WO2016184795A1 - Novel peptides - Google Patents

Abstract

An isolated HMGB1 inhibitory peptide comprising a Box-A domain and absent a Box-B domain characterized in that it is resistant to oxidation

Description

Novel Peptides

Field of the invention

This invention relates to novel peptide inhibitors of HMGB1 bioactivity and their use in therapy Background to the invention

Injury can trigger an acute inflammatory response, even in the absence of concomitant infection. "Sterile" inflammation is also associated with several types of cancer. Two events are key for the development of sterile inflammation: the recruitment of leukocytes, especially neutrophils and monocytes, and their activation to release proinflammatory cytokines.

High Mobility Group Box 1 (HMGB1 ) is a nuclear protein that signals tissue damage when released into the extracellular medium, and thus works as a Damage Associated Molecular Pattern (DAMP) (Bianchi et al, 2007). Extracellular HMGB1 can act both as a chemoattractant for leukocytes and as a proinflammatory mediator to induce both recruited leukocytes and resident immune cells to release TNF, IL-1 , IL-6 and other cytokines. Notably, immune cells secrete HMGB1 when activated by infection or tissue damage (Andersson et al, 2012); mesothelioma and other cancer cells secrete HMGB1 constitutively (Jube et al., 2012).

HMGB1 belongs to the HMG family of chromatin binding proteins. Based on their structural properties, HMG proteins have been renamed and subdivided into three different families named HMGA, HMGB, and HMGN. Most HMG proteins contain DNA binding domains named HGM boxes, L-shaped domains of about 80 aminoacids which bind the DNA minor groove: some HMG proteins recognize specific DNA sequences, others, like HMGB proteins, have little or no sequence specificity. The HMGB1 molecule has a tripartite structure composed of two HMG box domains called Box-A and Box-B, separated by a short hinge region, and a negatively charged acidic carboxyl terminus, linked to the boxes by a basic region of about 20 residues.

The Box-A and Box-B domains are involved in the protein's function as non-sequence-specific architectural DNA-binding elements, conferring the ability to bind DNA into recognized distorted DNA structures and stabilizing nucleosome assembly, remodeling and sliding. Both Box-A and Box-B of HMGB1 are strongly positively charged and are arranged in three a-helices having a similar L-shaped fold. The long arm of the "L" contains the N-terminal extended strand and helix III (Andersson et al. 2002; Agresti et al., 2003; Thomas, J. O. 2001 ), while the short arm comprises helices I and II. Structure function analysis demonstrated that the Box-A domain is a weak agonist of the inflammatory cytokine release triggered by HMGB1 and competitively inhibits the pro- inflammatory activities of the whole protein. Therefore, from a pharmacological point of view, Box- A based peptides act as antagonists of the pathological conditions induced and/or sustained by HMGB1.Box-B on the other hand has been shown to act as HMGB1 agonist (see Degryse et al, 2001 ).

HMGB1 proteins are highly conserved throughout evolution, with the highest sequence conservation within the Box-A and Box-B domains, and a slight variability within the carboxy- terminal tail (described for example in Sessa et al, 2007). Murine and rat HMGB1 , for instance, differ from the human form by only two substitutions that are located in the carboxy-terminal acidic domain. Recent studies have shown that the proinflammatory cytokine-stimulating activity of HMGB1 depends on the redox state of three cysteines: C23 and C45 must form a disulfide bond within the first HMG-box domain of HMGB1 , Box-A, whereas the unpaired C106 within Box-B must be in the thiol state (Yang et al, 2012). Both terminal oxidation of these cysteines to sulfonates (CyS03^~) with Reactive Oxygen Species (ROS) and their complete reduction to thiols (CySH) abrogates the cytokine-stimulating activity.

It has previously been shown that the redox state of the three cysteines in HMGB1 is very important for HMGB1 bioactivity (Venerau et al., 2012). In particular, when the protein is fully reduced it acts as chemoattractant, when the protein contains a disulfide bond between the first two cysteines it induces cytokine transcription and when the three cysteines are terminally oxidated to sulfonates, upon accumulation of ROS and Reactive Nitrogen Species (RNS), HMGB1 is inactive.

Box-A is one of the highly conserved DNA binding domains of the HMGB1 protein.

The in vivo and in vitro activity of Box-A based peptides has been studied in the past decade with the aim of finding suitable therapeutic approaches involving the use of HMGB1 antagonists.

Box-A peptides have been shown to act as competitive inhibitors for HMGB1 and appear to inhibit all its activities, both in vivo and in vitro.

It has been demonstrated that the peptide Box-A functions as a competitive inhibitor of HMGB1 proinflammatory activity (see WO2002092004).

Box-A peptides have been demonstrated to play a protective role against sepsis in a mouse peritonitis model (Yang et al., 2004), and against hepatitis in a mouse model of HBV infection (Sitia et al., 2007). Moreover, Box-A peptides have been shown to elicit anticonvulsant effects in epilepsies (Maroso et al., 2010). Furthermore, Box-A peptides inhibit maturation, survival and Th1 differentiation of dendritic cells and T cell proliferation (Dumitriu et al., 2005). Box-A peptides also inhibit the chemotactic activity of HMGB1 on fibroblasts (Palumbo et al, 2004) and have been shown to inhibit metastasis formation (Bald et al., 2014)

Mutant Box-A peptides have been engineered in order to obtain longer acting molecules for therapy.

WO2006024547, for instance, discloses mutant Box-A peptides wherein systematic mutations of single amino acids of the wild-type HMGB1 Box-A domain yielded peptides with an increased resistance to proteases and more favourable pharmacokinetic and pharmacodynamic profiles.

WO2008031612 discloses an alternative strategy to improve the therapeutic effect of Box A peptides by means of conjugation to polymers, a well-known strategy to increase the

pharmacodynamics and pharmacokinetic profile of peptides. In particular, Box A peptides disclosed therein are covalently linked to polyethyleneglycol (PEG).

The present inventors and others have discovered that the HMGB1 protein is susceptible to inactivation upon oxidation of its cysteine residues. Hence, therapeutic efficacy of Box-A peptides is hindered by the presence of an oxidative environment at disease target sites.

The problem underlying the present invention is therefore the provision of novel HMGB1 -derived inhibitory peptides which are resistant to oxidation for the treatment and/or prevention of HMGB1 associated pathologies.

The inventors have engineered HMGB1 -derived peptides which inhibit HMGB1 bioactivity, are resistant to oxidative damage, and maintain a prolonged inhibitory activity.

Summary of the invention

The invention provides peptide inhibitors of HMGB1 bioactivity.

The present invention relates to the use of peptides containing certain sequences of the HMGB1 protein and variants, derivatives and fragments thereof in the treatment and prevention of disease, for example a disease associated with activity or overactivity of HMGB1 (hereinafter referred to as "an HMGB1 associated pathology") including but not limited to cancer (including metastasis thereof), mesothelioma, inflammation, sterile inflammation, sepsis, neuroinflammation and epilepsy.

It is known that certain peptides derived from the HMGB1 protein, notably from the Box-A domain of the HMGB1 protein, act as inhibitors of HMGB1 bioactivity, both in vitro and in vivo.

The present inventors have found novel HMGB1 peptides with an inhibitory activity and which are resistant to oxidative stress, therefore maintaining the ability to inhibit HMGB1 in an oxidative environment, for instance at a site of inflammation upon accumulation of ROS and RNS. Said peptides therefore have applications in the treatment of diseases where the target tissue displays high concentrations of inflammatory mediators which result in a highly oxidative environment which could lead to inactivation of HMGB1 inhibitory peptides.

Thus, the present invention relates to HMGB1 inhibitory peptides which comprise a Box-A domain and are absent a Box-B domain and are resistant to oxidation.

The invention also relates to polynucleotides encoding such HMGB1 inhibitory peptides, to vectors comprising such polynucleotides and to host cells transformed with such vectors.

Furthermore, the present invention also relates to a process for preparing HMGB1 inhibitory peptides.

Brief description of the figures

Figure 1 : HMGB1 and three HMGB1 inhibitory peptides (Peptides 1 , 2 and 3).

Figure 2: Inhibition of HMGB1 -induced migration of mouse 3T3 fibroblasts by Peptides 1 , 2 and 3.

Figure 3: Comparison of the efficiency of inhibition of HMGB1 -induced cell migration of mouse 3T3 fibroblasts by Peptides 1 , 2 and 3.

Figure 4: Sequence alignment among Box-A containing proteins: Human homologs of HMGB1

Figure 5: Sequence alignment among Box-A containing proteins: HMGB1 across species.

Figure 6: Proliferation curves of ABI-B/c-LUC cells in the presence of control, recombinant HMGB1 0,1 nM and recombinant HMGB1 10nM.

Figure 7: Proliferation curves of ABI-B/c-LUC cells in the presence of control and recombinant HMGB1 0,1 nM (with and without Peptide 1 10nM).

Figure 8: Proliferation curves of ABI-B/c-LUC cells in the presence of control and recombinant HMGB1 0, 1 nM (with and without Peptide 2 10nM).

Figure 9: Proliferation curves of ABI-B/c-LUC cells in the presence of control and recombinant HMGB1 0, 1 nM (with and without Peptide 3 10nM).

Brief Description of the Sequences in the Sequence listing

SEQ ID No. 1 Human HMGB1 aa Sequence (Accession Number P09429, U51677)

SEQ ID No. 2 HMGB1 Inhibitory Peptide 1 WT aa Sequence

SEQ ID No. 3 HMGB1 Inhibitory Peptide 1 WT, nt sequence SEQ ID No. 4 HMGB1 Inhibitory Peptide 2 (C23-S; C45-S) aa sequence SEQ ID No. 5 HMGB1 Inhibitory Peptide 2 (C23-S; C45-S) nt sequence SEQ ID No. 6 HMGB1 Inhibitory Peptide 3 (C23-A; C45-A) aa sequence SEQ ID No. 7 HMGB1 Inhibitory Peptide 3 (C23-A; C45-A) nt sequence SEQ ID No. 8 HMGB1 Inhibitory Peptide 4 (C23-L; C45-L); aa sequence SEQ ID No. 9 HMGB1 Inhibitory Peptide 4 (C23-L; C45-L); nt sequence SEQ ID No. 10 HMGB1 Inhibitory Peptide 5 (C23-G; C45-G); aa sequence SEQ ID No. 1 1 HMGB1 Inhibitory Peptide 5 (C23-G; C45-G); nt sequence SEQ ID No. 12 HMGB1 Inhibitory Peptide 6 (C23-V; C45-V); aa sequence SEQ ID No. 13 HMGB1 Inhibitory Peptide 6 (C23-V; C45-V); nt sequence SEQ ID No. 14 HMGB1 Inhibitory Peptide 7 (C23-I; C45-I); aa sequence SEQ ID No. 15 HMGB1 Inhibitory Peptide 7 (C23-I; C45-I); nt sequence SEQ ID No. 16 HMGB1 Inhibitory Peptide 8(C23-A; C45-L) aa Sequence SEQ ID No. 17 HMGB1 Inhibitory Peptide 8 (C23-A; C45-L) nt Sequence SEQ ID No. 18 HMGB1 Inhibitory Peptide 9 (C23-L; C45-A); aa Sequence SEQ ID No. 19 HMGB1 Inhibitory Peptide 9 (C23-L; C45-A); nt Sequence SEQ ID No. 20 HMGB1 Inhibitory Peptide 10 (C23-A;C45-G); aa Sequence SEQ ID No. 21 HMGB1 Inhibitory Peptide 10 (C23-A;C45-G); nt Sequence SEQ ID No. 22 HMGB1 Inhibitory Peptide 11 (C23-G;C45-A); aa Sequence SEQ ID No. 23 HMGB1 Inhibitory Peptide 1 1 (C23-G;C45-A); nt Sequence SEQ ID No. 24 HMGB1 Inhibitory Peptide 12 (C23-L;C45-G); aa Sequence SEQ ID No. 25 HMGB1 Inhibitory Peptide 12 (C23-L;C45-G); nt Sequence SEQ ID No. 26 HMGB1 Inhibitory Peptide 13 (C23-G;C45-L); aa Sequence SEQ ID No. 27 HMGB1 Inhibitory Peptide 13 (C23-G;C45-L); nt Sequence SEQ ID No. 28 HMGB1 Inhibitory Peptide 14 (C23-V; C45-S); aa Sequence SEQ ID No. 29 HMGB1 Inhibitory Peptide 14 (C23-V; C45-S); nt Sequence SEQ ID No. 30 HMGB1 Inhibitory Peptide 15 (C23-V; C45-A); aa Sequence SEQ ID No. 31 HMGB1 Inhibitory Peptide 15 (C23-V; C45-A); nt Sequence SEQ ID No. 32 HMGB1 Inhibitory Peptide 16 (C23-V; C45-L); aa Sequence SEQ ID No. 33 HMGB1 Inhibitory Peptide 16 (C23-V; C45-L); nt Sequence SEQ ID No. 34 HMGB1 Inhibitory Peptide 17 (C23-V; C45-G); aa Sequence SEQ ID No. 35 HMGB1 Inhibitory Peptide 17 (C23-V; C45-G); nt Sequence SEQ ID No. 36 HMGB1 Inhibitory Peptide 18 (C23-V; C45-I); aa Sequence SEQ ID No. 37 HMGB1 Inhibitory Peptide 18 (C23-V; C45-I); nt Sequence SEQ ID No. 38 HMGB1 Inhibitory Peptide 19 (C23-S; C45-V); aa Sequence SEQ ID No. 39 HMGB1 Inhibitory Peptide 19 (C23-S; C45-V); nt Sequence SEQ ID No. 40 HMGB1 Inhibitory Peptide 20 (C23-A; C45-V); aa Sequence SEQ ID No. 41 HMGB1 Inhibitory Peptide 20 (C23-A; C45-V); nt Sequence SEQ ID No. 42 HMGB1 Inhibitory Peptide 21 (C23-L; C45-V); aa Sequence SEQ ID No. 43 HMGB1 Inhibitory Peptide 21 (C23-L; C45-V); nt Sequence SEQ ID No. 44 HMGB1 Inhibitory Peptide 22 (C23-G; C45-V); aa Sequence SEQ ID No. 45 HMGB1 Inhibitory Peptide 22 (C23-G; C45-V); nt Sequence SEQ ID No. 46 HMGB1 Inhibitory Peptide 23 (C23-I; C45-S); aa Sequence SEQ ID No. 47 HMGB1 Inhibitory Peptide 23 (C23-I; C45-S); aa Sequence SEQ ID No. 48 HMGB1 Inhibitory Peptide 24 (C23-I; C45-A); aa Sequence SEQ ID No. 49 HMGB1 Inhibitory Peptide 24 (C23-I; C45-A); nt Sequence SEQ ID No. 50 HMGB1 Inhibitory Peptide 24 (C23-I; C45-L); aa Sequence SEQ ID No. 51 HMGB1 Inhibitory Peptide 24 (C23-I; C45-L); nt Sequence SEQ ID No. 52 HMGB1 Inhibitory Peptide 25 (C23-I; C45-G); aa Sequence SEQ ID No. 53 HMGB1 Inhibitory Peptide 25 (C23-I; C45-G); nt Sequence SEQ ID No. 54 HMGB1 Inhibitory Peptide 26 (C23-I; C45-V); aa Sequence SEQ ID No. 55 HMGB1 Inhibitory Peptide 26 (C23-I; C45-V); nt Sequence SEQ ID No. 56 HMGB1 Inhibitory Peptide 27 (C23-S; C45-I); aa Sequence SEQ ID No. 57 HMGB1 Inhibitory Peptide 27 (C23-S; C45-I); nt Sequence SEQ ID No. 58 HMGB1 Inhibitory Peptide 28 (C23-A; C45-I); aa Sequence

SEQ ID No. 59 HMGB1 Inhibitory Peptide 28 (C23-A; C45-I); nt Sequence

SEQ ID No. 60 HMGB1 Inhibitory Peptide 29 (C23-L; C45-I); aa Sequence

SEQ ID No. 61 HMGB1 Inhibitory Peptide 29 (C23-L; C45-I); nt Sequence

SEQ ID No. 62 HMGB1 Inhibitory Peptide 30 (C23-G; C45-I); aa Sequence

SEQ ID No. 63 HMGB1 Inhibitory Peptide 30 (C23-G; C45-I); nt Sequence

SEQ ID No. 64 HMGB1 Inhibitory Peptide 31 (C23-D;C45-D); aa Sequence

SEQ ID No. 65 HMGB1 Inhibitory Peptide 31 (C23-D; C45-D); nt Sequence

SEQ ID No. 66 HMGB1 Inhibitory Peptide 32 (C23-T;C45-T); aa Sequence

SEQ ID No. 67 HMGB1 Inhibitory Peptide 32 (C23-T; C45-T); nt Sequence

Detailed description of the invention

Definitions and Embodiments

As used herein, "HMGB1 protein" is a substantially pure and isolated polypeptide described for instance in GenBank accession number U51677, recombinantly produced or isolated from natural sources, which increases the release of proinflammatory cytokines from a cell and/or increases inflammation. The term embraces the human protein and homologues thereof.

The human HMGB1 protein sequence is provided as SEQ ID No 1.

As used herein, a Box-A domain is a sequence having the DNA binding function of an HMG Box-A domain, comprising at least residues 23 to 45, e.g. at least residues 2 to 45, e.g. at least residues 2 to 78 e.g. at least residues 2 to 89 or at least residues 6 to 74 of SEQ ID No 1 or the

corresponding residues of a variant thereof.

The Box-B domain is a sequence of an HMG Box-B domain comprising residues 90 to 165 of SEQ ID NO 1 or the corresponding residues of a variant thereof.

The acidic carboxyl terminus consists of aminoacids 185-204 of SEQ ID NO 1 or the

corresponding residues of a variant thereof.

The hinge region refers to aminoacids 83 to 89 of SEQ ID NO 1 or the corresponding residues of a variant thereof. .

As used herein, a "HMGB1 inhibitory peptide" is a peptide which acts as an inhibitor of HMGB1 bioactivity. Variants of sequences includes homologues. A "homologue" can be a corresponding HMGB1 sequence found in another species such as a non-human mammalian sequence e.g. a murine sequence, a non mammalian vertebrate sequence, or a non vertebrate sequence including but not limited to e.g. Mus Musculus , Gorilla gorilla, Gecko japonica, Heterocephalus glab, Pteropus alecto, Equus caballus, Sus scrofa, Oryctolagus cunicul, Otolemur garnettii, Sorex

araneus.Rhinolophusferrumeq Papio anubis.Bos Taurus, Callithrix jacchus, Callicebus moloch, Canis familiaris, Cricetulus griseus, Bos mutus, Camelus ferus, Myotis brandtii, Heterocephalus glab, Anas platyrhynchos, Tupaia chinensis , Crotalus horridus, Xenopus laevis, Gallus gallus, Micrurus fulvius , Ambystoma mexicanum, Danio rerio , Oncorhynchus mykiss, Salmo salar , Ctenopharyngodon i., Anopheles gambiae (see sequences shown in Figure 5). In the context of the present invention, homologues of the HMGB1 proteins defined by SEQ ID No 1 include vertebrate and non vertebrate HMGB1 proteins. Preferred non vertebrate HMGB1 proteins are, for instance, the Anopheles gambia (XP_31 1154) HMGB1 protein.

Homologues also include proteins belonging to the HMG family of proteins other than HMGB1 , e.g. HMGB1 , HMGB2, HMGB3, HMG3M, HMGB4, HMG-4L or/and SP100 (see sequences shown in Figure 4) in humans and other species e.g. other mammalian species.

A variant of a HMGB1 inhibitory peptide also includes a peptide which has an aminoacid sequence having at least 60%, preferably at least 70%, 80%, 85%, 90%, and most preferably at least 95% sequence identity to the HMGB1 inhibitory peptide sequence referred to, as determined by the BLAST sequence comparison algorithm using default settings and which acts as an inhibitor of HMGB1 bioactivity. Where the difference is an insertion or a replacement, the different amino acid may be "another amino acid" as defined herein.

Sequence deviations preferably have a minimal impact on tertiary peptide structure. A variant may contain e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions by comparison to the reference sequence (a substitution will be understood to be a replacement, insertion or deletion of an amino acid in the sequence when optimally aligned e.g. using the above BLAST program).

Suitably, a variant may contain conservative aminoacid substitutions by comparison to the reference sequence which are expected to preserve the biological activity of an HMGB1 peptide as an inhibitor of HMGB1 bioactivity.

Conservative substitutions of amino acids can be grouped according to the chemical properties of the side chains. For example, aminoacids with neutral and hydrophobic side chains (e.g. A, V, I, L, P, F, M) or those having neutral or polar side chains (G, S, T, Y, C, N, Q); basic side chains (K, R, H); acidic side chains (D; E); aliphatic hydroxyl side chains (S, T); amine-containing side chains (N, Q, K, R, H); aromatic side chains (F, Y, W); sulfur-containing side chains (C, M).

Preferred conservative substitutions are: R-K; E-D; Y-F; L-M; V-L; Q-H.

Suitably, a variant may be obtained by substituting an aminoacid with the aminoacid present at the homologous position in a homologous HMGB protein, for instance HMGB2, HMGB3 or HMGB4 proteins, for example, at the homologous position within the Box-A domain thereof. Given the high homology among HMGB proteins and especially among Box-A domains, it is expected that an aminoacid which is functional in a Box-A domain can retain the same function in a different Box-A domain. Alternatively, a conservative substitution of said homologous aminoacid is expected to yield a functional peptide.

Alternatively, a variant may be obtained by substituting an aminoacid with the aminoacid present at the homologous position in a HMG protein, for example, at the homologous position within a HMG-box domain protein, for instance Sp100. Given the high homology among HMG-box domains, it is expected that an aminoacid which is functional in a HMG-box domain can retain the same function in a different HMG-box domain. Alternatively, a conservative substitution of said homologous aminoacid is expected to yield a functional peptide.

In the context of the present invention, a "derivative" of a HMGB1 inhibitory peptide includes a chemical modification to the peptide made for the purpose of improving its properties, especially its pharmakinetic, pharmakodynamic, chemical or physical properties.

For example, a derivative may be a chemical modification made to the peptide for the purpose of increasing its half life.

Strategies to increase half life include incorporating increased resistance to protease degradation by N-terminal acetylation or C-terminal amidation, insertion of non natural aminoacids at labile sites, cyclization using disulfide bonds, incorporation onto microspheres or nanoparticles;

alternative strategies to increase half life include increasing size and hydrodynamic volume to prevent kidney clearance, for instance by multimerization, polymer conjugation, e.g.

polyethyleneglycol (PEG) conjugation. Additional strategies involving increase in size and hydrodynamic volume to prevent kidney clearance in addition to attachment to proteins with long half life include attachment to Fc (natural antibody constant region), or human serum albumin. More generally, derivatives include fusion proteins formed by linking of the HMGB1 inhibitory peptide to a heterologous protein or peptide. Thus in a preferred embodiment the invention includes an isolated HMGB1 inhibitory peptide which is a derivative formed by conjugation to a polymer, for example the HMGB1 inhibitory peptide is pegylated.

Particularly preferred PEG polymers for peptide conjugation have a molecular weight ranging from 2kDa to 40 kDa, and may be linear or branched PEG.

A derivative can also include a chemical modification made for the purpose of improving resistance of the peptide to protease degradation. Thus the invention includes an isolated HMGB1 inhibitory peptide which is a derivative formed by acylation at the N-terminus and/or amidation at the C-terminus.

Particularly preferable strategies to increase resistance to protease of HMGB1 inhibitory peptides include point mutations which have been described in WO2006024547, which is incorporated herein by reference.

Further preferred strategies to extend circulation half-life of HMGB1 inhibitory peptides include covalent binding to PEG, as described in WO2008031612, incorporated herein by reference.

Further, a combination of PEG conjugation and point mutations are described in WO2008031612.

As used in the context of the present invention, "HMGB1 bioactivity" includes but is not limited to cell migration (e.g. chemotaxis), cytokine release, interaction and communication between for instance T cells and dendritic cells. Thus a HMGB1 peptide which inhibits HMGB1 bioactivity will inhibit one or more of HMGB1 protein's activities of cell migration, cytokine release, interaction and cell-cell communication (e.g. between for instance T cells and dendritic cells) and preferably will inhibit cell migration. Inhibition of cell migration can be measured using the "Cell Migration Assay" set out in the General Methods section in the Examples. Exemplary results for test peptides are shown in Figure 2. An alternative measure of inhibition HMGB1 bioactivity is anti- proliferative activity in HMGB1 expressing cancer cell lines, for example anti-proliferative activity in the AB1-B/c-LUC Cell Proliferation Assay set out in the General Methods section in the Examples.

As used herein, the expression "or a variant or derivative thereof means "or a variant thereof or a derivative thereof or a derivative of a variant thereof.

As intended herein, the terms "inhibit" or "decrease" encompass a measurable reduction by at least 20%, 50%, 70%, 75%, or 80% over untreated controls.

As used herein "oxidation" refers to the chemical modification of the oxidative state of atoms within a molecule, particularly the loss of electrons or an increase in oxidation state of a molecule, atom or ion. As used herein "resistant to oxidation" or "oxidation resistance" means having the ability to inhibit HMGB1 bioactivity following prolonged treatment with an oxidizing agent, including but not limited to H2O2 or other ROS or NOS; specifically oxidation resistance may be measured using the Oxidation Resistance Assay described in the General Methods section in the Examples.

Measurement of the oxidative state of Cysteine residues present on HMGB1 inhibitory peptides can be carried out as detailed in, inter alia, Veneraux et al., 2012. Essentially, liquid

chromatography and tandem mass spectrometry (LC-MS/MS) is used to determine the presence of different oxidized forms of the amino acid cysteine. The presence of SOH residues in cysteines was investigated with a 20-min incubation with 0.1 mM dimedone (5,5-dimethyl-1 ,3- cyclohexanedione). SO2H and SO3H oxidative modifications were sought by looking for mass increases of 32 and 48 amu, respectively, on each particular cysteine residue. The peptides were subjected to tryptic digestion and characterized by LC-MS/MS

Several amino acids are sensitive to oxidation, namely cysteine, methionine and tryptophan.

The thiol side chain in cysteine is susceptible to oxidation to give the disulfide derivative cystine, which serves an important structural role in many proteins. Furthermore, more aggressive oxidants convert cysteine to the corresponding sulfinic acid and sulfonic acid. The term "mutation" as used in the context of the present invention can be understood as substitution, deletion and/ or addition of a single amino acid in the target sequence. Preferably, the mutation of the target sequence in the present invention is a substitution. The substitution can occur with different genetically encoded amino acid or by non-genetically encoded amino acids. Furthermore, the substitution can occur with different non natural aminoacids.

References to "another amino acid" include other amino acids which are genetically encoded natural amino acids, non-genetically encoded natural amino acids and non-natural amino acids.

A "naturally encoded amino acid" refers to the 20 naturally encoded amino acids i.e. glycine, alanine, valine, leucine, isoleucine, proline, tyrosine, tryptophan, phenylalanine, cysteine, methionine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagines and glutamine, all of these amino acids being in their L-forms.

A non-naturally encoded acid means any amino acid which is not a naturally encoded amino acid, typically an amino acid of formula H₂N-CH(R)-COOH in which R is a sidechain not found in one of the naturally encoded amino acids. Suitably non-naturally encoded amino acids are L-amino acids.

Non-genetically encoded natural amino acids are for instance homocysteine, hydroxyproline, ornithin, hydroxylysine, citrulline, carnitine In an embodiment, there is provided an isolated HMGB1 inhibitory peptide comprising a Box-A domain and absent a Box-B domain characterized in that it is resistant to oxidation.

In a preferred embodiment, the invention provides an isolated HMGB1 inhibitory peptide as previously defined, comprising residues 23 to 45 of SEQ ID NO 1 or a variant or derivative, thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 1 are substituted by another aminoacid thereby to increase the oxidation resistance thereof.

Suitably both cysteines at positions 23 and position 45 of SEQ ID NO 1 are substituted by another aminoacid which may be the same or different. In one preferred embodiment the amino acids substituting at position 23 and position 45 of SEQ ID NO 1 are the same. Alternatively they are different.

Suitably, the isolated HMGB1 inhibitory peptide of the present invention comprises SEQ ID NO 2 or a variant or derivative thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 2 are independently substituted by aminoacids selected from the group consisting of Serine, Alanine, Leucine, Valine, Glycine, Isoleucine and Threonine and more suitably selected from the group consisting of Serine, Alanine, Leucine, Valine, Glycine and Isoleucine.

Alternatively, the isolated HMGB1 inhibitory peptide of the present invention comprises SEQ ID NO 2 or a variant or derivative thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 2 are independently substituted by aminoacids selected from the group consisting of Alanine, Leucine, Valine, Glycine and Isoleucine.

Preferably, the isolated HMGB1 inhibitory peptide of the present invention comprises SEQ ID NO 2 or a variant or derivative thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 2 are independently substituted by Alanine and preferably both are substituted by Alanine.

According to another preferred embodiment, the isolated HMGB1 inhibitory peptide of the present invention comprises SEQ ID NO 2 or a variant or derivative thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 2 are independently substituted by Serine and preferably both are substituted by Serine.

In a further preferred embodiment, the invention provides an isolated HMGB1 inhibitory peptide as defined herein, comprising an aminoacid sequence selected from SEQ ID NO 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG-4L or/and SP100, as described in Figure 4. Said sequence may also be selected from SEQ ID NO 66 or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG-4L or/and SP100, as described in Figure 4.

Suitably, the isolated HMGB1 inhibitory peptide of the present invention comprises an aminoacid sequence selected from SEQ ID NO 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, ,HMGB4, HMG-4L or/and SP100, as described in Figure 4. In another embodiment, isolated HMGB1 inhibitory peptide of the present invention comprises an aminoacid sequence selected from SEQ ID NO 4 or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG-4L or/and SP100, as described in Figure 4. In another embodiment, isolated HMGB1 inhibitory peptide of the present invention comprises an aminoacid sequence selected from SEQ ID NO 6 or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG-4L or/and SP100, as described in Figure 4. A corresponding sequence containing one or more amino acid differences may (for example and without limitation) contain 1 , 2, 3, 4 or 5 differences such as 1 , 2, 3 or 4 e.g. 1 , 2 or 3 e.g. 1 or 2 such as 1 difference.

In a preferred embodiment, the invention provides an isolated HMGB1 inhibitory peptide as defined herein comprising SEQ ID No 2 or a variant having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity to said sequence, wherein residues at positions 23 and 45 are not cysteines, or a derivative thereof.

In an alternative embodiment, the isolated HMGB1 inhibitory peptide according to the present invention is a derivative formed by acylation at the N-terminus and/or amidation at the C-terminus. In a further embodiment, the isolated HMGB1 inhibitory peptide of the present invention is a derivative formed by conjugation to a polymer.

In one embodiment the isolated HMGB1 inhibitory peptide is pegylated. In another embodiment the isolated HMGB1 inhibitory peptide is not pegylated.

Suitably the isolated HMGB1 inhibitory peptide is recombinant. According to a further aspect of the present invention, there is provided an isolated polynucleotide encoding the HMGB1 inhibitory peptide of the present invention.

Preferred polynucleotides encoding HMGB1 Peptides of the invention are SEQ ID No. 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, or a corresponding polynucleotide which is codon optimized for a host cell, for example SEQ ID No. 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59 or 61 or a corresponding polynucleotide which is codon optimized for a host cell and in particular SEQ ID No. 5 or 7 or a corresponding polynucleotide which is codon optimized for a host cell. A further polynucleotide is SEQ ID No. 67, or a corresponding polynucleotide which is codon optimized for a host cell.

An embodiment of the present invention provides an expression vector comprising a

polynucleotide encoding the HMGB1 inhibitory peptide of the present invention.

Suitably, preferred vectors comprising polynucleotides of the present invention are pT7.7 vectors. In a further embodiment, the present invention provides a host cell comprising the vector vector comprising a polynucleotide encoding the HMGB1 inhibitory peptide of the present invention.

Suitably, preferred host cells according to the present invention are E.Coli cells, particularly BL21 cells.

Pegylation methodology

As noted herein, HMGB1 inhibitory peptides according to the invention may optionally be pegylated in order to extend circulation half-life.

A number of pegylation technologies are known in the art. According to one method, illustrated in WO2008031612 (herein incorporated by reference), the PEG group is incorporated by reductive amination of the N-terminus of the HMGB1 inhibitory peptide. Thus the HMGB1 inhibitory peptide is reacted with a PEG-aldehyde and reduced under conditions well known to a skilled person. According to another method, illustrated in WO2007130453 (herein incorporated by reference), the PEG group is incorporated via one (or more) non-natural amino acids introduced into the peptide sequence. The substitution of one or more of the amino acids of the peptide for a non- natural amino acid should ideally have minimal impact on the inhibitory effect of the peptide on HMGB1 activity. Suitable non-natural amino acids to be incorporated contain azide or alkyne groups (such as the amino acids L-azidohomoalanine and L-homopropargylglycine). These amino acids may be reacted with a PEG group linked to an alkyne or azide group thereby to link the PEG group to the peptide via a triazole moiety. The reaction conditions to be used are those well known to a skilled person for the Huisgen reaction. For example, a Met amino acid might be substituted for the non-natural amino acid. Medical uses

According to a further aspect of the present invention, there is provided an isolated HMGB1 peptide according to the present invention for use as medicament

HMGB1 inhibitory peptides are suitable for treating or preventing HMGB1 associated pathologies.

Suitably, HMGB1 associated pathologies according to the present invention are pathologies associated with HMGB1 bioactivity, including but not limited to cell migration, cytokine release, cell-cell interaction and cell-cell communication (e.g. between T cells and dendritic cells) , acute and chronic inflammation.

Non limiting examples of conditions which can be usefully treated using HMGB1 inhibitory peptides of the present invention include the broad spectrum of pathological conditions induced by the HMGB1-chemokine and by the HMGB1-induced cascade of inflammatory cytokines grouped in the following categories: restenosis and other cardiovascular diseases, reperfusion injury, inflammation diseases such as inflammatory bowel disease, systemic inflammation response syndrome, e. g. sepsis, adult respiratory distress syndrome, etc. autoimmune diseases such as rheumatoid arthritis and osteoarthritis, obstetric and gynaecological diseases, infectious diseases, atopic diseases, such as asthma, eczema, etc. tumor pathologies, e.g. solid or non-solid tumor diseases associated with organ or tissue transplants, so such as reperfusion injuries after organ transplantation, organ rejection and graft-versus-host disease, congenital diseases,

dermatological diseases such as psoriasis or alopecia, neurological diseases, ophthalmological diseases, renal, metabolic or idiopathic diseases and intoxication conditions, e.g. iatrogenic toxicity, wherein the above diseases are caused by, associated with and/or accompanied by HMGB1 protein bioactivity.

In particular, the pathologies belonging to inflammatory and autoimmune diseases include rheumatoid arthritis/seronegative arthropathies, osteoarthritis, inflammatory bowel disease, Crohn's disease, intestinal infarction, systemic lupus erythematosus, iridoeyelitis/uveitis, optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis, Vegener's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures. Systemic inflammatory response includes sepsis syndrome (including gram positive sepsis, gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urosepsis, septic conjunctivitis), meningococcemia, trauma

hemorrhage, hums, ionizing radiation exposure, acute and chronic prostatitis, acute and chronic pancreatitis, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, ulcerative,

pseudomembranous, acute and ischemic cholitis, diverticulitis, achalasia, cholangitis,

cholecystitis, enteritis, adult respiratory distress syndrome (ARDS). Reperfusion injury includes post-pump syndrome and ischemia-reperfusion injury. Cardiovascular disease includes cardiac stun syndrome, myocardial infarction and ischemia, atherosclerosis, thrombophlebitis, endocarditis, pericarditis, congestive heart failure, cardiac inflammatory disease, restenosis. Obstetric and gynecologic diseases include premature labour, endometriosis, miscarriage, vaginitis and infertility. Infectious diseases include HIV infection/HIV neuropathy, meningitis, B-and C-hepatitis, herpes simplex infection, septic arthritis, peritonitis, E. cold 0157:H7,

pneumonia, epiglottitis, haemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, candidiasis, filariasis, amebiasis, malaria, Dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis,

mycobacterium avium intracellulare, Pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epidydimitis, legionella, Lyme disease, influenza A, Epstein-Barr Virus,

Cytomegalovirus, viral associated hemophagocytic syndrome, viral encephalitis/aseptic meningitis. Allergic and atopic disease include asthma, allergy, anaphylactic shock, immune complex disease, hay fever, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis. Malignancies (liquid and solid tumor pathologies) include mesothelioma, e.g. pleural, peritoneal and pericardial mesothelioma, melanoma, ALL, AML, CML, CLL, Hodgkin's disease, non Hodgkin's lymphoma, Kaposi's sarcoma, colorectal carcinoma, nasopharyngeal carcinoma, malignant histiocytosis and paraneoplastic

syndrome/hypercalcemia of malignancy. Transplant diseases include organ transplant rejection and graft-versus-host disease. Congenital disease includes cystic fibrosis, familial

hematophagocytic lymphohistiocytosis and sickle cell anemia. Dermatologic disease includes psoriasis, psoriatic lo arthritis and alopecia. Neurologic disease includes neurodegenerative diseases (multiple sclerosis, migraine, headache, amyloid-associated pathologies, prion diseases/Creutzfeld-Jacob disease, Alzheimer and Parkinson's diseases, multiple sclerosis, amyotrophic emilateral sclerosis) and peripheral neuropathies, migraine, headache. Renal disease includes nephrotic syndrome, hemodialysis and uremia. Iatrogenic intoxication condition includes OKT3 therapy, Anti-CD3 therapy, Cytokine therapy, Chemotherapy, Radiation therapy and chronic salicylate intoxication.

Metabolic and idiopathic disease includes Wilson's disease, hemochromatosis, alpha-1 antitrypsin deficiency, diabetes, weight loss, ao anorexia, cachexia, obesity, Hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis evaluation and primary biliary cirrhosis.

Ophthalmological disease include glaucoma, retinopathies and dry-eye. A miscellanea of other pathologies comprehends: multiple organ dysfunction syndrome, muscular dystrophy, septic meningitis, atherosclerosis, epiglottitis, Whipple's disease, asthma, allergy, allergic rhinitis, organ necrosis, fever, septicaemia, endotoxic shock, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, urethritis, emphysema, rhinitis, alveolitis, bronchiolitis, pharyngitis, epithelial barrier dysfunctions,

pneumoultramicropicsilicovolcanoconiosis, pleurisy, sinusitis, so influenza, respiratory syncytial virus infection, disseminated bacteremia, hydatid cyst, dermatomyositis, burns, sunburn, urticaria, warst, wheel, vasulitis, angiitis, myocarditis, arteritis, periarteritis nodosa, rheumatic fever, celiac disease, encephalitis, cerebral embolism, Guillame-Barre syndrome, -25 neuritis, neuralgia, iatrogenic complications/peripheral nerve lesions, spinal cord injury, paralysis, uveitis, arthriditis, arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, synovitis, myasthenia gravis, Goodpasture's syndrome, Babcets's syndrome, ankylosing spondylitis,

Barger's disease, Retier's syndrome, bullous dermatitis (bullous pemphigoid), pemphigous and pemphigous vulgaris and alopecia.

Particularly relevant HMGB1 related diseases, in the context of the present invention, are epilepsy, especially acute and chronic seizures and in the treatment of drug resistant epilepsies, Alzheimer's disease, systemic Lupus erythematosus, cardiac inflammatory disease, restenosis, liver fibrosis, sepsis, ischemia-reperfusion injury, arthritis, cancer, particularly malignant mesothelioma, e.g. pleural, peritoneal, pericardial mesothelioma, ovarian cancer, melanoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, metastatic cancer.

Thus, in a further embodiment, there is provided the use of an HMGB1 inhibitory peptide according to the present invention for the manufacture of a medicament for the treatment of an HMGB1 associated pathology such as one of the aforementioned conditions or diseases.

Suitably, the HMGB1 related disease is selected from epilepsy, especially acute and chronic seizures and in the treatment of drug resistant epilepsies, Alzheimer's disease, systemic Lupus erythematosus, cardiac inflammatory disease, restenosis, liver fibrosis, sepsis, ischemia- reperfusion injury, arthritis, cancer, particularly malignant mesothelioma, e.g. pleural, peritoneal, pericardial mesothelioma, ovarian cancer, melanoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, metastatic cancer.

There is also provided a method of treating an HMGB1 associated pathology comprising administering a therapeutically effective amount of an HMGB1 peptide of the present invention to a subject in need thereof.

In a further embodiment, there is provided an HMGB1 inhibitory peptide of the present invention for use in the treatment of an HMGB1 associated pathology A therapeutically effective amount of an HMGB1 peptide of the invention will suitably be approximately 600 mg and may be administered between twice a week and once per month (however these doses and frequencies are purely illustrative and non-limiting).

More generally, a dose of 0.001 to 20 mg/kg, for instance 0.2-20mg/kg may be suitable.

Compositions

The invention provides an HMGB1 inhibitory peptide of the invention together with one or more pharmaceutically acceptable diluents or carriers.

A composition, e.g. for injection, will suitably comprise the HMGB1 inhibitory peptide of the invention together with water for injection and appropriate buffering salts (e.g. citrate, Tris, phosphate salts) and substances (e.g. salts or polyols) to modify the tonicity of the composition. The pH of an aqueous composition may be adjusted for optimal protein stability or physiological comfort and may, for example, be around 6.5 to 8.5 e.g. 7-7.4. Other possible composition components include complexing agents (e.g. EDTA), anti-oxidants and preservatives.

A composition of the invention may be provided in lyophilised form suitable for reconstitution with water for injection. Lyophilised compositions may contain bulking agents such as mannitol and lyoprotectants such as polyols e.g. trehalose or sucrose.

As well as by the parenteral route, the HMGB1 Peptide may also be administered by other routes, for example intranasally, by inhalation or by epicutaneous administration.

Exemplary compositions may be gleaned by reference to Remington's Pharmaceutical Sciences (18^th Ed, A R Gennaro, ed, Mack Publishing Company, 1990).

Combinations

The HMGB1 inhibitory peptides of the invention may be administered in combination with other active ingredients.

HMGB1 inhibitory peptides of the invention might be administered in association with

anticonvulsant drugs for the treatment and prevention of epilepsy. Anticonvulsant drugs might for instance include , Gamma-Aminobutyric Acid Type A (GABAA) Receptor Agonist s(e.g.

Benzazepines, Pyrimidines, Xanthines), Gamma-Aminobutyric Acid Transaminase (GABA-T) Inhibitors (Pentanoic Acids ), Carbonic Anhydrase (CA) Inhibitors (Azoles, Thiazoles), Alpha- Amino-3-Hydroxy-5-Methyl-4-lsoxazolepropionic Acid (AMPA) Receptor Antagonists (Hexoses), Sodium Channel Blockers (Dibenzazepines, Azoles, Imidazoles. HMGB1 inhibitory peptides of the invention might be administered in association with cholinesterase inhibitors or NMDA Receptor antagonists for the treatment of Alzheimer's disease.

HMGB1 inhibitory peptides of the invention might be administered in association with Nonsteroidal anti-inflammatory drugs (NSAIDs), Corticosteroids, Inosine Monophosphate Dehydrogenase (IMPDH) Inhibitors, ), lnterleukin-6 (IL-6) Receptor Antagonists, for the treatment of systemic Lupus erythematosus;

HMGB1 inhibitory peptides of the invention might be administered in association with Beta-Tubulin Inhibitors , Mammalian Target of Rapamycin (mTOR) Inhibitors, 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG-CoA) Reductase Inhibitors , Thromboxane A2 (TXA2) Synthesis Inhibitors, Retinoic Acid Receptor (RAR) Agonists, Glucocorticoid Receptor (GR) Agonists for the treatment of cardiac inflammatory disease and restenosis;

HMGB1 inhibitory peptides of the invention might be administered in association with Interferon- Gamma Receptor 1 (IFNGR1 ) Agonist for the treatment of liver fibrosis;

HMGB1 inhibitory peptides of the invention might be administered in association with antibiotics, Arginine Vasopressin Receptor 1A and/or 1 B and/or 2 (AVPR1A/ AVPR1 B/ AVPR2) Agonists, Sodium Channel Blockers, Glucocorticoid Receptor (GR) Agonists, Bacterial Topoisomerase II Inhibitors, Alpha-1 A Adrenergic Receptor (ADRA1 A) Agonists, Thrombin Receptor Antagonists, Tumor Necrosis Factor (TNF) Inhibitor, Toll-Like Receptor 4 (TLR4) Antagonist, for the treatment of sepsis;

HMGB1 inhibitory peptides of the invention might be administered in association with

Mitochondrial Permeability Transition Pore (mPTP) Modulators for the treatment of ischemia- reperfusion injury; HMGB1 inhibitory Peptides might be administered in association with Xanthine Oxidase (XO) Inhibitors, Cyclooxygenase-1 (COX-1 ) and/or Cyclooxygenase-2 (COX-2)lnhibitors, CD28 Receptor Antagonists, 15-Lipoxygenase (15-LO) Inhibitors, lnterleukin-1 (IL-1 ) Inhibitors, Dihydroorotate Dehydrogenase (DHODH) Inhibitors, Organic Anion Transporter 3 (OAT3) Inhibitors for the treatment of arthritis;

HMGB1 inhibitory peptides of the invention might be administered in association with anticancer drugs, for example small molecule anticancer drugs, cytotoxic drugs, radioactive isotopes, therapeutic vaccines, autologous or heterologous cell therapy, for the treatment of cancer.

Such combinations may be administered separately or simultaneously and may be administered in the same composition or in different compositions and may be administered by the same route or different routes Processes

General molecular biology techniques

General texts which describe molecular biological techniques, which are applicable to the present invention, such as cloning, mutation, cell culture and the like, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. ("Berger"); Sambrook et al., Molecular Cloning— A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2002) ("Ausubel"), all of which are hereby incorporated by reference in their entireties). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., cloning, expression and isolation of peptides.

DNA encoding HMGB1 inhibitory peptides may also be obtained by chemical synthesis (see Engels et al (1989) Angew Chem, Intl Ed 28, 716-634).

Variants of HMGB1 inhibitory peptides may be prepared by means of site directed mutagenesis, PCR amplification or other technique where the primers have point mutations. (see Sambrook and Ausubel supra for mutagenesis techniques).

Oligonucleotides, e.g., for use in site-directed mutagenesis of HMGB1 inhibitory peptides, are typically synthesized chemically, for example, according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts. 22(20): 1859-1862, (1981 ) e.g., using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic Acids Res., 12:6159-6168 (1984), or as described by Tang and Tirrell J. Am. Chem. Soc. (2001 ) 123: 1 1089-1 1090 and Tang, et al. Angew. Chem. Int. Ed. (2001 ) 40:8, all of which are hereby incorporated by reference in their entireties.

Codon usage is suitably optimised for the host.

Codon alterations to optimize codon usage for the given host may be carried out by a number of methods. For example codon usage tables are published by University of Wisconsin, see e.g. "Ecohigh._Cod".

DNA encoding the HMGB1 inhibitory peptides is introduced into the host cell by means of a vector. Typical vectors contain, inter alia, transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular transgene (open reading frame). The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence; sequences permitting replication of the cassette in eukaryotes, prokaryotes or both (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes or both. (See, for example, Giliman & Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider, B., et al., Protein Expr. Purif. 6435: 10 (1995), all of which are hereby incorporated by reference). Additionally, a catalogue of Bacteria and Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds.) published by the ATCC.

Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY.

The vectors may be introduced into host cells by standard methods including electroporation (From et al., PNAS. USA 82, 5824 (1985)), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)), treatment with calcium chloride or the DEAE-dextran method. Berger, Sambrook, and Ausubel provide a variety of appropriate transformation methods.

Promoters that may be used in vectors include strong promoters heterologous to the host, such as viral promoters including CMV promoters. Exemplary promoters include pol II and pol III promoters. One commonly used and highly specific promoter is the bacteriophage T7 promoter.

Vectors will include an origin of replication. For example, the origin of replication of plasmid pBR322 (New England Biolabs) is suitable for most Gram negative bacteria.

Vectors will typically contain a selectable marker gene to select for transformed host cells. Typical selection genes include those coding for proteins that confer resistance to antibiotics or other toxins (e.g. ampicillin, tetracycline, kanamycin), complement auxotrophic deficiencies, or supply critical nutrients not available in complex media.

A ribosome binding site is normally necessary for translation initiation of mRNA and is

characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the peptide to be expressed.

The Shine-Dalgarno sequence is variable but typically has a high A-G content. A tag to aid protein purification such as a poly His tag (e.g. containing 5 or 6 His residues) may be encoded, typically at the 5' end of the coding sequence for the HMGB1 inhibitory peptides

The purification tag may be a component of the vector or it may be part of the HMGB1 inhibitory peptides DNA that is inserted into the vector.

Preferably, no purification tag is present.

General techniques of protein isolation and purification may be gleaned by reference to R Scopes "Protein Purification: Principles and Practice" 3rd Ed Sprinter-Verlag, NY (1993) and Walker "The Protein Protocols Handbook" Humana Press NJ (1996) and Ausubel, supra.

General cell culture techniques and media that may be suitable are disclosed inter alia in

Sambrook, supra, and Phelan, M. C. 2001. "Basic Techniques for Mammalian Cell Tissue Culture". Current Protocols in Cell Biology.

Sequence comparison tools

The terms "identical" or percentage "identity," in the context of two or more polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues that are the same (i.e., 90% identity, optionally 95%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the compliment of a test sequence. Suitably, the comparison is performed over a window corresponding to the entire length of the reference sequence (as opposed to the derivative sequence).

For sequence comparison, one sequence acts as the reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percentage sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window", as used herein, references to a segment in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981 ), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerised implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

Examples of algorithm that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al., supra). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1 , an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001.

Examples of the invention

General methods:

Cell Migration Assay:

Modified Boyden chambers are used with filters (8 μηι pores; Neuro Probe) coated with 50 μg/ml fibronectin (Roche). 50,000 cells (mouse 3T3 fibroblasts) in 200 μΙ are added to the upper chamber.

HMGB1 and Test peptides are added to the lower chamber, and then cells are left to migrate for 3 h at 37°C. Non-migrating cells are removed with a cotton swab, and migrated cells are fixed with ethanol and stained with Giemsa. All assays are done in triplicate, and migrated cells are counted in 10 random fields/filter.

Oxidation Resistance Assay:

Test peptides are exposed to H2O2 (10 mM) for 1 h. The reaction is stopped by adding β- mercaptoethanol (10mM). Thereafter their ability to block HMGB1-induced Cell Migration is measured using the Cell Migration Assay.

Murine malignant mesothelioma cell line (Mezzapelle R.et al., 2016):

Murine malignant mesothelioma (MM) AB1 cells were obtained from Cell Bank Australia and cultured in RPMI 1640 (Life Technologies) supplemented with 5% v/v fetal bovine serum (Life Technologies), 2 mM L-glutamine and 100 U/ml penicillin/streptomycin.

Each cell line was intraperitoneally (i.p.) injected in BALB/c mice to obtain tumours. The masses were explanted and mechanically disaggregated; the resulting cells were cultured as above and named ABI-B/c, AB12-B/C and AB22-B/C.

Luciferase-expressing cells were obtained by infecting the above cells with a 3rd generation lentiviral vector carrying the luciferase gene (pLenti PGK V5-LUC Neo (w623-2); Addgene).

Infected cells were selected with geneticin and maintained in culture as above. Cells generated from the original strains were named: AB1-LUC. Cells generated from the masses in BALB/c mice were named AB1-B/c-LUC. AB1-B/C-LUC Cell Proliferation Assay:

AB1-B/C-LUC cells were plated in MW6, 5x10⁴ cells/well in RPMI supplemented with FBS (5%), L- glutamine and antibiotics.

The proliferations were performed in triplicate. After 24h, the cells were treated with the different peptides using RPMI 1 % FBS; in the meantime the cells were counted at Oh time point.

Cells were counted every 24h until the end time point. The cells were resuspended in RPMI with FBS (5%).

The cells were stained with Trypan Blue to discriminate dead cells and were counted with an automated cell counter (Countess, Life Technologies).

Example 1

In order to test the effect of oxidative damage on the bioactivity of HMGB1 inhibitory peptides, HMGB1 Inhibitory Peptides 1 , 2 and 3 were treated as per the Oxidation Resistance Assay and their ability to block the HMGB1 -induced cell migration was measured using the Cell Migration Assay.

As shown in Figure 2, HMGB1 protein induced the migration of 3T3 fibroblasts.

When Peptide 1 , which retains the two Cysteines at positions 23 and 45, is added to the cells migrating towards HMGB1 it inhibits the HMGB1 -induced cell migration. Upon treatment of Peptide 1 with H2O2 the ability to inhibit HMGB1-induced cell migration is lost.

HMGB1 Inhibitory Peptide 2 where the two Cysteines at positions 23 and 45 have been substituted by Serine, surprisingly is not very efficient in inhibiting HMGB1-induced migration compared to the WT HMGB1 Peptide 1 ; however, its bioactivity is not affected by exposure to

HMGB1 Inhibitory Peptide 3, where the two cysteines at positions 23 and 45 have been substituted by Alanine, retains the ability to inhibit HMGB1 -induced migration of mouse 3T3 fibroblasts comparable to the WT peptide and its bioactivity is not affected by exposure to H2O2.

Figure 3 illustrates the relative inhibitory efficiency, calculated as the ratio between the number of cells per field treated with HMGB1 Inhibitory Peptide 1 and the two mutants, HMGB1 Inhibitory Peptide 2 and HMGB1 Inhibitory Peptide 3, multiplied by 100 (cell number exposed to HMGB1 Inhibitory Peptide 1 / cell number exposed to HMGB1 Inhibitory Peptide 2 X 100 and cell number exposed to HMGB1 Inhibitory Peptide 1 / cell number exposed to HMGB1 Inhibitory Peptide 3 X 100]). HMGB1 Inhibitory Peptide 1 , HMGB1 Inhibitory Peptide 2 and HMGB1 Inhibitory Peptide 3 were compared, before and after treatment with H2O2 10 mM per the Oxidation Resistance Assay.

In order to test whether Peptide 1 may act as weak agonist or as competitive inhibitor of HMGB1 the activity of Peptides 1 , 2 and 3 was tested at two concentrations: 1 nM and 0,2 nM. The peptides were used at a concentration of 1 nM and 0,2 nM.

Relative inhibitory efficiencies clearly show that HMGB1 Inhibitory Peptide 3 has inhibitory efficiency comparable to the WT peptide (HMGB1 Inhibitory Peptide 1 ) before treatment with H2O2, and retains such efficiency upon treatment with H2O2 (Figure 3) demonstrating that HMGB1 Inhibitory Peptide 3 is stable and active upon exposure to oxidative conditions. HMGB1 Inhibitory Peptide 2, although less potent than HMGB1 Inhibitory Peptides 1 and 3, was nevertheless also stable and active upon exposure to oxidative conditions.

Example 2

The effect of exogenous HMGB1 and Peptides 1 , 2 and 3 on proliferation of ABI-B/c-LUC cells was tested in the AB1-B/c-LUC Cell Proliferation Assay.

It has been shown that HMGB1 is involved in the onset and progression of malignant

mesothelioma (Jube et al., 2012), an aggressive tumour characterized by chronic inflammation.

The mesothelioma cell line ABI -B/c-LUC was treated with two different concentrations of recombinant HMGB1 (concentration 0,1 nM or 10 nM) or control (FBS 2%). As shown in Figure 6, the AB1 cells have increased proliferation in the presence of recombinant HMGB1.

In order to test the effect of HMGB1 inhibitory peptides (HMGB1 Peptides 1 , 2 and 3) on cell proliferation, these peptides were added at a concentration of 0,1 nM. The results are shown in Figures 7, 8 and 9. The three curves shown in Figures 7, 8 and 9 are: (i) control (FBS 2%) (ii) HMGB1 (concentration 0,1 nM) and (iii) HMGB1 (concentration 0,1 nM) plus peptide

(concentration 1 nM).

As shown in Figures 7 and 9, Peptide 1 and Peptide 3 inhibit ABI-B/c-LUC cell proliferation until day 5, then the inhibitory effect disappears. Peptide 2 appears to inhibit for a longer period -it inhibits cell proliferation until day 7 (Figure 8).

From the examples it may be concluded:

Peptide 1 is active as an inhibitor of migration of 3T3 fibroblast, but this activity is lost in an oxidative environment. Peptides 2 and 3 are active as inhibitors of migration of 3T3 fibroblast, and this activity is not lost in an oxidative environment. Peptide 3 is more potent than Peptide 2.

Peptides 1 , 2 and 3 are active as inhibitors of ABI-B/c-LUC cell growth. The inhibitory activity of Peptide 2 was longer lasting than that of Peptides 1 and 3.

Abbreviations

HMGB1 : High Mobility Group Box 1

DAMP: Damage Associated Molecular Pattern

ROS: Reactive Oxygen Species

RNS: reactive Oxygen Species

aa: amino acid

nt: nucleotide

References

1. Bianchi et al, Immunol Rev. 2007 Dec;220:35-46.

2. Agresti et al, Biochem Biophys Res Commun. 2003 Mar 7;302(2):421 -6.

3. Andersson et al, J Leukoc Biol. 2002 Dec;72(6): 1084-91.

4. Andersson et al, Mol Med. 2012 Mar 30; 18:250-9.

5. Bald et al, Nature. 2014 Mar 6;507(7490):109-13.

6. Degryse et al, JCB 2001 152;1 197

7. Dumitriu et al, J Immunol. 2005 Jun 15;174(12):7506-15.

8. Jube et al, Cancer Res. 2012 Jul 1 ;72(13):3290-301.

9. Maroso et al, Nat Med. 2010 Apr;16(4):413-9.

10. Mezzapelle R.et al., Sci. Rep. 2016 6:22850

11. Palumbo et al., 2004 JCB 164:441

12. Sessa et al, Gene. 2007 Jan 31 ;387(1-2): 133-40.

13. Sitia et al, J Leukoc Biol. 2007 Jan;81 (1 ):100-7.

14. Thomas JO et al, Biochem Soc Trans. 2001 Aug;29(Pt 4):395^101.

15. Venerau et al, J Exp Med. 2012 Aug 27;209(9): 1519-28

16. Yang et al, Proc Natl Acad Sci U S A. 2004 Jan 6;101 (1 ):296-301.

17. Yang et al, Mol Med. 2012 Mar 30; 18:250-9. Sequence listing

SEQ ID No. 1 Human HMGB1 aa Sequence (Accession Number P09429)

MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKF EDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGL SIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGWKAEK SKKKKEEEEDEEDEEDEEEEEDEEDEDEEEDDDD

SEQ ID No. 2 HMGB1 Peptide 1 WT aa Sequence

MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 3 HMGB1 Peptide 1 WT, nt sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCTGCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGTGCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 4 HMGB1 Peptide 2 (C23-S; C45-S) aa sequence

MGKGDPKKPRGKMSSYAFFVQTSREEHKKKHPDASVNFSEFSKKSSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 5 HMGB1 Peptide 2 (C23-S; C45-S) nt sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCTCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CC AAG AAGTCCTCAG AGAG GTG GAAG ACCATGTCTGCTAAAG AAAAG GG G AAATTTG AAG AT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC SEQ ID No. 6 HMGB1 Peptide 3 (C23-A; C45-A) aa sequence

MGKGDPKKPRGKMSSYAFFVQTAREEHKKKHPDASVNFSEFSKKASERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 7 HMGB1 Peptide 3 (C23-A; C45-A) nt sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGCCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 8 HMGB1 Peptide 4 (C23-L; C45-L); aa sequence

MGKGDPKKPRGKMSSYAFFVQTLREEHKKKHPDASVNFSEFSKKLSERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 9 HMGB1 Peptide 4 (C23-L; C45-L); nt sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCCTGCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CC AAG AAGCTGTCAGAG AG GTG GAAGACC ATGTCTGCTAAAG AAAAG G GG AAATTTG AAG AT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 10 HMGB1 Peptide 5 (C23-G; C45-G); aa sequence

MGKGDPKKPRGKMSSYAFFVQTGREEHKKKHPDASVNFSEFSKKGSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 1 1 HMGB1 Peptide 5 (C23-G; C45-G); nt sequence ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGGCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTC TCCAAGAAGGGCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGA TATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAG G G GAG ACCAAAAAG AAGTTC

SEQ ID No. 12 HMGB1 Peptide 6 (C23-V; C45-V); aa sequence

MGKGDPKKPRGKMSSYAFFVQTVREEHKKKHPDASVNFSEFSKKVSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 13 HMGB1 Peptide 6 (C23-V; C45-V); nt sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGTCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGTCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 14 HMGB1 Peptide 7 (C23-I; C45-I); aa sequence

MGKGDPKKPRGKMSSYAFFVQTIREEHKKKHPDASVNFSEFSKKISERWKTMSAKEKGKFEDMA KADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 15 HMGB1 Peptide 7 (C23-I; C45-I); nt sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCATCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGATCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 16 HMGB1 Peptide 8(C23-A; C45-L) aa Sequence MGKGDPKKPRGKMSSYAFFVQTAREEHKKKHPDASVNFSEFSKKLSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 17 HMGB1 Peptide 8 (C23-A; C45-L) nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CC AAG AAGCTGTCAGAG AG GTG GAAGACC ATGTCTGCTAAAG AAAAG G GG AAATTTG AAG AT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 18 HMGB1 Peptide 9(C23-L; C45-A); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTLREEHKKKHPDASVNFSEFSKKASERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 19 HMGB1 Peptide 9(C23-L; C45-A); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCCTGCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CC AAG AAGGCCTCAG AGAG GTG GAAG ACCATGTCTGCTAAAGAAAAG G GG AAATTTG AAG AT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 20 HMGB1 Peptide 10(C23-A;C45-G); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTAREEHKKKHPDASVNFSEFSKKGSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 21 HMGB1 Peptide 10(C23-A;C45-G); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA

AACCGCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT

CCAAGAAGGGCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 22 HMGB1 Peptide 1 1 (C23-G;C45-A); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTGREEHKKKHPDASVNFSEFSKKASERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 23 HMGB1 Peptide 1 1 (C23-G;C45-A); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGGCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTC TCCAAGAAGGCCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGA TATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAG G G GAG ACCAAAAAG AAGTTC

SEQ ID No. 24 HMGB1 Peptide 12 (C23-L;C45-G); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTLREEHKKKHPDASVNFSEFSKKGSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 25 HMGB1 Peptide 12 (C23-L;C45-G); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCCTGCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGGCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 26 HMGB1 Peptide 13 (C23-G;C45-L); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTGREEHKKKHPDASVNFSEFSKKLSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF SEQ ID No. 27 HMGB1 Peptide 13 (C23-G;C45-L); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGGCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTC TCCAAGAAGCTGTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGA TATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAG G G GAG ACCAAAAAG AAGTTC

SEQ ID No. 28 HMGB1 Peptide 14 (C23-V; C45-S); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTVREEHKKKHPDASVNFSEFSKKSSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 29 HMGB1 Peptide 14 (C23-V; C45-S); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGTCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGTCCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 30 HMGB1 Peptide 15 (C23-V; C45-A); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTVREEHKKKHPDASVNFSEFSKKASERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 31 HMGB1 Peptide 15 (C23-V; C45-A); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGTCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGCCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 32 HMGB1 Peptide 16 (C23-V; C45-L); aa Sequence MGKGDPKKPRGKMSSYAFFVQTVREEHKKKHPDASVNFSEFSKKLSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 33 HMGB1 Peptide 16 (C23-V; C45-L); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGTCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CC AAG AAGCTGTCAGAGAG GTG GAAGACCATGTCTGCTAAAG AAAAG G G GAAATTTG AAG AT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 34 HMGB1 Peptide 17 (C23-V; C45-G); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTVREEHKKKHPDASVNFSEFSKKGSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 35 HMGB1 Peptide 17 (C23-V; C45-G); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGTCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CC AAG AAGGGCTCAG AGAG GTG GAAGACCATGTCTGCTAAAG AAAAG G GG AAATTTG AAG AT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 36 HMGB1 Peptide 18 (C23-V; C45-I); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTVREEHKKKHPDASVNFSEFSKKISERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 37 HMGB1 Peptide 18 (C23-V; C45-I); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA

AACCGTCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT

CCAAGAAGATCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 38 HMGB1 Peptide 19 (C23-S; C45-V); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTSREEHKKKHPDASVNFSEFSKKVSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 39 HMGB1 Peptide 19 (C23-S; C45-V); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCTCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGTCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 40 HMGB1 Peptide 20 (C23-A; C45-V); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTAREEHKKKHPDASVNFSEFSKKVSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 41 HMGB1 Peptide 20 (C23-A; C45-V); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGTCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 42 HMGB1 Peptide 21 (C23-L; C45-V); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTLREEHKKKHPDASVNFSEFSKKVSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF SEQ ID No. 43 HMGB1 Peptide 21 (C23-L; C45-V); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGTGCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGTCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 44 HMGB1 Peptide 22 (C23-G; C45-V); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTGREEHKKKHPDASVNFSEFSKKVSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 45 HMGB1 Peptide 22 (C23-G; C45-V); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGGCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTC TCCAAGAAGGTCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGA TATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAG G G GAG ACCAAAAAG AAGTTC

SEQ ID No. 46 HMGB1 Peptide 23 (C23-I; C45-S); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTSREEHKKKHPDASVNFSEFSKKSSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 47 HMGB1 Peptide 23 (C23-I; C45-S); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCATCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGTCCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 48 HMGB1 Peptide 24 (C23-I; C45-A); aa Sequence MGKGDPKKPRGKMSSYAFFVQTIREEHKKKHPDASVNFSEFSKKASERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 49 HMGB1 Peptide 24 (C23-I; C45-A); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCATCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGCCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 50 HMGB1 Peptide 24 (C23-I; C45-L); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTIREEHKKKHPDASVNFSEFSKKLSERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 51 HMGB1 Peptide 24 (C23-I; C45-L); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCATCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CC AAG AAGCTGTCAGAG AG GTG GAAGACC ATGTCTGCTAAAG AAAAG G GG AAATTTG AAG AT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 52 HMGB1 Peptide 25 (C23-I; C45-G); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTIREEHKKKHPDASVNFSEFSKKGSERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 53 HMGB1 Peptide 25 (C23-I; C45-G); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA

AACCATCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT

CCAAGAAGGGCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 54 HMGB1 Peptide 26 (C23-I; C45-V); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTIREEHKKKHPDASVNFSEFSKKVSERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 55 HMGB1 Peptide 26 (C23-I; C45-V); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCATCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGGTCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 56 HMGB1 Peptide 27 (C23-S; C45-I); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTSREEHKKKHPDASVNFSEFSKKISERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 57 HMGB1 Peptide 27 (C23-S; C45-I); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCTCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGATCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 58 HMGB1 Peptide 28 (C23-A; C45-I); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTAREEHKKKHPDASVNFSEFSKKISERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF SEQ ID No. 59 HMGB1 Peptide 28 (C23-A; C45-I); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGATCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 60 HMGB1 Peptide 29 (C23-L; C45-I); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTLREEHKKKHPDASVNFSEFSKKISERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 61 HMGB1 Peptide 29 (C23-L; C45-I); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGCCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGATCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 62 HMGB1 Peptide 30 (C23-G; C45-I); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTGREEHKKKHPDASVNFSEFSKKISERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 63 HMGB1 Peptide 30 (C23-G; C45-I); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGGCCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTC TCCAAGAAGATCTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGA TATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAG G G GAG ACCAAAAAG AAGTTC

SEQ ID No. 64 HMGB1 Peptide 31 (C23-D;C45-D); aa Sequence MGKGDPKKPRGKMSSYAFFVQTDREEHKKKHPDASVNFSEFSKKDSERWKTMSAKEKGKFED MAKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 65 HMGB1 Peptide 31 (C23-D; C45-D); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCGACCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CC AAG AAGGACTC AG AG AG GTG G AAG ACCATGTCTGCTAAAG AAAAG G G G AAATTT G AAG AT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

SEQ ID No. 66 HMGB1 Peptide 31 (C23-T;C45-T); aa Sequence

MGKGDPKKPRGKMSSYAFFVQTTREEHKKKHPDASVNFSEFSKKTSERWKTMSAKEKGKFEDM AKADKARYEREMKTYIPPKGETKKKF

SEQ ID No. 67 HMGB1 Peptide 31 (C23-T; C45-T); nt Sequence

ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCCTCATATGCATTCTTTGTGCA AACCACTCGGGAGGAGCACAAGAAGAAGCACCCGGATGCTTCTGTCAACTTCTCAGAGTTCT CCAAGAAGACTTCAGAGAGGTGGAAGACCATGTCTGCTAAAGAAAAGGGGAAATTTGAAGAT ATGGCAAAGGCTGACAAGGCTCGTTATGAAAGAGAAATGAAAACCTACATCCCCCCCAAAGG G GAG ACCAAAAAG AAGTTC

Claims

Claims

An isolated HMGB1 inhibitory peptide comprising a Box-A domain and absent a Box-B domain characterized in that it is resistant to oxidation

An isolated HMGB1 inhibitory peptide according to Claim 1 , comprising residues 23 to 45 of SEQ ID NO 1 or a variant or derivative, thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 1 are substituted by another aminoacid thereby to increase the oxidation resistance thereof.

An isolated HMGB1 inhibitory peptide according to Claim 2, comprising SEQ ID NO 2 or a variant or derivative thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 2 are independently substituted by aminoacids selected from the group consisting of Serine, Alanine, Leucine, Valine, Glycine, Isoleucine and

Threonine.

An isolated HMGB1 inhibitory peptide according to Claim 2, comprising SEQ ID NO 2 or a variant or derivative thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 2 are independently substituted by aminoacids selected from the group consisting of Alanine, Leucine, Valine, Glycine and Isoleucine.

An isolated HMGB1 inhibitory peptide according to Claim 2, comprising SEQ ID NO 2 or a variant or derivative thereof, wherein one or both cysteines at positions 23 and position 45 of SEQ ID NO 2 are independently substituted by Alanine.

An isolated HMGB1 inhibitory peptide according to Claim 1 , comprising an aminoacid sequence selected from SEQ ID NO 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG- 4L or/and SP100 .

An isolated HMGB1 inhibitory peptide according to Claim 6, comprising an aminoacid sequence selected from SEQ ID NO 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG- 4L or/and SP100.

An isolated HMGB1 inhibitory peptide according to Claim 1 , comprising the aminoacid sequence of SEQ ID NO 4, or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG-4L or/and SP100 .

9. An isolated HMGB1 inhibitory peptide according to Claim 1 , comprising the aminoacid sequence of SEQ ID NO 6, or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG-4L or/and SP100 .

10. An isolated HMGB1 inhibitory peptide according to Claim 1 , comprising an aminoacid sequence selected from SEQ ID NO 66 or a derivative thereof, or a corresponding sequence containing one or more amino acid differences, wherein each amino acid difference is a difference found in the sequence of a homologue, and wherein the homologue is selected from HMGB1V, HMGB2, HMGB3, HMG3M, HMGB4, HMG-4L or/and SP100 .

1 1. An isolated HMGB1 inhibitory peptide according to Claim 2 comprising SEQ ID No 2 or a variant having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity to said sequence, wherein residues at positions 23 and 45 are not cysteines, or a derivative thereof.

12. An isolated HMGB1 inhibitory peptide according to Claim 1 to 10 which is a derivative formed by acylation at the N-terminus and/or amidation at the C-terminus.

13. An isolated HMGB1 inhibitory peptide according to Claim 1 to 10 which is a derivative formed by conjugation to a polymer.

14. An isolated HMGB1 inhibitory peptide according to Claim 13 which is pegylated

15. An isolated polynucleotide encoding the HMGB1 inhibitory peptide of claims 1 to 1 1.

16. An expression vector comprising a polynucleotide according to Claim 15.

17. A host cell comprising the vector of claim 16.

18. An isolated HMGB1 peptide according to any one of Claims 1 to 14 for use as

medicament

19. A pharmaceutical composition comprising an isolated HMGB1 inhibitory peptide

according to Claims 1 to 14 together with one or more pharmaceutically acceptable diluents or carriers.

20. Use of an HMGB1 inhibitory peptide according to any one of claims 1 to 14 for the

manufacture of a medicament for the treatment of an HMGB1 associated pathology

21. An HMGB1 inhibitory peptide of any one of claims 1 to 14 for use in the treatment of an HMGB1 associated pathology.

22. Use of the HMGB1 inhibitory peptide according to claim 20 or the HMGB1 inhibitory peptide for use according to claim 21 wherein the HMGB1 associated pathology is selected from epilepsy, especially acute and chronic seizures and in the treatment of drug resistant epilepsies, Alzheimer's disease, systemic Lupus erythematosus, cardiac inflammatory disease, restenosis, liver fibrosis, sepsis, ischemia-reperfusion injury, arthritis, cancer, particularly malignant mesothelioma, e.g. pleural, peritoneal, pericardial mesothelioma, ovarian cancer, melanoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, metastatic cancer.

23. A method of treating an HMGB1 associated pathology comprising administering a

therapeutically effective amount of an HMGB1 peptide of any one of claims 1 to 14 to a subject in need thereof.

24. The method of claim 23 wherein the HMGB1 associated pathology is selected from

epilepsy, especially acute and chronic seizures and in the treatment of drug resistant epilepsies, Alzheimer's disease, systemic Lupus erythematosus, cardiac inflammatory disease, restenosis, liver fibrosis, sepsis, ischemia-reperfusion injury, arthritis, cancer, particularly malignant mesothelioma, e.g. pleural, peritoneal, pericardial mesothelioma, ovarian cancer, melanoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, metastatic cancer.