the chronic inflammatory response.

54. An agent as described in any of paragraphs 50-53 wherein the agent is selected from the group consisting of antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, small inhibitor compounds, a domain of tenascin-C or variant thereof, polypeptides and proteins, compounds with binding affinity for tenascin-C, short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides.

55. An agent described in any of paragraphs 1-17, 31-43 and 50-54 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation.

56. An agent as described in paragraphs 1-17, 31-43 and 50-55 wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis and cardiovascular disease.

57. A composition comprising an agent as defined in any of paragraphs 11-17, 31-43 and 50-56 and a pharmaceutically acceptable carrier, excipient and/or diluent; optionally further comprising at least one other agent; optionally wherein the at least one other agent is an anti-inflammatory agent, a statin, a biological agent (biologicals), an immunosuppressive agent, a salicylate and/or a microbicidal agent; optionally wherein the anti-inflammatory agent is selected from the group consisting non-steroidal anti-inflammatories (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs) or immunosuppressants.

58. An agent or composition as defined in paragraphs 1-17, 31-43 and 50-57 for use as a medicament.

59. An agent or composition as defined in paragraphs 1-17, 31-43 and 50-57 for use in the treatment of a chronic inflammatory condition.

60. Use of an agent or composition as defined in paragraphs 1-17, 31-43 and 50-57 in the manufacture of a medicament for the treatment of a chronic inflammatory condition.

61. An agent, composition or use as described in paragraphs 58-60 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation.

62. An agent, composition or use as described in claims 58-60 wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis and cardiovascular disease.

63. A method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an agent or composition as defined in paragraphs 11-17, 31-43 and 50-57.

64. A kit of parts for performing the method of paragraphs 18 and 20-49 comprising:

(i) one or more cells;

(ii) a control sample of one or more cells

(iii) a sample of one or more of SEQ ID NOs: 10, 11, 12, 17 and 18; and

(iv) instructions for their use; and optionally comprising:

(v) a candidate agent; optionally further comprising:

65. A kit of parts for performing the method of paragraphs 19-49 comprising:

(i) TLR4;

(ii) a control sample of TLR4;

(iii) a sample of one or more of SEQ ID NOs: 11-15 and 17-18; and

(iv) instructions for their use; and optionally comprising:

(v) a candidate agent; optionally further comprising:

66. A kit of parts as defined in paragraph 64 or 65 wherein (i) and (ii) are provided together (as one) and split before use.

67. A kit of parts comprising:

(i) an agent or composition as defined in paragraphs 1-17, 31-43 and 50-57; (ii) administration means; and

(iii) instructions for their use; and optionally comprising:

(iv) at least one other agent.

68. An agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with the P domain within the FBG domain of tenascin-R.

69. An agent as defined in paragraph 68 wherein the agent modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

70. An agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with one or more portions of the FBG domain of tenascin-R, wherein the one or more portions are selected from SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

71. An agent as defined in paragraph 70 wherein the agent modulates the biological activity associated with SEQ ID NO: 21, or an agent as defined in claims 68 or 69 wherein the agent modulates the biological activity associated with SEQ ID NO: 22.

72. An agent as defined in paragraph 71 wherein the agent additionally modulates the biological activity associated with one or more of SEQ ID NOs: 23, 24, 25, 26 and 29.

73. An agent as defined in any of paragraphs 68-70 wherein the agent modulates the biological activity associated with one or more of SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

74. An agent as defined in paragraph 73 wherein the agent additionally modulates the biological activity associated with one or more of SEQ ID NOs: 21, 22, 26 and 29.

75. An agent as defined in any one of paragraphs 68-74 wherein the agent modulates the biological activity associated with the positively charged amino acids R, K, R from the sequence RNLYKLR (SEQ ID NO: 27) of the FBG domain of tenascin-R.

76. An agent as defined in any one of paragraphs 68-75 wherein the agent modulates the biological activity associated with the sequence RNLYK (SEQ ID NO: 28) of the FBG domain of tenascin-R.

77. An agent as defined in any one of paragraphs 68-76 wherein the agent modulates the biological activity associated with the sequence RNLYKLR (SEQ ID NO: 27) of the FBG domain of tenascin-R.

78. An agent as defined in any one of paragraphs 68-77 wherein the agent modulates the biological activity associated with one or more of residues 152, 157, 158, 159, 160, 161 and 162 (as defined in SEQ ID NO: 2) of the FBG domain of tenascin-R.

79. An agent according to any of any one of paragraphs 68-78 wherein the agent is selected from the group consisting of antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, small inhibitor compounds, a domain of tenascin-C or variant thereof, polypeptides and proteins, compounds with binding affinity for tenascin-C, short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides; optionally wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fv fragments, scFv fragments, Fab, single variable domains and domain antibodies; optionally wherein the antibody or antigen-binding fragment thereof is human or humanised.

80. A method of identifying an agent that modulates the activity of tenascin-R comprising:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with tenascin-R or FBG-R or one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 and the one or more candidate agents;

(iii) contacting one or more cells with tenascin-R or FBG-R or one or more of SEQ ID NOs: 20, 21, 22, 27 and 28 and no candidate agent;

(iv) determining whether said candidate agent modulates the effect of tenascin-R or FBG-R or SEQ ID NOs: 20, 21, 22, 27 and 28 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

81. A method of identifying an agent that modulates the activity of tenascin-R comprising:

(i) providing one or more candidate agents;

(ii) contacting TLR4 with tenascin-R or FBG-R or one or more of SEQ ID NOs: 10-15 and 17-18 and the one or more candidate agents;

(iii) contacting TLR4 with tenascin-R or FBG-R or one or more of SEQ ID NOs: 10-15 and 17-18 and no candidate agent;

(iv) determining whether said candidate agent modulates the binding of TLR4 to the tenascin-R or FBG-R or one or more of SEQ ID NOs: 10-15 and 17-18 in step (ii) in comparison to the binding of TLR4 to tenascin-R or FBG-R or one or more of SEQ ID NOs: 10-15 and 17-18 in step (iii).

82. The agent or method as defined in any one of paragraphs 68-81 wherein the agent binds to or within tenascin-R, preferably to the FBG domain of tenascin-R.

83. The agent or method as defined in paragraph 82 wherein the agent binds to or within the P domain within the FBG domain of tenascin-R.

84. The agent or method as defined in paragraph 83 wherein the agent binds to or within one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

85. The agent or method as defined in paragraph 82 wherein the agent binds to or within one or more portions of the FBG domain of tenascin-R, wherein the one or more portions are selected from SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

86. The agent or method as defined in any of paragraphs 82-85 wherein the agent binds to or within SEQ ID NO: 21 or SEQ ID NO: 22.

87. The agent or method as defined in paragraph 86 wherein the agent additionally binds to or within one or more of SEQ ID NOs: 23, 24, 25, 26 and 19.

88. The agent or method as defined in any of claims 82-85 wherein the agent binds to or within one or more of SEQ ID NO: 23; SEQ ID NO: 24; and SEQ ID NO: 25.

89. The agent or method as defined in paragraph 88 wherein the agent additionally binds to or within one or more of SEQ ID NOs: 21, 22, 26 and 29. 90. The agent or method as defined in any one of paragraphs 68-89 wherein the agent binds to the positively charged amino acids R, K and R from the sequence SEQ ID NO: 27.

91. The agent or method as defined in any one of paragraphs 68-90 wherein the agent binds to the sequence SEQ ID NO: 27.

92. The method as claimed in any of paragraphs 80-91 wherein the activity of tenascin-R which is modulated is the activity associated with one or more of the domains or portion(s) of the FBG domain of tenascin-R as defined for the agents of claims 68-78.

93. A method of identifying an agent that modulates a chronic inflammatory response by conducting the method of paragraphs 80-92.

94. A method as claimed in paragraph 93 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation, such as rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, and diseases associated with neurological inflammation such as alzheimer's disease and parkinson's disease.

95. An agent identified according to the method as claimed in any of paragraphs 80-94.

96. An agent as claimed in paragraph 95 wherein the agent identified modulates a chronic inflammatory response

97. An agent claimed in any of paragraphs 68-79, 82-91 and 95-96 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation, such as rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, nonhealing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, and diseases associated with neurological inflammation such as alzheimer's disease and parkinson's disease.

98. A composition comprising an agent as defined in any of paragraphs 68-79, 82-91 and 95-97 and a pharmaceutically acceptable carrier, excipient and/or diluent; optionally further comprising at least one other agent; optionally wherein the at least one other agent is an anti-inflammatory agent, a statin, a biological agent (biologicals), an immunosuppressive agent, a salicylate and/or a microbicidal agent; optionally wherein the anti-inflammatory agent is selected from the group consisting non-steroidal anti-inflammatories (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs) or immunosuppressants.

99. An agent or composition as defined in paragraphs 68-79, 82-91 and 95-98 for use as a medicament.

100. An agent or composition as defined in paragraphs 68-79, 82-91 and 95-98 for use in the treatment of a chronic inflammatory condition. 101. Use of an agent or composition as defined in paragraphs 68-79, 82-91 and 95-98 in the manufacture of a medicament for the treatment of a chronic inflammatory condition.

102. An agent, composition or use as defined in paragraphs 99-101 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation. 103. An agent, composition or use as defined in paragraph 99-102 wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease, tumours, and diseases associated with neurological inflammation such as alzheimer's disease and parkinson's disease.

104. A method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an agent or composition as defined in paragraphs 68-79, 82-91 and 95-98.

105. A kit of parts for performing the method of paragraphs 80 and 82-94 comprising:

(i) one or more cells;

(ii) a control sample of one or more cells

(iii) a sample of tenascin-R or FBG-R or one or more of SEQ ID NOS: 20, 21, 22, 27 and 28; and

(iv) instructions for their use; and optionally comprising:

(v) a candidate agent; optionally further comprising:

106. A kit of parts for performing the method of paragraphs 81-94 comprising:

(i) TLR4;

(ii) a control sample of TLR4;

(iii) a sample of tenascin-R, FBG-R, or one or more of SEQ ID NOs: 21-25 and 27-28; and

(v) instructions for their use; and optionally comprising:

(vi) a candidate agent; optionally further comprising:

(vii) means of determining the effect of a candidate agent on either tenascin-R activity or chronic inflammation.

107. A kit of parts comprising:

(i) an agent or composition as defined in paragraphs 68-79, 82-91 and 95-98;

(ii) administration means; and

(iii) instructions for their use; and optionally comprising:

(iv) at least one other agent.

108. An agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with the P domain within the FBG domain of tenascin-W. 109. An agent as defined in paragraph 108 wherein the agent modulates the biological activity associated with one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

110. An agent for modulation of a chronic inflammatory response wherein the agent modulates the biological activity associated with one or more portions of the FBG domain of tenascin-W, wherein the one or more portions are selected from SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

111. An agent as defined in paragraph 110 wherein the agent modulates the biological activity associated with SEQ ID NO: 31, or an agent as defined in claims 108 or 109 wherein the agent modulates the biological activity associated with SEQ ID NO: 32.

112. An agent as defined in paragraph 111 wherein the agent additionally modulates the biological activity associated with one or more of SEQ ID NOs: 33, 34, 35, 36 and 39.

113. An agent as defined in any of paragraphs 108-110 wherein the agent modulates the biological activity associated with one or more of SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35. 114. An agent as defined in paragraph 113 wherein the agent additionally modulates the biological activity associated with one or more of SEQ ID NOs: 31, 32, 36 and 39.

115. An agent as defined in any one of paragraph 108-114 wherein the agent modulates the biological activity associated with the positively charged amino acids K, R, K from the sequence KERYKLT (SEQ ID NO: 37) of the FBG domain of tenascin-W.

116. An agent as defined in any one of paragraphs 108-115 wherein the agent modulates the biological activity associated with the sequence KERYK (SEQ ID NO: 38) of the FBG domain of tenascin-W.

117. An agent as defined in any one of paragraphs 108-116 wherein the agent modulates the biological activity associated with the sequence KERYKLT (SEQ ID NO: 37) of the FBG domain of tenascin-W.

118. An agent as defined in any one of paragraph 108-117 wherein the agent modulates the biological activity associated with one or more of residues 154, 159, 160, 161, 162 and 164 (as defined in SEQ ID NO: 3) of the FBG domain of tenascin-W.

119. An agent according to any one of paragraphs 108-118 wherein the agent is selected from the group consisting of antibodies (polyclonal or monoclonal) and antigen-binding fragments thereof, small inhibitor compounds, a domain of tenascin-C or variant thereof, polypeptides and proteins, compounds with binding affinity for tenascin-C, short interfering RNA (SiRNA) molecules, short hairpin RNA molecules (shRNA), antisense oligonucleotides; optionally wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fv fragments, scFv fragments, Fab, single variable domains and domain antibodies; optionally wherein the antibody or antigen-binding fragment thereof is human or humanised.

120. A method of identifying an agent that modulates the activity of tenascin-W comprising:

(i) providing one or more candidate agents;

(ii) contacting one or more cells with tenascin-W or FBG-W or one or more of SEQ ID NOs: 30, 31, 32, 37 and 38 and the one or more candidate agents; (iii) contacting one or more cells with tenascin-W or FBG-W or one or more of SEQ ID NOs: 30, 31, 32, 37 and 38 and no candidate agent;

(v) determining whether said candidate agent modulates the effect of tenascin-W or FBG-W or SEQ ID NOs: 30, 31, 32, 37 and 28 on the one or more cells in step (ii) in comparison to the cell(s) of control step (iii).

121. A method of identifying an agent that modulates the activity of tenascin-W comprising:

(i) providing one or more candidate agents;

(ii) contacting TLR4 with tenascin-W or FBG-W or one or more of SEQ ID NOs: 30-35 and 37-38 and the one or more candidate agents;

(iii) contacting TLR4 with tenascin-W or FBG-W or one or more of SEQ ID NOs: 30-35 and 37-38 and no candidate agent;

(iv) determining whether said candidate agent modulates the binding of TLR4 to the tenascin-W or FBG-W or one or more of SEQ ID NOs: 30-35 and 37-38 in step (ii) in comparison to the binding of TLR4 to tenascin-W or FBG-W or one or more of SEQ ID NOs: 30-35 and 37-38 in step (iii).

122. The agent or method as defined in any one of paragraphs 108-121 wherein the agent binds to or within tenascin-W, preferably to the FBG domain of tenascin-W.

123. The agent or method as defined in paragraph 122 wherein the agent binds to or within the P domain within the FBG domain of tenascin-W.

124. The agent or method as defined in paragraph 123 wherein the agent binds to or within one or more portions within the P domain, wherein the one or more portions are selected from SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

125. The agent or method as defined in paragraph 122 wherein the agent binds to or within one or more portions of the FBG domain of tenascin-W, wherein the one or more portions are selected from SEQ ID NO: 31; SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

126. The agent or method as defined in any of paragraphs 122-125 wherein the agent binds to or within SEQ ID NO: 31 or SEQ ID NO: 32.

127. The agent or method as defined in paragraph 126 wherein the agent additionally binds to or within one or more of SEQ ID NOs: 33, 34, 35, 36 and 39.

128. The agent or method as defined in any of paragraphs 121-125 wherein the agent binds to or within one or more of SEQ ID NO: 33; SEQ ID NO: 34; and SEQ ID NO: 35.

129. The agent or method as defined in paragraph 128 wherein the agent additionally binds to or within one or more of SEQ ID NOs: 31, 32, 36 and 39.

130. The agent or method as defined in any one of paragraphs 108-129 wherein the agent binds to the positively charged amino acids K, R and K from the sequence SEQ ID NO: 37.

131. The agent or method as defined in any one of paragraphs 108-130 wherein the agent binds to the sequence SEQ ID NO: 37.

132. The method as claimed in any of paragraphs 120-131 wherein the activity of tenascin-W which is modulated is the activity associated with one or more of the domains or portion(s) of the FBG domain of tenascin-W as defined for the agents of claims 108-118. 133. A method of identifying an agent that modulates a chronic inflammatory response by conducting the method of paragraphs 120-132.

134. A method as claimed in paragraph 133 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation, such as rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease and tumours.

135. An agent identified according to the method as defined in any of paragraphs 120-134.

136. An agent as defined in paragraph 135 wherein the agent identified modulates a chronic inflammatory response.

137. An agent defined in any of paragraphs 108-119, 122-131 and 135-136 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation, such as rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, nonhealing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease and tumours.

138. A composition comprising an agent as defined in any of paragraphs 108-119, 122-131 and 135-137 and a pharmaceutically acceptable carrier, excipient and/or diluent; optionally further comprising at least one other agent; optionally wherein the at least one other agent is an antiinflammatory agent, a statin, a biological agent (biologicals), an immunosuppressive agent, a salicylate and/or a microbicidal agent; optionally wherein the anti-inflammatory agent is selected from the group consisting non-steroidal anti-inflammatories (NSAIDs), corticosteroids, disease- modifying antirheumatic drugs (DMARDs) or immunosuppressants.

139. An agent or composition as defined in paragraphs 108-119, 122-131 and 135-138 for use as a medicament.

140. An agent or composition as defined in paragraphs 108-119, 122-131 and 135-138 for use in the treatment of a chronic inflammatory condition.

141. Use of an agent or composition as defined in paragraphs 108-119, 122-131 and 135-138 in the manufacture of a medicament for the treatment of a chronic inflammatory condition.

142. An agent, composition or use as defined in paragraphs 139-141 wherein the chronic inflammatory response is associated with a condition characterised by inappropriate inflammation. 143. An agent, composition or use as defined in paragraphs 139-142 wherein the chronic inflammatory response is associated with rheumatoid arthritis (RA), autoimmune conditions, inflammatory bowel diseases, non-healing wounds, multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupus erythrematosus (including systemic lupus erythrematosus), asthma, fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UV damage, psoriasis, ankylosing spondylitis, cardiovascular disease and tumours. 144. A method of treating a chronic inflammatory condition comprising administering to a subject an effective amount of an agent or composition as defined in paragraphs 108-119, 122-131 and 135-138.

145. A kit of parts for performing the method of paragraphs 120 and 122-134 comprising:

(i) one or more cells;

(ii) a control sample of one or more cells;

(iii) a sample of tenascin-W or FBG-W or one or more of SEQ ID NOS: 30, 31, 32, 37 and 38; and

(iv) instructions for their use; and optionally comprising:

(vi) a candidate agent; optionally further comprising:

(vii) means of determining the effect of a candidate agent on either tenascin-W activity or chronic inflammation.

146. A kit of parts for performing the method of paragraphs 121-134 comprising:

(i) TLR4;

(ii) a control sample of TLR4;

(iii) a sample of tenascin-W, FBG-W, or one or more of SEQ ID NOs: 31-35 and 37-38; and

(iv) instructions for their use; and optionally comprising:

(v) a candidate agent; optionally further comprising:

147. A kit of parts comprising:

(i) an agent or composition as defined in paragraphs 108-119, 122-131 and 135-138;

(ii) administration means; and

(iii) instructions for their use; and optionally comprising:

(iv) at least one other agent.

148. A method substantially as described herein with reference to the Examples and Figures.

149. A composition substantially as described herein with reference to the Examples and Figures.

150. An agent substantially as described herein with reference to the Examples and Figures.

151. A method substantially as described herein with reference to the Examples and Figures.

152. A composition substantially as described herein with reference to the Examples and Figures.

153. A kit of parts substantially as described herein with reference to the Examples and Figures.

As used herein, the term "comprising" in context of the present specification should be interpreted as "including".

Embodiments and preferences may be combined as technically appropriate.

The disclosure herein describes embodiments comprising certain integers. The disclosure also extends to the same embodiments consisting or consisting essentially of said integers. Positive recitations of embodiments disclosed herein may also be employed as basis to exclude said embodiment.

The number employed for the loops of the FBG domain allocate the first amino acid residue in the FBG domain as one, for example position 2115 in the full length Tenascin-C protein equates to residue 142 of the FBG domain.

Examples embodying an aspect of the invention will now be described with reference to the following figures in which:

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

(a) Paw swelling in wild type (+/+] (white bars) and tenascin-C null (-/-) (black bars) mice over time after injection of zymosan. Data are shown as the mean increase in paw diameter compared to paw diameter before injection +/-SEM (n = 24 mice per genotype). **= p<0.01. (b-e) Representative sections of the ankle joint from wild type (b, c) and tenascin-C null (d, e) mice 4 days after zymosan injection, stained with hemotoxylin and eosin (b, d) and safranin-0 (c, e). Boxes highlight the joint synovium (s) and cartilage proteoglycan (cp). Magnification xlO. Quantification of joint inflammation (f) and chondrocyte death (g) in knee joints 4 days after injection with zymosan from wild type mice (white bars) and tenascin-C null mice (black bars). Data are expressed as the mean (+/- SD) (n = 24 mice per genotype). *= p <0.05.

Figure 2. Synovial inflammation is induced in tenascin-C deficient mice upon injection of antigen, (a-b, g) Representative sections of the knee joint of sham injected wild type mice, (c-f, h-i) Representative sections of the knee joint of wild type (c, d, h) or tenascin-C null (e, f, i) mice 24 hours after intra-articular injection of mBSA. Inflammatory cell infiltration in the capsule, meniscus and the joint space of both wild type and tenascin-C null mice is highlighted by (cap), (M) and (J) respectively. (S) highlights the healthy synovium of sham injected mice that is no more than 1-3 cells thick along the entire bone surface and (ST) highlights the synovia of wild type and tenascin-C null mice which are both significantly thickened. Sections are stained with hemotoxylin and eosin (a, c, e, g, h, i) and safranin-0 (b, d, f).

Magnification xlO (a-f) or x40 (g-i). (n = 5 mice per genotype).

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

Representative sections of the knee joint of wild type (a, b, f) or tenascin-C null (c, d, e) mice 3 days after intra-articular injection of mBSA. (a, c) The line highlights increased inflammation of the capsule in wild type mice compared to tenascin-C null mice, (b, d) (cp) highlights increased cartilage proteoglycan loss in wild type mice compared to tenascin-C null mice, (e, f) Significant synovial hyperplasia (line), cell and fibrin deposits in the joint space (arrow) and pannus invasion (arrow heads) are observed in wild type mice compared to tenascin-C null mice. Sections are stained with hemotoxylin and eosin (a, c, e, f) and safranin-0 (b, d) Magnification xlO (a-d) or x20 (e-f). (n = 5 mice per genotype).

Figure 4. tenascin-C deficient mice are protected from tissue destruction during antigen induced arthritis, (a-b) Representative sections of the knee joint of wild type mice 7 days after intra-articular injection of mBSA, stained with hemotoxylin and eosin (a) and safranin-0 (b). Magnification xlO. (n = 24 mice per genotype). Arrowhead highlights area of bone erosion. Arrow highlights pannus invasion into articular cartilage, (c-d) Representative sections of the knee joint of tenascin-C null type mice 7 days after intra-articular injection of mBSA, stained with hemotoxylin and eosin (c) and safranin-0 (d). Magnification xlO. (n = 24 mice per genotype). J highlights the joint space and AC the intact articular cartilage, (e) Histological score of knee joint inflammation 24 hours, 3 days and 7 days after injection with mBSA from wild type mice (white bars) and tenascin-C null mice (black bars). Data represent the mean +/- SD (n = 5 per genotype (24h, 3d) or 24 per genotype (7d)). (f) Quantification of chondrocyte death, cartilage surface erosion and bone erosion after injection with mBSA in knee joints from 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 7d. Data represent the mean +/- SD (n = 5 per genotype (24h, 3d) or 24 per genotype (7d)).

Figure 5. tenascin-C induces TNF-a, IL-6 and IL-8 synthesis in primary human macrophages and RA synovial fibroblasts, (a-b) Primary human macrophages (a) and RA synovial fibroblasts (b) were unstimulated (no addition) or stimulated with LPS (1 ng/ml (a) or 10 ng/ml (b)) or recombinant tenascin-C (1.0 μΜ - 1.0 nM) for 24h. Data shown are the mean of triplicate values (+/- SD) from one of three representative experiments, (c) Primary human macrophages were unstimulated (no addition) or stimulated with LPS (1 ng/ml) or recombinant tenascin-C (1.0 μΜ) for 24h. (-) indicates cells were pre-incubated with medium alone. (P) Cells were pre-incubated with 25 μg/ml polymyxin B for 30 min before stimulation. (H) Cells were incubated with medium with no addition or containing LPS or tenascin-C that was boiled for 15 minutes before addition to cells. Data shown are the mean of triplicate values (+/- 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) Primary human macrophages were unstimulated (no addition) or stimulated with LPS (1 ng/ml), recombinant tenascin-C (TNC) or 1.0 μΜ tenascin-C domains (TA, EGF-L, TNIII1-5, TNIII1-3, TNIII3-5, TNIII5-7, TNIII6-8 and FBG) for 24h. Data shown are the mean of triplicate values (+/- SD) from one of three representative experiments, (b) RA synovial membrane cells were unstimulated (no addition) or stimulated with LPS (10 ng/ml) or recombinant FBG (1.0 - 0.01 μΜ) for 24h. Data shown are the mean % change in cytokine levels compared to unstimulated cells (+/- SEM) from five different patients, (c-h) Representative sections of the knee joint of wild type mice 3 days after intraarticular injection of PBS (c-e) or 1 μg FBG (f-h). Sections are stained with hemotoxylin and eosin (c,d,f,g) or Safranin-0 (e, h). Magnification xlO (c, f) or x25 (d,e,g,h) (n = 5 mice per genotype), (i) Quantification of joint inflammation, bone erosion, cartilage surface erosion and chondrocyte death in the knee joints of wild type mice 3 days after intra-articular injection of PBS (black bars) or 1 μg FBG (white bars). Data represent the mean +/- SD (n = 5 per genotype).

Figure 7. FBG mediated cytokine synthesis is MyD88 dependent, (a) Human RA synovial fibroblasts were either uninfected, infected with adenovirus expressing GFP alone (AdGFP) or infected with adenovirus expressing dominant negative MyD88 (AdMyD88dn). Cells were unstimulated, stimulated with LPS (10 ng/ml) or stimulated with FBG (ΙμΜ) for 24h. Data shown are the mean of three independent experiments (+/- SEM). (b) Mouse embryonic fibroblasts isolated from wild type (+/+) or MyD88 deficient (-/-) mice were unstimulated (-) or stimulated with PAM3 (100 ng/ml), LPS (100 ng/ml), TNFa (100 ng/ml), IL-1 (5 ng/ml) and FBG (1 μΜ) for 24h. Data shown are the mean of three independent experiments (+/-SEM).

Figure 8. FBG mediated cytokine synthesis is TLR4 dependent but does not require

CD 14 or MD-2. (a) Primary human macrophages were pre-incubated with medium alone or medium containing function blocking antibodies to TLR2 (10 μg/ml), TLR4 (25 μg/ml) or isotype control antibodies (25 μg/ml) for 30 min before stimulation. Cells were unstimulated, or stimulated with LPS (1 ng/ml), FBG (1 μΜ) or PAM3 (10 ng/ml) for 24h. Data shown are the mean of three independent experiments (+/-SEM). (b) Mouse embryonic fibroblasts isolated from wild type, TLR2 (TLR2 -/-) or TLR4 (TLR4 -/-) deficient mice were unstimulated or stimulated with PAM3 (100 ng/ml), LPS (100 ng/ml), IL-1 (5 ng/ml) and FBG (1 μΜ) for 24h. Data shown are the mean of three independent experiments (+/-SEM). (c) Bone marrow derived macrophages isolated from wild type, TLR2 (TLR2 -/-) or TLR4 (TLR4 -/-) deficient mice were unstimulated or stimulated with PAM3 (100 ng/ml), LPS (100 ng/ml) or FBG (1 μΜ) for 24h. Data shown are the mean of three independent experiments (+/-SEM). (d) Human macrophages were pre-incubated with no inhibitor, ^g/ml msbB LPS or 10μg/ml anti-CD14 antibody for 30 min before stimulation with LPS (1 ng/ml), FBG (1 μΜ) or PAM3 (10 ng/ml) for 24h.

Data shown are the mean of three independent experiments (+/-SEM).

Figure 9. Paw swelling over time after injection of zymosan. Representative images of the paws of non-injected tenascin-C null mice (a, e) (diameter 1.6 mm), tenascin-C null mice 24h (d, f) (diameter 2.5 mm) and 4d (b, h) (diameter 1.7 mm) after zymosan injection and from wild type mice 4d after zymosan injection (c, g) (diam. 2.1 mm). Figure 10. Synthesis of recombinant proteins, (a) Domain structure of the tenascin-C monomer comprising different domains, including the assembly domain (TA), 14 and a half EGF-like repeats (EGF-L), 17 fibronectin type Ill-like repeats (TNIII) (8 constitutively expressed (1-8) and 9 that can be alternatively spliced, and a fibrinogen-like globe (FBG). (b) The regions covered by the recombinant proteins that were synthesized, the corresponding amino acid residues and the molecular weight of each protein.

Figure 11. Analysis of protein purity. Silver stained gel showing 1 μg of each recombinant protein analysed by SDS-PAGE under reducing conditions. Lanes: 1 (TA), 2 (EGF-L),

3 (TNIII1-5), 4 (TNIII5-7), 5 (TNIII6-8), 6 (TNIII1-3), 7 (TNIII3-5) and 8 (FBG). Figure 12. FBG-mediated joint inflammation in vivo requires expression of TLR4.

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

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

Figure 14. Nucleotide sequence of human tenascin-C

Figure 15. TNF synthesis in response to specific FBG peptides. TNF synthesis by RA membrane cultures incubated for 24h with no addition or 100 μΜ of each FBG peptide (PI, P3-P9).

Figure 16. TNF and IL8 synthesis in response to varying concentrations of specific FBG peptides. TNF & IL8 synthesis by RA membrane cultures incubated for 24h with no addition or 25, 100 or 250 μΜ of FBG peptide.

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

IL8 synthesis by macrophages after 24h incubation with no addition, 1 ng/ml LPS, 1 μΜ whole FBG domain (FBG) or 1 or 20 μΜ of FBG peptides (PI, P3-P9).

Figure 18. IL8 and TNF synthesis in response to LPS and FBG following pre-incubation with FBG peptides. TNF and IL8 synthesis by macrophages after 24h incubation with no addition, 1 ng/ml LPS or 1 μΜ whole FBG domain (FBG), either with or without pre-incubation with 20 μΜ of FBG peptides.

Figure 19. IL8 and TNF synthesis in response to tenascin-C targeted siRNAs. Tenascin-C mRNA levels in RA fibroblasts transfected with luciferase specific siRNA (control), or with tenascin-C targeted siRNAs: oligo 1 (si 1), oligo 2 (si 2) or a combination of oligos 1+2 (si 1+2). IL6 synthesis in RA fibroblasts transfected with luciferase siRNA (control) or with a combination of tenascin-C targeted oligos 1+2 (siRNA) in the presence or absence of 10 ng/ml LPS for 24h.

Figure 20. FBG-C, -R and -W can induce the activation of the Nf-kB activation and cytokine synthesis. A. ThPl Nf-kB cells were stimulated with different doses of

FBG-C, -R, -W and -X for 24h and Nf-kB activation was measured using QUANTI- Blue™. B. Primary human macrophages were stimulated with different doses of FBG-C,-R, -W and -X for 24h and cytokine synthesis was measured by ELISA. Data shown as mean + SEM N=3 independent donors or 3 independent experiments. One-way ANOVA vs non-stimulated, *p<0.05, **p<0.01, ***p<0.001.

Figure 21. The FBG domains of the tenascin family members show a high % of homology.

Multiple alignment analysis was used to determine the conserved regions between the FBG domains of the tenascin family members. The regions corresponding to the A, B and P domains are indicated by a solid line, dots or long dashes respectively. Figure 22. Peptide mapping of FBG-C active site. A. ThPl Nf-kB cells were stimulated with

LPS (0.5 ng/mL), FBG-C (0.5 μΜ) or 20, 50 or 100 μΜ of peptides 1-9 for 24h and Nf-kB activation was measured using QUANTI-Blue™ . B. Primary human macrophages were stimulated with LPS (Ing/mL), FBG-C (1 μΜ) or 20 μΜ of peptides 1-9 for 24h and cytokine synthesis was measured by ELISA. Data shown as mean + SEM N=3 independent donors or 3 independent experiments. One-way

ANOVA vs non-stimulated, *p<0.05, **p<0.01, ***p<0.001.

Figure 23. The overlapping region of peptides 5 and 6 correspond to loop 5 of the FBG domain. A. Sequence of peptide 5 and 6 highlighting overlapping amino acids in bold. B. Sequence alignment of the FBG domains of the tenascin family members showing amino acid composition of loop 5 (bold). Positive charge amino acids are in italic. C. Models of the FBG domain of tenascin-C, -R, -W and -X highlighting the amino acid composition in loop 5.

Figure 24. Positive charges in loop 5 are important for the induction of the immune response by FBG-C. A. ThPl Nf-kB cells were stimulated with 20, 50 and 100 μΜ of peptides loop 5-4, loop 5-deletion, loop 5-mutation 1, loop 5-mutation 2 and loop 5 mutation-3. Nf-kB activation was measured after 24h using QUANTI-Blue™. B. Primary human macrophages were stimulated for 24h with 20, 50 and 100 μΜ of peptides P5, loop 5-4, loop 5-mutation 1, loop 5-mutation 2 and loop 5 mutation-3. IL-8 synthesis was measured by ELISA. C. ThPl Nf-kB cells were stimulated for 24h with increasing doses of FBG-C, FBG-C mutant 1, FBG-C mutant 2 and FBG-C mutant 3. Nf-kB activation was measured using QUANTI-Blue™. D. Primary human macrophages were stimulated for 24h with increasing doses of FBG-C, FBG-C mutant 1, FBG-C mutant 2 and FBG-C mutant 3. Cytokines synthesis was measured by ELISA. Data shown as mean + SEM. N=3 independent donors or 3 independent experiments. Paired t-test vs FBG-C, *p<0.05, **p<0.01, ***p<0.001.

Figure 25. Cytokine induction by FBG-C, -R and -W is TLR4 dependent. Primary human macrophages were pre-incubated for 6 h with TLR4 inhibitor TAK 242 (A) or 30 min with TLR4 polyclonal antibody (B) prior stimulation with LPS (Ing/mL) or FBG-C, -R and -W (1 μΜ) for 24h. Data shown as mean + SEM; N=3 independent donors or 3 independent experiments. Paired t-test vs non-treated with TAK242, *p<0.05, **p<0.01, ***p<0.001.

Figure 26. TLR4 binds directly to FBG-C, -R and W in a solid phase binding assay. 96 well plates were coated with FBG-C, -R, -W or -X and TLR4 was added in a dose dependent manner. Curves were fitted in GraphPad Prism using one binding site hyperbola analysis. Data shown as mean + SEM; N=4.

Figure 27. T LR4 shows decreased binding to FBG-C mutants in a solid phase binding assay.

96 well plates were coated with FBG-C, FBG-C mutant 1, FBG-C mutant 2, FBG-C mutant 3, and TLR4 was added in a dose dependent manner. Curves were fitted in GraphPad Prism using one binding site hyperbola analysis. Data shown as mean + SEM; N=4.

Figure 28. Mutation in loop 5 of FBG-X partially recover protein activity. A. ThPl Nf-kB cells were stimulated for 24h with increasing doses of FBG-C, FBG-X, FBG-X mutant

1 and FBG-X mutant 2. Nf-kB activation was measured using QUANTI-Blue™. B. Primary human macrophages were stimulated for 24h with increasing doses of FBG-C, FBG-X, FBG-X mutant 1 and FBG-X mutant 2. Cytokines synthesis was measured by ELISA. C. TLR4 doesn't bind to FBG-X mutant 1 and binds with low affinity to FBG-X mutant 2 in a solid phase binding assay. Plate was coated with

FBG-C, FBG-X, FBG-X mutant 1 or FBG-X mutant 2 and TLR4 was added in a dose dependent manner. Data shown as mean + SEM. N=3 independent donors or 3 independent experiments. One-way ANOVA vs non-stimulated, *p<0.05, **p<0.01, ***p<0.001.

Figure 29. Amino acids composition of loop 10 of the FBG of the tenascin family members. A. Sequence alignment of the FBG domains of the tenascin family members showing amino acid composition of loop 10 (bold). Positive charge amino acids are in italic. B. Models of the FBG domain of tenascin-C, -R, -W and -X highlighting amino acids composition in loop 10.

Figure 30. Mutations in loop 10 have a modest effect in FBG-C protein activity. A. ThPl

Nf-kB cells were stimulated with 20, 50 and 100 μΜ of peptides loop 5-4, P9 and P9 mutant. Nf-kB activation was measured after 24h using QUANTI-Blue™. B. Primary human macrophages were stimulated for 24h with 20, 50 and 100 μΜ of peptides P5, loop 5-4, P9 and P9 mutant. IL-8 synthesis was measured by ELISA. C. ThPl Nf- kB cells were stimulated for 24h with increasing doses of FBG-C, FBG-C mutant 4, FBG-C mutant 5 and FBG-C mutant 3. Nf-kB activation was measured using QUANTI-Blue™. D. Primary human macrophages were stimulated for 24h with increasing doses of FBG-C, FBG-C mutant 4, FBG-C mutant 5 and FBG-C mutant 3. Cytokines synthesis was measured by ELISA. Data shown as mean + SEM. N=3 independent donors or 3 independent experiments. Paired t-test vs FBG-C, *p<0.05, **p<0.01, ***p<0.001.

Figure 31. TLR4 shows decreased binding to FBG-C mutants in a solid phase binding assay. 96 well plates were coated with FBG-C, FBG-C mutant 3, FBG-C mutant 4, FBG-C mutant 5, and TLR4 was added in a dose dependent manner. Curves were fitted in GraphPad Prism using one binding site hyperbola analysis. Data shown as mean + SEM; N=3.

Figure 32. Peptides 5, 6, 7 and 9 partially block TLR4-FBG-C in vitro interaction. 96 well plates were coated with FBG-C, and TLR4 was pre-incubated with 200 μΜ of peptides before added in a dose dependent manner. Curves were fitted in GraphPad Prism using one binding site hyperbola analysis. Data shown as mean + SEM; N=3. Figure 33. Amino acids composition of peptide 7. A. Sequence alignment of the FBG domains of the tenascin family members showing amino acid composition of peptide 7. B.

Models of the FBG domain of tenascin-C, -R, -W and -X highlighting P7 amino acids composition.

Figure 34. Forward and Reverse primer sequences of tenascin -R, -X and -W FBG. Each sequence has a His-tag (underlined) and a restriction site in the forward primer for Ndel (bold) and in the reverse primer for Xhol (bold).

Figure 35. Biophysical characterization of FBG-C, -R, -W -X. A. Protein purity was verified by silver staining of 1 ug of FBG-C, -R, -W and -X. B. Anti-His tag western blot of 1 ug of FBG-C, -R, -W and -X. C. Circular dichroism (CD) spectra in the far UV region of FBG-C, -R, -W and -X. TN buffer signal in black solid line and FBG-C, -R, -W and -X negative peaks in black dashes.

Figure 36. FBG-C, -R and -W activity is not due to LPS contamination. ThPl Nf-kB cells were stimulated with 0.5 ng/mL of LPS or 0.5 μΜ of FBG-C, -R, -W and -X previously incubated for 30 min with polymixyn B or boiled for 15 min. Nf-kB activation was measured after 24h using QUANTI-Blue™ (A). Primary human macrophages were stimulated with 1 ng/mL of LPS (B) or 1 μΜ of FBG-C, -R, -W and -X (C) for 24h. Samples were previously incubated for 30 min with polymixyn B or boiled for 15 min. Cytokine synthesis was measured by ELISA. Data shown as mean + SEM N=3 independent donors or 3 independent experiments. Un-pair t-test vs non-treated, *p<0.05, **p<0.01, ***p<0.001.

Figure 37. Modelling the structure of FBG-C, -R, -W and -X. A. FBG-C, -R, -W and -X models based on fibrinogen y chain crystal structure. Sub-domain protein organisation is highlighted: A-subdomain in black at the N-terminus, B sub-domain in medium grey in the middle and P subdomain in light grey at the C-terminus. Proteins were modelled using Swiss Pro modeller software. B. Alignment of FBG-C, -R, -W and -X models highlighting in black conserved amino acids. C. Alignment of FBG-C, -R, -W and -X models highlighting in black conserved structure.

Figure 38. Peptide variations containing long version of loop 5 can activate Nf-kB. ThPl

Nf-kB cells were stimulated with 20, 50 or 100 μΜ of peptide variations for 24 h and Nf-kB activation was measured using QUANTI-Blue™. Data shown as mean + SEM N=3 independent donors or 3 independent experiments. One-way ANOVA vs non- stimulated, *p<0.05, **p<0.01, ***p<0.001.

Figure 39. Biophysical characterization of FBG-C mutant 1, 2, 3, 4 and 5. A. Protein purity was verified by silver staining of 1 ug of protein sample. B. Anti-His tag western blot of 1 ug of each protein. C. CD spectra in the far UV region of FBG-C mutant 1 (grey line signal), FBG-C mutant 2 (black dashes signal) compared to FBG-C (grey dots signal). TN buffer signal in solid black line (Upper panel) CD spectra of FBG-C mutant 3 (black dashes signal), compared to FBG-C (grey dots signal). TN buffer signal in solid black line (Lower panel). D. CD spectra in the far UV region of FBG-C mutant 4 (upper panel) and FBG-C 5 (lower panel) in black dashes signal compared to FBG-C (grey dots signal). TN buffer signal in solid black line.

Figure 40. Biophysical characterization of FBG-X mutant 1 and 2. A. Protein purity was verified by silver staining of 1 ug of protein sample. B. Anti-His tag western blot of 1 ug of FBG-X mutant 1. C. CD spectra in the far UV region of FBG-X mutant 1 (black dashes signal) compared to FBG-X (grey dots signal). TN buffer signal in solid black line. D. CD spectra in the far UV region of FBG-X mutant 2 (black dashes signal) compared to FBG-X (grey dots signal). TN buffer signal in solid black line.

Figure 41. Hydrophobic and polar amino acids in loop 7 contribute to TLR4 binding, a.

Sequence of FBG-C mutants 6 and 7. b. ThPl NF-kB cells were stimulated for 24h with increasing doses (μΜ) of FBG-C, FBG-C mutant 6, FBG-C mutant 7 and 1 μΜ of FBG-C mutant 3. NF-kB activation wasmeasured using QUANTI-Blue™. Data shown as mean ± SEM. 600 N=3 independent experiments. Paired t-test vs FBG-C, *p<0.05, **p<0.01, ***p<0.001. c. Primary human macrophages were stimulated for 24h with increasing doses (μΜ) of FBG-C, FBG-C mutant 6, FBG-C mutant 7 and 1 μΜ FBG- CMutant 3. Cytokine synthesis was measured by ELISA. Data shown as mean ± SEM. N=3 independent donors. Paired t-test vs FBG-C, *p<0.05, **p<0.01, ***p<0.001. d. 96 well plates were coated with 1 μg/mL of FBG-C, FBG-C mutant 3, FBG-C mutant 6, FBG-C mutant 7, and TLR4 wasadded in a dose dependent manner. Curves were fitted in GraphPad Prism using one binding sitehyperbola equation. Data shown as mean + SEM; N=3.

Figure 42. Mutations in FBG-X recover protein activity, a. FBG-X chimeric proteins were designedto introduce the amino acids found in FBG-C to activate and bind to TLR4 (highlighted) into the FBG-Xsequence (bold), b. ThPl NF-kB cells were left unstimulated (-) or stimulated for 24h with 0.5 ng/mLof LPS or increasing doses (μΜ) of FBG-C, FBG-X, FBG-X mutant 1, 2, 3 and 4. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean + SEM. N=3 independent experiments.One-way ANOVA vs FBG-C. c. Primary human macrophages were left unstimulated (-) or stimulatedfor 24h with Ing/mL of LPS or increasing doses (μΜ) of FBG-C, FBG-X, FBG-X mutant 1, 2, 3 and 4.Cytokine synthesis was measured by ELISA. Data shown as mean + SEM. N=3 independent donors.One-way ANOVA vs FBG-C. d. 96 wells plate was coated with 1 μg/mL of FBG-C, FBG-X, FBG-X mutant 1, 2, 3 and 4 and TLR4 was added in a dose dependent manner. Data shown as mean ± SD.N=4 independent experiments, e. Model of FBG-C showing the regions involved in binding and activation of TLR4. Loop 5 is shown in dark grey, loop 7-8 in medium grey and loop 10 in light grey. The amino acids mutated are highlighted in a darker colour.

Figure 43. Structures of direct and indirect binders of TLR4. The structure of TLR4 (PDB code4g8a) is shown in dark grey together with a. activators of TLR4 that interact with MD2 (PDB code 4g8a -light grey) : LPS (PDB code 4g8a) and HMGB1 (PDB code 2yrq); b. activators of TLR4 thatrequire but do not bind M-2: Ni2+ (dark grey circles mapped onto critical histidine 456 and 458 of TLR4) and diCi4-amidine; c. activators of TLR4 that do not require MD2 but exhibit structural homology to MD2:DERP-2 (PDB code lktj superimposed upon MD2 of PDB code 4g8a - light grey); and d. activators ofTLR4 that can bind directly to TLR4 but exhibit no structural homology to MD2: biglycan (bovine structure, PDB code 2ft3), surfactant protein d (PDB code lpwb; carbohydrate region marked in light grey), tenascin-C FBG-like domain (homology model - grey; loop 5 - dark grey; loop 7 -dark grey at bottom right of structure) with the TLR4 structure annotated with a high frequency of observed interactions in ICM protein:protein docking with loops 5 and 7 (light grey - TLR4 monomer 1; very dark grey - TLR4 monomer 2).

Figure 44. Conservation of the FBG-C active epitope across species and in other fibrinogen- related proteins, a. Multiple sequence alignment of the FBG domain of tenascin-C of different species, b. Multiple sequence alignment of all of the human FBG domain related proteins, c. Multiple sequence alignment of human FBG-C and fibrinogen related domains from ancient invertebrates. The amino acids involved in TLR4 activation and binding in loop 5, 7 and 10 are highlighted in a black box.

EXAMPLE 1 -GENERAL METHODS

Reagents

Zymosan, methylated BSA and Freund's complete adjuvant, anti-FLAG M2 antibody (mouse monoclonal antibody), blasticidin, and isotype control antibodies (Mouse IgG2a, IgGl) were from Sigma-Aldrich (Dorset, UK). Hypnorm was from VetaPharma Ltd. (Leeds, UK). The Limulus amaebocyte lysate assay was from Associates of Cape Cod (Liverpool, UK). Wild type human embryonic kidney (HEK293-EBNA) cells were from Invitrogen (Groningen, Netherlands). M-CSF and murine IL-Ιβ were from PeproTech (Neuilly-Sur-Seine, France). DMEM, RPMI 1640, fetal bovine serum (FBS), penicillin/streptomycin, antibiotic-antimycotic solution PSA and β- Mercaptoethanol were from PAA Laboratories (Yeovil, UK). HEK293 cell lines stably expressing human TLR2 and TLR4/CD14/MD-2, polymyxin B, msbB LPS and the function blocking TLR2 [Clone: TL2.1 Isotype: Mouse IgG2a) and TLR4 antibodies [Clone: HTA125 Isotype: Mouse IgG2a) were from Invivogen (Calne, UK). Phenol-chloroform-purified Escherichia coli LPS (rough and smooth) and Pam3Cys-Ser-Lys4 (Pam3C) were from Alexis (Birmingham, UK). Murine TNF-a and IL-1 receptor antagonist (IL-lra-IL-lF3) were from R&D Systems (Abingdon, UK). Function blocking anti-CD14 antibodies [Isotype: Mouse IgGl) were from Abeam (Cambridge, UK). Human and murine TNF-a, 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 recombinant full-length human tenascin-C from the conditioned medium of the mammalian cell line HEK293 transfected with his-tagged human tenascin-C in the pCEP-pu vector as described (Lange (2007)). Tenascin-C was purified to homogeneity as described (Lange (2007) and determined to be free of LPS contamination using the Limulus amaebocyte lysate assay according to the manufacturer's instructions.

Synthesis of recombinant proteins

Proteins corresponding to each domain of tenascin-C were synthesized (TA, EGF-L, various

TNIII repeats and FBG) and purified. See Example 2.

Measurement of LPS Contamination in Recombinant Proteins To ascertain the levels of LPS in each recombinant protein the Limulus amaebocyte lysate assay was used according to the manufacturer's instructions (sensitivity ~0.7 ± 0.5 pg LPS per mg protein). All recombinant proteins used in this study had levels of LPS that were less than lOpg/ml. Adenoviral Vectors and Their Propagation

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

Animals

Homozygous tenascin-C deficient mice from the original stock described by Saga (1992) on a 129/sv an inbred strain of mice with a white bellied and agouti appearance background were provided by Prof. Charles French-Constant (University of Edinburgh, UK). Age matched congenic inbred wild type 129/sv mice were obtained from Charles River (Margate, UK). All tenascin-C deficient and wild type 129/sv mice were male and between 8-10 weeks of age at the time of experimentation.

Homozygous TLR2 and TLR4 deficient mice on a C57BL/6 background (an inbred strain of mice with a black coat) were obtained from B&K Universal (Hull, UK) Hoshino (1999) and Takeuchi (1999). Homozygous MyD88 deficient mice on a C57BL/6 background were provided by the Sanger Institute (Cambridge, UK). Age matched congenic inbred wild type C57B/L6 mice were obtained from Charles River (Margate, UK). For isolation of mouse embryo fibroblasts one female aged 8-10 weeks was mated with two males aged 8-10 weeks. For isolation of bone marrow derived macrophages mice were female and between 10-12 weeks of age at the time of experimentation.

All animals were fed standard rodent chow and water ad libitum, and were housed (<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). Multiple group means were analyzed by one-way analysis of variance, followed by the Dunnett Multiple Comparisons test, where appropriate. Unpaired t-test was used for experiments involving only two groups.

EXAMPLE 2 - SYNTHESIS OF RECOMBINANT PROTEINS

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

Pfu Turbo polymerase was from Stratagene (Amsterdam, Netherlands). Easy mix 50 PCR tubes were from Molecular Bioproducts (Lutterworth, UK). RNeasy kits and Ni²⁺-NTA-agarose columns were from Qiagen (Crawley, UK). pCR Blunt vector, pCEP4 plasmid vector, human embryonic kidney (HEK293-EBNA) cells and 4-12% Bis-Tris gradient gels were from Invitrogen (Groningen, Netherlands). pET32b vector and BL21 (DE3) Rosetta cells were from Novagen (Kent, UK). HiTrap Q columns, HiTrap S columns, Sephacryl S500 HR column and heparin sepharose columns were from Amersham (Buckinghamshire, UK).

Restriction enzymes were obtained from New England BioLabs (Hitchin, UK). DMEM, fetal bovine serum (FBS) and penicillin/streptomycin were from PAA laboratories (Yeovil, UK). FuGENE6 transfection reagent 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, UK). Anti-tetra-his antibody (mouse monoclonal antibody) was from Qiagen (Crawley, UK). Alkaline phosphatase-conjugated goat anti-(mouse IgG) IgG and Western Blue stabilized substrate for alkaline phosphatase were from Promega (Southampton, UK). Precision Protein Standards for SDS-PAGE were from BioRad (Hemel Hempstead, UK).

Primer design

Domain boundaries were determined using alignments published in the human tenascin-C sequence (Siri (1991) accession number P24821 (Swiss-Prot)). To clone each domain we designed PCR primers where both the forward and reverse primers contained 18-21 bases corresponding to the 5' and 3' terminal sequences of the requisite coding sequence. The forward primer contained an Ndel restriction site, followed by an N terminal his tag, immediately before the coding sequence. The final 3 bases of the Ndel site form the ATC methionine initiation code. The reverse primer included a TTA stop codon immediately after the coding sequence, followed by a BamHl or a Kpnl site to allow unidirectional cloning into pET32b expression vectors.

Table 1

PCR2

FW: GACTAGAAGGACGACGATGACAAGTGCTGTCTCCAGCC

TGCCAC (SEQ ID NO: 45)

RV: GACAGCGGvlTCCTTAATGATGATGATGATGATGTGAGCA

GTCTTCTCCGCTGTAGC (SEQ ID NO: 46)

TN1-5 FW: ATACvlTvlTCCATCATCATCATCATCATGAGGTGTCTCCTCC

CAAAGA (SEQ ID NO: 47)

RV: GCCCCMCCTTAAGTGGATGCCTTCACACGTGC (SEQ ID NO: 48)

TN1-3 FW: ATAC TA TCCATC ATC ATC ATC ATC ATGAGGTGTCTCCTC

CCAAAGA (SEQ ID NO: 49)

RV: GCCCCMCCTTATGTTGTGAAGGTCTCTTT GGC (SEQ ID NO: 50)

TN3-5 FW: ATAG4 TJ4 TCCATCATCATCATCATCATCGCTTGGATGCC

CCCAGCCAGAT (SEQ ID NO: 51)

RV: GCCCCMCCTTAAGTGGATGCCTTCACACGTGC (SEQ ID NO: 52)

TN5-7 FW: ATACvlTvlTCCATCATCATCATCATCATGAGTTGGACACG

CCCAAGGAC (SEQ ID NO: 53)

RV: GCCGGvlTCCTTATGTTGTGAACTTGGCAGTGATGGTTG (SEQ ID NO: 54)

TN6-8 FW: ATACvlTvlTGCATCATCATCATCATCATGCCATGGGCTCCCC

AAAGGAA (SEQ ID NO: 55)

RV: GCCGGvlTCCTTATGTGGTGAAGATGGTCTGGATCAT (SEQ ID NO: 56)

FBG FW: ATAG4 TJ4 TCCATCATCATCATCATCATATTGGACTCCTGTAC

CCCTTCC (SEQ ID NO: 57)

RV: GCCGGvlTCCTTATGCCCGTTTGCGCCTGCCT TCAA (SEQ ID NO: 58)

All primers above are written 5' to 3'. Flag sequences are in bold, His tags (CATCATCATCATCATCAT SEQ ID NO: 132) are underlined, and restriction enzyme cleavage sites (CATATG = Ndel site, GGATCC = BamHl, GGTACC = Kpnl site) are in bold italics.

PCR

PCR amplification was carried out using 10 pmol/μΐ of each primer, ^g template, 5μ1 DMSO, and 1.25 units Pfu Turbo polymerase in a final volume of 25μ1. This was added to buffer and dNTPs 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 cycled 40 times with denaturing, annealing and elongation temperatures of 95°C, 55-65°C (depending on melting temperature (Tm) of primers) and 72°C respectively.

Cloning

PCR products were ligated into pCR Blunt vectors and sequenced to ensure no errors had been introduced by PCR. Clones were selected that had no errors or silent mutations. Inserts were then ligated into pET32b using Ndel and BamHl restriction sites engineered into primers (TN5-7 and TN6-8). Human tenascin-C has internal BamHl sites within the TA domain (position 494) and TNIII2 (position 2509). TA and TN1-8 were therefore cloned using the Ndel site in the FW primer and the Kpnl site in the cloning site of pCRBlunt. Human tenascin-C contains no internal Kpnl sites. TN1-5, TN1-3 and TN3-5 were cloned using Ndel and Kpnl sites in the primers. FBG contains an internal Ndel site (position 6439) and was therefore cloned using a two step ligation of Ndel and BamHl digestion, followed by Ndel digestion. (Positions refer to sites within the full length nucleotide sequence of tenascin-C, given in figure 14)

Bacterial growth, induction and lysis

The plasmids were transformed into BL21 (DE3) Rosetta cells, cultured in 3 L of Luria-

Bertani medium containing 50 μg/ml carbenicillin and induced with 1 mM isopropyl- -D- thiogalactopyranoside. After 3 hours, the cells were harvested by centrifugation at 4,000 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 lysed with a French press. Inclusion bodies were collected by centrifugation at 12,000 rpm for 20 min at 4 °C. With the exception of TA and FBG the proteins were located entirely 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.

Purification of bacterial proteins

The solution containing recombinant protein was applied to a Ni²⁺-NTA-agarose column 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 washing and elution buffer contained 6 M guanidine hydrochloride. Following Ni chromatography TA and FBG required no subsequent purification. TN1-3 and TN6-8 were further purified by anion exchange chromatography using a HiTrap Q column, TN1-5, TN3-5 and TN5-7 by cation exchange chromatography using a HiTrap S column, and TN1-8 using a HiTrap S column followed by gel filtration using a Sephacryl S500 HR column.

Refolding of insoluble proteins

TA and FBG were refolded by diluting to 20 μg/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. The solution was then dialyzed twice against 15 volumes of 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl, 10 mM CaC , 5 mM β-mercaptoethanol, and 1 mM 2-hydroxyethyl disulfide for 24 hours at 4 °C, twice against 20 mM Tris-HCl, pH 8.0 for 8 hours at 4 °C and then centrifuged at 12,000 rpm for 30 min at 4 °C. Refolding was assessed by size shifts using SDS PAGE under reducing and non reducing conditions. Protein activity was confirmed by TA domain polymerization and FBG binding to heparin sepharose columns. Synthesis ofEGF-L domain using mammalian cells

Initial attempts to express and purify the EGF-L repeats region using an E.coli expression system were unsuccessful. This is most likely to be attributable to difficulty in achieving protein folding due to a total of 91 cysteines in this region. Therefore, the EGF-like domains of TN-C were expressed using HEK293 cells.

Two PCR reactions were carried out. The first PCR product consisted of a restriction enzyme Kpn\ site, a Kozak sequence followed by the TN-C signal sequence. The second PCR product consisted of a FLAG peptide, the EGF-like domain sequence, followed by a histidine tag and a BamHl restriction enzyme sequence.

The two PCR products were ligated together as described by Ho (1989). PCR reactions were carried out as described above. The entire construct was cloned into the PCR blunt vector and sequenced. It was then subcloned into the pCEP4 vector. The DNA was transfected into HEK293 cells using Fugene and cells were selected for hygromycin resistance (200 μg/ml) in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum, penicillin (100 units/ml) and streptomycin (100 units/ml). 2 litres conditioned medium (collected after cells have been cultured in medium) from stably transfected cells was collected and pooled. The pooled conditioned medium (2 litres) was centrifuged at 3000 rpm to separate cell debris from the medium.

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

Analysis of protein purity

Each protein was dialysed against 1000 volumes of 150 mM NaCl and 50mM Tris pH 7.5.

Protein purity was analyzed by SDSPAGE under reducing conditions. To do this 1 μg of each purified recombinant protein was run on a 4-12% Bis-Tris gradient gel and the gel was subsequently silver stained to demonstrate a single band (figure 10). Western blotting analyses were also carried out. Proteins separated by SDS-PAGE were electrotransferred to polyvinylidene difluoride membranes. The 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 blot was then incubated with secondary antibody conjugated to alkaline phosphatase and the protein bands visualized using Western Blue stabilized substrate whereby the gels show a single specific band recognised by each antibody at the expected Mw (not shown)

EXAMPLE 3 - ANIMAL MODELS

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

Control mice received an injection of 10 μΐ PBS alone or were not injected. For macroscopic assessment of arthritis, the thickness of each hind paw was measured daily with microcalipers (Kroeplin, Schluchlem, Germany) and the diameter expressed as an average for each inflamed hind paw per mouse.

Following completion of the experiment (day=4), mice were euthanized and hind paws fixed in 10% (v/v) buffered formalin, decalcified with 10% EDTA and processed to paraffin.

Antigen-induced arthritis

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

At day 7, arthritis was induced by intra-articular injection of mBSA (100 μg in 10 μΐ of sterile PBS) into the right knee joint using a sterile 33-gauge microcannula. Control mice received an injection of 10 1 PBS alone or were not injected.

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

Injection ofFBG

Wild type mice were anesthetized by intraperitoneal injection of 150 μΐ of Hypnorm diluted 1: 10 in sterile water and then injected with 100 ng, 1 or 3 μg FBG in 10 μΐ of sterile PBS into the right knee joint using a sterile 33-gauge microcannula. Control mice received an injection of 10 μΐ PBS alone or were not injected.

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

Histology of knee joints

Coronal tissue sections (4 μηι) were cut at 7 depths throughout the joint; 80 μηι apart and stained with hematoxylin and eosin or Safranin-0 to assess joint pathology. Histopathologic changes were scored using the following parameters as described in Van Lent (2006).

Inflammation (the influx of inflammatory cells into synovium (infiltrate) and the joint cavity (exudates), was graded using an arbitrary scale from 0 (no inflammation) to 3 (severe inflammation). Chondrocyte death was determined as the percentage of cartilage area containing empty lacunae in relation to the total area. Cartilage surface erosion was determined as the amount of cartilage lost in relation 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 of 0 (no damage) to 3 (complete loss of bone structure). Histological analysis was performed by an investigator who was blinded to the experimental groups. The mean score for each animal in an experimental group was calculated by averaging the histopathologic scores in at least 5 section depths per joint.

Results

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

Zymosan injection into the footpad was used to induce acute synovitis in mice. Wild type mice exhibited rapid paw swelling reaching maximal paw diameter by 24 hours (2.56mm, an increase of 62% of the starting paw diameter). This was maintained for a further 24 hours. After 2 days paw diameter decreased but paws remained swollen by 4 days (2.08mm, an increase of 32%) (figure la). tenascin-C deficient mice exhibited a similar degree of paw swelling to wild type mice 24 hours post injection (2.41mm, an increase of 57% of starting paw diameter). However, swelling in the tenascin-C null mice subsided faster than in the wild type mice; paw diameter was significantly reduced at 2 days and had declined to 1.7mm (an increase of only 11%) by 4 days (figure la). By day 4 post injection the paws of wild type mice were still visibly swollen and red, whereas the paws of tenascin-C null mice were not visibly swollen or red and resembled non- injected paws (Figure 9).

This difference was reflected histologically at 4 days. The synovia of wild type mice were significantly inflamed and exhibited cellular infiltration and cartilage proteoglycan loss was observed (Figure lb, c). In contrast, the synovium of tenascin-C deficient mice exhibited no synovitis, cellular infiltrate or cartilage proteoglycan loss (figure Id, e) and resembled the joints of sham injected and non injected mice (not shown). Quantification of joint inflammation revealed whilst there was little exudate (cellular mass in the joint cavity) in either wild type or tenascin-C null mice, levels of infiltrate (cellular mass in the synovial layer) were significantly reduced in tenascin-C null mice (figure If). No erosion of cartilage or bone occurred in mice of either genotype (not shown), however a low level of chondrocyte death occurred in wild type mice, that was not observed in tenascin-C null mice (figure lg). Thus tenascin-C expression appears to promote the maintenance of acute inflammation.

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

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

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

By 7 days wild type mice exhibited persistent inflammatory cell infiltration and joint space exudate, extensive synovitis and pannus formation and destruction of articular cartilage and bone erosion (figure 4a, b). Sham injected knees and knees from mice that had undergone no injection were healthy and exhibited no inflammation or joint destruction (not shown). tenascin-C deficient mice also had healthy joints that exhibited only mild inflammatory cell infiltration, with no joint space exudate, synovitis, pannus formation, destruction of articular cartilage or bone erosion (figure 4c, d). Joints from tenascin-C deficient mice that had been sham injected and or that had undergone no injection were also healthy (not shown).

These histological data are reflected upon scoring of joint disease as described in materials and methods. Levels of cellular infiltrate and exudate observed in both wild type mice and tenascin-C null mice 24 hours post injection were not significantly different. However, whilst cellular 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). Increasingly high levels of chondrocyte death occurred in the cartilage of wild type mice over time, but no significant death was observed in tenascin-C null mice (figure 4f). No cartilage surface erosion and bone erosion was evident in wild type mice at 24 hours or 3 days (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 whilst the initiation of joint inflammation (cell influx into the synovium and joint space) is unaffected in tenascin-C null mice, unlike in wild type mice disease does not progress to tissue destruction and cell death. These results demonstrate that expression of tenascin-C is required for persistent synovial inflammation and joint destruction in this model.

EXAMPLE 4 - CELL CULTURE

Patient Specimens

Human monocytes were isolated from peripheral blood (London Blood Bank) and macrophages were derived from monocytes after differentiation for 4 days with 100 ng/ml of M- CSF as previously described (Foxwell (1998)). 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 previously described (Brennan(1989)). The study was approved by the local Trust ethics committee (Riverside NHS Research Committee), and waste tissue (synovium after joint replacement surgery) was obtained only after receiving signed informed consent from the patient and anonymyzing the tissue to protect patient identity.

Immediately after isolation, RA membrane cells and macrophages were cultured at lxlO⁵ cells/well in RPMI 1640 containing 10% (v/v) FBS and 100 U/ml (Units/ml) penicillin/streptomycin in 96-well tissue culture plates for 24 hours before stimulation. Synovial fibroblasts (used only at either passage number 2 or 3) were cultured at lxlO⁴ cells/well in DMEM containing 10% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates for 24 hours before stimulation.

Mouse embryonic fibroblasts (MEFs) and bone marrow derived macrophages (BMDMs)

MEFs express high levels of mRNA of all 9 murine TLRs and are specifically and highly responsive to TLR ligand activation. MEFs from mice with targeted deletions of TLR2, TLR4 and MyD88 demonstrate profound defects in their IL-6 response to specific ligands (Kurt-Jones (2004)). MEFs were isolated from dl3 embryos harvested from age-matched, pregnant female wild type, TLR2, TLR4 and null mice (as described in Todaro (1963)). Fibroblasts were cultured at 2xl0⁴ cells/well in DMEM containing 10% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96- well tissue culture plates for 24 hours before stimulation.

BMDMs were derived by aspirating the femurs of age matched female wild type, TLR2 and TLR4 null mice as described in Butler (1999)) and culturing the cells for 7 days in DMEM, 20% (v/v) FBS, lOml/L (v/v) antibiotic-antimycotic solution PSA, 50μΜ β-Mercaptoethanol and lOng/ml M-CSF. Macrophages were then cultured at lxlO⁵ cells/well in DMEM, 20% (v/v) FBS, lOml/L (v/v) antibiotic-antimycotic solution PSA, 50μΜ β-Mercaptoethanol in 96-well tissue culture plates for 24 hours before stimulation.

HEK293 cell lines

HEK293 cell lines expressing TLR2 and TLR4/CD14/MD-2 were cultured at lxlO⁴ cells/well in DMEM containing 10% (v/v) FBS and 10 μg/ml blasticidin in 96-well tissue culture plates for 24 hours before stimulation.

Cell stimulation and assessment of cytokine synthesis

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 for human macrophages, lOng/ml for human fibroblasts, RA membrane cells and HEKs, lOOng/ml for MEFS and BMDMs and lOng/ml for HEKS), PAM3 (10 ng/ml for human macrophages, human fibroblasts, and HEKs, lOOng/ml for MEFs and BMDMs), murine IL-1 (5ng/ml for MEFS) and murine TNF-a (lOOng/ml for MEFS). Unless specifically stated otherwise rough LPS was used for in vitro studies.

For adenoviral gene transfer experiments, human RA synovial fibroblasts were incubated with adenoviral vectors at a multiplicity of infection of 100, washed after 2 hours, cultured in complete medium for 24 hours, then stimulated for 24 hours, after which time supernatants were collected.

Where stated, cells were pre-incubated with 10μg/ml anti-CD14 antibody, 10μg/ml IL1 receptor antagonist, 10μg/ml anti-TLR2 antibody, 25μg/ml anti-TLR4 antibody, 10 or 25μg/ml isotype control antibody, 25μg/ml polymyxin B, or ^g/ml msbB LPS, for 30 minutes at 37°C before stimulation. Where stated, recombinant tenascin-C and FBG, and LPS were boiled for 15 minutes before addition to cells

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

Supernatants were subsequently examined for the presence of the cytokines TNF-a, IL-6, and IL-8 by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Absorbance was read on a spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic, Vantaa, Finland) and analyzed using the Ascent software program (Thermo Labsystems, Altrincham, UK).

Results

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

We next investigated whether tenascin-C might activate the innate immune response. tenascin-C was used to stimulate primary human macrophages and RA synovial fibroblasts and the production of the pro-inflammatory cytokines TNF-a, IL-6 and IL-8 examined. The bacterial cell wall component LPS was used as a positive control. tenascin-C induced a cell type specific cytokine profile which was significantly different from LPS. It dose dependency stimulated the production of TNF-a, IL-6 and IL-8 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 TNF-a synthesis in fibroblasts (data not shown). tenascin-C stimulation of IL-6 (Figure 5c), IL-8 and TNF-a by human macrophages and IL-6 by synovial fibroblasts (not shown) was heat sensitive and unaffected by the LPS inhibitor, polymyxin B. Together these results provide strong evidence that cytokine induction by tenascin-C is not due to LPS contamination.

The fibrinog en-like globe (FBG) mediates tenascin-C activation of cells.

Tenascin-C is a large hexameric molecule, each domain of which binds to different cell surface receptors (reviewed in Orend (2005)). Understanding the mechanism of action of tenascin-C will require 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 made in E.coli, purified (figure 11), and found to contain <10 pg/ml LPS by subjecting neat protein to the Limulus amaebocyte lysate assay. Only one domain of tenascin-C was active. The fibrinogen-like globe (FBG) stimulated TNF-a 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 equal extent 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 expression of inflammatory cytokines in synovial membranes from RA patients. This tissue model of RA (comprising a mixed population of all synovial cell types) spontaneously produces high levels of IL-6, IL-8 and TNF-a (Brennan (1989)) (figure 6b). FBG further enhanced synthesis of all these cytokines (figure 6b). To determine whether FBG could induce inflammation in vivo, wild type mice were injected intra- articularly with FBG. We observed a transient and dose dependent stimulation of joint inflammation. No inflammation or proteoglycan loss occurred in non-injected mice or in mice injected with PBS (figure 6c-e) or lOOng FBG (data not shown). In mice injected with 1 μg FBG inflammatory cell infiltration (figure 6f), mild synovitis, pannus formation (figure 6g) and proteoglycan loss (figure 6h) was observed. A similar response was seen in mice injected with 3 μg FBG (data not shown). Upon histological quantification, high levels of cellular infiltrate and exudate and chondrocyte death were observed in mice injected with FBG, together with a modest amount of cartilage surface erosion and bone damage (figure 6i).

FBG mediated cytokine synthesis is dependent on Myd88

Many DAMPs, including fibrinogen (Smiley (2001)), have been shown to stimulate the innate immune response by activation of TLRs. Therefore, we investigated whether TLRs might also mediate tenascin-C induced cytokine production. Myeloid differentiation factor 88 (MyD88) is required for signalling by all TLRs, except TLR3 (O'Neill (2008)). Infection of synovial fibroblasts with adenovirus expressing dominant negative MyD88, but not GFP control virus, abolished FBG induction of 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 as addition of IL-1 receptor antagonist did not inhibit induction of cytokines (data not shown). To confirm that FBG action is MyD88 dependent we demonstrated that FBG does not stimulate cytokine synthesis in embryonic fibroblasts isolated from mice with targeted deletions in the MyD88 gene. The TLR2 ligand PAM3, TLR4 ligand LPS and IL-1 all signal via MyD88. Stimulation with these was also abolished in MEFs from deficient mice. However, TNF-a, which does not signal via MyD88, was unaffected (figure 7b). Re-transfection of wild type MyD88 restored the responsiveness 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 recognised by one or both of TLR2 and 4 (reviewed in O'Neill (2008)). Neutralising antibodies to TLR4 inhibited both FBG and LPS induced IL-6, IL-8 and TNF-a synthesis in human macrophages and IL-6 synthesis in RA synovial fibroblasts but had no effect on the function of the TLR2 ligand, PAM3. Antibodies to TLR2 inhibited PAM3 mediated cytokine synthesis but had no effect on LPS or FBG induced cytokine synthesis. Isotype matched controls had no effect on cytokine synthesis induced by any ligand (TNF-a synthesis by human macrophages is shown in figure 8a). To confirm that FBG action is TLR4 dependent we demonstrated that FBG does not stimulate cytokine synthesis in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR4 gene. FBG mediated cytokine synthesis was unaffected in embryonic fibroblasts or macrophages isolated from mice with targeted deletions in the TLR2 gene. Cells isolated from TLR2 deficient mice were unresponsive to PAM3 but responsive to LPS and IL-1. Cells isolated from TLR4 deficient mice were unresponsive to LPS but did respond to PAM3 and IL-1 (figure 8b, c). In addition, expression of TLR4 was required for the 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 co-receptor requirements for FBG and LPS

LPS signalling via TLR4 is mediated by a receptor complex including the soluble protein

MD-2 and GPI-linked cell surface or soluble CD14 (reviewed in Fitzgerald (2004)). We next examined whether CD14 and MD-2 are required for FBG activation of TLR4. As a positive control here we examined the activity of smooth glycosylated LPS which requires both MD-2 and CD14 (Jiang (2005)). LPS mediated IL-6, IL-8 and TNF-a synthesis by human macrophages and IL-6 synthesis by RA synovial fibroblasts was inhibited by anti-CD14 antibodies and an antagonistic LPS derived from the msbB mutant E.coli which competes for LPS binding to MD-2 (Coats(2007)). Conversely, both PAM3, which does not require these co-receptors for activation of TLR2, and FBG- mediated cytokine synthesis was unaffected by anti CD14 antibodies or msbB mutant LPS (figure 8d shows TNF-a synthesis by human macrophages). These data suggest that neither CD14 nor MD-2 is required for FBG mediated cytokine synthesis. Therefore, whilst LPS and FBG both signal via activation of TLR4, they may have different co-receptor requirements.

EXAMPLE 5 - INHIBITION OF TENASCIN-C ACTION AND SYNTHESIS IN HUMAN TISSUE

This example studies the effect of (1) prevention of the pro-inflammatory action of tenascin-C and (2) inhibition of tenascin-C expression in the human RA synovium.

Methods

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

The facility was unable to synthesize peptide 7, presumably due to the formation of secondary structure 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

Patient specimens 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 (GIBCO) containing 5% fetal calf serum (FCS) (GIBCO), 5 mg/ml collagenase type IV (Sigma) and 0 15 mg/ml DNAse type I (Sigma) and 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). RA synovial fibroblasts were isolated from the mixed population of RA membrane cells by selection in DMEM (Bio-Whittaker) supplemented with 10% FBS, 1 μΜ glutamine, 100 U/ml penicillin, and streptomycin. Human monocytes were isolated from peripheral blood (London Blood Bank) and macrophages were derived from monocytes after differentiation for 4 days with 100 ng/ml of M- CSF.

The study was approved by the local Trust ethics committee, and waste tissue (synovium after joint replacement surgery) was obtained only after receiving signed informed consent from the patient and anonymyzing the tissue to protect patient identity.

Cell stimulation and assessment of cytokine synthesis Immediately after isolation, RA membrane cells were cultured at lxlO⁵ cells/well in RPMI 1640 containing 10% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates. Cells were incubated for 24h at 37°C with no addition, buffer control (PBS, 1% BSA, 0.01% NaNs), or with 25 μηι, 100 μΜ or 250 μΜ of each FBG spanning peptide.

Synovial fibroblasts (used only at either passage number 2 or 3) were seeded at a concentration of 5 ^χ 10⁴ cells in a 3.5-cm dish. siRNA was transfected at a final concentration of 10 nM using Lipofectamine 2000 (Invitrogen) for 4 h in serum-free OptiMEM I. Two different siRNAs against human tenascin-C were used (s7069 and s229491) (Applied Biosystems).

siRNA sequences of s7069 are: (sense 5' CGCGAGAACUUCUACCAAAtt 3' (SEQ ID NO: 68), antisense 5' UUUGGUAGAAGUUCUCGCGtc 3' (SEQ ID NO: 69)) and of s229491 are (5' GGAAUAUGAAUAAAGAAGAtt 3' (SEQ ID NO: 70), antisense 5' UCUUCUUUAUUCAUAUUCCgg 3' (SEQ ID NO: 71)). siRNA against luciferase (Dharmacon) was transfected as a non-targeting control.

Four hours after transfection, medium was changed with pre-equilibrated Dulbecco's modified Eagle's medium containing 10% FBS (v/v) and cells were incubated for a further 48h and 72h. Cells were then stimulated with 10 ng/ml LPS for 24h at 37°C. Tenascin-C mRNA and protein levels were quantitated by PCR and western blotting respectively. Total RNA was extracted from cells using a QiaAmp RNA Blood mini kit (Qiagen, Germany). cDNA was synthesised from equivalent amounts of total RNA using Superscript® III Reverse Transcriptase (Invitrogen) and 18-mer oligo dTs (Eurofins MWG Operon).

Gene expression was analysed by delta-delta ct methods based on quantitative real-time PCR with TaqMan primer set human tenascin-C (Hs01115663-ml) and human ribosomal protein endogenous control (RPLPO) (4310879E) (Applied Biosystems) in a Corbett Rotor-gene 6000 machine (Corbett Research Ltd). Tenascin-C protein was detected in cell supernatants and cell lysates by by SDS PAGE and western blotting using antibody MAB1908 (Millipore).

Macrophages were cultured at lxlO⁵ cells/well in RPMI 1640 containing 5% (v/v) FBS and 100 U/ml penicillin/streptomycin in 96-well tissue culture plates for 24h before stimulation. Cells were incubated for 24h at 37°C with no addition, 1.0 μΜ FBG, 1 ng/ml LPS or 1 or 20 μΜ FBG peptide. Where stated, cells were pre-incubated with 20 μΜ FBG peptides for 15 min.

The viability of the cells was not significantly affected throughout the experimental time period when examined by the MTT cell viability assay (Sigma, Poole, UK). Supernatants were examined for the presence of the cytokines TNF-a, IL-6, and IL-8 by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&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, UK).

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

Results

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

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

We synthesized a series of 8 overlapping peptides of ~30 amino acids that span the entire sequence of FBG (Table 2). Peptides were tested for the ability to block spontaneous cytokine synthesis in RA synovial membrane cultures. TNF and IL8 synthesis was inhibited by peptides 3 and 8, but not by any other peptide (TNF shown in figure 15). Peptides 3 and 8 dose dependency inhibited cytokine synthesis with the highest concentrations achieving 95% and 56% inhibition respectively (figure 16). Whilst peptide 5 had no effect on TNF synthesis, it dose dependently blocked IL8 synthesis in RA membrane cells with a maximal inhibition of 81% (figure 16).

To map the active domain within FBG responsible for inducing cytokine production we stimulated primary human macrophages with each FBG peptide. Peptides 1, 5 and 6 all induced cytokine synthesis in a dose dependent manner. (Figure 17).

To determine if any peptide could block FBG induced cytokine synthesis in human macrophages, cells were pre-incubated with each FBG peptide before stimulation with either whole FBG or LPS. Peptide 5 specifically blocked FBG mediated cytokine synthesis, whilst peptide 8 blocked cytokine synthesis in response to both LPS and FBG (figure 18).

Peptide 8 therefore non-specifically blocks cytokine production induced by any stimuli. This domain is the integrin binding domain of FBG that mediates cell adhesion and thus may be acting to prevent cell attachment to tissue culture plates. Peptide 5 specifically blocks FBG- induced cytokine synthesis suggesting that targeting this domain may be useful in preventing tenascin-C induced inflammation.

Silencing tenascin-C gene expression inhibits cytokine synthesis in RA synovial fibroblasts

Examination of the effect of inhibiting tenascin-C expression in the human RA synovium has identified synovial fibroblasts as the major source of tenascin-C in RA (figure 1C) (in Goh 2010).

siRNA mediated knockdown of tenascin-C expression in these cells has been shown with a maximal efficiency between 94-96% (figure 19). In cells transfected with tenascin-C siRNA, both the basal level of cytokine synthesis and LPS induced cytokine production was inhibited by 38% and 44% respectively compared to control cells (figure 19)

These data reveal that silencing tenascin-C in RA synovial fibroblasts reduces the synthesis of pro-inflammatory cytokines and suggest that ablation of tenascin-C expression is a viable strategy to inhibit inflammation in the synovium. This work has established that blocking tenascin-C activity (with peptides) and tenascin-C expression (with siRNA) reduces inflammatory cytokine synthesis in human RA synovia. These data shows that tenascin-C blockade is of potential clinical benefit in treating RA and other inflammatory diseases.

EXAMPLE 6 - HOW THE FBG DOMAIN OF TENASCIN-C (FBG-C) ACTIVATES TLR4

Here is established how the FBG domain of tenascin-C (FBG-C) activates TLR4 in order to be able to develop specific inhibitors for the pro-inflammatory action of this matrix glycoprotein.

Using the whole tenascin family to determine how the FBG domain of tenascin-C acts

In addition to tenascin-C, the tenascin family also includes tenascin-R, -W and -X (Table 3).

FBG Domain Accession Amino Molecular No. of pi

number acids Weight amino

position (kDa) acids

FBG-C human P24821 1974-2202 26.1 229 8.78

FBG-X human P22105 4013-4243 26.1 231 5.54

FBG-R human Q92752 1128-1359 27.0 232 8.63

FBG-W human Q9UQP3 1060-1300 27.6 240 9.18

Table 3. Features of the FBG domains of tenascin family members

Each has a distinct pattern of expression in adults: tenascin-R is mainly expressed in the brain and the central nervous system; tenascin-W is expressed in smooth muscle and bone; and tenascin-X is expressed in loose connective tissue. Only tenascin-C is expressed at universally sites of inflammation. The tenascin family have a similar domain organisation and all contain an FBG domain, which is highly homologous amongst all members (Table 4). However, it is not known if the FBG of the other tenascins can induce an immune response. Assessing the activity of these FBG domains allows the comparison among their sequences based on their function and determine which regions of the protein might be involved in inducing an inflammatory response.

FBG -C FBG -X FBG -R

FBG-X 52.89 %

FBG-R 59.65 % 54.67 %

FBG-W 53.02 % 49.56 % 55.74 %

Table 4. % of identity among the FBG domain of the tenascin family members. Pairwise alignment analysis with ClustalW software was used to compare the sequences of the FBG domain of the tenascin family members. The amino acid sequences of FBG-C, FBG-R and FBG-W are shown above as SEQ ID NOs: 1, 2 and 3, respectively. The amino acid sequence of FBG-X is shown in SEQ ID NO: 4

The FBG domains of tenascin-R, -W and -X (FBG-R, -W and -X) were made by cloning their DNA sequence into pET32b vector (Figure 34) and proteins were expressed in E.coli BL21 DE3. FBG-R, -W and -X were purified using nickel chromatography and biophysically characterized to check protein secondary structure, purity, correct folding and identity using SDS page silver staining, western blot and circular dichroism (Figure 35).

The inflammatory activity of FBG-R, -W and -X was assessed using a monocytic cell line ThPl Nf-kB, which have an Nf-kB reporter that is activated downstream of TLR4; and in primary human macrophages where cytokine synthesis was measured by ELISA. FBG-R and -W could induce the activation of Nf-kB and the synthesis of IL-6, IL-8 and TNF in primary human macrophages to an extent similar to FBG-C. However, the ability of FBG-X to induce an immune response was significantly reduced, showing little Nf-kB activation and induction of proinflammatory cytokines (Figure 20). Protein samples were treated with polymyxin B or boiling to verify that the protein immune activity was not due to LPS contamination (Figure 36).

In silico analysis comparing the protein sequences of the FBG domain of the tenascin family members revealed multiple regions of high conservation (Figure 21). As a consequence, it was difficult to determine a specific region for protein activity. Peptide mapping was therefore used to determine which amino acids of FBG-C could be involved in its immune function. 9 peptides of 30 amino acids long encompassing the complete sequence of FBG-C (see Table 2, Example 5) were tested in ThPl Nf-kB cells and primary human macrophages. Only peptides 5 and 6 could induce an inflammatory response in both cell types (Figure 22).

To determine the location of the overlapping region in the FBG structure, structural analysis was performed. Because the protein structure of the tenascin family members is not known, homology modelling was used to determine it. Proteins were modelled based on the structure of C-terminus fibrinogen y chain, as all FBG domain containing proteins have a similar pattern of folding and high % of conservation (Table 5). They all comprise 3 sub-domains: A, B and P. The results showed high homology in the protein structure among the FBG domain of the tenascin family members and similar pattern of folding compared to the structure of other FBG containing proteins (Figure 37)

Table 5. Identity between the FBG domains of human tenascin-C, -R, -X and -W and fibrinogen Y chain. Pairwise alignment analysis with ClustalW was used to determine the % of identity of the tenascin family members against C-terminus fibrinogen Y chain.

The overlapping region among P5 and P6 corresponds to a surface loop (loop 5) in the P- subdomain of FBG-C. Loop 5 contains a specific epitope that is conserved also in FBG-R and -W, but not in FBG-X (Figure 23). Scrambled and shorter versions of P5, P6 and loop 5 (Table 6) were designed and it was found that the activity of the peptides was due to this specific sequence (Figure 38). Thus, these amino acids were selected for mutagenesis studies.

KGVFLTRYVTDARDVHFDKYGASRELEAKD (SEQ ID NO: 72)

P6S YKGTSDHFRVGSNSRYETSMGKGAATLKY (SEQ ID NO: 73)

P9S AFERHMKWKRKLRGAGHREPELHSNSNRIFQF (SEQ ID NO: 74)

Loop 5-1 KTRYK (SEQ ID NO: 18)

Loop 5-2 GDAKTRYKLKVEG (SEQ ID NO: 75)

Loop 5-3 DKFSVGDAKTRYKLKVEGYSG (SEQ ID NO: 76)

Table 6. Amino acid sequences of PIS, P5S and P6S and variations of loop 5. Scramble peptides were designed as controls to determine sequence specificity of peptide 1, 5 and 6. 3 peptides were designed incorporating peptide 5 and 6 overlapping sequence (bold) and varying the number of contiguous amino acids

Mutagenesis studies

New peptides were designed introducing mutations to the positive charged amino acids in loop 5 (Table 7).

Loop 5-4 AFAVYDKFSVGDAKTRYKLKVEGYSGTAGD (SEQ ID NO: 77)

Loop 5-deletion GETAFAVYDKFSVGDAVEGYSGTAGDSMAY (SEQ ID NO: 78)

Loop 5 -mutant 1 AFAVYD KF SVG D AAT AYKLKVEG YSGTAG D (SEQ ID NO: 79)

Loop 5 -mutant 2 AFAVYDKFSVGDAKTRYALAVEGYSGTAGD (SEQ ID NO: 80)

Loop 5 -mutant 3 AFAVYDKFSVGDAATAYALAVEGYSGTAGD (SEQ ID NO: 81)

Table 7. Amino acid sequences of loop 5 mutant peptides. The overlapping sequence of peptide 5 and 6 correspond to loop 5. 2 peptides were designed incorporating or deleting this sequence (bold). 3 peptides were designed containing alanine substitutions (bold) to positive charged amino acids in this sequence.

In addition, 3 mutant proteins were made using the Quick-Change mutagenesis kit to alter their amino acid sequence. Mutant protein 1 and 2 included double mutations in the amino acid sequence of loop 5 and in mutant protein 3, 4 mutations were introduced, similar to the mutation peptides (Table 8).

FBG-C loop 5 AFAVYDKFSVGDAKTRYKLKVEGYSGTAGD (SEQ ID NO: 77) FBG-C mutant 1 AF AVYD KFS VG D AATAYKL KVE G YSGT AG D (SEQ ID NO: 79)

FBG-C mutant 2 AFAVYDKFSVGDAKTRYALAVEGYSGTAGD (SEQ ID NO: 80)

FBG-C mutant 3 AF AVYD KFS VG D AATAYALAVE G YSGT AG D (SEQ ID NO: 81)

Table 8. FBG-C mutant protein sequences. Mutant proteins were made in loop 5 where positive charged amino acids (bold) were substitute by alanine (bold).

These proteins were expressed in E.coli, purified using nickel chromatography and biophysically characterised (Figure 39). Assessing protein and peptide activity showed a reduction of Nf-kB activation in ThPl cells and a lower induction of cytokine synthesis in primary human macrophages by FBG-C mutant 1 and 2 and loop 5- mutant 1, 2 and 3 compared to FBG-C WT or loop 5. In contrast, mutant protein 3 showed a complete loss in protein activity in ThPl Nf-kB cells and in primary human macrophages (Figure 24). These results indicate that positive charged amino acids in loop 5 are important for TLR4 activation by FBG-C.

In addition, it was examined if the activity of FBG-R and -W was TLR4 dependent. The use of a TLR4 antibody or a TLR4 inhibitor (TAK 242) was able to block cytokine induction in primary human macrophages by FBG-R and -W showing that these proteins also signal through this receptor (Figure 25). The interaction among FBG-C, -R and -W with TLR4 was corroborated using a solid phase binding ELISA that showed that these proteins can bind to TLR4 (Figure 26), with a KD ~ 50-60nM. FBG-X didn't show binding to this receptor consistent with little protein activity observed in the cell assays (Table 9).

Protein KD (nM) ± SD

FBG-C 58.21 ± 5.24

FBG-R 50.56 ± 14.44

FBG-W 57.74 ± 9.91

FBG-X nd

Table 9. Kd values of FBG-C, -R, -W and -X binding to TLR4 based on solid phase binding assay. Data shows mean ± standard deviation, N=4. nd: non determined.

Binding of FBG-C mutant proteins to TLR4 was also assessed using solid phase binding. A reduction in binding was observed of FBG-C mutant 1, 2 and 3 to TLR4 compared to FBG-C (Figure 27). However, a modest but significant reduction in the KD affinity values in the FBG-C mutant 1 and 3 was shown (Table 10), indicating that they might retain some binding to TLR4 that is not affected by these mutations.

Protein KD (nM) ± SD p value

FBG-C 58.21 ± 5.24

FBG-C mut l 81.01 ± 15.45 * 0.031 FBG-C mut 2 75.92 ± 15.58 0.074

FBG-C mut 3 121.10 ± 15.29 ***

0.0002

Table 10. Kd values of FBG-C, FBG-C mutant 1, FBG-C mutant 2, FBG-C mutant 3 binding to TLR4 based on solid phase binding assay. Data shows mean ± standard deviation, N=4. Un-paired t-test vs FBG-C.

To further analyse the importance of the positive charged amino acids in loop 5, another mutant proteins were made to recover the protein activity of FBG-X. FBG-X mutant 1 have 2 hydrophobic amino acids substituted for the positive charged amino acids that are particularly important to stimulate an immune response by the other tenascin family members; FBG-C mutant 2 have the complete sequence of loop 5 changed to the sequence of FBG-C (Table 11).

FBG-C AFAVYDKFSVGDAKTRYKLKVEGYSGTAGD (SEQ ID NO: 77)

FBG-X GDEAVFAQYDSFHVDSAAEYYRLHLEGYHG (SEQ ID NO: 82)

FBG-X mutant 1 GDEAVFAQYDSFHVDSAKAKYRLHLEGYHG (SEQ ID NO: 83)

FBG-X mutant 2 G D E AVF AQYD S F H VGD AKTKYRLKL EG YHG (SEQ ID NO: 84)

Table 11. FBG-X mutant protein sequence. FBG-X chimeric protein was designed to add positive charged amino acids missing from FBG-X sequence and important for FBG-C activity.

FBG-X mutant 1 and mutant 2 were made and expressed in E.coli, purified using nickel chromatography and biophysically characterised (Figure 40). Assessment of the protein activity in ThPl NF-kB cells and primary human macrophages showed that FBG-X mutant 1 cannot activate the NF-kB transcription factor or induce the synthesis of pro-inflammatory cytokines. However, a modest recovery was observed with FBG-X mutant 2 which could induce NF-kB transcription factor activation and cytokine synthesis at higher concentrations compared to FBG-C. Furthermore, in a solid phase binding assay, FBG-X mutant 1 didn't bind to TLR4 similarly to FBG-X, but FBG-X mutant 2 showed low binding affinity to TLR4 (KD=173.09 nM) compared to FBG-C (Figure 28, Table 12).

Protein KD (nM) ± SD

FBG-C 59.77 ± 7.08

FBG-X nd

FBG-X mutant 1 nd

FBG-X mutant 2 173.09 ± 56.66

Table 12. Kd values of FBG-C, -R, -W and -X binding to TLR4 based on solid phase binding assay. Data shows mean ± standard deviation, N=4. nd: non determined. These data suggest that changing only 3 amino acids is not enough to recover FBG-X protein activity; however changing the entire amino acids composition of FBG-X loop 5 conferred a modest recovery in protein activity indicating that adjacent amino acids give the structure to this loop necessary for receptor activation. However, we observed a partial recovery of the protein activity of FBG-X suggesting that other possible site might be necessary for TLR4 binding but not activation.

Another region that has a different amino acid composition among the tenascin family members is the sequence of peptide 9. FBG-X lacks the positive charged amino acids at the C- terminus of this peptide that corresponds to loop 10 (Figure 29). New peptides were designed introducing mutations to the positive charged amino acids in loop 10 (Table 13).

P9 FHWKGHEHSIQFAEMKLRPSNFRNLEGRPvKRA (SEQ ID NO: 67)

P9 mutant 1 FHWKGHEHSIQFAEMKLRPSNFRNLEGGG (SEQ ID NO: 85)

Table 13. Amino acid sequences of P9 mutant peptides. Positive charged amino acids at the C terminus of P9 (bold) were mutated to glycines (bold).

In addition, 2 mutant proteins were made: FBG-C mutant 4 has a mutation in loop 5 and also in loop 10, while FBG-C mutant 5 has only positive charged amino acids substitutions in loop 10 (Table 14).

Table 14. FBG-C mutant protein sequences. Mutant proteins were made in loop 10 where positive charged amino acids (bold) were substitute by glycine (bold) or deleted.

These proteins were expressed in E.coli, purified using nickel chromatography and biophysically characterised (Figure 39). Peptide 9, peptide mutant 9 and FBG-C mutant 4 had no effect activating the NF-kB transcription factor or inducing cytokine synthesis in primary human macrophages. However, FBG-C mutant 5 showed only a modest but significant decrease in the activation of NF-kB transcription factor and no significant effect was observed in cytokine induction in primary human macrophages (Figure 30). In the binding assay, FBG-C mutant 5 presented a non-significant reduction in the affinity to TLR4. Conversely, FBG-C mutant 4 showed a significant reduction in the affinity to TLR4, greater than FBG-C mutant 3, suggesting that loop 10 is involved in FBG-C interaction with TLR4 (Figure 31, Table 15).

Protein KD (nM) ± SD p value

FBG-C 53.4 ± 7.93 FBG-C mut 4 336.4 ± 103.12 ** 0.009 FBG-C mut 5 63.03 ± 8.92 0.234

Table 15. Kd values of FBG-C, FBG-C mutant 4 and FBG-C mutant 5 binding to TLR4 based on solid phase binding assay. Data shows mean ± standard deviation, N=3. Un-paired t-test vs FBG-C.

Another approach taken to establish possible regions of interaction among FBG-C and TLR4 was a blocking binding assay where TLR4 was pre-incubated with the peptides prior addition to FBG-C coated plates. It was found that P5 and P6 could interfere in the interaction of FBG-C and TLR4; however the greatest effect was shown by P7, which could significantly blocked the binding of TLR4 to FBG-C (Figure 32, table 16).

Protein KD (nM) ± SD p value

TLR4 only 68.85 ± 7.79

TLR4 + PI 78.75 ± 11.33 0.280

TLR4 + P2 83.29 ± 5.24 0.056

TLR4 + P3 64.45 ± 15.67 0.672

TLR4 + P4 42.63 ± 10.06 *

0.023

TLR4 + P5 241.22 ± 89.15 *

0.028

TLR4 + P6 370.82 ± 141.64 *

0.021

TLR4 + P7 383.71 ± 113.31 **

0.008

TLR4 + P8 31.87 ± 8.22 *

0.048

TLR4 + P9 86.26 ± 16.57 0.17

Table 16. Kd values of FBG-C binding to TLR4 only or TLR4 pre-incubated with peptides P1-P9. Data shows mean ± standard deviation, N=3. Un-paired t-test vs TLR4 only.

P7 sequence corresponds to loop 7, a-helix and loop 8 in the P-subdomain of the proteins, which are regions of high conservation among the FBG domain of the tenascin family members (Figure 33). Further analyses were therefore performed to investigate the role of loop 7.

Peptide 7, which blocked TLR4 binding by intact FBG-C, comprises loops 7-8 of FBG-C. This region is highly conserved in FBG-R and -W and in the inactive FBG-X, with the exception of 3 distinct residues (Fig. 33). FBG-C mutant 6 was created in which these 3 amino acids were substituted for those present in FBG-X: D157P, I160L, N162S. FBG-C mutant 7 contained these three substitutions plus mutation of the four positively charged amino acids in loop 5 (Fig. 41a). Mutant 6 exhibited a modest decrease in NF-kB activation and cytokine synthesis, whilst mutant 7 was not active in these assays, as expected due to the loss of positive charge in loop 5 (Fig. 41b, c). FBG-C mutant 6 also exhibited a modest reduction in binding to TLR4, while mutant 7 showed a more significant reduction in the affinity to TLR4, similar to FBG-C mutant 3 (Fig. 41c and Table 17).

Protein KD (nM) i SO p value

FBG-C 60.48 * 11.43

FBG-C mut 6 86.69 ± 2125 0.135

FBG-C milt 7 123.78 + 28.33 0.023 ^** Table 17. Affinity of TLR4 binding to FBG-C mutants. 96 well plates were coated with FBG-C or FBG-C mutatns and TLR4 was addead in a dose dependent manner. The affinity of each FBG variant for TLR4 was calculated using GraphPad Prism. N=3 for FBG-C and FBG-C mutants 6 and 7. Un- paried t-test vs FBG-C, *= p<0.05, **= p<0.01, ***= p<0.001.

These data suggest that loop 7 contributes to FBG-C binding to TLR4, specifically mediating polar and hydrophobic interactions, and that cooperation between loops 5 and 7 mediates high affinity binding to this receptor.

Creating an active FBG-X

To confirm if the amino acids identified in FBG-C are sufficient to create an FBG domain that is capable of binding to and activating TLR4, we generated variants of the inactive FBG-X in which sequences from loops 5 and 7, and the C terminal region of FBG-C were substituted into the corresponding region of FBG-X. FBG-X mutant 1 has 3 amino acids changed to create a positively charged 'KAKYR' (SEQ ID NO: 88) sequence in loop 5 analogous to the KTRYK (SEQ ID NO: 18) sequence in FBG-C, FBG-X mutant 2 has the complete sequence of FBG-C loop 5 inserted, FBG-X mutant 3 includes the mutations made

in FBG-X mutant 2 plus addition of the positively c 212 harged amino acids at the C-terminus, and FBG-X mutant 4 includes mutations in loop 5 and the C terminus, plus the three amino acids substitution in loop 7: P161D, L165I and S167N (Fig. 42a). Similarly to wild type FBG-X, FBG-X mutant 1 was unable to activate NF-kB and induce cytokine synthesis, and did not bind to TLR4, whilst FBG-X mutant 2 exhibited a modest increase in activity at high doses and a low affinity for TLR4 (Fig. 42b, c, d and Table 18). Protein KO (nM) i SD p value

FBG-C 59.77 ± 708

FBG-X ND

FBG-X mutant 1 ND

FBG-X mutant 2 173 09 ± 56.66 ^** 0 007

FBG-X mutant 3 154 96 + 46.74 " 0.006

FBG-X mutant 4 78.33 + 18.41 0.109

Table 18. Affinity of TLR4 binding to FBG-X mutants. 96 well plates were coated with FBG-C, FBG- X, FBG-X mutants 1, 2, 3 or 4, and TLR4 was added in a dose dependent manner. The affinity of FBG-C or FBG-X mutants to TLR4 was calculated using GraphPad Prism. N=4. Un-paired t-test vs FBG-C. ** = p<0.01.

These data indicate that whilst the positive charges in the KTRYK (SEQ ID NO: 18) sequence are essential for TLR4 activation they are not sufficient, and that adjacent amino acids in this loop are also required. These data also confirm that additional sites outside loop 5 are required to activate and bind to TLR4 effectively. Both FBG-X mutant 3 and mutant 4 activated NF- kB and induced cytokine synthesis at similar levels to FBG-C. However, mutant 3 bound to TLR4 with a low affinity similar to mutant 2, while mutant 4 bound TLR4 with a high affinity comparable to FBG-C (Fig. 42b-d and Table 18). These results confirm that loop 7 and the C-terminus of FBG-C are necessary to assist loop 5 in optimal binding to TLR4, and highlight specific amino acids that are essential for effective receptor activation. Together these data demonstrate the cooperative action of 3 distinct regions in FBG-C that work together to bind to and activate TLR4 (Fig. 42e).

Discussion

Tenascin-C is a large (~1.5 million Da) multi-modular, extracellular matrix protein that is specifically up-regulated at sites of tissue injury, where it triggers an immune response, inducing the synthesis of pro-inflammatory cytokines via activation of TLR4 by its FBG domain. Whilst transiently

expressed during physiological tissue repair, persistent tenascin-C expression is associated with sterile pathological inflammation, where it contributes to disease chronicity in a variety of conditions.

Here the present inventors have identified three sites within the FBG domain of tenascin-C that bind to TLR4 to drive its activation, mapping key residues at each site. Together these data reveal a unique mode of pattern recognition by TLR4, and highlight regions of tenascin-C that might be specifically targeted for the amelioration of chronic inflammation. The FBG domains of tenascin-R and -W activated NF-kB in ThPl cells and induced cytokine synthesis in human macrophages similarly to the FBG domain of tenascin-C. Despite a high level of sequence identity and structural homology, FBG-X was not active in these assays. Tenascin-R is constitutively expressed in the perineuronal nets of the CNS, but is also up-regulated at sites of inflammation after murine spinal cord injury and optic nerve lesion. Tenascin-R null mice recover better after spinal cord injury compared to wild type mice, suggesting that tenascin-R helps drive inflammation during CNS injury. The fibronectin type III repeats (FN6-8) and the EGF-L domain of tenascin-R induce microglial synthesis of chemokine-induced cytokine 3 and TNFa in vitro, although the receptors involved are not known, and the FBG domain enhances microglial adhesion and migration in vitro. Our data indicate that the latter domain may also contribute to pro inflammatory cytokine synthesis following neural trauma via activation of TLR4 expressed by microglia and astrocytes; key cellular players in CNS repair. Constitutive tenascin-R expression raises the question of how the inflammatory activity of tenascins is regulated. Within the complex insoluble structure of the perineural nets, the FBG domain of tenascin-R may be physically constrained in a way that precludes TLR4 binding under normal circumstances. However, these nets are degraded upon injury, which could release FBG-containing fragments capable of TLR4 activation.

Alternatively, up-regulation of tenascin-R expression upon injury may create tissue concentrations above the threshold of TLR4 detection, or soluble de novo tenascin-R may be more readily accessible to cell surface TLR4. Tenascin-W is the most recently discovered family member and its expression in healthy tissue is predominantly restricted to stem cell niches. It is significantly up-regulated in the stroma of solid tumours, and both local and systemic levels can be reliable markers of tumor detection and prognosis. Tenascin-W expression is induced by TNFa in mouse embryonic fibroblasts, suggesting that, like tenascin-C, it may be responsive to inflammatory stimuli. TLR4 up-regulation in cancers including colorectal, liver, pancreatic and breast has been linked to increased tumor cell migration, invasion, survival and proliferation. Our data suggest that the FBG-W could promote disease progression either by activation of tumor cell surface TLR4 resulting in direct effects on cell phenotype, and/or by activation of immune cells resident in the tumor environment.

Tenascin-X is constitutively expressed in connective tissues, such as the dermis and glomeruli, where its role in maintaining tissue architecture, for example by regulating collagen fibril formation and elastic fiber stability, has been best studied. Tenascin-X expression is regulated upon tissue injury, but unlike the other family members, which appear in the early inflammatory stages, tenascin-X appears after the resolution of inflammation during tissue rebuilding. Consistent with these reports, whilst FBG-X does not activate TLR4, this domain can influence epithelial-to- mesenchymal transition via interaction with the small latent complex of TGF-β, exposing active TGF- 40, suggesting that this family member mediates tissue remodelling during repair, rather than inflammation.

The pro-inflammatory activity of the FBG domains of tenascin-C, -R and -W was TLR4 dependent, and each could bind directly to the extracellular domain of TLR4, whilst the inactive FBG-X did not. A number of modes of TLR4 activation are emerging. The prototypic TLR4 agonist LPS has an absolute requirement for the co-receptor MD2; it exhibits negligible binding to TLR4 alone, but binds with high affinity to the TLR4/MD2 complex (Table 19).

Protein com lex d Method used

LFS-MD2 2.33 yM SPR

LPS-MD2-TLR4 3 nM ^H-lipM A binding assay

TLR -MB2 12 nM Sold phase binding assay

HMGB1- MD2 12 nM SPR

Surfactant protein D-TLR4 15.8 nM Solid phase ttnding assay

FBO-C, - and -W - TLR4 50-60 nM Solid phase binding assay

Table 19. Affinity constants (Kd) for protein complexes including TLR4.

The molecular nature of this complex is well defined, with both the ligand interaction site and the TLR4 dimerization site experimentally validated. Like LPS, the endogenous TLR4 agonist HMGB1 requires MD2 for activity; it also cannot bind TLR4 directly but binds with high affinity to MD2 (Table 19). This interaction requires one cysteine in HMGB1 and binding, as well as activity in vitro and in vivo, can be blocked by the peptide tetramer FSSE which residues 105-108 in HMGBl are predicted to bind inside the hydrophobic pocket of MD2. These data suggest a similar mode of TLR4 activation, despite obvious structural differences, with both HMGBl and LPS inserting into the hydrophobic core of MD2 (Fig. 43a). A second mode of TLR4 activation has been reported for nickel and the cationic lipid diC14-amidine. Like LPS and HMGBl, both require MD2 for their activity, however each binds to TLR4 some distance from the MD2 binding site, close to the TLR4 dimerization domain (Fig. 43b).

Here, we show that the interaction of purified FBG-C, -R and -291 W with the purified extracellular domain of TLR4 can occur in the absence of MD2. Two other endogenous TLR4 agonists, surfactant protein D and biglycan, also bind to TLR4 in the absence of any co-factors, with affinities comparable to the tenascin FBG domains, and to the affinity of MD2, for TLR4 (Table 19). Like these endogenous ligands, the house dust mite allergen Der p2 also binds to the extracellular domain of TLR4 in the absence of MD2. However, Der p2 is a member of the MD2-like lipid binding family, exhibiting a pleated beta-sheet structure enclosing a hydrophobic cavity almost identical to MD2, enabling it to substitute for MD2 in LPS recognition, driving allergic TLR4 activation upon pathogen sensitization. Based on their structural homology Der p2 has been proposed to house LPS in its hydrophobic pocket and bind to TLR4 in the same location as MD2 (Fig. 43c), although participating residues have not been experimentally interrogated.

This molecular mimicry doesn't explain how TLR4 ligands biochemically distinct to MD2 can activate TLR4. The tenascin FBG domains, and bigylcan and surfactant protein D, exhibit a very different structure to MD2 and Der p2, and to each other (Fig. 43d). FBG domains do not possess a hydrophobic cavity, consistent with them being able to activate TLR4 independently of LPS and with their inability to enhance LPS-mediated TLR4 activation; conversely cytokine synthesis is inhibited when cells are stimulated with tenascin-C and LPS simultaneously, suggesting competition for the same or overlapping binding sites within TLR4.

Here, we mapped specific regions within FBG-C involved in TLR4 signalling, identifying loop 5 as essential for TLR4 activation, and loops 5 and 7, and the C-terminal tail (loop 10) as contributors to TLR4 binding. These regions are conserved in the FBG domains of tenascin-R and - W, but absent in the inactive FBG domain from tenascin-X, the latter becoming active only upon transplantation of these sites from FBG-C. Whilst TLR4 ligands exhibit diverse structures, we examined whether some degree of sequence conservation in TLR4 binding sites is feasible. MD2 binds to each TLR4 of the receptor homodimer via ionic and polar interactions (TLR4-1: R68, K109, R106, D101, S103, S98, D99, G123; TLR4-2: K125, R90, M85, L87); and the same has been predicted for Der p2. Our data indicate several common themes: firstly multiple binding sites participate in mediating a high affinity interaction with TLR4, and secondly similar types of interactions appear to be involved with clusters of charged residues and of hydrophobicity driving ionic and hydrophobic bonds respectively. Whilst we found no obvious sequence or structural homology of loops 7 and 10 of FBG-C with any other known TLR4 ligands, residues K319, T320 and K323 in the non-carbohydrate-binding loop region of surfactant protein D form a triad which is reminiscent of K123, T124 and K127 in loop 5 of FBG-C and it is possible that this may mediate interactions with TLR4.

In order to study the structural possibility of direct binding modes of FBG domains to

TLR4, we used FBG-C homology models within protein:protein docking procedures, retaining only models in whichthe residues of loops 5 and 7 contact TLR4 (we excluded loop 10 based on the lack of a structuraltemplate for this region of FBG-C, rendering its modelling less accurate than for other regions). Three distinct areas in one TLR4 monomer (TLR4-1) of a homodimer complex exhibited a highfrequency of predicted interaction with FBG in docking studies, as well 331 as one region in the second TLR monomer 2 (TLR4-2) (Fig. 43d).

These theoretical data suggest that the FBG-C domain may have some binding sites in common with MD2; for example the A and the B patches found at the C terminus and mid region of the extracellular domain of TLR4-1 respectively and which make up the primary MD2 binding interface, and regions in the dimerization interface in TLR-2. However these models indicate that FBG, which is larger in volume than MD2, may bind at additional sites towards the membrane proximal part of the extracellular domain of TLR4-1, regions that are not occupied by MD2. These models also imply that FBG, like MD2, may be able to mediate cross-TLR4-dimeric interactions and thus activate TLR4 signalling independently of accessory molecules, but the locations of FBG binding sites within TLR4 and the impact of FBG binding on TLR4 dimerizationshould be experimentally examined going forward. Moreover, the role of MD2 in endogenousactivation of TLR4 remains unresolved. Whilst FBG-C activation of TLR4 is not inhibited by anantagonistic LPS variant which competes for LPS binding to MD-2, and the sites that mediate thebinding of purified FBG-TLR4 independently of MD2 are involved in FBG-mediated macrophageactivation, it is still not clear what role MD2 plays in the cellular context. For example, whilst bigylcancan bind to purified TLR4 directly, high complexes containing, biglycan, TLR4 and MD2 can also bedemonstrated by gel filtration47, and ligands such as HMBG1 and S100A12 do bind to TLR4/MD2complexes with high affinity (Table 19), leaving the nature of the receptor complexes that senseendogenous stimuli in vivo unclear.

Evolutionary analysis of the FBG domain of tenascin-C shows that it's architecture is relatively ancient, and highly conserved. The sequences of loops 5 and 7, and the C-terminus show some divergence but are extraordinarily well conserved in higher eukaryotes (Fig. 44a). In addition to the tenascin family, a number of other proteins contain an FBG domain. First described in mammalian fibrinogen, fibrinogen related proteins (FRePs) are defined as proteins that contain an FBG domain. There are 2515 FRePs in eukaryotes, all in multicellular organisms, with the sea sponge (Phylum Porifera) containing the first complete example. In mammals there are 541 FRePs, with 21 human FRePs including fibrinogen, tenascins, ficolins, angiopoietins, angiopoietin-related proteins, fibroleukin and FIBCD-1. Each FReP contains an FBG domain that exhibits ~30-40 % sequence identity, but which also contains unique sequences that enable diversification of function. For example, Tie2 binding sites in the FBG domains of angiopoietin-1 and -2 enables their regulation of angiogenesis, and sugar binding sites in H- and L-ficolin FBG domains mediate their recognition of pathogenic glycans upstream of complement activation.

Analysis of the sequence of the human FRePs reveals that only the fibrinogen y chain possesses an extended C-terminal sequence akin to the active tenascin FBG domains (Fig. 44b). These data are consistent with the ability of fibrinogen to activate TLR449 and imply that this domain may do so via a similar mode of action to tenascin-C. Furthermore, whilst invertebrates such as the sea cucumber [P. parvimensis), freshwater snail [B. galabratd), horseshoe crab (T. t dentatus) and sea sponge (5. domunculd) each possess FRePs that mediate defence against infection (reviewed in 50), the FBG domain of B. galabrata might also be predicted to activate TLR4 directly, based on the similarity of loops 5 and 7 to FBG-C (Fig. 44c). This emergence of a pathogen-independent means of activating immunity 371 suggests a relatively early evolutionary pressure to react to threat other than infection.

Conclusions

In conclusion, for the first time it is shown that in addition to the FBG domain of tenasin-C, this domain in other tenascin family members (FBG-R and -W) can induce an inflammatory response and that this effect is specific, as FBG-X doesn't have an immune function. The pro- inflammatory epitope of this family has consequently been mapped to loop 5 in the P-subdomain, where positive charged amino acids are necessary for the activation of TLR4, whilst positive and hydrophobic residues in loops 10 and 7 respectively assist in mediating high affinity binding to TLR4. This epitope is essential for detection by TLR4 and its ectopic expression can convert immunologically inactive proteins into TLR agonists. Together these data reveal how endogenous molecules can be marked for innate immune recognition, paving the way for the design of specific inhibitors of FBG domain containing TLR stimuli.

EXAMPLE 7 - METHODOLOGY

Cloning tenascin-R, -W, -X FBG into pET32b vector

PCR was performed as described in table 9, using 400 nM of each primer (table 20), 1 μg DNA template, 200 μΜ dNTPs, IX Phusion HF buffer and 0.02 units^L of Phusion Hot Start II DNA Polymerase in a final volume of 50 μί,. The templates used were IMAGE consortium cDNA clones containing full length sequence of tenascin-R (ID: 40114168), tenascin-W (ID: 9020609) and tenascin-X (ID: 5179997) (Source Bioscience).

Table 20. Forward and Reverse primer sequences of tenascin -R, -X and -W FBG. Each sequence has a His-tag (underlined) and a restriction site in the forward primer for Ndel (bold) and in the reverse primer for Xhol (bold).

Table 21. Touch down PCR steps. This PCR protocol was used for amplifying tenascin-R, -W, -X FBG genes using IMAGE consortium clones as templates

Step Temperature Time Cycles

Denaturation 94^QC 10 min 1

1 94^QC 1 min 3

69^QC

72^QC

2 94^QC 1 min 6

67^QC

72^QC

3 94^QC lmin 20

65^QC

72^QC

4 94^QC lmin 3

62^QC

72^QC

5 94^QC lmin 3

60^QC

72^QC

Elongation 72^QC 10 min 1 PCR products were ligated into pCRBlunt vector using T4 DNA ligase and rapid ligation buffer (Thermo Scientific) for lh at room temperature. The clones were sequenced (Eurofins, UK) to ensure no errors had been introduced by PCR. Inserts that had no mutations were ligated into pET32b using Ndel and Xhol restriction sites. The inserts were verified by enzyme restriction digest. DNA was visualised in 1.5 % agarose gels stained with ethidium bromide.

Expression of FBG-R, FBG-W and FBG-X in E.coli

E.coli BL21 (DE3) cells containing pET32b vector were inoculated in 20 mL of LB media + 1% glucose and 100 μg/mL carbenicillin and incubated overnight at 37 °C, 220 rpm. The culture was centrifuged for 10 min, 3000 rpm and resuspended in 10 mL of fresh LB media. This was used to inoculate 1 L of LB + 100 μg/mL carbenicillin. The culture was incubated at 37 °C, 220 rpm until bacterial growth reached an OD600 between 0.6-0.8. ImM of IPTG was added to induce protein expression for 3h at 37 °C, 220 rpm.

Bacterial culture was centrifuged for 15 min, 3000 rpm and the pellet was washed with TBS buffer (50 mM Tris pH 7, 150 mM NaCl). Bacterial pellet was resuspended in 40 mL TBS and lysed with French Press at 1500 PSI four times. Bacterial solution was centrifuged at 14000 rpm for 15 min at 4°C.

Purification of FBG-C, FBG-R, FBG-W and FBG-X using Ni²⁺ chromatography

Protein solubilisation

Inclusion bodies were resuspended in 8M urea, 20mM Tris pH8.0, 5mM β- mercaptoethanol. The solution was left overnight on a rotator at 4°C to solubilize the protein. Ni²⁺ column chromatography under denaturing conditions

10 mL of Ni²⁺ beads (Bio-Rad) were placed in a Poly-Prep chromatography column (Bio- Rad). Beads were washed with 5 column volumes of ultrapure water and equilibrated with 5 column volumes of urea buffer (8M urea, 20mM Tris pH8.0). 40 mL of solubilised protein pool was loaded into the column and the flow through was collected. The column was washed with 5 column volumes of urea buffer followed by 150 mL of 0.1 % triton-X114 in urea buffer and 100 mL of urea buffer. Finally, the FBG protein was eluted using 300 mM imidazole in urea buffer. 5 fractions of 5 mL were collected. 10 xL of each sample + 10 xL of loading buffer were loaded into 12% SDS-PAGE gel to visualised protein yields.

Protein refolding

After purification, the protein fractions were pooled together and the concentration measured at O.D.28o by nanodrop. The protein solution was diluted until OD280nm < 0.2 in 8M urea, 20mM Tris pH8.0, 20mM cystamine to avoid protein precipitation during dialysis. The diluted protein solution was dialyzed for 4 days:

Day 1 and 2: Dialysed 24h against TN buffer (50 mM Tris pH 8, 150 mM NaCl) + 5mM β-ΜΕ, ImM 2-hydroxyethyldisulphide at 4C.

Day 3 and 4: Dialyse 24h against TN buffer at 4C. After refolding, the protein solution was filtered through 0.45 μηι Corning® Vacuum Filter (CN Membrane) to remove any protein precipitation.

Ni²⁺ column chromatography under native conditions

1.5 mL of Ni²⁺ beads (Bio-Rad) were placed in a Poly-Prep chromatography column (Bio-Rad). Beads were washed with 5 column volumes of ultrapure water and equilibrated with 5 column volumes of TN buffer. The refolded protein solution was loaded into the column and the flow through was collected. The column was washed with 5 column volumes of TN buffer followed by 50 column volumes of 0.1 % triton-X114 in TN buffer and 30 column volumes of TN buffer. 20 mM imidazole (in TNC buffer) wash was performed to remove any unspecific binding protein. Finally, the FBG protein was eluted using 1 mL of 300 mM imidazole in TN buffer. 6 fractions were collected in total and were dialyzed against TN buffer to remove imidazole. The concentration of the protein was measured at O.D.28o by nanodrop and by Bradford assay (Bio-rad).

Tenascin-C, R, -W, -X protein characterization

LPS test

The LPS content was measured using the Endpoint Chromogenic LAL Assays (Lonza) according to manufacturer's instructions.

Silver staining

1 μg of each protein was separated by SDS-PAGE and samples were fixed with 50 % methanol, 5% acetic acid solution. Gel was stained with 0.1 % silver nitrate solution and bands were developed using 0.04% formalin and 2 % sodium carbonate solution. The reaction was stopped using 5 % acetic acid.

Western blot

Proteins were separated by SDS-PAGE and were electrotransferred using Trans- Blot® Turbo™ Transfer System to Trans-Blot® Turbo™ Mini Nitrocellulose membranes (BioRad). Membranes were blocked with 5% BSA in phosphate buffered saline, 0.05% tween (PBST) and then incubated with primary antibody tetra-his (Qiagen) (1:10000 dilution). Membranes were then incubated with secondary antibody anti mouse IgG-HRP (Biorad) and the protein bands visualized using ECL plus (Amersham).

CD spectra

200 of each sample in TN buffer were analysed using the J-815 Circular Dichroism

Spectrometer (Jasco). The CD spectrum was measured in the "far UV" spectral region (190-250 nm) at 20°C or 85°C. Three repeated measured were taken and the average plotted and analysed using Spectra Manager™ II software. FBG proteins and peptides activity

FBG proteins and peptides activity in ThPl Nf-kB cells

THPl-Blue™ NF- Β Cells (Invivogen) have an Nf-kB-inducible reporter gene (a soluble embryonic alkaline phosphatase -SEAP) that is released in the media upon Nf-kB activation. Cells were culture in RPMI 1640, 10% FBS, 10 μg/ml blasticidin and 100 μg/ml Normocin™ (Invivogen). Cells were plated (10^s cells/mL) in 96 wells plate and stimulated with different doses of LPS, recombinant FBG proteins or peptides. As a control for LPS contamination, the different stimuli were pre-incubated for 30 min with 10 μg/mL polymyxin B (BioChemika) or boiled for 15 min prior cell stimulation. After 24 h, supernatant was collected and the presence of SEAP was detected using QUANTI-Blue™ (I nvivogen). Absorbance was read at OD 620 nm on FluoStar Omega plate reader.

FBG proteins and peptides activity in primary human macrophages

Human monocytes were isolated from peripheral blood (London Blood Bank) by ficoll gradient and counterflow centrifugation. Cells were plated (1 million cells/mL) in RPMI 1640, 5% (v/v) FBS, 1% penicillin/streptomycin and differentiated into macrophages for 5 days with 100 ng/mL of M-CSF.

Macrophages were plated (10^s cells/mL) in RPMI 1640, 3% (v/v) FBS and 1% penicillin/streptomycin for 24 h before stimulation with different doses of LPS (InvivoGen), recombinant FBG proteins or peptides. As a control for LPS contamination, the different stimuli were pre-incubated for 30 min with 10 μg/mL polymyxin B (BioChemika) or boiled for 15 min prior cell stimulation. Macrophages were stimulated for 24h, the media was collected and cytokines levels detected by ELISA (BD Biosciences) accordi ng to the manufacturer's instructions. Absorbance was read at 450 nm on FluoStar Omega plate reader and data analysed usi ng Omega software.

Co-stimulation of ThP l NF-kB cells with FBG-C and peptides

THPl-Blue™ NF- Β Cells (Invivogen) were cultured in RPMI 1640, 10% FBS, 10 μg/ml blasticidin and 100 μg/ml Normocin™ (Invivogen). Cells were plated (10^s cells/mL) in 96 wells plate and stimulated with 100 μΜ of different peptides for 30 min at 37^QC. Then, the cells were stimulated with FBG-C for 24 h at 37^QC. Supernatant was collected and the presence of SEAP was detected using QUANTI-Blue™ (I nvivogen). Absorbance was read at OD 620 nm on FluoStar Omega plate reader.

TLR4 protein activity dependency

Primary human macrophages were obtained and cultured as explain above. Cells were incubated with PAb hTLR4 antibody or IgG isotype control (Inivogen) for 1 h at 37^QC or with 3 μΜ of CLI-095 TLR4 inhibitor (Invivogen) for 6 h at 37^QC prior to cell stimulation. Macrophages were stimulated with 1 ng/mL of LPS or 1 μΜ of FBG proteins. After 24 h, the media was collected and cytokines levels detected by ELISA (BD Biosciences) according to the manufactu rer's instructions. Absorbance was read at 450 nm on FluoStar Omega plate reader and data analysed using Omega software.

Solid phase binding assay

Nunc, Maxisorp 96 well plates (Thermo Scientific) were coated with lug/ml solution of recombinant FBG proteins in PBS and incubated with shaking overnight at 4°C. Unspecific binding was blocked with 10 % BSA in PBST (0.05% tween) for 2 h at room temperature with shaking. TLR4 was added at a maximum concentration of 27 μg/ml followed by a series dilution 1:3 in 2% BSA/PBST and incubated with shaking for 2h at room temperature. MAb hTLR4 (Invivogen) was added at a final concentration of lug/ml in 2% BSA/PBST and incubated with shaking for 2h at 4°C. Secondary antibody anti mouse IgG-HRP (Biorad) 1:1000 dilution in PBST was incubated with shaking for lh at room temperature. Colorimetric reaction was started adding TMB substrate and incubating for 10 min in the dark. Colour development was stopped using 6% sulphuric acid solution and plates were read at OD 450 nm in FluoStar Omega plate reader.

In-silico analysis of homology and protein modelling.

The protein sequences of tenascin-C, -R, -W and -X FBG and fibrinogen β and y were obtained from UniProt. Pairwise alignment and multiple protein alignment analysis were performed using Clustal W and Jalview software. Protein models were made using SWISS-MODEL software based on the crystal structure of c-terminus fibrinogen y chain (PDB: 1FID). Protein models were coloured and structurally analysed using Pymol Molecular Graphics System, Version 1.7.4 Schrodinger, LLC.

Mutagenesis

Protein mutants were made using QuikChange II XL Site- Directed Mutagenesis Kit (Agilent Technologies). Specific primers were designed for each mutation and PCR was run according to manufacturer's instructions (Table 22).

Table 22. Primer sequences used to insert specific mutations in FBG-C and FBG-X The PCR products were transformed into XLIO-Gold ultracompetent cells. Plasmids were isolated using QIAprep Spin Miniprep Kit (Qiagen) and were sequenced (Eurofins, UK) to validate the insertion of the mutations. The clones with the correct mutant sequences were transformed into BL21 DE3 cells for protein over-expression and purification as described above. Proteins were also characterised using SDS-PAGE gels, western blots, CD spectroscopy and the LPS content was measured.

Blocking TLR4-FBG-C in vitro binding with peptides

Nunc, Maxisorp 96 well plates (Thermo Scientific) were coated with lug/ml solution of recombinant FBG proteins in PBS and incubated with shaking overnight at 4°C. Unspecific binding was blocked with 10 % BSA in PBST (0.05% tween) for 2 h at room temperature with shaking. TLR4 was pre-incubated with different peptides concentration for 1 h at room temperature. Then, it was added at a maximum concentration of 27 μg/ml followed by a series dilution 1 :3 in 2% BSA/PBST and incubated with shaking for 2h at room temperature. MAb hTLR4 (Invivogen) was added at a final concentration of lug/ml in 2% BSA/PBST and incubated with shaking for 2h at 4°C. Secondary antibody anti mouse IgG-HRP (Biorad) 1: 1000 dilution in PBST was incubated with shaking for lh at room temperature. Colorimetric reaction was started adding TMB substrate and incubating for 10 min in the dark. Colour development was stopped using 6% sulphuric acid solution and plates were read at OD 450 nm in FluoStar Omega plate reader.

Statistics

The data mean ± SEM and statistical analysis were calculated usi ng Graph Pad Prism 6 software. Multiple group means were analysed by one-way analysis of variance, followed by the Dunnett multiple comparisons test, where appropriate. Two-tailed, unpaired t test was used for experiments involving only two groups. P values less than 0.05 were considered significant.

In-silico analysis of homology and protein modelling.

Sequence alignment: All protein sequences were obtained from the UniProt Knowledge database and the accession numbers are presented in Table 23, with the exception of the FBG domain of B.flo dae and C. savignyi, which predicted sequences were taken from. Pairwise alignment and multiple protein alignment analysis were performed using Clustal Omega. Alignments were coloured using

Jalview software. Multiple sequence alignments were performed using Clustal Omega 1.2.1 and coloured according to the Clustal colour scheme. FBG Domain Accession number

FBG-C 5. scmfa

FBG-C NLmuscutus

FBG-C R. norvegicus

FBG-C S. Taurus

FBG-C G. gallus

FBG-C D. Reno

FBG-C T. rubripes

Angiopoietin 1

Angiopoietin 2

Angiopoietin 4

Angiopoietin-f elated protein 1

Angiopoietin-related protein 2

Angiopoietin-related protein 3

Angiopoietin-related protein 4

Angiopoietin-related protein 5

Angiopoietin-related protein S

Angiopoietin-related protein 7

Fibrinogen β chain C-termtnus

Fibrinogen Y chain C -terminus

Fibroteukjn

FfBGD-1

Ficolin-H

Fieoltn-L

Ficolin-M

Tenascin-C

Tenascin-R

Tenascfn-W Q9UQP3 Tenascin-X P225Q5 Frtf* P. parvimensls P19477

FrtP ft §lab ta G3LHF9

Fr»P T. t ^'fU iaius Q9U8W8

FrtP $, domuncula Q5W4Y8

Table 23. FBG domains examined by multiple sequence alignment analysis.

Homology Modelling

Homology models of the FBG domains of 480 the tenascin family members were built using two different methods and two different templates. The homology modelling functionality of the ICM platform was used as follows. The sequence of FBG-C was used to query the PDB for structural homologues using the "PDB Search by Homology" functionality (See Abagyan, R.A. & Batalov, S. Do aligned sequences share the same fold? / Mol Biol 273, 355-68 (1997). Matches were triaged by their sequence identity, pP values (a predictive measure of how structurally dissimilar the match is likely to be compared to the query sequence and the resolution of the structure itself. From this analysis the structure of fibrinogen-like recognition domain of FIBCDl, PDB code 4M7H was chosen, presenting a sequence identity of 43% and resolution of 2.0 A. Sequence alignment of this structure and the tenascin FBG domains was generated using ICM and optimised by hand. Homology modelling was performed within ICM using default parameters. Resultant possible models were scored using an energy-based function and the top ten superimposed revealing no significant structural difference between them; as such the model with the best score was taken for further analysis. The majority of the sequence was fully built with reference to the template however the C493 terminal ten residues NFRNLEGRRKR extended beyond the C-terminus of the template structures, hence their structure in the model was not be considered to be predictive. Similarly, SWISSMODEL (see Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42, W252-8 (2014).495) was used to generate models based on the structure of the C-terminus fibrinogen y chain (PDB: 496 1FID). Models generated by either method or template exhibited no significant structural difference. Protein models were coloured and structurally analysed using Pymol Molecular Graphics System, Version 1.7.4 Schrodinger, LLC and ICM-Pro.

TLR4:FBG-like proteimprotein docking

The homology model of the FBG-C domain was docked into the TLR4 dimer using the fourier transform protein:protein docking method within ICM using standard parameters and with post- prediction refinement enabled. The structure of TLR4/MD2/LPS (PDB code 4G8A) was used as a basis for protein:protein docking with the tenascin FBG domain homology model generated within ICM. The MD2/LPS components of this structure were removed. The glycosylations of the TLR4 molecules were retained but all waters and crystallography condition molecules were removed. 5000 models were generated and those with loop 5 and 7 interactions within 3.5A with TLR4 were retained.

Claims

1. A binding domain comprising two variable domains wherein each variable domain comprises 3 CDRs and a framework wherein the binding domain is specific to an FBG domain of a tenascin, and binds to at least residues 123 and 127, and at least one of the residues 125 or 129 of SEQ ID NO: 1, 2 or 3.

2. A binding domain according to claim 1, wherein the domain binds a further, 1, 2, 3, 5, 6, 7, 8, 9 or more residues in range 116 to 132 of said sequences.

3. A binding domain according to claim 1 or 2, which binds SEQ ID NO: 17 and/or 18, in particular a binding domain which binds specifically to one or both of said sequences.

4. A binding domain according to any one of claims 1 to 3, which binds SEQ ID NO: 27 and/or 28, in particular a binding domain which binds specifically to one or both of said sequences.

5. A binding domain according to any one of claims 1 to 4, which binds SEQ ID NO: 37 and/or 38, in particular a binding domain which binds specifically one or both of said sequences.

6. A binding domain according to claim 1 or 2, which is human.

7. A binding domain according to claim 1 or 2, which is humanised.

8. A binding domain with affinity in the rnage of 5pM to 500nM.

9. A binding domain according to any one of claims 1 to 5, which is specific to loop 5 of tenascin-C.

10. An antibody or binding fragment thereof comprising a binding domain according to any one of claims 1 to 9.

11. An antibody according to claim 10 which is a full length antibody.

12. An antibody or binding fragment according to claim 10, which is selected from a Fab, modified Fab, Fab', modified Fab', F(ab') 2, Fv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies.

13. An antibody according to any one of claims 10 or 12, wherein the antibody or binding fragment is neutralising.

14. A chimeric antigen receptor comprising a binding domain according to any one of claims 1 to 9.

15. A polynucleotide sequence encoding a binding domain according to any one of claims 1 to 9 or an antibody according to any one of claims 10 to 13 or a chimeric antigen receptor according to claim 14.

16. A vector comprising a polynucleotide according to claim 15.

17. A cell comprising a polynucleotide according to claim 15 or a vector according to claim 16.

18. A pharmaceutical formulation comprising an antibody according to any one of claims 10 to 13.

19. A method of treating a patient compring administering a therapeutically effective amout of an antibody or binding according to any one of claims 10 to 13, a chimeric antigen receptor according to claim 14 or a pharmaceutical formulation accordinig to claim 18.

20. An antibody or binding fragment according to any one of claims 10 to 13, a chimeric antigen receptor according to claim 14 or a pharmaceutical formulation accordinig to claim 18, for use in treatment, in particular for use in the treatment of a chronic inflammatory response.

21. Use of an antibody or binding fragment according to any one of claims 10 to 13, a chimeric antigen receptor according to claim 14 or a pharmaceutical formulation accordinig to claim 18 for the manufacture of a medicament for the treatment of a chronic inflammatory response.

22. An epitope of no more than 18 amino acids from the P domain of a tenacin comprising at least residues 129 and 127, and at least one of the residues 125 or 123 of SEQ ID NO: 1, 2 or 3.

23. An epitope according to claim 22 further comprising 1, 2, 3, 5, 6, 7, 8, 9 or more residues in range 116 to 132 of said sequences.

24. An epitope according to claim 22 or 23, which is SEQ ID NO: 17 or 18.

25. An epitope according to claim 22 or 23 which is SEQ ID NO: 27 or 28.

26. An epitope according to claim 22 or 23 which binds SEQ ID NO: 37 or 38,

27. An epitope according to any one of claims 23 to 26 conjugated to a carrier.

28. Use of an epitope to according to any one of claims 24 to 27, to immunize a host animal or to interrogate a library.