WO2013034736A1 - Use of staphylococcal superantigen- like 3 (ssl3) as an tlr2 inhibitor - Google Patents

Abstract

The present invention relates to the treatment of medical conditions involving the activation of toll-like receptor 2 (TLR2), and especially to immune-modulation for use in the therapy for immune-mediated- and inflammatory diseases. In particular the invention relates to a novel TLR2 inhibitor: Staphylococcal superantigen-like 3 (SSL3) protein, or a homolog, or a derivative thereof, for use in the treatment of medical conditions involving the activation of TLR2. The invention also relates to compositions and methods for use in the treatment of medical conditions involving the activation of TLR2.

Description

USE OF STAPHYLOCOCCAL SUPERANTIGEN- LIKE 3 (SSL3) AS AN TLR2 INHIBITOR

The present invention relates to the treatment of medical conditions involving the activation of toll-like receptor 2 (TLR2), and especially to immune-modulation for use in the therapy for immune-mediated- and inflammatory diseases.

Next to an acquired immune system, humans and most animals also have an innate immunity, which is available for immediate response to threats, by activation of type 1 interferons and pro-inflammatory cytokines such as: interleukin (IL-)l beta, IL6, IL8, IL12, and tumour necrosis factor alpha (TNFa). As more became known of the innate immune system, initial assumptions that this was a simple or primitive system, were soon set aside; the innate immune system turns out to be highly complex, with specific receptors and a multitude of factors with agonist or antagonist activity. Also, the primary innate immune response is the indispensable basis for the secondary acquired immune response

Central to the innate immune response is the recognition of conserved molecular signatures from pathogens, by pattern recognition receptors (PRR). An important group of such PRRs are the so-called Toll-like receptors (TLR). TLRs have evolved to recognize highly conserved structures of viral (TLR 3, 7, 8, and 9) and bacterial (TLR1 , 2, 4, 5, 6, 7, and 9) origin. This specificity allows TLRs to rapidly detect the presence of an invading micro-organism and subsequently induce activation of inflammatory and antimicrobial immune responses. In addition, TLRs expressed on dendritic cells and on B-lymphocytes initiate antigen-specific adaptive immune responses in the secondary immune response. (Botos et al., 201 1 , Structure, vol. 19, p. 447; Jin & Lee, 2008, Immunity, vol. 29, p. 182).

Ligands for TLRs range from bacterial lipoproteins (TLR2), lipopolysaccharide (TLR4) and flagellin (TLR5) to bacterial CpG-rich DNA (TLR9) and double stranded RNA (TLR3) or single stranded RNA (TLR7 and 8). TLRs are type I transmembrane

glycoproteins characterized by an extracellular leucine-rich repeat domain and an intracellular Toll/IL-1 receptor domain. Most TLRs use MyD88 as a universal adapter protein via a cascade of intracellular signalling to activate the transcription factor NFkB. The activation of TLRs is the ligand-induced dimerisation of a TLR; the subsequent interaction of the two TI R domains is the event that initiates the recruitment of MyD88 and IRAK proteins. The TLR-dimers can be heterodimers of different TLRs, this is considered to contribute to broadening of the receptors' repertoire. It is widely accepted that TLRs are highly relevant as therapeutic targets, because of their central role, at a very early time in immune-mediated- and inflammatory diseases.

Approaches to modulate the functioning of TLRs by specific binding molecules, such as agonistic or antagonistic ligands, antibodies or fragments thereof, proteins/peptides, or small molecules, are widely being investigated, for a large number of immune-mediated- and inflammatory diseases.

In spite of the differences in such medical conditions, it is believed that the common mechanism at their basis is an over-reaction of the immune reaction. The initiation can be an infection by an external agent, such as a virus, bacterium, or parasite; alternatively, the initiation may also be from an internal, non-infectious source, so-called 'sterile

inflammation'. Nevertheless the resulting activation of the immunesystem, with TLRs at the start, leads to inflammatory reactions which subsequently run out of control, as the resulting damage to cells and tissues exacerbates the (over-) reaction. This can lead to the disease becoming a chronic condition. It is generally believed that down-regulating TLR activity would break this vicious circle by slowing down the over-reaction, and normalising the immune response. Therefore there is a clear and unmet need for factors that can act as inhibitor of TLR function. (Hennessy et al., 2010, Nature Rev. Drug Disc, vol. 9, p. 293; Lome et al., 2010, Intens. Care Med., vol. 36, p. 1826).

An important requirement for any immuno-modulatory intervention, and especially one that would target a central molecule as a TLR, is its specificity; the general or aspecific inhibition of such receptors could lead to severe systemic defects.

A further requirement is to have control over the level of the immune-modulation applied; an over-inhibition is clearly undesirable, as this would only introduce the next disease condition. Therefore close control of the level of inhibition is important.

One of the TLRs for which ways to modulate its activity is actively investigated is TLR 2, which is classified as CD282. TLR2 is expressed on a variety of immune cells such as neutrophils, macrophages and dendrocytes. This surface exposure, is both necessary for its function, but also conveniently makes it accessible for therapy with a variety of molecules.

Because of its specificity for lipoproteins as ligands, TLR2 is involved in the innate immunity against bacteria. One example is the involvement of TLR2 in the process leading to Gram-positive shock syndrome, as this could be prevented by an antibody (T2.5) that bound to TLR2 and inhibited its activation (Meng et al., 2004, The J. of Clin. Invest., vol. 1 13, p. 1473). Like for many other bacteria, TLR2 is involved in the innate immunity to Staphylococcus aureus. This was demonstrated when S. aureus bacterial infection increased in number and severity both in TLR2 knockout mice infected with wildtype S. aureus, and in normal mice infected with an S. aureus strain defective in lipoprotein production. (Schmaler et al., 2010, Int. J. of Med. Microbiol., vol. 300, p. 155). TLR2 has also been implicated to have a role in a wide variety of allergic- and immune-mediated inflammatory diseases: sepsis, ischemia / reperfusion injury to heart or kidneys, cardiovascular disease and atherosclerosis, allergies, asthma, atopy, atopic dermatitis, rheumatoid arthritis, systemic lupus erymathosis (SLE), and diabetes. (O'Neill et al., 2009, Pharmacol. Rev., vol. 61 , p. 177).

In such diseases the cause for TLR2 activation may be derived, not from bacterial infection, but from an endogenous TLR2 ligand; many such possible ligands are known (Erridge, 2010, J. of Leukoc. Biol., vol. 87, p. 989). Several potential TLR2 inhibitors are being investigated, these are antibodies, peptides, and small molecules. (Hennessy, 2010; O'Neill 2009; both supra). No candidate has yet lead to a successful therapeutic product.

TLR activation is initiated by the binding of agonistic ligands, which induces dimerisation and interaction of both TIR domains. In this activated state, the intracellular TIR domain of the TLR can recruit the other factors more downstream in the cascade, such as MyD88, MAL, and IRAK. For TLR2 the dimers are commonly formed as heterodimers with TRL1 or TRL6. In this way, the diacylated lipoproteins from Gram-positive bacteria are bound by a TLR 2 - TLR 6 heterodimer, and triacylated lipoproteins from Gram-negative bacteria by a TLR 1 - TLR 2 heterodimer. TLR2 homodimers can recognise the artificial lipopeptide

Pam2Cys. TLR1/2 uses CD14 as co-factor, and TLR2/6 uses CD36 as cofactor. (Jin & Lee, 2008, supra).

Most studies on the structure and function of TLRs have been done with cells from human and mouse origin. However, the structures of TLRs in other mammals have been found to be highly conserved. In birds, some differences to the TLR system were found, but TLR2 structure and function was mainly conserved (Brownlie & Allan, 201 1 , Cell Tissue Res., vol. 343, p. 121 ). Interestingly, in chickens one TLR2 heterodimer combined the functions of TLR1/2 and TLR2/6 of mammals: the chicken TLR2type2/TLR16 heterodimer was capable of binding both diacylated and triacylated peptides (Keestra et al. 2007, The J. of Immunol., vol. 178, p. 71 10).

In nucleotide databases such as NCBI's GenBank™, a wide variety of TLR2 nucleotide sequences are available, both from humans and from a wide variety of animals: mouse and several species of rodents, chimpanzee, bovines, goat, sheep, antelope, dog, horse, swine, chicken, several species of fish, etc..

Staphylococci are nonmotile, nonspore forming, Gram positive, facultative anaerobic cocci, belonging to the Firmicutes. Colonies on blood agar are round convex, with golden colour. Staphylococcus aureus (S. aureus) is a commensal of the skin and mucous membranes in humans and animals. Those Staphylococci that are pathogenic have acquired certain additional genetic elements that allow the expression of virulence factors. These mobile genomic elements are so-called 'pathogenicity islands' (PI); for S. aureus: SaPI. (Feng et al., 2008, FEMS Microbiol. Rev., vol. 32, p. 23).

S. aureus has several SaPIs and can therefore express a wide arsenal of virulence factors; these include: adhesins, stress factors, and exoproteins. The exoproteins are enzymes, toxins and immunomodulators. The toxins include the well known toxic-shock syndrome toxin, which is a 'superantigen'. Such superantigens are able to activate subsets of T-lymphocytes without antigenic specificity by interacting directly with MHC class II molecules on macrophage's and with the Vb chain of T-cell receptors. This causes a cytokine release leading to major systemic shock effects.

The immunomodulators that S. aureus secretes in different stages of infection assist the establishment and expansion of the bacterial infection; they reduce or evade the detection and the clearance of S. aureus by the immune- or the complement system, and the mobilisation of phagocytes, such as neutrophils, monocytes and macrophages. Some are for example: the chemotaxis inhibitory protein (CHIPS), the Staphylococcal complement inhibitor (SCIN), and the formyl peptide receptor-like 1 inhibitory protein (FLIPr). (Veldkamp & van Strijp, 2009, Adv. Exp. Med. Biol., vol. 666, p. 19).

A group of 14 genes has been identified that potentially encode proteins that resemble superantigens, but they lack the MHC binding capacity. Hence their name: staphylococcal superantigen-like (SSL) proteins. Previously these proteins were known as staphylococcal exotoxin-like (SET) proteins (Arcus et al., 2002, J. of Biol. Chem., vol. 277, p. 32274), but nomenclature was disorderly for SETs from various S. aureus strains. These have now been renamed to SSL 1- 14 (Lina et al., 2004, J. of Infect. Dis., vol. 189, p. 2334), whereby the SSL proteins are named in the order in which their encoding gene occurs on the S. aureus genome. (Smyth et al., 2007, J. of Med. Microbiol., vol. 56, p. 418). SSL1 -1 1 are on SaPI2 (previously named: vSa alpha), and 12-14 on cluster IEC-2 of the S. aureus genome. Not every SSL gene is present in every S. aureus isolate, and alternatively, for some SSL genes there exist some allelic variants.

SSLs are polymorphic paralogs of the superantigens, which have elements of sequence and structure in common. However the few SSLs that have been characterised, were found to each have very different functions: SSL5 binds to P-selectin glycoprotein ligandl (PSGL1 ) on neutrophils, thereby blocking their mobilisation to a site of infection; SSL7 binds to human IgA and to complement factor C5; SSL10 inhibits CXCR4; and SSL1 1 binds to the myeloid receptor FcaRI (CD 89). (Fraser & Proft, 2008, Imm. Reviews, vol. 225, p. 226; Bestebroer et al., 2009, Blood., vol. 1 13, p. 328; Walenkamp et al., 2009, Neoplasia, vol. 1 1 , p. 333; Langley et al., 2010, Crit. Rev. in Immunol., vol. 30, p. 149).

Based on these findings the SSL proteins have been suggested to be immune evasion proteins, but most SSLs have thus not yet been studied or characterised. Many SSL gene- and putative protein sequences are available in databases, but such

publications are merely based on in silico analyses of S. aureus genomic data. Recently the regulation of SSL gene expression was analysed (Benson et al., 201 1 , Molec. Microbiol., vol. 81 , p. 659). SSLs have been described for use in targeting of a chosen antigen to antigen-presenting cells (WO 2005/092918), although only the use of SSL7 and 9 was disclosed in detail.

The many SSL sequences published, are derived from S. aureus isolates from humans but also from a variety of animal species: cow, goat, sheep, rabbit, and chicken (Smyth et al., 2007, J. of Med. Microbiol., vol. 56, p. 418).

It is an object of the present invention to accommodate to the need in the field for an effective inhibitor of TLR2 activity, for use in the treatment of a variety of disorders involving TLR2 activation. It was surprisingly found that this objective could be met through the use of a

Staphylococcal superantigen-like 3 (SSL3) protein, or a homolog of said SSL3 protein, or a derivative of either protein, for use in the treatment of medical conditions involving the activation of toll-like receptor 2 (TLR2). The crucial discovery made by the inventors was the finding that SSL3 binds to the extracellular domain of TLR2, and potently inhibits the activation of TLR2 and thereby its capability to initiate an innate immune response. SSL4 was found to have the same inhibitory effect on TLR2, albeit to a lesser extent; as SSL4 is highly identical to SSL3, it is considered a homolog of SSL3. The inhibition of TLR2 by SSL3, or by a homolog was also possible by using a fragment of either of the two proteins, comprising the C-terminal part of SSL3, or of the homolog.

Although they do not wish to be bound by theory, the inventors suggest that S. aureus expresses and secretes SSL3 and SSL4 upon infection of a host to inhibit the normal activation of TLR2. This provides a blockade of the innate immune response that would otherwise occur when the native TLR2 would recognise lipoproteins from S. aureus, and would initiate the production of cytokines, and the mobilisation of phagocytes. The inhibition of TLR2 provides S. aureus with a clear path to establish its infection undisturbed, and create tolerance once infection is established.

During natural selection, S. aureus has thus developed a natural inhibitor of TLR2, to circumvent (one branch of) the host's immune system, and facilitate infection of and tolerance in human or animal hosts.

The advantageous utility of this discovery is in the use of SSL3, or its homologs, or derivatives, for the therapy of medical conditions involving the activation of TLR2, and specifically for immune-mediated- and inflammatory diseases.

This was not at all straightforward: even though TLR2 is an important factor in the innate immunity, there was no indication in the prior art that any one of the many exoproteins of S. aureus would interact with this receptor, let alone inhibit its activation directly. Also, it was in no way evident that an SSL protein could interact with a TLR receptor, as the SSL proteins of which the function was known, all have very different activities; indeed: of the SSL1-1 1 , none of the others was found to have any (similar) activity towards TLR2.

Petzl et al. (2008, Vet. Res., vol. 39, p. 18), and Yang et al. (2008, Molec. Immunol., vol. 45, p. 1385), have speculated on the role of TLR2 and TLR4 in subclinical S. aureus infection in bovine mastitis. However, their working hypothesis presumed an increase of TLR2 abundance after S. aureus infection, and no molecular mechanism could be found to explain why NFkB levels did not increase. They concluded that the S. aureus infection mechanism posed a paradox.

SSL3 and SSL4 are the first non-antibody proteins that are now known to inhibit the activation of TLR2 by directly binding to TLR2, i.e. there is a molecular interaction between SSL3 or SSL4 and TLR2, and this interaction itself inhibits the activation of TLR2. The only other protein of which a similar binding and inhibition of activation of TLR2 is known, is the T2.5 antibody (Meng et al., 2004, supra). In the prior art other proteins and factors have been described that bind TLR2 and inhibit its functioning. However, these actually inhibit the factors 'downstream' of TLR2 in the signalling cascade of the innate immune system, not the activation of TLR2 itself. For example:

Pathak et al. (2007, Nature Immunol., vol. 8, p. 610), described a direct interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2. However, the binding of ESAT-6 to the extracellular domain of TLR2 activated the intracellular signalling molecule Akt and this prevented the interaction between the adaptor MyD88 and its downstream kinase IRAK4, which both are active downstream of TLR2 activation. Therefore, ESAT-6 inhibited the signalling by TLR2 once it was activated, not the activation (ligand binding followed by dimerisation) of TLR2 itself. Similarly, the small molecule compound E567 is an inhibitor of the signalling by (activated) TLR2, not of the activation of TLR2 per se; E567 targets the adapter proteins MyD88 and MyD88 adapter-like, which are both involved in the signalling pathways downstream in the cascade of TLR2 and TLR4 (Zhou et al., 2010, Antiviral Res., vol. 87, p. 295).

Also, the 'viral-derived peptide' OPN-401 , as developed by Opsona Therapeutics™ (as described in Hennessy et al., 2010, supra) is entirely unrelated to SSL3 protein, or a homolog, or derivative, and acts by an entirely different mechanism. Therefore in one aspect the invention relates to a Staphylococcal superantigen-like 3

(SSL3) protein, or a homolog of said SSL3 protein, or a derivative of either protein, for use in the treatment of medical conditions involving the activation of toll-like receptor 2 (TLR2).

According to the prior art, an "SSL3 protein" is a protein that is encoded by the gene on the genome of S. aureus that is named SSL3, because of its relative location in the order of SSL genes (Smyth, 2007, supra). In addition, an SSL3 protein for the invention has the characterising feature that it is capable of direct binding to TLR2, and thereby inhibiting the activation of the TIR domain of said TLR2 by a TLR2 ligand such as a bacterial lipoprotein. Methods to determine such binding, and such inhibition are described and exemplified in detail herein.

The amino acid sequence of a reference SSL3 protein for use according to the invention is SSL3 from S. aureus strain NCTC 8325, and is represented as SEQ ID NO: 1. Examples of further SSL3 proteins for use according to the invention are displayed in Table 1 . This displays the details of a representative number of SSL3 proteins from S. aureus strains, from humans and animals, and from regular S. aureus strains, or MRSA type strains. Most of these are derived from a public database, with the exception of a number of SSL3 proteins from bovine isolates of S. aureus, that were analysed in house. Their amino acid sequences are presented in SEQ ID NO's: 2-5.

The SSL 3 proteins for use according to the invention, that are listed in Table 1 were compared by multiple amino acid sequence alignment, a picture of a specific grouping emerged: amongst them the SSL3 protein were very conserved, and none had an amino acid sequence identity to any of the others, or to the reference SSL3 protein sequence (SEQ ID NO: 1 ), that was less than 90%; Table 2 presents the % identity of the mutual alignment results for SSL3 proteins, and Figure 8, presents these results in a

dendrographic tree. Therefore, in a preferred embodiment the invention relates to the SSL3 protein for use according to the invention, wherein the SSL3 protein is a protein comprising an amino acid sequence having at least 90 % amino acid sequence identity to the amino acid sequence of SEQ ID NO. 1.

The percentage of amino acid sequence identity between two or more proteins can conveniently be calculated using a computer algorithm, and suitable software packages for protein sequence analysis are commercially available. A preferred method is to use the 'Blast' algorithm which is available on the internet website of the NCBI, by selecting protein blast (Blastp), and select 'Align 2 (or more) sequences'. Default parameters should be used, and an alignment to the full length of SEQ ID NO: 1.

This definition of SSL3 proteins for use according to the invention, by the minimal level of amino acid sequence identity, in addition with the requirement for TLR2 inhibition as described, sets the said SSL3 proteins clearly apart from any protein in the prior art; the best match of SEQ I D NO: 1 to any other amino acid sequences of unrelated proteins in the public databases was 55 % identity or less; whereby an 'unrelated' protein is one of which the annotation indicated it was not an SSL3 or an SSL4 protein. This also applies to the other SSL proteins from S. aureus; an example is presented in Table 5, and is described below.

In a preferred embodiment, the SSL3 protein for use according to the invention, has at least 91 % amino acid sequence identity to the amino acid sequence of SEQ ID NO. 1 , more preferably, 92, 93, 94, 95, 96, 97, 98, 99, or even 100 % sequence identity to the amino acid sequence of SEQ ID NO. 1 , in that order of preference.

For the invention, the term "comprising" (as well as variations such as "comprise",

"comprises", and "comprised") as used herein, refer(s) to all elements, and in any possible combination conceivable for the invention, that are covered by or included in the text section, paragraph, claim, etc., in which this term is used, even if such elements or combinations are not explicitly recited; and not to the exclusion of any of such element(s) or combinations. Consequently, any such text section, paragraph, claim, etc., can also relate to one or more embodiment(s) wherein the term "comprising" (or its variants) is replaced by terms such as "consist of", "consisting of", or "consist essentially of". Table 1 : List of SSL3 and SSL4 amino acid sequences, used to make alignments

(1 ) Isolate sequenced by Broad Institute Sequencing Genomic Platform - no information available

(2) Isolate sequenced by Craig Venter Institute - no information available Table 2: Multiple alignment scores for SSL3 proteins in % amino acid sequence identity

SSL3 sequences representative for others:

NCTC8325for21189 Mu50forN315, Mu3, A9763, A9299, A8115, ED98, A8117, ECT-R2 and 21318 MW2forATCC51811and TCH70 COL for FPR-3757, Newman, TCH1516, 132, ATCC BAA-39, TW20, JKD6008, 011 for 046 CGS01, MRSA131 and T0131

JH9 for JH1, A9717, A6224, A5937, A10102, A8819, A8796, CGS03 and 21172 31193 for 21305

Table 3: Multiple alignment scores for SSL4 proteins in % amino acid sequence identity

Therefore, in a more preferred embodiment, the SSL3 protein for use according to the invention, consists of the amino acid sequence of any one SEQ ID NO. selected from the group consisting of SEQ ID NO. 1 through SEQ ID NO: 5.

For the invention, the term "protein" refers to any molecular chain of amino acids. A protein is not necessarily of a specific length, structure or shape and can, if required, be modified in vivo or in vitro, by, e.g. glycosylation, amidation, carboxylation,

phosphorylation, pegylation, or changes in spatial folding. The protein can be a native or a mature protein, a pre- or pro-protein, or a functional fragment of a protein. A protein can be of biologic or of synthetic origin, and may be obtained by isolation, purification, assembly etc. A protein may be a chimeric- or fusion protein, created from fusion by biologic or chemical processes, of two or more proteins protein fragments. Inter alia, peptides, oligopeptides and polypeptides are included within the term protein.

"Staphylococcus aureus" and 'S. aureus' for the invention are terms used to refer to the bacterial organism that is currently known by this name. However, in respect of the precise taxonomic classification of S. aureus, the skilled person will realise this may change over time as new insights can lead to reclassification into new or other taxonomic groups. However, as this does not change the characteristics or the protein repertoire of the organism involved, only its classification, such re-classified organisms are considered to be within the scope of the invention.

In that respect the invention intends to encompass all bacteria sub-classified from S. aureus for the invention, either as a sub-species, strain, isolate, genotype, serotype, variant or subtype and the like.

A "homolog" for use according to the invention is a protein that is homologous to, and has the essential characteristics of, an SSL3 protein for use according to the invention. In particular this regards being capable of direct binding to TLR2 and thereby inhibit the activation of the TIR domain of said TLR2 by a TLR2 ligand such as a bacterial lipoprotein.

As described above, no unrelated protein had more than 55 % amino acid sequence identity to the SSL3 protein for use according to the invention. Therefore, in a preferred embodiment, the homolog for use according to the invention, is a protein that is capable of direct binding to TLR2 and thereby inhibit the activation of the TIR domain of said TLR2 by a TLR2 ligand such as a bacterial lipoprotein, and wherein said protein comprises an amino acid sequence having at least 56 % amino acid sequence identity to the amino acid sequence of SEQ ID NO. 1.

"Direct binding" for the invention has been described above, and involves a direct molecular interaction, without intermediate molecules being involved. More preferably, the homolog for use according to the invention has at least 60 % amino acid sequence identity with SEQ ID NO: 1 , even more preferably 65, 70, 75, 80, 85, 86, 87, 88, or even 89 % sequence identity to the amino acid sequence of SEQ ID NO. 1 , in that order of preference. The inventors noted that in SaPI2 on the genome of S. aureus bacteria isolated from some animal species, specifically bovine S. aureus isolates, no copy of an SSL3 gene was present, in stead there was a copy of an SSL4 gene. (Smyth et al., 2007, supra). When tested, the SSL4 proteins were found to share with SSL3 the capability for inhibiting TLR2, only to a lesser extent. Therefore, the inventors propose that an SSL4 protein is a natural homolog for SSL3, and appears in a number of S. aureus strains.

The amino acid sequence of a reference SSL4 protein for use according to the invention, is SSL4 from S. aureus strain NCTC 8325, and is represented as SEQ ID NO: 6.

SEQ ID NO: 1 and SEQ ID NO: 6 have 62 % amino acid sequence identity.

Examples of further SSL4 proteins for use according to the invention are displayed in Table 1. This displays the details of a representative number of SSL4 proteins from S. aureus strains, from humans and animals, and from regular S. aureus strains, or MRSA type strains. Most of these are derived from a public database, with the exception of a number of SSL4 proteins from bovine isolates of S. aureus, that were analysed in house. Their amino acid sequences are presented in SEQ ID NO's: 7-8.

The SSL4 proteins for use according to the invention, that are listed in Table 1 were compared by multiple amino acid sequence alignment. Table 3 presents the % identity of the mutual alignment results for SSL4 proteins, and Figure 10, presents these results in a dendrographic tree. Although quite well conserved amongst them, the SSL4 proteins were not so conserved as SSL3 proteins; their mutual amino acid sequence identity was between 57 and 98 % (Table 3). Amino acid sequence identity with the reference SSL4 protein (SEQ ID NO: 6) was between 59 and 99 %. The reason being that SSL 4 genes were found to appear in different allelic variants, named set2 and set9. This makes that the group of SSL4 proteins differs amongst themselves in length and in sequence.

Therefore in a preferred embodiment, the homolog for use according to the invention is a protein, comprising an amino acid sequence having at least 59 % amino acid sequence identity to the amino acid sequence of SEQ ID NO. 6.

More preferably, the homolog for use according to the invention has at least 60 % amino acid sequence identity with SEQ ID NO: 6, even more preferably 62, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or even 99 % sequence identity to the amino acid sequence of SEQ ID NO. 6, in that order of preference.

In an even more preferred embodiment, the homolog for use according to the invention comprises the amino acid sequence of any one SEQ ID NO. selected from the group consisting of SEQ ID NO. 6 through SEQ ID NO: 8.

To compare SSL3 and SSL4 proteins, a number of representative of SSL3 and SSL4 proteins from the various subgroups seen in the dendrographic trees (Figures 9 and 10), were compared by multiple amino acid sequence alignment. This is presented in Figure 1 1 , as a textual output; Table 4 presents the corresponding amino acid sequence identity levels between SSL3 and SSL4 proteins, and correlates these to SEQ ID NO: 1 and 6.

This demonstrates that in spite of the variance in SSL4 proteins, the SSL3 and SSL4 are still within the definition of homologs of SSL3 for use according to the invention, which uses a cut off of more than 55% amino acid sequence identity to SEQ ID NO: 1 . When comparing SSL3 and SSL4 proteins in detail, it was apparent that SSL4 proteins are generally shorter, lacking a section of sequence in the N-terminal half as compared to SSL3. Nevertheless, the C-terminal halves of SSL3 and SSL4 were found to be highly conserved. The inventors therefore speculate that the active site of SSL3 and SSL4 for binding to TLR2 is in the C-terminal half of the proteins. Table 4: Pairwise alignments of SSL3 and SSL4 proteins

SEQ ID NO: 1

SEQ ID NO: 6

A "derivative" for the invention is a molecule that is derived from an SSL3 protein for use according to the invention, or from a homolog of said SSL3 protein for use according to the invention. Said derivative for the invention is still capable of direct binding to TLR2 and thereby inhibit the activation of the TIR domain of said TLR2 by a TLR2 ligand such as a bacterial lipoprotein.

A test for determining whether a particular fragment is a fragment for use according to the invention, can for example be performed using TLR2 expressing cells, as exemplified herein. When using primary cells of the immune system, the read-out usually employs IL8 production or NFkB expression. When used on recombinant cells expressing a

heterologous TLR2, often the expression of a reporter gene is used. Such a system can indicate the activation of TLR2 by a TLR2 ligand such as a bacterial lipoprotein for example by detection of a reduction in luciferase or GFP expression as compared to uninhibited TLR2 expressing cells. In this type of assay a fragment for use according to the invention can block the expression of such a reporter gene, so that inhibition of TLR2 is detected routinely.

The fragment for use according to the invention preferably achieves at least 50% inhibition of the activation of the TIR domain of TLR2 by a TLR2 ligand such as a bacterial lipoprotein, compared to an uninhibited culture. More preferably, 60, 70, 80, 90, or even 100 % inhibition, in this order of preference.

Bacterial lipoproteins for use in such a test are commonly known and available; conveniently synthetic peptides are used such as: Pam2Cys, Pam3Cys, or MALP-2.

A derivative for use according to the invention can for example be a fragment from an SSL3 protein for use according to the invention, or be a fragment from a homolog for use according to the invention. In that case the fragment is itself a protein. For example the fragment for use according to the invention can be a mature or processed form of an SSL3 protein or of a homolog, both for use according to the invention, i.e. without a 'leader', 'anchor', 'signal' or 'tail' sequence.

In a preferred embodiment of a fragment as a derivative for use according to the invention, the fragment is a part of a SSL3 protein, or of a homolog, both for use according to the invention, which comprises the C-terminal region of said SSL3 protein or homolog. This region was found to contain the TLR2 binding activity.

Examples of fragments for use according to the invention are: the region from amino acid numbers 127 to 326 of SEQ ID NO: 1 , or the region from amino acids 79 - 278 of SEQ ID NO: 6, both 200 amino acids in length.

In a preferred embodiment, the derivative for use according to the invention is a fragment of a protein, whereby the fragment is taken from the C-terminal side from an SSL3 protein for use according to the invention, or from the C-terminal side from a homolog for use according to the invention. More preferably, said fragment is at least 175, 150, 100, 90, 80, 70, 60, or even 50 amino acids in length, taken from the C-terminal side of the SSL3 protein, or the homolog, both for use according to the invention.

The capability of such a preferred fragment to inhibit TLR2 activation, is demonstrated in Figure 1 1 : this compares the capacity to inhibit TLR2 activation by SSL3 and by a C- terminal fragment of SSL3, the amino acids 127-326 of SEQ ID NO: 1. Both are almost equally effective.

This is also established when comparing the C-terminal regions of SSL3 and SSL4 with the other SSL proteins of S. aureus; SSL 1 , 2, and 5-14 are all about 200 amino acids in length. When aligning the amino acid sequences of the other SSLs (from S. aureus strain NCTC 8325) to the C-termini of SSL3 and of SSL4, the results show that although there is conservation, this does not exceed 46 % amino acid sequence identity (for SSL1 1 ) to the C-terminal region of SSL3 (amino acid numbers 127-326 of SEQ ID NO: 1 ), see Table 5. Surprisingly the sequence identity between SSL3 and SSL4 in this region is 76 %.

Therefore the inventors speculate that this region holds the capability for inhibiting TLR2. Table 5: List of pairwise alignments of the C-terminal ends of amino acid sequences from SSL3 and SSL4 with the full length of the other SSL proteins; all SSL amino acid sequences are from S. aureus strain NCTC 8325. pairwise alignment smade using Alignplus™ (Scientific Educational Software), using default parameters.

The fragment for the derivative for use according to the invention can be comprised in a fusion protein, or a chimeric protein, whereby the resulting fusion or chimeric proteins have maintained the capacity to bind directly to TLR2 and thereby inhibit the activation of said TLR2 by a ligand such as a bacterial lipoprotein.

The design and selection of such protein fragments is well within the routine capabilities of the skilled person, and these types of manipulations can conveniently be done by using molecular biological techniques, whereby a nucleic acid sequence encoding the fragment as a derivative for use according to the invention, is manipulated to encode the desired fusion or chimeric protein.

Routinely a nucleic acid as described above, is manipulated in the context of a vector, such as a DNA plasmid, enabling the amplification in e.g. bacterial cultures, and the manipulation in a variety of molecular biological techniques. A wide variety of suitable plasmid vectors is available commercially.

The relevant molecular biological techniques are explained in great detail in standard text-books like Sambrook & Russell: "Molecular cloning: a laboratory manual" (2001 , Cold Spring Harbour Laboratory Press; ISBN: 0879695773); Ausubel et al., in: Current Protocols in Molecular Biology (J. Wiley and Sons Inc, NY, 2003, ISBN:

047150338X); C. Dieffenbach & G. Dveksler: "PCR primers: a laboratory manual" (CSHL Press, ISBN 0879696540); and "PCR protocols", by: J. Bartlett and D. Stirling (Humana press, ISBN: 0896036421 ).

A derivative for use according to the invention can also be a modified protein, which is a protein that is a modified version of an SSL3 protein for use according to the invention, or of a homolog of said SSL3, for use according to the invention. Typical protein engineering techniques are well known, to make a protein more stable e.g. in storage or by in vivo half-life; more effective in terms of bio-availability; or change or improve other

pharmacological qualities. For example, it can be advantageous to change certain amino acids containing exposed side-chains to another amino acid residue, in order to provide for greater chemical stability.

For the present invention modified proteins have a further advantage, in that they can be made to be less immunogenic than the native S. aureus proteins, such as SSL3 or SSL4. The inventors have observed that in the serum of healthy human volunteers, antibodies against SSL3 and SSL4 were detectable, consequently these proteins have a certain antigenicity. While this may be favourable to reduce unwanted over-inhibition, under certain conditions it may also affect in vitro half-life. Methods to make modified proteins, and to make proteins less immunogenic are well known in the art, e.g. De Groot & Scott (2007, Trends in Immunol., vol. 28, p. 482). Such methods comprise for example the removal of T-cell epitopes, the addition of a poly-ethylene glycol moiety, or the attachment of an Fc domain. This approach is referred to as "deimmunization".

These manipulations are conveniently done by molecular biological techniques, as described above. This way modifications can be made to the inserted nucleic acid e.g. insertions, deletions, or mutations, using common techniques of restriction enzyme digestion or by PCR. A derivative for use according to the invention can also be an antibody that has an antigen binding site that mimics the binding site of SSL3 for TLR2. The principle of mimicking antibodies is well known in the art. Preferably these are monoclonal antibodies, and preferably their Fc part has been adapted to match the target species, so-called speciesation, such as humanisation, bovinisation, caninisation, etc. All well known in the art.

In order to determine what the binding site of SSL3, its homolog, or derivative, all for use according to the invention, exactly is, crystallisation experiments can be performed of a TLR2 bound by SSL3 protein, or homolog, or derivative. Analysis of the crystal structure of the complex will reveal details on the exact molecular interaction between the two molecules. Such techniques are well known in the art (e.g. Baker et al., 2007, J. of Mol. Biol., vol. 374, p. 1298).

The mimicking antibody for the invention is not the same as the T2.5 antibody as described by Meng et al; (2004, supra), or the OPN-305 antibody as developed by as developed by Opsona Therapeutics™ (as described in Hennessy et al., 2010, supra).

A derivative for use according to the invention can also be a molecule that is not itself a protein, but has a similar structure and the same function of an SSL3 protein for use according to the invention, or of a homolog of said SSL3, for use according to the invention TLR2. This is the so-called 'peptidomimetics' approach, well known in the art. Herein a part, or the whole of a template molecule is rebuild into copy molecules using chemical building blocks that mimic the structure of the template, but are not the same. Special care is of course taken to mimic carefully those regions of the template that have the binding- or interacting activity with the receptor. Through an iterative routine process, the copied molecules can be further improved, until the desired activity and physiological qualities are obtained. A recent review is: Gentilucca et al. (2006, Curr. Med. Chem., vol. 13, p. 2449).

The use of a peptidomimetic, in stead of a protein may be favourable in cases where a protein would not be physiologically acceptable, or would not be stable or active enough.

Therefore, in a preferred embodiment the derivative for use according to the invention is a fragment of the SSL3 protein, or of the homolog, or said derivative is a mimicking antibody, or said derivative is a peptidomimetic, all for use according to the invention.

One of the advantages achieved through the SSL3 protein, or the homolog of said SSL3 protein, or a derivative of either protein, all for use according to the invention, is that these proteins are highly specific, so that only TLR2 is inhibited. Also, the level of inhibition achieved can be manipulated so that no over-inhibition occurs; for example, if inhibition by SSL3 protein would be too strong, a homolog such as SSL4 could be used for inhibiting TLR2, which has a lower inhibiting effect.

For the invention, 'inhibition' (and similars such as 'inhibiting' and 'inhibitor') has a meaning as is common in the field. Consequently, an inhibitor for the invention is a compound that can decrease, reduce, block, prevent, inactivate, or delay, the activation or the activity of its target receptor: TLR2

To determine a level of inhibition, in vitro or in vivo experiments can routinely be done. In vitro experiments have been described above to illustrate how to test whether a particular fragment is a fragment for use according to the invention,

Inhibition of TLR2 for the invention is achieved when the reduction of activity is at least 50%, preferably 60, 80, 90, or even 100 % reduction, in this order of preference.

For the invention 'activation' (and similars such as 'activity' or 'activator') has a meaning as is common in the field. Consequently, the activation of TLR2 for the invention is the process of obtaining TLR2 in an activated state, which is the state wherein ligand is bound and dimers have formed, and the intracellular domain of TLR2 can signal to activate the factors downstream in the cascade, such as MyD88, MAL, and IRAK.

As a further advantage, the SSL3 protein, or the homolog of said SSL3 protein, or a derivative of either protein, all for use according to the invention, enable the study and the development of other therapeutic molecules, through research or experimental use. For example when investigating molecules to enhance TLR2 activity, the SSL3 protein, the homolog, or the derivative, all for use according to the invention, can be used to establish levels of minimal TLR2 activation. Also, when investigating other targets in the innate immune system, or in the signalling cascade up- or downstream of TLRs, the SSL3 protein, the homolog, or the derivative, all for use according to the invention, can effectively be used to establish conditions wherein an effect from TLR2 activation is ruled out. This makes other effects stand out more.

The protein components for the invention: the SSL3 protein, the homolog of said SSL3 protein, and the derivative of either protein (when in the embodiment of a protein), all for use according to the invention, can be obtained in variety of ways: e.g. the SSL3 protein and the homolog by isolation from an in vitro culture of S. aureus, or from an animal infected with S. aureus.

However most conveniently the proteins are produced through the use of a recombinant expression system, by the expression of a nucleotide sequence that encodes these protein components. Recombinant expression systems for this purpose commonly employ a host cell, which is cultured in vitro. Well known in the art are host cells from bacterial, yeast, fungal, insect, or vertebrate cell expression systems. In particular the host cell may be a cell of bacterial origin, e.g. from E. coli, Bacillus subtilis, Lactobacillus sp. or Caulobacter crescentus, possibly in combination with the use of bacteria-derived plasmids or bacteriophages for expressing a protein component for the vaccine according to the invention.

The host cell may also be of eukaryotic origin, e.g. yeast-cells in combination with yeast-specific vector molecules (WO 2010/099186); or higher eukaryotic cells, like insect cells (Luckow et al., 1988, Bio-technology, vol. 6, p. 47) in combination with vectors or recombinant baculoviruses; or plant cells in combination with e.g. Ti-plasmid based vectors or plant viral vectors (Barton et al., 1983, Cell, vol. 32, p. 1033); or mammalian cells like Hela cells, Chinese Hamster Ovary cells, or Madin-Darby canine kidney-cells, also with appropriate vectors or recombinant viruses.

Next to these expression systems, plant cell, or parasite-based expression systems are attractive expression systems. Parasite expression systems are e.g.

described in the French Patent Application, number FR 2,714,074. Plant cell expression systems for polypeptides for biological application are e.g. discussed by Fischer et al. (1999, Eur. J. of Biochem., vol. 262, p. 810), and Larrick et al. (2001 , Biomol. Engin., vol. 18, p. 87). Also genetically modified animals may be generated which can express such proteins; preferably mammalians expressing the proteins in their milk, from which they can be isolated, or which may be used directly. This is well known for rabbits, and goats.

Expression may also be performed in so-called cell-free expression systems. Such systems comprise all essential factors for expression of an appropriate recombinant nucleic acid, operably linked to a promoter that will function in that particular system. Examples are an E. coli lysate system (Roche, Basel, Switzerland), or a rabbit reticulocyte lysate system (Promega corp., Madison, USA).

As is well known in the art, a consequence of the choice for a specific expression system is the level of post-translational processing that is provided to the expressed protein; e.g. a prokaryotic expression system will not attach any glycosylation signals to the

polypeptide produced, whereas insect, yeast or mammalian systems do attach N- and/or O-linked glycosylation, of increasing complexity. Also, levels of lipidation, and amidation may vary; as well as type of protein processing, depending on the proteases present. The skilled person can readily make the proper choice based on selection of the system giving the best balance of protein amount and pharmacological effectiveness.

Alternatively, when the derivative for use according to the invention is not a protein, common chemical- and biochemical techniques can be used for synthesis, coupling, and purification; all well known to a skilled person. An even more effective inhibition of TLR2 can be achieved by using more than one of the elements for inhibiting TLR2, in a combination for use according to the invention. For example: the SSL3 protein and the homolog (e.g. an SSL4 protein) combined in one formulation for a very strong inhibition. Alternatively, for a very safe inhibition: a modified protein and a peptidomimetic, etc.

To devise and select such combinations, and to test the level of improvement obtained, is well within the routine capabilities of the skilled person. Advantageous improvements of such a combination over use of single components can for instance relate to: level of inhibition, half-life in vivo, onset till effect, duration of effect, level and kind of side-effects, etc. For most of these parameters both an increase or a decrease can be an improvement, depending on the circumstances of the application, the human or animal target and its condition, the final formulation used, etc.

The SSL3 protein, the homolog of said SSL3 protein, and the derivative of either protein, all for use according to the invention, are preferably applied in a therapy for immune- mediated- or inflammatory disease.

Such therapy provides clear advantages to the human or animal target treated; the effect can vary from relieving a minor inconvenience to preventing life-threatening organ failure or systemic conditions. Therefore in a preferred embodiment the invention relates to the SSL3 protein, the homolog of said SSL3 protein, and/or the derivative of either protein, for use in the treatment of medical conditions involving the activation of TLR2, wherein the medical condition is an immune-mediated disease or an inflammatory disease. In a further preferred embodiment, the immune-mediated- or inflammatory disease are one or more selected from the group consisting of: auto-immune disease, allergy, asthma, atopy, atopic dermatitis, atherosclerosis, arthritis - especially rheumatoid arthritis,

Alzheimer disease, cardio-vascular disease, diabetes, immune senescence, ischemia / reperfusion injury of the heart or of kidneys, feline infectious peritonitis, mastitis, psoriasis, sepsis, systemic lupus erymathosis, tumour metastasis, and visceral or cutaneous Leishmaniasis.

For the effective treatment of such immune-mediated- or inflammatory diseases, the SSL3 protein, the homolog of said SSL3 protein, and/or the derivative of either protein, all for use according to the invention, are conveniently formulated into a pharmaceutical composition for use according to the invention.

Such composition enables the inhibition of TLR2 in a therapy for immune- mediated- or inflammatory disease. Consequently, the use and application of said pharmaceutical composition can effectively be applied in immune-modulation for the treatment of medical conditions involving the activation of TLR2.

Therefore, in a further aspect, the invention relates to a pharmaceutical composition comprising an SSL3-like protein, a homolog of said SSL3 protein, a derivative of either protein, or a combination thereof, for use according to the invention. A "pharmaceutical composition" is intended to aid in the effective administration of a pharmaceutically active compound, without causing (severe) adverse effects to the health of the target human or animal to which it is administered. A pharmaceutical composition can for instance be sterile water or a sterile physiological salt solution. In a more complex form the composition can e.g. be a buffer, which can comprise further additives, such as stabilisers or conservatives. Details and examples are for instance described in well- known handbooks e.g.: such as: "Remington: the science and practice of pharmacy" (2000, Lippincot, USA, ISBN: 683306472); "Veterinary vaccinology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681 ); and the Merck Index, Merck & Co., Rahway, NJ, USA.

The pharmaceutical composition according to the invention is preferably formulated as a depot or sustained release formulation. Furthermore, one may administer the composition in a targeted delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, an arthritic joint or inflamed lesion characterized by immunopathology. The liposomes will be targeted to and taken up selectively by the afflicted tissue. Preferably the components for the pharmaceutical composition according to the invention are serum free (without animal serum); protein free (without animal protein, but may contain other animal derived components); animal compound free (ACF; not containing any component derived from an animal); or even 'chemically defined', in that order of preference.

The composition according to the invention may additionally comprise carriers or excipients common in the field, provided they are supportive of the intended therapeutic application for a human or animal target .

In a preferred embodiment the pharmaceutical composition according to the invention is characterised in that it comprises at least one additional therapeutic component.

The additional therapeutic component(s) may be an immune enhancing substance e.g. a chemokine, or an immunostimulatory nucleic acid, e.g. a CpG motif.

The preparation of the pharmaceutical composition according to the invention is performed by routine means well known to the skilled person, using methods and materials common in the field. Such preparation will in general comprise the steps of admixing and formulation of the components of the composition with pharmaceutically acceptable carriers and excipients, followed by apportionment into appropriate sized containers or dosage forms.

The various stages of the manufacturing process will need to be monitored by adequate tests, for instance by immunological tests for the quality and quantity of the active components; by micro-biological tests for sterility and absence of extraneous agents; and ultimately by test in laboratory animals for efficacy and safety. When such extensive tests for quality, quantity and sterility were all found to be compliant with the prevailing regulations, the pharmaceutical products are released for sale.

Therefore, in a further aspect the invention relates to a method for the preparation of the pharmaceutical composition according to the invention, comprising the admixing of the SSL3 protein, the homolog of said SSL3 protein, the derivative of either protein, or a combination thereof, for use according to the invention, with a pharmaceutically acceptable carrier or excipient. The method of preparation according to the invention may comprise the admixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions; all referred to in common handbooks, as described.

The pharmaceutical composition according to the invention, in order to be effective in a therapy for a human or animal target for use according to the invention as described, requires the application by a method of treatment.

Therefore in a further aspect, the invention relates to a method for the treatment of medical conditions involving the activation of TLR2, comprising the application to a human or animal target of the pharmaceutical composition according to the invention.

In a preferred embodiment of the method for treatment according to the invention, the medical condition is an immune-mediated disease or an inflammatory disease. "Treatment " (or "treating") means to administer a pharmaceutical composition according to the invention, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease, for which the said composition has therapeutic activity. Typically, the composition is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of, or inhibiting the progression of such symptom(s) by any clinically measurable degree.

The amount of the pharmaceutical composition that is effective to alleviate any particular disease symptom (also referred to as the "therapeutically effective amount") may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the active component to elicit a desired response in the subject.

Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.

While individual results may vary, the said treatment for the present invention must alleviate the target disease symptom(s) in at least a statistically significant number of subjects.

For the method of treatment according to the invention, the mode of administration can vary; suitable routes of administration include: oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial.

Alternatively, it can be advantageous to administer the pharmaceutical composition according to the invention in a local rather than systemic manner, for example, via injection of the antibody directly into an arthritic joint or inflamed lesion characterized by immunopathology, such as a skin lesion, a tumour, an inflamed organ, etc.

The administration regimen depends on several factors, including the serum- or tissue turnover rate of the SSL3 protein, the homolog of said SSL3 protein, or the derivative of either of these proteins, for use according to the invention, as well as the level of symptoms, the immunogenicity of the active components, and the accessibility of the target cells in the biological matrix. The regimen is for example: three times a day, once daily, or once weekly. The determination of the appropriate dose to be applied to the target, is typically done in the clinic, based on relevant parameters indicating therapeutic effectiveness. Generally, the dose will initially be somewhat less than the optimum dose, and may be increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.

For the invention, the pharmaceutical composition, the method of preparation of said composition, and the method of treatment, all according to the invention, including relevant parameters such as the formulation, dose, and administration regimen, are all balanced with respect to an optimised pharmaceutical effectiveness versus level of undesired side effects. A measure for the balance between therapeutic and toxic effects is the 'therapeutic index': LD₅₀/ ED_50.

Such considerations can be made by a person of skill in the art, in consultation with a clinician. A "therapeutically effective amount", (or effective dose) for the invention, refers to an amount of the SSL3 protein, the homolog of said SSL3 protein, the derivative of either protein, or a combination thereof, all for inhibition of TLR2, that causes the desired 'effectiveness': a measurable improvement in one or more symptoms of a disease or condition, or in the progression of such disease or condition. A therapeutically effective dose further refers to that amount of the SSL3 protein, homolog, derivative, or

combination that is sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.

The therapeutically effective dose relates to the individual ingredient or to the combination of ingredients, whatever is appropriate.

An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10% or more

An effective amount can also result in an improvement in a subjective measure, in cases where subjective measures are used to assess disease severity.

The effective amount may be a preferred amount in the target's serum, e.g.: 0.1 , 0.3, 1 , 3, 10, 30, 100, 300 μg ml or more; or a concentration in the target's serum, e.g.: below 1 mM, 10 μΜ, 10 nM, or below 10 pM; or an amount received per dose, e.g.: 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/dose, etc.

In a further aspect, the invention relates to the use of an SSL3 protein, or a homolog of said SSL3 protein, or a derivative of either protein, or a combination thereof, for use according to the invention.

In a further aspect, the invention relates to the use of an SSL3 protein, or a homolog of said SSL3 protein, or a derivative of either protein, or a combination thereof, for use in the manufacture of a medicament for use according to the invention.

The invention will now be further described with reference to the following, non-limiting, examples.

Examples

1. Characterisation of SSL3 and SSL4 from S. aureus as inhibitors of the activity of TLR2

1.1. Materials and methods

1.1.1 Antibodies

FITC-conjugated mAbs directed against CD9, CD1 1 a, CD31 , CD46, CD62L, CD66, and phycoerythrin (PE)-conjugated mAbs directed against CD35, CD44, CD47, CD49b, CD54, CD58, CD87, CD1 14, CDw1 19, CD162, and CD321 , allophycocyanin (APC)-conjugated mAbs directed against CD1 1 b, CD1 1 c, CD13, CD14, CD29, CD45, CD50, CD55, and

Alexa-647-conjugated mAb directed against CD16 were purchased from BD Bioscience. FITC-labelled mAbs against CD120a, and CD120b, and an APC-conjugated mAb against Siglec-9 were from R&D Systems. Anti-CD43-FITC was from Santa Cruz Biotechnology. Anti-LTB4R-FITC, anti-CD32-PE, and anti-CD89-PE were from AbD Serotec. Anti-CD88- PE was from Biolegend. Anti-CD282-PE was from Ebioscience. Anti-CD63-PE was purchased from Immunotech. Fluorescent formylated peptide (fluorescein conjugated of the hexapeptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys) to detect formyl peptide receptor 1 and anti-CD10-APC were purchased from Invitrogen. 1.1.2 Cloning, expression and purification of SSL3 and SSL4

For expression of recombinant SSL3, the SSL3 gene of S. aureus strain NCTC 8325 (SAOUHSC_00386), except for the signal sequence, was cloned into the pRSETB vector (Invitrogen) as described (Bestebroer et al., 2007, Blood vol. 109, p. 2936). After verification of the correct sequence, the pRSETB/SSL3 expression vector was

transformed in Rosetta-Gami(DE3)pLysS E. coli (Novagen). Expression of histidine (His)- tagged SSL3 was induced with 1 mM isopropyl-3-D-thiogalactopyranoside (IPTG; Roche Diagnostics) for 4 h at 37°C in LB containing 20 mM glucose. His-tagged SSL3 was isolated under denaturing conditions on a HiTrap™ chelating column, according to the manufacturer's description. Elution was performed in 50 mM EDTA under denaturing conditions. Renaturation of His-SSL3 was performed by dialysis, after which the His-tag was removed by enterokinase cleavage according to the manufacturer's instructions (Invitrogen).

Finally, the purity of SSL3 was checked by SDS-PAGE and protein was stored in PBS at -20°C. Cloning and expression of SSL 1 , 2, 4, and 5 to 1 1 from S. aureus strains NCTC 8325 and SSL4 from strain MRSA252 was performed as described for SSL3 with minor modifications. The N-terminal histidine tag of the pRSETB vector, contains besides the histidine tag and enterokinase cleavage site also an Xpress epitope, which was replaced by a 6 residue histidine tag just downstream the enterokinase cleavage site. After enterokinase cleavage, an additional Glycine residue remains at the N-terminus of the SSL4 proteins.

1.1.3 Cells

Human neutrophils and peripheral mononuclear cells (PBMCs) were isolated as described (Bestebroer et al., 2007, supra). Human embryonal kidney cells expressing TLR2 (HEK- TLR2) and TLR2 in combination with TLR1 (HEK-TLR1/2) and TLR6 (HEK-TLR2/6) were obtained from Invivogen. HEK-TLR cell lines were maintained in DMEM, containing 10 μg ml gentamicin, 10 μg ml blasticidin and 10% FCS. Mouse macrophage cell line

RAW264.7 was cultured in DMEM, containing 10 μg ml gentamicin and 10% FCS.

1.1.4 SSL3 binding to cells

To determine binding of SSL3 to different leukocyte populations, SSL3 was labelled with fluorescein isothiocyanate (FITC). Therefore, 1 mg/ml SSL3 was incubated with 100 μg/ml FITC in 0.1 M sodium carbonate buffer (pH 9.6) for 1 hour at 37°C. A HiTrap desalting column (GE healthcare) was used to separate FITC-labelled SSL3 from unbound FITC.

For binding of SSL3-FITC to leukocytes, human neutrophils (5 x 10⁶ cells/ml) and PBMCs (1 x 10⁷ cells/ml) were incubated on ice for 30 min with increasing concentrations of SSL3- FITC in RPMI (Gibco), containing 0.05% human serum albumin (Sanquin). After washing, fluorescence was measured on a flow cytometer (FACSCalibur; Becton Dickinson).

1.1.5 Competition for TLR2 binding between SSL3 and antibody T2.5

To determine a putative receptor for SSL3, a mixture of neutrophils (5 * 10⁶ cells/ml) and PBMCs (1 x 10⁷ cells/ml) were incubated with either SSL3 (10 Mg/ml) or RPMI/HSA and incubated 30 min on ice. Subsequently, 39 different FITC-, PE-, or APC-conjugated monoclonal antibodies (mAbs) directed against various cell-surface receptors were added to the cell mixture and incubated for 45 min on ice. After washing, fluorescence was measured using flow cytometry. Neutrophils, monocytes and lymphocytes were selected by gating. In another experiment, leukocytes were incubated with increasing

concentrations of SSL3 for 30 min at 4°C. Subsequently, the cells were incubated with anti-TLR2 antibody T2.5 (anti-CD282-PE; 1 :100 dilution) using the same conditions as in the screening assays.

1.1.6 TLR2 ligand-induced IL-8 production

To test the effect of SSL3 on TLR2 ligand-induced IL-8 production, HEK-TLR2, HEK- TLR1/2, HEK-TLR2/6, PBMC, neutrophils, and RAW264.7 cells were used. HEK and

RAW264.7 cells were seeded in 96 wells culture plates until confluency. Freshly isolated PBMC and neutrophils were added to 96 wells culture plates (2.5 ^χ 10⁶ cells/well). To avoid activation of TLR4 on PBMC and neutrophils by endotoxin, SSL3 was pretreated with 20 μg ml polymyxin B sulphate (Sigma) for 1 hour. Additionally, PBMC were preincubated with 10 μg ml blocking anti-TLR4 mAb (clone HTA125; Bioconnect) for 30 minutes. Next, the cells were preincubated for 30 minutes at 37°C with increasing concentrations of SSL3. Then, cells were stimulated with different, increasing

concentrations of Pam2Cys, Pam3Cys (both from EMC microcollections), MALP-2 (Santa Cruz), or recombinant flagellin of P. aeruginosa (Chapter 2), as indicated in the Results section (Example 2).

After overnight incubation in a 37°C incubator, culture supernatants were tested for presence of IL-8 using a specific ELISA following the manufacturer's instructions

(Sanquin). Culture supernatants of RAW264.7 cells were tested for the presence of mouse TNFa using a specific ELISA kit (R&D systems). IL-8 production experiments with PBMC and neutrophils were performed in RPMI/10% FCS. Experiments with HEK and RAW264.7 cells were performed in DMEM/10% FCS. Cytotoxic effect of SSL3 on cells was tested using the lactate dehydrogenase (LDH) cellular cytotoxicity detection kit following the manufacturer's description (Roche Diagnostics). In some experiments, next to SSL3 and SSL4, the other SSLs of SaPI2 were tested for IL-8 production by MALP-2- activated HEK-TLR2/6 cells, as described above.

1.1.7 Cloning and expression of human and mouse TLR2

The recombinant extracellular domain of human TLR2 (hTLR2) was cloned in HEK293 cells (U-Protein Express, The Netherlands). The recombinant extracellular domain of mouse TLR2 (mTLR2) was cloned and expressed by a different department (Crystal and Structural Chemistry, University Utrecht, The Netherlands) in HEK293 cells. Both hTLR2 and mTLR2 contain an N-terminal 6 residues histidine tag, a 3x streptavidin tag and a TEV cleavage site. 1.1.8 ELISA

To test binding of SSL3 to the recombinant extracellular domains of human and mouse TLR2, the TLR2 proteins were coated to an ELISA plate (Nunc maxisorp™) at 10 μg ml. Wells were blocked with 4% skimmed milk in PBS/0.05% Tween. His-tagged SSL3 was allowed to bind to the coated TLR2 proteins for 1 hour at 37°C. Bound His-SSL3 was detected with anti-Xpress™ mAb (Invitrogen) and subsequent binding of peroxidase- labeled goat anti-mouse IgG and visualized as described (Haas et al, 2004, J. of

Immunol., vol. 173, p. 5704).

1.2. Results

1.2.1 SSL3 binds to TLR2 on neutrophils and on monocytes.

To investigate its role in immune evasion, SSL3 of S. aureus strain NCTC 8325 was cloned in E. coli. The protein was pure according to SDS-PAGE and fluorescently-labelled to study the interaction with human leukocytes. SSL3 specifically interacted with human neutrophils (Fig. 1A) and monocytes (Fig. 1 B), whereas almost no binding was observed for lymphocytes (Fig. 1 C).

To verify that the molecular target for SSL3 was exclusively TLR2 on phagocytes, the binding of SSL3 to other receptors that are expressed on neutrophils and monocytes, with crucial functions in innate immunity (e.g. chemotaxis, activation, adhesion, and phagocytosis), was investigated, using a panel of monoclonal antibodies (mAb) recognizing these receptors.

It was found that SSL3 specifically inhibited binding of the function-blocking TLR2 monoclonal antibody T2.5 to neutrophils and monocytes (Fig. 2A). Inhibition of other tested cell-surface receptors was not observed.

The expression of TLR2 differed between cell-types; monocytes (Fig. 2B) expressed higher levels compared to neutrophils (Fig. 2C), whereas TLR2 was absent on lymphocytes (data not shown). SSL3 dose-dependently blocked binding of anti-TLR2 to monocytes (Fig. 2B) and neutrophils (Fig. 2C). The IC50 for monocytes was around 0.05 μg/ml SSL3 and for neutrophils around 0.02 μg/ml (Fig. 2D). This slightly lower half maximal inhibitory concentration corresponds with the lower expression of TLR2 on neutrophils. These data indicate that SSL3 efficiently, and specifically, blocks a domain of

TLR2 that is important for its function. 1.2.2 SSL3 inhibits the activation of TLR2

To test whether SSL3, next to binding, could also inhibit TLR2 function, HEK cells expressing TLR2 (HEK-TLR2) were stimulated with the synthetic lipopeptides Pam2Cys and MALP-2, and the production of interleukin-8 (IL-8) was measured. SSL3 was found to potently inhibited TLR2 activation by both agonists in a dose-dependent manner (Fig. 3A and 3B), confirming that SSL3 functionally inhibits TLR2. At 1 g/ml SSL3, IL-8 production was abolished even when stimulated with 100 ng/ml Pam2Cys or MALP-2. Since TLR2 can dimerise with either TLR1 or TLR6 and thereby can discriminate between di- and tri- acylated lipoproteins and augment the cellular cytokine response, SSL3 inhibition was also tested on HEK-TLR2/6 or HEK-TLR1/2 cells activated with their specific synthetic ligands, MALP-2 (Fig. 3C) and Pam3Cys (Fig. 3D), respectively. SSL3 inhibited the IL-8 production of HEK-TLR1/2 cells, however inhibition was less potent in comparison with HEK-TLR2/6 cells.

The effect of SSL3 on TLR2 activation was also tested in primary human neutrophils and monocytes. In contrast to HEK-TLR2 cells, neutrophils and monocytes also express TLR4, which can be activated in by lipopolysaccharide that is present in recombinant proteins generated in E. coli. To prevent IL-8 production via TLR4, we pretreated SSL3 with 20 μg ml polymyxin-B to inactivate the lipopolysaccharide contamination. Additionally, PBMCs were pretreated with 10 μg ml blocking anti-TLR4 mAb to prevent TLR4 activation. These precautions were sufficient to block TLR4 activation in both cell types, as even the highest concentration of SSL3, without addition of MALP-2, did not induce IL-8 production (Fig. 4A and 4B).

In addition to HEK cells overexpressing TLR2, SSL3 also efficiently inhibited TLR2 activation by MALP-2 of both neutrophils (Fig. 4A) and PBMCs (Fig. 4B), as a source for monocytes.

SSL3 was not cytotoxic for cells, as verified by a lactate dehydrogenase (LDH) cytotoxicity assay performed on PMBCs and HEK-TLR2/6 cells after overnight incubation with SSL3 (Fig. 4C and 4D). SSL3 did not affect the IL-8 ELISA, as no difference in IL-8 standard curve was observed in the presence of 10 μg ml SSL3 (data not shown).

The inhibition of TLR2 activation could also be obtained using a C-terminal fragment of SSL3, the fragment from amino acids 127-326 of SEQ ID NO:1 , see Figure 12.

1.2.3 SSL3 recognizes both human TLR2 and mouse TLR2

These results thus strongly suggest that SSL3 is a specific TLR2 inhibitor. It was further investigated whether SSL3 binds to the extracellular domain of TLR2 since this domain is crucial for ligand recognition and TLR2 activation. Therefore, the extracellular domains of human and mouse TLR2, expressed in HEK293 cells, were purified and tested for binding to SSL3. ELISA studies showed that SSL3 effectively and dose-dependently bound to the extracellular domains of both human and mouse TLR2 (Fig. 5A). As SSL3 efficiently bound to human as well as mouse TLR2, it was tested whether SSL3 could also inhibit the activation of TLR2 in the mouse macrophage cell line RAW264.7.

Indeed, SSL3 also functionally inhibited mouse TLR2. SSL3 potently inhibited binding of the function-blocking anti-TLR2 to RAW264.7 cells (95.6 ± 0.95% inhibition at 0.1 μg ml (data not shown). In addition, SSL3 completely blocked TLR2 activation by MALP-2, as measured by inhibition of TNFa production (Fig. 5B). Altogether we have shown that SSL3 is a specific and potent inhibitor of human and murine TLR2, which makes in vivo testing in mouse models feasible. 1.2.4 SSL3 exclusively targets TLR2

TLRs, including TLR5, induce intracellular signalling via the common adaptor protein MyD88. To exclude an effect of SSL3 on this common TLR signalling pathway

downstream of TLR2, we tested whether SSL3 could inhibit TLR5 activation. Therefore, HEK-TLR5 cells were activated with flagellin a TLR5-specific ligand. Isolation of flagellin and AprA has been described (Bardoel et al., 201 1 , PLoS Pathog. vol. 7: e1002206. doi:10.1371/journal.ppat.1002206). Briefly, flagellin was obtained by expression of the flic gene (Swiss-prot acc. nr. P72151 ) of P. aeruginosa strain PA01 in E. coli. AprA was obtained by expression of the aprA gene (Swiss-prot acc. nr. Q03023) of P. aeruginosa strain PA01 in E. coli. Both proteins were expressed with a N-terminal 6x his-tag and purified using a His trap™ column (GE Healthcare)

SSL3 could not inhibit flagellin-induced IL-8 production of neutrophils (Fig. 6). In contrast, AprA, which degrades flagellin and thereby prevents TLR5 activation, abolished flagellin mediated IL-8 production (Fig. 6). Polymyxin B was added to prevent TLR4 dependent IL- 8 production as a result of endotoxin contamination of SSL3. Addition of only Polymyxin B to flagellin did not change the flagellin-induced activation of TLR5. As control, IL-8 production by MALP-2 was inhibited by SSL3. These results exclude that SSL3 inhibits the common MyD88-mediated intracellular signalling cascade, and confirm that SSL3 specifically acts on TLR2 itself. 1.2.5 Lack of affinity of other SSLs for TLR2

SSLs present in pathogenicity island SAPI2 share some sequence and structural elements. It was therefore tested whether SSL1 to 1 1 , all from S. aureus strain NCTC 8325 could, could inhibit TLR2 activation, as observed for SSL3. However, none of the other SSLs, except for SSL4, inhibited the MALP-2 induced IL-8 production by HEK-TLR2 cells using a concentration of 10 μg ml (Fig. 7A).

To check the TLR2 inhibiting activity of both SSL4 variants, we analyzed the effect of both proteins on HEK-TLR2/6 cells activated with MALP-2. SSL4-MRSA was about 10- fold more active then SSL4-8325, which correlates with the higher homology to SSL3 in the amino acid sequence alignment (Fig. 7B). However, SSL4-8325 (Fig. 7B) was still about 30-fold less active then SSL3-8325 (Fig. 3B). In conclusion, the TLR2 inhibiting properties of SSL3 reside within its C-terminal domain. See Table 5.

Legend to the figures

Figure 1 : Binding of SSL3-FITC to leukocytes

Leukocytes were incubated with 0, 1 , 3 or 10 μς ηιΙ FITC-labeled SSL3 for 30 min at 4°C. Neutrophils (A), monocytes (B), and lymphocytes (C) were gated according to forward- and side-scatter properties.

Figure 2: SSL3 competes with antibody T2.5 for TLR2 binding

(A) Leukocytes were preincubated with 10 μg ml SSL3 for 30 min at 4°C, and

subsequently incubated with a panel of different monoclonal antibodies directed against cell-surface receptors for 30 min at 4°C. Fold inhibition was calculated by dividing the fluorescence of untreated cells by that of treated cells. Data represent mean ± SEM of three independent experiments.

(B-D) Leukocytes were incubated with various concentrations of SSL3 for 30 min at 4°C. Next, cells were incubated with PE-labeled anti-TLR2 for 30 min at 4°C.

Histograms depict binding of TLR2 to neutrophils (B) and monocytes (C). Relative fluorescence (D) of anti-TLR2 binding to neutrophils and monocytes to calculate the IC50.

Data represent mean ± SEM of three independent experiments. Figure 3: SSL3 inhibits the activation of TLR2 on HEK-TLR2 cells

(A, B) HEK cells transfected with TLR2 were incubated with 0, 0.1 , 0.3 and 1 pg/ml SSL3 for 30 min. Cells were subsequently stimulated with increasing concentrations Pam2Cys (A) or MALP-2 (B).

(C) HEK-TLR1/2 were preincubated with 0, 0.1 , 1 , and 10 g/ml SSL3 for 30 min, and subsequently stimulated with various concentrations Pam3Cys.

(D) HEK-TLR2/6 were preincubated with different concentrations SSL3 for 30 min, and subsequently stimulated with various concentrations MALP-2.

All stimulations were performed overnight and cell supernatant was collected to measure produced IL-8 levels by ELISA.

(A, B) IL-8 production is expressed as OD 450 nm.

(C) The IL-8 production relative to cells stimulated with 1 μg ml Pam3Cys was calculated and expressed as mean ± SD of triplicate experiments.

(D) The IL-8 production relative to cells stimulated with 30 ng/ml MALP-2 was calculated and expressed as mean ± SEM of three independent experiments. Figure 4: SSL3 inhibits the activation of TLR2 on human leukocytes

(A, B) SSL3 was preincubated with 20 μg/ml polymyxin B and PBMCs were preincubated with 10 g/ml anti-TLR4. Neutrophils (A) and PBMCs (B) were isolated from healthy donors and incubated with SSL3 for 30 min. Next, cells were stimulated with increasing concentrations of MALP-2. After overnight incubation, cell supernatant was harvested and IL-8 levels were determined by ELISA. Data are expressed as IL-8 production relative to stimulation with 30 ng/ml MALP-2. For neutrophils data represent mean ± SEM of three independent experiments and for PBMCs a representative experiment is shown. (C, D) Analysis of cytotoxic effects of SSL3 on PBMCs (C) and HEK-TLR2/6 cells (D). Cells were incubated overnight with SSL3 and toxicity was tested using the lactate dehydrogenase (LDH) cellular cytotoxicity detection kit. LDH is depicted relative to the positive control (lysed cells).

Figure 5: SSL3 binds to mouse TLR2 and functionally inhibits its activity

(A) A 96-wells plate was coated with the recombinant extracellular domain of mouse or human TLR2 (10 g/ml). Coated wells were blocked with 4% skimmed milk, and subsequently increasing concentrations of His-SSL3 was added for 1 h at 37°C. Binding of SSL3 was detected with an anti-Xpress mAb, followed by a peroxidase-labeled goat anti-mouse IgG. (B) Mouse macrophage cells (RAW264.7) were preincubate with SSL3 for 30 min. Next, cells were stimulated with increasing concentrations MALP-2. After overnight incubation, cell supernatant was collected and TNFa levels were determined by ELISA. Data are expressed as TNFa production relative to cells stimulated with 1 ng/ml MALP-2 and represent the mean ± SEM of three independent experiments. Figure 6: TLR5 activation is not bound, and not inhibited by SSL3

Flagellin of P. aeruginosa was preincubated with polymyxin B (PMX-B; 20 g/ml), PMX-B + AprA (10 Mg/ml) or PMX-B + SSL3 (3 Mg/ml) for 30 min at 37 °C. Neutrophils were stimulated overnight with treated flagellin at 37°C. In addition, neutrophils were stimulated with MALP-2 +/- SSL3 in the presence of PMX-B. Next, cell supernatant was collected and IL-8 production was measured by ELISA. Data are expressed as absorbance at 450 nm. Figure 7: Effect of other SSLs on inhibition of TLR2 activation

(A) HEK-TLR2/6 cells were preincubated with 10 g/ml SSL1 -1 1 for 30 min at 37°C, and subsequently stimulated with 3 ng/ml MALP-2. After overnight incubation, cell supernatant was harvested to determine IL-8 production by ELISA. IL-8 production is expressed relative to cells treated with MALP-2 only.

(B) HEK-TLR2/6 cells were preincubated with increasing concentrations of SSL4- 8325 and SSL4-MRSA252 for 30 min, and subsequently stimulated with 30 ng/ml MALP- 2. After overnight incubation, cell supernatant was collected and IL-8 production was determined by ELISA. Data are expressed as absorbance at 450 nm.

Figure 8: S. aureus SSL3 protein multiple alignment - graphic version

Most SSL3 amino acid sequences were retrieved from the public NCBI protein database, and some from non-pulic sequenced bovine S. aureus isolates. Partial SSL3 sequences were omitted from the further analysis, and for highly identical SSL3 proteins, only one representative sequence was used (see Table 2).

Sequences were aligned using the CLUSTALW™ program. The phylogenetic tree was constructed using the neighbor-joining method (with bootstrap 500) and evaluated using the interior branch test method with MEGA™ version 5 software (Tamura, Peterson, Stecher, Nei, and Kumar, 201 1 ).

Figure 9: S. aureus SSL4 protein multiple alignment - graphic version

See legend to Figure 8, whereby Figure 9 deals with SSL4 amino acid sequences (see Table 3). Figure 10: Multiple alignment of a representative number of S. aureus SSL3 and

SSL4 proteins - text version.

Results from multiple amino acid sequence alignment using the ClustalW™ algorithm on the amino acid sequences from a representative selection of SSL3 and SSL4 proteins, each from 4 S. aureus isolates.

The protein sequences were derived from the NCBI database or from an in house sequencing program. The conserved amino acid residues are indicated by a dot; gaps in the sequence are indicated by a horizontal bar.

SSL3 is from strains: 21269, acc. no. EGS84524; LGA251 , acc. no. CCC87131 ;

COL, acc. no. YP_185360; and A6300 acc. no. ZP_05693238.

SSL4 is from strains: s1444, in house; COL, acc. no. YP_185362; ST398, acc. no.

CAQ48930; and D139, acc. no. ZP_06323515. Figure 11 : Inhibition of TLR2 by SSL3 and C-terminal fragment of SSL3

Similar to the results in Figure 4, and performed according to Example 1.1 .6, the inhibition of TLR2 activation, as detected by IL8 production, could be inhibited both by SSL3 (panel A) and by a C-terminal fragment of SSL3, the amino acids 127-326 of SEQ ID NO:1 (panel B).

Claims

Claims

1. Staphylococcal superantigen-like 3 (SSL3) protein, or a homolog of said SSL3 protein, or a derivative of either protein, for use in the treatment of medical conditions involving the activation of toll-like receptor 2 (TLR2).

2. The SSL3 protein for use in the treatment according to claim 1 , wherein the SSL3 protein is a protein comprising an amino acid sequence having at least 90 % amino acid sequence identity to the amino acid sequence of SEQ ID NO. 1.

3. The homolog for use in the treatment according to claim 1 , wherein said homolog is a protein that is capable of direct binding to TLR2 and thereby inhibit the activation of the TIR domain of said TLR2 by a TLR2 ligand such as a bacterial lipoprotein, and wherein said protein comprises an amino acid sequence having at least 56 % amino acid sequence identity to the amino acid sequence of SEQ ID NO. 1.

4. The derivative for use in the treatment, according to claim 1 , wherein said derivative is a fragment of the SSL3 protein for use in the treatment according to any one of claims 1 or 2, or said derivative is a fragment of the homolog for use in the treatment according to any one of claims 1 or 3, or said derivative is a mimicking antibody, or said derivative is a peptidomimetic.

5. The SSL3 protein, or the homolog of said SSL3 protein, or the derivative of either protein, according to anyone of claims 1 - 4, for use in the treatment of medical conditions involving the activation of TLR2, wherein the medical condition is an immune-mediated disease or an inflammatory disease.

6. Pharmaceutical composition comprising an SSL3-like protein, a homolog of said SSL3 protein, a derivative of either protein, or a combination thereof, for use in the treatment of medical conditions involving the activation of TLR2.

7. Method for the preparation of the pharmaceutical composition according to claim 6, comprising the admixing of the SSL3 protein, the homolog of said SSL3 protein, the derivative of either protein, or a combination thereof, for use in the treatment of medical conditions involving the activation of TLR2, with a pharmaceutically acceptable carrier or excipient.

8. A method for use in the treatment of medical conditions involving the activation of TLR2, comprising the application to a human or animal target of the pharmaceutical composition according to claim 6.

9. The method for treatment according to claim 8, wherein the medical condition is an immune-mediated disease or an inflammatory disease.

10. Use of an SSL3 protein, or a homolog of said SSL3 protein, or a derivative of either protein, or a combination thereof, for use in the treatment of medical conditions involving the activation of TLR2.

1 1. Use of an SSL3 protein, or a homolog of said SSL3 protein, or a derivative of either protein, or a combination thereof, for use in the manufacture of a medicament for use in the treatment of medical conditions involving the activation of TLR2.