ES2362244T3 - HMGB1 BOX A POLYMER CONJUGATES AND HMGB1 BOX A VARIANTS. - Google Patents

Abstract

Polymer conjugate of the high affinity binding domain Box A of HMGB1 wild-type human and / or non-human (Box A of HMGB1) or a biologically active fragment of Box A of HMGB1.

Description

The present invention relates to novel polymer conjugates of the HMGB1 Box A high affinity binding domain (HMGB1 Box A) or a biologically active fragment of the HMGB1 Box A. In addition, the invention relates to novel polymer conjugates of polypeptide variants of the high affinity binding domain Box A of HMGB1 (Box A of HMGB1) or of the biologically active fragments of Box A of HMGB1. Furthermore, the present invention relates to the use of said polymer conjugates to diagnose, prevent, alleviate and / or treat pathologies associated with extracellular HMGB1 and / or associated with an increased expression of RAGE.

The HMGB1 protein belongs to the family of high mobility group proteins (HMG). HMG proteins, so named because of their high electrophoretic mobility in polyacrylamide gels, are the most ubiquitous proteins other than histones associated with isolated chromatin in eukaryotic cells. These proteins play a generalized "architectural" role in flexing, looping, folding and enveloping DNA, since they distort, flex or modify DNA structures and complexes with transcription factors.

or histones (Andersson et al., 2000; Agresti et al., 2003; Degryse et al., 2003). The high mobility group 1 protein (HMGB1) is usually a nuclear factor, in particular a transcriptional regulatory molecule that causes DNA flexion and facilitates the binding of several transcriptional complexes.

Structurally, the HMGB1 protein is an approximately 25 kDa protein with a highly conserved sequence among mammals, whereby 2 out of every 214 amino acids have conservative substitutions in all mammalian species. HMGB1 is ubiquitously present in all vertebrate nuclei, and in particular it can be found in fibroblasts, neurons, hepatocytes, gliocytes and in cells derived from hematopoietic stem cells, including monocytes / macrophages, neutrophils and platelets. The HMGB1 molecule has a tripartite structure composed of three distinct domains: two DNA binding domains, called HMG Box A and Box B, and an acidic carboxyl term, which causes it to be bipolarly charged.

The two boxes of HMGB are involved in the protein's function as nonspecific architectural DNA binding elements of the sequence, conferring the ability to bind to DNA in recognized distorted DNA structures and stabilizing the assembly, remodeling and slippage of nucleosomes. . Both boxes A and B of HMG are formed by 84 highly conserved amino acid residues, are strongly positively charged, and are arranged in three hé propellers that have a similar L-shaped folding. The long branch of the “L” contains the strand extended N-terminal and helix III (Andersson et al., 2002; Agresti et al., 2003; Thomas, JO 2001), while the short branch comprises helices I and II. The analysis of the structural function reveals that the proinflammatory cytosine domain of HMGB1 is Box B, and in particular the sequence of its first 20 amino acids. Box A is an extremely weak agonist of the release of proinflammatory cytosine activated by HMGB1, and competitively inhibits the proinflammatory activities of Box B and the entire protein. Therefore, from the pharmacological point of view, Box A acts as an antagonist of the pathologies induced and / or sustained by Box B and HMGB1.

The third domain, the term carboxyl or acid tail, is very negatively charged, since it contains 30 repetitive residues of aspartic and glutamic acid, and is linked to the boxes by a basic region of about 20 residues. Mouse and rat HMGB1 differ in human form only in two substitutions, which are located in this continuous C-terminal stretch.

In addition to its nuclear location and its role as a regulator of transcription factors, HMGB1 has also been found in the extracellular environment, actively released by cells activated from the immune system (monocytes and macrophages), or passively released by damaged or necrotic cells (Andersson et al., 2002; Scaffidi et al., 2002; Bonaldi et al., 2002; Taniguchi et al., 2003; Friedman et al., 2003; Palumbo et al., 2004).

Extracellularly released HMGB1 acts as a potent cytokine and as an extremely potent macrophage stimulating factor. HMGB1 acts directly by binding to the cell membrane, inducing signaling and chemotaxis, having a chemokine-like function (Yang et al., 2001) and also acting indirectly increasing the expression and secretion of pro-inflammatory cytokines. This makes the extracellular HMGB1 protein a potent chemotactic and immunoregulatory protein that promotes an effective inflammatory immune response. Additionally, other proteins belonging to the HMG family of proteins, and which are capable of flexing the DNA, are released together with HMGB1 in the extracellular medium. These proteins are, among others, HMGB2, HMGB3, HMG-1L10, MHG-4L and SP100-HMG. They share highly homologous amino acid sequences with HMGB1. Like HMGB1, they trigger / sustain inflammatory pathologies that interact with the same receptors, leading to the same downstream interaction pathways.

In healthy cells, HMGB1 migrates to the cytoplasm by both passive and active transport. However, all cultured cells and resting monocytes contain the highest proportion of HMGB1 in the nucleus, indicating that, at baseline, importation is much more efficient than export. The cells could transport HMGB1 out of the nucleus by acetylating lysine residues, which are abundant in HMGB1, thereby neutralizing their basic charge and making them unable to function as nuclear localization signals. The nuclear hyperacetylation of nuclear HMGB1 determines the relocation of this protein from the nucleus to the cytoplasm (in fibroblasts, for example), or its accumulation in secretory endolysosomes (in monocytes and activated macrophages, for example), and subsequent redirection towards release by a non-classical secretory route mediated by vesicles. The secretion of HMGB1 by already activated monocytes is then triggered by bioactive lysophosphatidylcholine (LPC), which is later generated at the site of inflammation from phosphatidylcholine by the action of sPLA2 secretory phospholipase produced by monocytes several hours after activation. . Therefore, it seems that the secretion of HMGB1 is induced by two signals (Bonaldi et al., 2003) and takes place in three stages: 1) initially, an inflammatory signal promotes the acetylation of HMGB1 and its relocation from the nucleus to the cytoplasm (stage 1) and storage in secretory cytoplasmic vesicles (stage 2); Next, a secretion signal (extracellular ATP or lysophosphatidylcholine) promotes exocytosis (third stage) (Andersson et al., 2002; Scaffidi et al., 2002; Gardella et al., 2002; Bonaldi et al., 2003; Friedman et al., 2003).

The released HMGB1 has been identified as one of the ligands that bind to the RAGE receptor. This receptor is expressed in most cell types, and at an elevated level, mainly in endothelial cells, in vascular smooth muscle cells, in monocytes and macrophages, and in mononuclear phagocytes. The recognition involves the C terminal of HMGB1. The interaction of HMGB1 and RAGE triggers a sustained period of cell activation mediated by the increase in RAGE and receptor-dependent signaling. In particular, the interaction of HMGB1 and RAGE activates several intracellular signal transduction pathways, which include mitogen-activated protein kinases (MAPK), Cdc-42, p21ras, Rac and the nuclear translocation factor B (NF-B) , the transcription factor classically linked to inflammatory processes (Schmidt et al., 2001).

According to several experimental tests, the released HMGB1 can also interact with receptors belonging to one or more subclasses of the family of Toll-like receptors. In addition, HMGB1 can also interact with the functional N-terminal lectin-like domain (D1) of thrombomodulin. Due to the ability of the functional D1 domain of thrombomodulin to intercept and bind to circulating HMGB1, interaction with RAGE receptors and Toll-like receptors is avoided.

When released in vivo, HMGB1 is an extremely potent cytokine and a potent macrophage stimulating factor. In fact, like other cytokine mediators of endotoxemia, HMGB1 activates in vitro a cascade of multiple proinflammatory cytokines (TNF, IL-1, IL-1, IL-1Ra, IL-6, IL-8, MIP- 1 and MIP-1) from human macrophages. Therefore, HMGB1 behaves as a late mediator during acute inflammation, and participates significantly in the pathogenesis of systemic inflammation after the response of early mediators has been resolved.

In addition, the increase in RAGE observed in proinflammatory environments and the demonstrated increased expression of this receptor in a variety of acute and chronic diseases provide support for RAGE as an attractive target for future medical interventions related to inflammation.

The observed pro-inflammatory effects of HMGB1 in vitro and the correlation between circulating HMGB1 levels and the development of the pathogenic sequence of systemic inflammation in vivo indicate that the selection as a therapeutic target of this cytokine-like molecule should be of clinical value. important, suggesting new therapeutic approaches for a "late" administration of antagonists / inhibitors (selective) of the extracellular activities of HMGB1.

Therefore, several attempts were made to block this extracellular chemo-cytokine HMGB1 protein. Several important approaches were directed to the administration of antibodies against HMGB1, of antibodies against fragments of HMGB1 (for example, Boxes of HMGB1), of antibodies to RAGE, of soluble RAGE (sRAGE), of ethyl pyruvate (Czura et al., 2003; Lotze et al., 2003) and the N-terminal lectin-like domain (D1) of thrombomodulin.

HMGB1 Box A, one of the two DNA binding domains in HMGB1, has been identified as a specific HMGB1 antagonist: Highly purified recombinant Box A protected mice against lethal experimental septicemia even when initial treatment was delayed 24 hours after pathology induction, further suggesting that HMGB1 antagonists can be successfully administered in a clinically relevant window wider than that used for other known cytokines (Yang et al., 2004).

Analysis of the structural function of truncated HMGB1 mutants revealed that the HMGB1 Box A domain competitively shifts the saturable binding of HMGB1 to macrophages, specifically antagonizing the activities of HMGB1. As seen with the protective activity of anti-HMGB1 antibodies, the administration of Box A restores mice from septicemia even when treatment has started as late as 24 hours after surgical induction of septicemia (Yang et al., 2004). HMGB1 antagonists or inhibitors selected from the group of antibodies or antibody fragments that bind to an HMGB1 protein, antisense sequences of HMGB1 genes and HMGB1 receptor antagonists are known from US 6,468,533, WO 02/74337 and US 2003/0144201.

A promising attempt to inhibit and / or antagonize the extracellular chemo-cytokine HMGB1 protein is therefore based on the experimental evidence that the two high affinity binding domains for DNA, i.e., Box A of HMGB1 and Box B of HMGB1, which are present in the HMGB1 molecule, have two opposite roles in the protein released in the extracellular space. The main activity of HMGB1 Box B is to mediate the proinflammatory activities attributed to the HMGB1 protein. On the other hand, Box A of HMGB1 acts as an antagonist, competing with the proinflammatory activity of the domain of Box B.

Patent application WO 2006/024547 describes polypeptide variants of HMGB1 Box A, or of biologically active fragments of HMGB1 Box A, which are obtained through systematic mutations of individual amino acids of HMGB1 Box A protein wild type Therefore, WO 2006/024547 provides new agents such as inhibitors and / or selective antagonists of extracellular HMGB1 and their use to prevent, alleviate and / or treat the broad spectrum of pathologies associated and induced by extracellular HMGB1 chemokine and / or by the cascade of multiple inflammatory cytokines caused by the extracellular release of the HMGB1 chemokine proteins.

The efficacy of the administration of synthetic protein drugs can be impeded in vivo by factors such as solubility at physiological pH, rapid elimination by glomerular filtration, clearance and cellular metabolism, as well as easy absorption. The effectiveness of oral administration is impeded, for example, since proteins are digested if taken orally. On the other hand, the effectiveness of systemic administration is impeded since proteins below 65-70 kDa are rapidly cleared from the body. In many cases, such disadvantageous effects lead to reduced patient compliance and reduced drug efficacy, preventing effective therapeutic use of such protein agents.

A successful strategy to improve the effectiveness and duration of the effects of protein agents and to reduce potential toxicological effects is the covalent binding of a biologically active protein agent to various polymers. One of the polymers that is most often used in the art to improve the pharmacological and toxicological properties of an active agent is polyethylene glycol, PEG briefly. Polyethylene glycol (PEG) polymers are amphiphilic, non-toxic and immunologically inert, and can be conjugated to drugs to manipulate many of the pharmacokinetic and toxicological properties.

Many covalent modifications of useful therapeutic proteins with polyethylene glycol (PEG) are disclosed in the art. The covalent binding of PEG to a protein ("pegylation") is useful in order to extend the circulation half-life of proteins, since it increases the effective size of proteins and reduces their rate of clearance of the organism. In addition, the modification of a protein with PEG increases the protein's solubility, stability, and decreases the immunogenicity of the protein.

The problem underlying the present invention was therefore the provision of novel therapeutically useful protein agents, acting as antagonists and / or selective inhibitors of extracellular HMGB1. Therefore, the scope of the present invention was to exploit the peculiar characteristics of some polymers, in particular PEG, in order to develop new ways of administering the high affinity binding domain Box A of HMGB-1 (Box A of HMGB1 ), which show the same if not an even higher pharmacological activity and also an improved pharmacokinetic and toxicological behavior compared to the unconjugated HMGB1 Box A polypeptide, and which allow to achieve the best availability of the HMGB1 Box A or a biologically active fragment thereof in various possible routes of administration.

Therefore, the present invention relates to a new polymer conjugate of the high affinity binding domain Box A of HMGB1 (Box A of HMGB1) of human and / or non-human wild type or of a biologically active fragment of Box A of HMGB1. SEQ ID NO: 1 shows the amino acid sequence of Box A of human HMGB1. A preferred non-human HMGB1 Box A is Anapheles gambia HMGB1 Box A, the sequence of which is shown in SEQ ID NO: 301.

A further aspect of the present invention relates to a polymer conjugate of a polypeptide variant of the high affinity binding domain Box A of human and / or non-human HMGB1 or of a biologically active fragment of Box A of HMGB1, whereby The amino acid sequence of said polypeptide variant differs from the amino acid sequence of wild-type HMGB1 Box A by the mutation of one or more individual amino acids.

In the context of the present invention, "HMGB1" includes the non-acetylated form or / and the acetylated form of HMGB1. Similarly, "HMGB1 homologous proteins" includes the non-acetylated form or / and the acetylated form of HMGB1 homologous proteins. Preferred HMGB1 homologous proteins are HMGB2, HMGB3, HMGB-1L10, HMG-4L or / and SP100-HMG.

The new polymer conjugates of the present invention show greater water solubility, improved pharmaceutical manageability, improved pharmacokinetics and bioavailability, and / or reduced toxicity and / or immunogenicity compared to unconjugated HMGB1 polypeptide or polypeptide variant. In addition, it was surprisingly found that the conjugation of the HMGB1 Box A polypeptide or polypeptide variant and / or fragment does not alter the biological activity of the protein.

The polymeric moiety according to the present invention must be biocompatible, can be of natural or semi-synthetic origin.

or synthetic, and can have a linear or branched structure. Exemplary polymers include without limitation polyalkylene glycols, poly (alkylene oxides), poly (acrylic acid), polyacrylates, polyacrylamide or N-alkyl derivatives thereof, poly (methacrylic acid), polymethacrylates, poly (ethylacrylic acid), polyethylacrylates, polyvinylpyrrolidone , poly (vinyl alcohol), poly (glycolic acid), poly (lactic acid), poly (lactic-co-glycolic acid), dextran, chitosan, polyamino acids.

In a very preferred embodiment of the present invention, the polymer is polyethylene glycol (PEG) or polyethylene glycol, in which the terminal OH group may be optionally modified, for example, with C1-C5 alkyl groups or C1-C5 acyl groups, preferably with C1, C2 or C3 alkyl groups or C1, C2 or C3 acyl groups. Preferably, the modified polyethylene glycol is methoxy polyethylene glycol (mPEG).

The polymer used according to the present invention has a molecular weight ranging from 100 to 100,000 Da, preferably 5,000 to 50,000 Da. In a very preferred embodiment of the invention, the polymer is PEG, which preferably has an OH and / or terminal methoxy group, with a molecular weight ranging from 10,000 to 40,000 Da, and preferably from 20,000 to 40,000 Da. In the most preferred embodiment, in the present invention a PEG is used which preferably has an OH and / or terminal methoxy group with an average molecular weight of 20,000 Da

or of 40,000 Da.

The polymeric moiety of the polymer conjugate of the invention is conjugated to the HMGB1 polypeptide or polypeptide variant by a covalent chemical bond, in order to provide a stable conjugate.

Preferred conjugation sites in the rest of HMGB1 Box A are selected from a lysine, cysteine, histidine, arginine, tyrosine, serine, threonine, aspartate and glutamate moiety, or from the N-terminal amino group of the protein moiety. The polymer conjugate of the present invention can be a mono-, di- and multi-phenylated conjugate. Preferably, the polymer conjugates of the invention are monopegylated.

The polymer moiety is usually covalently linked to the rest of the HMGB1 Box A through a linker group. In particular, in the context of the present invention, the term "linker group" means a group that is obtained by the chemical reaction between the polypeptide moiety and the polymer moiety. The linker group may be any moiety known to those skilled in the art of polymer conjugation, obtained by reacting the active group in the amino acid residue of the rest of the HMGB1 Box A and the polymer or polymer activated by a reactive group . Exemplary linker groups include, without limitation, alkylene, amine, amide, carbamate, carboxylate, carbonyl, ester, ether, thioether and disulfide groups. Preferably, the linker group is an amine bond, which is obtained by reacting the N-terminal amino acid residue with the activated polymeric moiety with an aldehyde reactive group and subsequent reduction.

In addition, the linker group may optionally contain one or more spacer groups. In the context of the present invention, a spacer group is defined as a bifunctional group which has in both terms a reactive functional terminal group. With the one reactive terminal group, the spacer reacts with the polymer moiety or with the reactive group in the polymer moiety. With the other functional group in the other term, the spacer group joins the functional group in the amino acid residue of the rest of the HMGB1 Box A. Suitable spacer groups are known to those skilled in the art. Examples of spacer groups include, but are not limited to, small hetero-, bifunctional or polymer molecules. For example, the spacer group may be represented by bifunctional C6-C12 alkyl groups or heterobifunctional alkyl groups containing 1-3 heteroatoms selected from N, S and O, or an intermediate short bifunctional PEG chain.

Covalent bonding of the polymer to the rest of the HMGB1 Box A to obtain the polymer conjugate of the invention can be achieved by known chemical synthesis techniques. For example, in an exemplary embodiment of the present invention, polymer conjugation can be achieved by reacting a Nhydroxysuccinimide polymer (for example NHS-PEG) with the free amine groups in the amino acid residues, preferably in the lysine residues, or in the N-terminal amino acid of the HMGB1 Box A polypeptide. Alternatively, polymer conjugation is achieved by reacting a PEG-aldehyde to the N-terminus of the polypeptide by reductive amination. In addition, polymer conjugates can also be obtained by reacting a PEG-maleimide to a Cys residue of the HMGB1 Box A polypeptide or polypeptide variant.

In the context of the present invention, the expression "HMGB1 Box A residue" indicates, within the polymer conjugate compound, the polypeptide moiety. Therefore, this expression refers to the wild-type HMGB1 Box A and its biologically active fragments, as well as polypeptide variants of the HMGB1 Box A and its biologically active fragments.

In the context of the present invention, when referring to the expression "HMGB1 Box A or amino acid sequence of HMGB1 Box A", it refers to both human and non-human HMGB1 Box A. In a preferred embodiment of the present invention, the rest of the HMGB1 Box A is derived from the wild type of the human HMGB1 Box A protein, and from the wild type of the HMGB1 Box A protein from Anopheles gambia.

"Biologically active fragments of the HMGB1 Box A", as used herein, means that it encompasses parts of the known wild type HMGB1 Box A protein, for which at least one of the biological activities of the corresponding mature protein when known assays are being used. Preferably, a fragment of the mature protein is considered as biologically active if the antagonistic activity with respect to the proinflammatory activity of Box B of HMGB1 and the HMGB1 protein as a whole can be determined. The biologically active fragments of the native HMGB1 Box A are fragments of at least 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75 or 80 amino acids. Preferred biologically active fragments of the native HMGB1 Box A used in the context of the present invention comprise fragments of at least 77 or at least 54 amino acids, respectively.

The term "mutation", as used in the context of the present invention, can be understood as substitution, deletion and / or addition of an individual amino acid in the target sequence. Preferably, mutation of the target sequence in the present invention is a substitution. Substitution can occur with different genetically encoded amino acids or by non-genetically encoded amino acids. Examples of non-genetically encoded amino acids are homocysteine, hydroxyproline, ornithine, hydroxylysine, citrulline, carnitine, etc.

In a more preferred embodiment of the present invention, the polypeptide variants of the HMGB1 Box A or a biologically active fragment thereof are obtained using a directed evolution process, the technology of which is widely described in WO 2004/022593 and in several additional patent applications (PCT / FR00 / 03503 PCT / FR01 / 01366, US 10 / 022,249, US 10 / 022,390, US 10 / 375,192, US 60 / 409,898, US 60 / 457,135, US 60 / 410,258 and US 60 /410.263), all in the name of Nautilus Biotech SA (Paris France).

The polypeptide variants of the present invention obtained using directed evolution technology are mutant proteins that differ from the amino acid sequence of the wild type HMGB1 Box A by the mutation of one or more individual amino acids. In a very preferred embodiment of the present invention, only one amino acid substitution occurs in the native protein sequence. However, the fact that the native protein can be further optimized by substituting a plurality, for example two or more, of amino acid substitutions is also encompassed by the object of the present invention. Therefore, modified polypeptide variants may differ from the wild-type protein sequence by amino acid substitutions in 1-10, preferably 2, 3, 4, 5 and 6 different amino acid target positions.

In particular, the highly preferred polypeptide variants of the HMGB1 Box A or a biologically active fragment thereof used as a remainder of the HMGB1 Box A of the polymer conjugates of the invention are those described in WO 2006/024547.

Accordingly, in a preferred embodiment of the invention, the remainder of the HMGB1 Box A of the polymer conjugate is derived from the Human HMGB1 Box A. In particular, a group of polypeptide variants derives from individual mutations introduced into the full-length amino acid sequence (84 amino acids) of Box A of human HMGB1 (SEQ ID NO: 1) (Fig. 1a). These preferred polypeptide variants are defined in the sequences SEQ ID NOS: 2-116 (Fig. 1b).

Other preferred polypeptide variants are obtained from biologically active fragments of the Human HMGB1 Box A of 77 amino acids (SEQ ID NO: 117) (Fig. 2a) and 54 amino acids (SEQ ID NO: 223) (Fig. 3a), respectively . Box A polypeptide variants of the 77 amino acid human HMGB1 fragment are defined in the sequences SEQ ID NOS: 118 to 222 (Fig. 2b). Box A polypeptide variants of the 54 amino acid human HMGB1 fragment are defined in the sequences SEQ ID NOS: 224 to 300 (Fig. 3b).

In a further preferred embodiment of the invention, the remainder of the HMGB1 Box A of the polymer conjugate is derived from the HMGB1 Box A of Anopheles gambia. In particular, a group of polypeptide variants derives from individual mutations introduced into the full-length amino acid sequence (84 amino acids) of Anopheles gambia HMGB1 Box A (SEQ ID NO: 301) (Fig. 4a). These polypeptide variants are identified in the sequences SEQ ID NOS: 302-418 (Fig. 4b). Other preferred polypeptide variants are generated from the biologically active fragments of the Anopheles gambia HMGB1 Box A of 77 amino acids (SEQ ID NO: 419) (Fig. 5a) and 54 amino acids (SEQ ID NO: 529) (Fig. 6a ), respectively. The polypeptide variants of Box A of the 77 amino acid HMGB1 fragment are defined in the sequences SEQ ID NOS: 420 to 528 (Fig. 5b). The polypeptide variants of Box A of the Anopheles gambia HMGB1 fragment (XP_311154) of 54 amino acids are defined in the sequences SEQ ID NOS: 530 to 610 (Fig. 6b).

In order to identify the most preferred polypeptide variants of the HMGB1 Box A used as a remainder of the HMGB1 Box A of the polymer conjugates of the invention, studies have been conducted to determine the polypeptide variants that show both a similar or even improved activity as an increased protease resistance compared to the wild type HMGB1 Box A protein. To this end, the activity of polypeptide variants of Box A of Box A of human HMGB1 of SEQ ID NO: 1 was determined by inhibiting the migration of NIH / 3T3 cells induced by HMGB1 in chemotaxis assays, as compared to antagonistic activity of the wild type of the human HMGB1 Box A itself (Example 1 and Figures 7.1 to 7.9). In addition, for those polypeptide variants, which show a similar or even greater antagonistic activity than the native HMGB1 Box A protein of SEQ ID NO: 1, in vitro resistance to protease digestion was determined by incubation of each of these polypeptide variants with a mixture of trypsin, -chymotrypsin, Asp-N endoproteinase and Glu-C endoproteinase (Sigma). This protease resistance test is described in Example 2, and the results of the protease resistance profile of said variants in comparison to the native HMGB1 Box A are shown in Figures 9.1 to 9.67.

From the results it can be assumed that the preferred polypeptide variants of Box A of HMGB1 useful as a remainder of Box A of HMGB1 of the polymer conjugate of the present invention are those variants that show similar or superior antagonistic activity together with a resistance increased to proteases. In particular, the preferred polypeptide variants are the polypeptides of SEQ ID NOS: 33, 35, 37-39, 42-45, 47-49, 52, 55, 57, 59, 62, 64, 67, 69 and 104. Between these preferred polypeptide variants, the most preferred variants are those defined in SEQ ID NOS: 45, 49, 52, 55, 59, 64 and 67. These highly preferred polypeptide variants show a greatly improved proteinase resistance profile compared to Box A of wild-type human HMGB1 of SEQ ID NO: 1 (see results of Example 2).

It is noted that the amino acids that appear in the various amino acid sequences that appear here are identified according to their known one-letter code abbreviations. It should also be noted that all sequences of the amino acid residues represented herein by their one letter abbreviated code have a left-to-right orientation in the conventional direction of the amino term to the carboxyl term.

In the present invention, it was surprisingly found that the polymer conjugates described above show improved pharmacokinetic and toxicological behavior, leading to improved bioavailability compared to the unconjugated HMGB1 Box A polypeptide moiety. A particular advantageous effect of the polymer conjugation of the HMGB1 Box A polypeptides and their variants, and in particular of the pegylation of these polypeptides, compared to the unconjugated form, is the increase in the hydrodynamic volume of the proteins. This leads to a significant and unexpected improvement of the pharmacokinetic properties of the conjugated compounds, because renal clearance is avoided, that is, the reduction of glomerular filtration.

In addition, the polymer conjugates of the invention have an increased resistance to the proteolytic activity of proteases and / or peptidases, in particular they have an increased resistance to the proteolytic activity of human proteases and / or peptidases, in particular human serum proteases and / or human gastrointestinal proteases or peptidases.

In particular, the proteolysis resistance is at least 10%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, 95% or greater compared to the unconjugated HMGB1 Box A. Protease resistance was measured at different time points (between 5 minutes and 8 hours) at 25 ° C after incubation of 20 g wild type of Box A or variants with a mixture of proteases at 1% w / w protein totals The protease mixture was recently prepared in each assay from stock solutions of Glu-C endoproteinase (SIGMA) 200 µg / ml; trypsin (SIGMA) 400 /g / ml and -chymotrypsin (SIGMA) 400 g / ml. After incubation with the proteases, the reaction was stopped by adding 10 µl of anti-protease solution (Roche), and the samples were stored at -20 ° C for the biological activity test.

As a consequence of the increased stability due to the increased resistance to protease activity, the polymer conjugates of the present invention also have a longer half-life in body fluids compared to the unconjugated HMGB1 Box A. In particular, the half-life in serum and / or in blood increases, thereby observing an increase of at least 10 minutes, 20 minutes, 30 minutes, 60 minutes

or even more, compared to the unconjugated HMGB1 Box A.

Due to the increase in the hydrodynamic volume of the proteins, and also due to a greater resistance to proteolysis and, thus, to the greater stability, the polymer conjugates of the invention also show improved therapeutic and biological properties and activity. In fact, they show a more favorable pharmacokinetic and pharmacological profile than the unconjugated HMGB1 Box A protein and protein variants.

Therefore, the invention relates to the use of the above-mentioned polymer conjugates of Box A of HMGB1 as an active agent in a medicament.

Still a further aspect of the invention is therefore the use of the polymer conjugates of the invention for the manufacture of a medicament for the prevention and / or treatment of pathologies associated with extracellular HMGB1.

or pathologies associated with proteins homologous to HMGB1. In particular, the pathologies associated with HMGB1 are pathologies that are mediated by a cascade of multiple inflammatory cytokines.

The broad spectrum of pathological conditions induced by the chemokine of HMGB1 and by the cascade of inflammatory cytokines induced by HMGB1 are grouped into the following categories: inflammatory disease, autoimmune disease, systemic inflammatory response syndrome, reperfusion injury after organ transplantation, cardiovascular conditions, obstetric and gynecological disease, infectious disease (viral and bacterial), allergic and atopic disease, pathologies of solid and non-solid tumors, transplant rejection diseases, congenital diseases, dermatological diseases, neurological diseases, cachexia, kidney diseases, conditions of iatrogenic intoxication, metabolic and idiopathic diseases.

The pathologies associated with HMGB1 according to the present invention are preferably pathological conditions mediated by activation of the inflammatory cytokine cascade. Non-limiting examples of conditions that can be treated in a useful manner using the present invention include the broad spectrum of pathological conditions induced by the chemokine of HMGB1 and by the cascade of inflammatory cytokines induced by HMGB1 grouped into the following categories: restenosis and other diseases cardiovascular, reperfusion injury, inflammatory diseases such as inflammatory bowel disease, systemic inflammatory response syndrome, for example septicemia, adult dysneic syndrome, etc., autoimmune diseases such as rheumatoid arthritis and osteoarthritis, obstetric and gynecological diseases, infectious diseases, atopic diseases, such as asthma, eczema, etc., tumor pathologies, for example diseases of solid and non-solid tumors associated with organ or tissue transplants, such as reperfusion injuries after organ transplantation, organ rejection and disease graft against the host, congenital diseases, dermatological diseases such as psoriasis or alopecia, neurological diseases, ophthalmic diseases, renal, metabolic or idiopathic diseases and intoxication states, for example iatrogenic toxicity and BehÇet disease, in which the above diseases are caused by, associated with and / or accompanied by the release of the HMGB1 protein.

In particular, pathologies pertaining to inflammatory and autoimmune diseases include rheumatoid arthritis / seronegative arthropathies, osteoarthritis, inflammatory bowel disease, Crohn's disease, intestinal infarction, systemic lupus erythematosus, iritis / uveitis, optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis / granulomatosis of Wegener, sarcoidosis, orchitis / vasectomy inversion procedures, systemic sclerosis and scleroderma. The systemic inflammatory response includes septicemic syndrome (including gram-positive sepsis, gram-negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urosepticemia, septic conjunctivitis), meningococcemia, traumatic hemorrhage, tinnitus, ionizing radiation exposure, acute prostatitis and chronic, acute and chronic pancreatitis, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, achalasia, cholangitis, cholecystitis, enteritis, adult dysneic syndrome (ARDS). The reperfusion injury includes post-pumping syndrome and ischemia-reperfusion injury. Cardiovascular disease includes heart stunning syndrome, myocardial infarction and ischemia, atherosclerosis, thrombophlebitis, endocarditis, pericarditis, congestive heart failure and restenosis. Obstetric and gynecological diseases include premature delivery, endometriosis, abortion, vaginitis and infertility. Infectious diseases include HIV infection / HIV neuropathy, meningitis, hepatitis B and C virus infection, herpes simplex infection, septic arthritis, peritonitis, E. coli 0157: H7, pneumonia, epiglottitis, hemolytic uremic syndrome / thrombolytic purpura thrombocytopenic, candidiasis, filariosis, amebiosis, malaria, Dengue hemorrhagic fever, leishmaniosis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterial tuberculosis, Mycobacterium avium intracellulare, Pneumocystis cariniiditisitisitis, epicellitis, oriniitis , legionella, Lyme disease, influenza A, Epstein-Barr virus, cytomegalovirus, virus-associated hemiafagocytic syndrome, viral encephalitis / aseptic meningitis. Allergic and atopic disease includes asthma, allergy, anaphylactic shock, immune complex disease, hay fever, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis. Cancers (pathologies of solid and liquid tumors) include ALL, AML, CML, CLL, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, colorectal carcinoma, nasopharyngeal carcinoma, malignant histiocytosis and paraneoplastic / hypercalcemia cancer syndrome. Transplant diseases include organ transplant rejections and graft versus host disease. Congenital diseases include cystic fibrosis, familial hematophagocytic lymphohistiocytosis and sickle cell anemia. Dermatological diseases include psoriasis, psoriatic arthritis and alopecia. Neurological diseases include neurodegenerative diseases (multiple sclerosis, migraine, headache, pathologies associated with amyloid, prion diseases / Creutzfeld-Jacob disease, Alzheimer's and Parkinson's diseases, multiple sclerosis, amyotrophic emilateral sclerosis) and peripheral neuropathies, migraine, headache. Kidney diseases include nephrotic syndrome, hemodialysis and uremia. Iatrogenic intoxication states include OKT3 therapy, anti-CD3 therapy, cytokine therapy, chemotherapy, radiotherapy and chronic salicylate poisoning. Metabolic and idiopathic diseases include Wilson's disease, hemachromatosis, alpha-1 antitrypsin deficiency, diabetes and diabetes complications, weight loss, anorexia, cachexia, obesity, Hashimoto's thyroiditis, osteoporosis, evaluation of the hypothalamus-pituitary-adrenal axis and primary biliary cirrhosis. Ophthalmological diseases include glaucoma, retinopathies and dry eye. A variety of other pathologies include: multiple organic dysfunction syndrome, muscular dystrophy, septic meningitis, atherosclerosis, epiglottitis, Whipple's disease, asthma, allergy, allergic rhinitis, organic necrosis, fever, septicemia, endotoxic shock, hyperpyrexia, eosinophilic granuloma, granulomatosis , sarcoidosis, septic abortion, urethritis, emphysema, rhinitis, alveolitis, bronchiolitis, pharyngitis, epithelial barrier dysfunctions, silicovolcanoconiosis pneumoultramicroscopic, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, disseminated bacteremia, hydatitis dermatitis, erythema, dermatitis solar, urticaria, warts, papules, vasculitis, angiitis, myocarditis, arteritis, periarteritis nodosa, rheumatic fever, celiac disease, encephalitis, cerebral embolism, Guillaume-Barre syndrome, neuritis, neuralgia, iatrogenic complications / peripheral nerve injuries, lesion of the spinal cord, paralysis, uve Tis, arthritis, arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, synovitis, myasthenia gravis, Goodpasture syndrome, BehÇet syndrome, ankylosing spondylitis, Barger's disease, Retier syndrome, bullous dermatitis (bullous pemphigoid), pemphigus and pemphigus vulgaris and alopecia.

In a further preferred embodiment, the polymeric compounds of the invention are used as active agents for the prevention, relief and / or treatment of pathologies related to RAGE. Pathologies related to RAGE are defined as pathological conditions associated with an increased expression of RAGE.

RAGE (receptor for advanced glycosylation end products) is a member of multiple ligands of the immunoglobulin superfamily of cell surface molecules. It is composed of three regions of the immunoglobulin type (an immunoglobulin domain of type V, followed by two immunoglobulin domains of type C), a transmembrane domain, and a very charged short cytosolic tail, which is essential for subsequent RAGE-dependent signaling . RAGE was first identified in 1992 as a binding target for AGEs, non-enzymatically oxidized and glycosylated proteins that accumulate in vascular tissue in aging, and at high speed in diabetes. RAGE is expressed in a broad set of cells, including endothelial cells, smooth muscle cells, mononuclear phagocytes and neurons. Although it is present in large quantities during development, especially in the central nervous system, its levels decrease during maturity.

As disclosed above, RAGE was the first receptor identified for extracellular HMGB1. Binding to HMGB1 on the cell surface induces the transcriptional increase of RAGE. Examples of pathologies related to RAGE are diabetes and disorders associated with diabetes, such as vasculopathy, diabetic neuropathy and retinopathy and other disorders, including Alzheimer's disease and immune / inflammatory reactions of the vessel walls. A very preferred example of pathologies related to RAGE in this context is type I and / or type II diabetes.

In a further aspect of the invention, the use of the HMGB1 Box A polymer conjugates described above is made in combination with an additional active agent.

The additional agent is preferably an agent capable of inhibiting an early mediator of the inflammatory cytokine cascade. Preferably, this additional agent is an antagonist or inhibitor of a cytokine selected from the group consisting of TNF, IL-1, IL-1, IL-Ra, IL-6, IL-8, IL-10, IL-13, IL-18, IFN-, MIP-1, MIF-1, MIP-2, MIF and PAF.

The additional agent used in combination with the polymer conjugate may also be a RAGE inhibitor, e.g. an antibody directed against RAGE, a nucleic acid or nucleic acid analog capable of inhibiting the expression of RAGE, e.g. an antisense molecule, a ribozyme or an RNA interference molecule, or a small synthetic molecule antagonistic of the interaction of HMGB1 with RAGE, preferably of the interaction of the non-acetylated and / or acetylated form of HMGB1 with RAGE, or soluble RAGE ( sRAGE). The antibody directed against RAGE is preferably a monoclonal antibody, more preferably a chimeric or humanized antibody or a recombinant antibody, such as a single chain antibody or an antigen-binding fragment of such antibody. The soluble RAGE analog may optionally be present as a fusion protein, e.g. with the Fc domain of a human antibody. The small synthetic molecular antagonist of the interaction of HMGB1 with RAGE preferably has a molecular weight of less than 1000 Daltons. The small synthetic molecular antagonist preferably inhibits the interaction of RAGE with the non-acetylated form or / and with the acetylated form of HMGB1, and with the non-acetylated form or / and with the acetylated form of proteins homologous to HMGB1, in particular HMGB2, HMGB3 , HMG-1L10, HMG-4L or / and SP100-HMG.

The additional agent used in combination with the polymer conjugate can also be an inhibitor of the interaction of a Toll-like receptor (TLR), e.g. of TLR2, TLR4, TLR7, TLR8 or / and TLR9, with HMGB1, inhibitor which is preferably a monoclonal or polyclonal antibody, a nucleic acid or nucleic acid analog capable of inhibiting the expression of TLR, e.g. an antisense molecule, a ribozyme or an RNA interference molecule,

or a synthetic molecule that is preferably less than 1000 Dalton in size. The inhibitor may be a known inhibitor of a Toll-like receptor, in particular TLR2, TLR4, TLR7, TLR8 or / and TLR9. The inhibitor preferably inhibits the interaction of the Toll type receptor with the non-acetylated form or / and the acetylated form of HMGB1. In yet another embodiment, the additional agent is the thrombomodulin N-terminal lectin-like domain (D1). The thrombomodulin D1 domain is capable of intercepting the non-acetylated form and / or the acetylated form of HMGB1 released and of homologous HMGB1 proteins released, in particular HMGB2, HMGB3, HMG1L10, HMG-4L or / and SP100-HMG, avoiding this way its interaction with RAGE and Toll type receptors. The D1 domain of thrombomodulin may be native or may be mutated, in order to make it resistant to proteases.

The additional agent may also be a synthetic nucleic acid or double-stranded nucleic acid analog molecule with a folded shape structure, particularly a double-stranded folded DNA, a PNA or a DNA / PNA chimera or hybrid or a double-stranded DNA structure cruciform, PNA or chimera or hybrid of DNA / PNA, capable of binding to the MHGB1 protein. Preferred nucleic acids and nucleic acid analog molecules are described in the international patent application of the same owners and also pending processing No. WO2006 / 002971 filed on July 4, 2005 (which claims the priority of the provisional US application. U.S. No. 60 / 584,678 filed July 2, 2004). The double-stranded synthetic nucleic acid or nucleic acid analog molecule with a folded shape structure is preferably capable of binding to the non-acetylated form or / and the acetylated form of HMGB1 and the non-acetylated form or / and the acetylated form of homologous proteins to HMGB1, in particular HMGB2, HMGB3, HMG-1L10, HMG-4L or / and SP100-HMG.

In yet another embodiment, the additional agent used in combination with the polymer conjugate is K-252a or / and a salt or derivative thereof, or a polymer conjugate of K-252a or / and a derivative thereof. The use of K-252a or polymer conjugates of K-252a and its derivatives is described in an international patent application of the same owners and also pending processing WO2007 / 02299, filed on August 25, 2005.

Therefore, a further aspect of the present invention is a pharmaceutical composition comprising an effective amount of at least one of the polymer conjugates of the HMGB1 Box A polypeptide or polypeptide variant or a biologically active fragment thereof, as active ingredient. for the treatment of pathologies associated with HMGB1, and pharmaceutically acceptable carriers, diluents and / or adjuvants. The pharmaceutical composition of the present invention is preferably suitable for the treatment of pathologies associated with the non-acetylated form or / and the acetylated form of HMGB1 and / or proteins homologous to HMGB1. In a further preferred embodiment, the pharmaceutical composition of the present invention comprising the at least one polymer conjugate also comprises an additional agent as defined above. The pharmaceutical composition of the present invention can be used for diagnostic or therapeutic applications.

The exact formulation, route of administration and dosage can be selected by the individual physician taking into account the patient's conditions. Administration can be done in a single dose or in repeated doses at intervals. The amount and dosage range can be adjusted individually in order to provide the therapeutic effect that results in improved symptoms or prolonged survival in a patient. The actual amount of composition administered will, of course, depend on the individual being treated, the weight of the individual, the severity of the affliction, the mode of administration and the judgment of the doctor prescribing the treatment. A suitable daily dose will be between 0.001 and 10 mg / kg, particularly between 0.1 and 5 mg / kg.

Administration can be carried out by known methods, e.g. by injection, in particular by intravenous, intramuscular, transmucosal, subcutaneous or intraperitoneal injection, and / or by oral, topical, nasal, inhalation, aerosol and / or rectal application, etc. Administration can be local or systemic.

Additionally, the polymer conjugates of the rest of the HMGB1 Box A object of this invention may be reversibly immobilized and / or adsorbed on the surface and / or within medical devices or drug delivery / transport systems (microspheres). The medical devices and microspheres may be reversibly charged with the polymer conjugates of this invention, through their bonding, impregnation, and / or adsorption on the surface of the medical device or the microsphere, or on a layer that covers its surface. When the medical device or microsphere comes into contact with body fluids, the reversed immobilized polymer conjugates are released. Therefore, the medical device and the microsphere act as drug release tools that elute the molecule object of this invention so that its release kinetics can be controlled, ensuring a controlled or sustained release, as treatment requires. Methods for coating / impregnating medical devices and for loading microspheres are well known to those skilled in the art.

Thus, a further aspect of the present invention is the use of the HMGB1 Box A polymer conjugates, in which the conjugated molecules are reversibly immobilized on the surface of medical devices or microspheres, or adsorbed onto it. Preferably, these medical instruments are surgical tools, implants, catheters or stents, e.g. vascular stents for angioplasty and, in particular, stents that elute the medicated drug.

Another aspect of the invention relates to a medical device reversibly coated with at least one polymer conjugate of the invention. Such a device can be selected from surgical instruments, implants, catheters or stents. Such a device can be useful for angioplasty.

The invention is further illustrated by the following figures:

Fig. 1a shows the amino acid sequence of Box A of native human HMGB1 formed by 84 amino acid residues (SEQ ID NO: 1).

Fig. 1b shows the type of amino acid substitution at the respective target positions selected to generate the full length human HMGB1 Box A polypeptide variant. In addition, in SEQ ID NOS: 2 to 116 the specific amino acid sequences of the generated polypeptide variant are presented.

Fig. 2a shows the amino acid sequence of the biologically active fragment of Box A of human HMGB1 formed by 77 amino acid residues (SEQ ID NO: 117).

Fig. 2b shows the type of amino acid substitution at the respective target positions selected to generate the polypeptide variant of the biologically active fragment of Box A of human HMGB1 formed by 77 amino acid residues. In addition, in SEQ ID NOS: 118 to 222 the specific amino acid sequences of the generated polypeptide variant are presented.

Fig. 3a shows the amino acid sequence of the biologically active fragment of Box A of human HMGB1 formed by 54 amino acid residues (SEQ ID NO: 223).

Fig. 3b shows the type of substituting amino acids at the respective target positions selected to generate the polypeptide variant of the biologically active fragment of Box A of human HMGB1 formed by 54 amino acid residues. In addition, in SEQ ID NOS: 224 to 300 the specific amino acid sequences of the generated polypeptide variant are presented.

Fig. 4a shows the amino acid sequence of the HMGB1 Box A of native Anopheles gambia formed by 84 amino acid residues (SEQ ID NO: 301).

Fig. 4b shows the type of amino acid substitution at the respective target positions selected to generate the full-length Anopheles gambia HMGB1 Box A polypeptide variant. In addition, SEQ ID NOS: 302-418 shows the specific amino acid sequences of the polypeptide variant generated.

Fig. 5a shows the amino acid sequence of the biologically active fragment of the HMGB1 Box A of Anopheles gambia formed by 77 amino acid residues (SEQ ID NO: 419).

Fig. 5b shows the type of substituting amino acids at the respective target positions selected to generate the polypeptide variant of the biologically active fragment of Box A of HMGB1 of Anopheles gambia formed by 77 amino acid residues. In addition, in SEQ ID NOS: 420 to 528 the specific amino acid sequences of the generated polypeptide variant are presented.

Fig. 6a shows the amino acid sequence of the biologically active fragment of the HMGB1 Box A of Anopheles gambia formed by 54 amino acid residues (SEQ ID NO: 529).

Fig. 6b shows the type of substituting amino acids at the respective target positions selected to generate the polypeptide variant of the biologically active fragment of Box A of HMGB1 of Anopheles gambia formed by 54 amino acid residues. In addition, SEQ ID NOS: 530 to 610 shows the specific amino acid sequences of the generated polypeptide variant.

Figures and Tables 7.1 to 7.9 show the results of the chemotaxis test described in Example 1 carried out on polypeptide variants of Box A of HMGB1 of SEQ ID NO: 2 to SEQ ID NO: 116 used as rest of Box A of HMGB1 of the polymer conjugates of the present invention. In each figure the activity of a set of polypeptide variants was tested in the inhibition of HMGB1-induced NIH / 3T3 cell migration, compared to the activity of the full-length fragment of the human wild type HMGB1 Box A of SEC ID NO: 1. Each figure shows a table that gives the numerical data of the statistical analysis, and a bar chart in columns that shows the results of the chemotaxis test.

Figure 7.1 and Table 7.1 show the bar graph and statistical data of the results of the chemotactic migration assay in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the human HMGB1 Box A of SEQ ID NO : 1 (CT500) and polypeptide variants of SEQ ID NO: 2 to 15 (identified in the Table and Figure with the code CT501, CT568, CT569, CT570, CT571, CT502, CT572, CT503, CT573, CT504, CT574, CT575, CT576 and CT505, respectively).

Figure 7.2 and Table 7.2 show the bar graph and statistical data of the results of the chemotactic migration assay in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the human HMGB1 Box A of SEQ ID NO : 1 (CT500) and polypeptide variants of SEQ ID NOS: 16-23 and 25-29 (identified in the Table and Figure with the code CT577, CT578, CT506, CT579, CT580, CT581, CT507, CT582, CT584, CT508, CT509, CT510 and CT585, respectively).

Figure 7.3 and Table 7.3 show the bar graph and statistical data of the results of the chemotactic migration assay in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the human HMGB1 Box A of SEQ ID NO : 1 (CT500) and polypeptide variants of SEQ ID NOS: 30-35 and 37-43 (identified in the Table and Figure with the code CT511, CT512, CT513, CT514, CT586, CT515, CT516, CT517, CT518, CT519, CT520, CT521 and CT522, respectively).

Figure 7.4 and Table 7.4 show the bar graph and statistical data of the results of the chemotactic migration test in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the Box

5 A of human HMGB1 of SEQ ID NOS: 44-57 (identified in Table and Figure with code CT523, CT524, CT525, CT526, CT527, CT528, CT588, CT529, CT530, CT589, CT590, CT531, CT591 and CT532 , respectively).

Figure 7.5 and Table 7.5 show the bar graph and statistical data of the results of the chemotactic migration assay in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the human HMGB1 Box A of SEQ ID NO : 1 (CT500) and polypeptide variants of SEQ ID NOS: 58-67 and 69-71 (identified in the Table and Figure with the code CT592, CT533, CT593, CT534, CT535, CT536, CT537, CT594, CT538, CT539, CT540, CT541 and CT542, respectively).

Figure 7.6 and Table 7.6 show the bar graph and statistical data of the results of the chemotactic migration test in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the Box

15 A of human HMGB1 of SEQ ID NO: 1 (CT500) and polypeptide variants of SEQ ID NOS: 72-85 (identified in the Table and Figure with code CT596, CT597, CT598, CT599, CT600, CT601, CT602, CT603 , CT543, CT544, CT545, CT546, CT547 and CT604, respectively).

Figure 7.7 and Table 7.7 show the bar graph and statistical data of the results of the chemotactic migration assay in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the human HMGB1 Box A of SEQ ID NO : 1 (CT500) and polypeptide variants of SEQ ID NOS: 86-99 (identified in the Table and Figure with the code CT548, CT549, CT605, CT606, CT607, CT608, CT609, CT610, CT550, CT551, CT611, CT552, CT553 and CT554, respectively).

Figure 7.8 and Table 7.8 show the bar graph and statistical data of the results of the chemotactic migration test in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the Box

25 A of human HMGB1 of SEQ ID NO: 1 (CT500) and polypeptide variants of SEQ ID NOS: 100-113 (identified in the Table and Figure with code CT555, CT556, CT557, CT558, CT559, CT612, CT560, CT561 , CT613, CT562, CT563, CT564, CT565 and CT566, respectively).

Figure 7.9 and Table 7.9 show the bar graph and statistical data of the results of the chemotactic migration assay in the inhibition of NIH / 3T3 cells induced by HMGB1 by the wild type of the human HMGB1 Box A of SEQ ID NO : 1 (CT500) and polypeptide variants of SEQ ID NOS: 114-116 (identified in the Table and Figure with the code CT567, CT614 and CT615, respectively).

Fig. 8 shows the image of the SDS-PAGE Tricine gel loaded with the wild type of the Human HMGB1 Box A of SEQ ID NO: 1 (CT500) at different time points after digestion with proteases of the resistance test to proteases described in Example 2. The wild-type protein of Box A tested for

35 determining protease resistance is a protein labeled with His. After 5 minutes of digestion, CT500 shows two main bands: one that corresponds to the original protein in the sample, and the second one that corresponds to the 84 amino acid protein without the His marker of the N term (indicated in the figure with a arrow). The profile of this second band shows resistance to proteases for 30 minutes. The small bands present in this and other gels of Fig. 9.1 to Fig. 9.67 correspond to digested fragments of Box A.

Fig. 9.1 to Fig. 9.67 shows the image of the SDS-PAGE Tricine gel loaded with the polypeptide variants of the rest of the HMGB1 Box A of the polymer conjugates of the present invention at different time points after digestion with proteases of the Protease resistance assay described in Example 2. The polypeptide variants of Box A tested for protease resistance are proteins

45 marked with His. After 5 minutes of digestion, the SDS-PAGE gel image of the polypeptide variants shows two main bands: one corresponding to the original protein variant in the sample, and the second corresponding to the 84 amino acid protein variant of the sample. Box A without the His marker of the term N.

Figure 9.1 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 2 (CT501) at different time points after digestion with proteases.

Figure 9.2 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 7 (CT502) at different time points after digestion with proteases.

Figure 9.3 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 9 (CT503) at different time points after digestion with proteases.

Figure 9.4 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 11 (CT504) at different time points after digestion with proteases.

Figure 9.5 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 15 (CT505) at different time points after digestion with proteases.

Figure 9.6 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 18 (CT506) at different time points after digestion with proteases.

Figure 9.7: Trichine gel SDS-PAGE of the polypeptide variant of SEQ ID NO: 22 (CT507) at different time points after digestion with proteases.

Figure 9.8 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 26 (CT508) at different time points after digestion with proteases.

Figure 9.9 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 27 (CT509) at different time points after digestion with proteases.

Figure 9.10 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 28 (CT510) at different time points after digestion with proteases.

Figure 9.11 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 30 (CT511) at different time points after digestion with proteases.

Figure 9.12 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 31 (CT512) at different time points after digestion with proteases.

Figure 9.13 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 32 (CT513) at different time points after digestion with proteases.

Figure 9.14 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 33 (CT514) at different time points after digestion with proteases.

Figure 9.15 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 35 (CT515) at different time points after digestion with proteases.

Figure 9.16 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 37 (CT516) at different time points after digestion with proteases.

Figure 9.17 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 38 (CT517) at different time points after digestion with proteases.

Figure 9.18 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 39 (CT518) at different time points after digestion with proteases.

Figure 9.19 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 40 (CT519) at different time points after digestion with proteases.

Figure 9.20 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 41 (CT520) at different time points after digestion with proteases.

Figure 9.21 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 42 (CT521) at different time points after digestion with proteases.

Figure 9.22 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 43 (CT522) at different time points after digestion with proteases.

Figure 9.23 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 44 (CT523) at different time points after digestion with proteases.

Figure 9.24 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 45 (CT524) at different time points after digestion with proteases.

Figure 9.25 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 46 (CT525) at different time points after digestion with proteases.

Figure 9.26 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 47 (CT526) at different time points after digestion with proteases.

Figure 9.27 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 48 (CT527) at different time points after digestion with proteases.

Figure 9.28 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 49 (CT528) at different time points after digestion with proteases.

Figure 9.29 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 51 (CT529) at different time points after digestion with proteases.

Figure 9.30 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 52 (CT530) at different time points after digestion with proteases.

Figure 9.31 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 55 (CT531) at different time points after digestion with proteases.

Figure 9.32 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 57 (CT532) at different time points after digestion with proteases.

Figure 9.33 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 59 (CT533) at different time points after digestion with proteases.

Figure 9.34 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 61 (CT534) at different time points after digestion with proteases.

Figure 9.35 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 62 (CT535) at different time points after digestion with proteases.

Figure 9.36 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 63 (CT536) at different time points after digestion with proteases.

Figure 9.37 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 64 (CT537) at different time points after digestion with proteases.

Figure 9.38 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 66 (CT538) at different time points after digestion with proteases.

Figure 9.39 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 67 (CT539) at different time points after digestion with proteases.

Figure 9.40 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 69 (CT540) at different time points after digestion with proteases.

Figure 9.41 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 70 (CT541) at different time points after digestion with proteases.

Figure 9.42 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 71 (CT542) at different time points after digestion with proteases.

Figure 9.43 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 80 (CT543) at different time points after digestion with proteases.

Figure 9.44 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 81 (CT544) at different time points after digestion with proteases.

Figure 9.45 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 82 (CT545) at different time points after digestion with proteases.

Figure 9.46 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 83 (CT546) at different time points after digestion with proteases.

Figure 9.47 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 84 (CT547) at different time points after digestion with proteases.

Figure 9.48 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 86 (CT548) at different time points after digestion with proteases.

Figure 9.49 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 87 (CT549) at different time points after digestion with proteases.

Figure 9.50 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 94 (CT550) at different time points after digestion with proteases.

Figure 9.51 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 95 (CT551) at different time points after digestion with proteases.

Figure 9.52 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 97 (CT552) at different time points after digestion with proteases.

Figure 9.53 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 98 (CT553) at different time points after digestion with proteases.

Figure 9.54 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 99 (CT554) at different time points after digestion with proteases.

Figure 9.55 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 100 (CT555) at different time points after digestion with proteases.

Figure 9.56 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 101 (CT556) at different time points after digestion with proteases.

Figure 9.57 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 102 (CT557) at different time points after digestion with proteases.

Figure 9.58 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 103 (CT558) at different time points after digestion with proteases.

Figure 9.59 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 104 (CT559) at different time points after digestion with proteases.

Figure 9.60 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 106 (CT560) at different time points after digestion with proteases.

Figure 9.61 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 107 (CT561) at different time points after digestion with proteases. ID NO: 106 (CT560) at different time points after digestion with proteases.

Figure 9.61 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 107 (CT561) at different time points after digestion with proteases.

Figure 9.62 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 109 (CT562) at different time points after digestion with proteases.

Figure 9.63 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 110 (CT563) at different time points after digestion with proteases.

Figure 9.64 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 111 (CT564) at different time points after digestion with proteases.

Figure 9.65 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 112 (CT565) at different time points after digestion with proteases.

Figure 9.66 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 113 (CT566) at different time points after digestion with proteases.

Figure 9.67 shows the SDS-PAGE Tricine gel of the polypeptide variant of SEQ ID NO: 19 (CT567) at different time points after digestion with proteases.

Fig. 10 shows a table that summarizes the results of the Tricine SDS-PAGE gel. A cross indicates the presence in the gel of the band corresponding to the long protein fragment of 84 wild-type amino acids of Box A of HMGB1 or of the polypeptide variant of Box A of HMGB1.

Fig. 11 shows the mean plasma concentration / time after a single subcutaneous administration of the wild type (WT) of Box A and variant number 64 (64) of Box A at a dose of 1 mg / kg.

Data representation: Medium  SEM.

Fig. 12 shows the mean plasma concentration / time after a single subcutaneous administration of the wild type (WT) of Box A and variant number 64 (64) of Box A at a dose of 1 mg / kg, and of the wild type of the PEGylated Box A (PEG-WT) and of the 64th variant (PEG-64) of the PEGylated Box A at a dose of 5 mg / kg. Data representation: Medium  SEM.

EXAMPLES

1. In vitro activity assay: NIH / 3T3 cell migration assay

The purpose of the present study was to evaluate the activity of each of the HMGB1 Box A polypeptide variants as defined in SEQ ID NOS: 2-116 and compare its activity with that of the full-length fragment of Box A of Human wild type HMGB1 of SEQ ID NO: 1 in order to select all variants with similar or better activity than the wild type.

The activity of HMGB1 Box A is evaluated in vitro as inhibition of HMGB1-induced NIH / 3T3 cell migration.

1.1 Materials  Wild type and variants of Box A of HMGB1 (Nautilus Biotech)  NIH / 3T3 cells (ATCC n. CRL-1658)  DMEM medium (GIBCO; catalog number. 31966-021)  Bovine fetal serum (GIBCO; catalog number 10270-106) Penicillin-streptomycin 10,000 U / ml (GIBCO; catalog number 15140-122) 200 mM L-glutamine (GIBCO; catalog number 25030-024)  TrypLE Select (GIBCO; catalog number 12563-011)  Phosphate buffered saline solution (0.138 M NaCl, 0.0027 M KCl, 0.01 M phosphate, pH 7.4)  PVP-free filters (pore sizes of 8 ;m; total diameter 13 mm) (Neuro Probe; catalog number

PFA8)  Human fibronectin (Roche; catalog number 1080938)  Blind Well Chemotaxis Chambers (Neuro Probe; catalog number BW25)  GIEMSA Stain Modified (Sigma; catalog number GS1 L)

1.2 Preparation of filters

The PVP-free filters of polycarbonate membranes (pore size of 8 µm, total diameter of 13 mm) were prepared about one hour before carrying out the experiment by coating them with 30 µl / filter of a 50 µg solution / ml of fibronectin dispensed on the opaque side of the filter. The stock solution of fibronectin is prepared by diluting the lyophilized fibronectin in ddH2O to a final concentration of 1 mg / ml, and keeping the solution about 1 hour at 37 ° C for total dissolution. This stock solution can be stored at -20 ° C.

The filters are then allowed to dry under the laminar flow of the hood (about an hour).

1.3 Preparation of the cells

The day before the experiment (approximately 22-24 hours before conducting the experiment), the NIH / 3T3 cells were seeded at 106 cells / plate.

When the filters were ready for use, the cells were detached with trypsin, counted and resuspended at 106 cells / ml in serum free culture medium.

1.4 Chemotaxia Assay

In each chemotaxis experiment, 14 different polypeptide variants of the full-length fragment of human HMGB1 Box A of SEQ ID NO: 1 were tested. As a negative control, representing spontaneous migration, growth cell medium was used without addition of serum (without FBS).

As a positive control, 1 nM of HMGB1 was used. The wild type of HMGB1 Box A or the 0.5 / 1 nM polypeptide variants tested were added to 1 mM of HMGB1 to inhibit the migration of NIH / 3T3 cells induced by HMGB1.

The negative control (without FBS) and the positive control (1 nM of HMGB1) were tested in triplicate in each experiment.

The wild-type activity of HMGB1 Box A (SEQ ID NO: 1) inhibiting HMGB1-induced cell migration was assayed in triplicate in each experiment.

Each of the HMGB1 Box A polypeptide variants (SEQ ID NOS: 2 to 116) was tested in duplicate.

Blind Well Chemotaxis Chambers chemotaxis cameras were used. The dry, clean bottom well of each chamber was filled with 50 µl of DMEM without FBS added with the appropriate chemotactic agent and inhibitors. A slightly positive meniscus should form when the well is full; This helps prevent air bubbles from being trapped when the filter is applied. With a small forceps, the filter is placed over the full well (the face facing up is treated with fibronectin), being careful not to trap air bubbles and not to touch the filter with your fingers. The filter retainer is screwed manually. A cell suspension (50,000 cells / 50 µl) is pipetted into the upper well, and 150 µl of serum free medium is added to fill the upper well of the chamber. The full chamber is incubated for 3 hours (37 ° C, 5% CO2) to allow cell migration. After incubation, the fluid is removed from the filter. The retainer is unscrewed and immersed in cold distilled water. The filter is lifted with a forceps, placed on a clean surface (solid paraffin) (with the migrated cells placed on the upper face) and fixed with a needle (placed in the border area).

1.5 GIEMSA staining of migrated cells

The filters were fixed with ethanol once, and then washed three times in running water. A working solution of GIEMSA Stain Modified was prepared which was diluted 1:10 in ddH2O just before use. After washing the filters, staining was added and allowed to incubate for 20 minutes. The staining was washed under running water. The filters were then placed on slides, with the migrated cells placed on the underside, and the side with the non-migrated cells was gently cleaned with a damp cotton wipe (cleaned twice, using two tails or both ends of a double pointed tip), being careful not to move the filter. After cleaning, a coverslip was placed on the filter and the cells were counted in a 40X microscope in 10 random fields / filter.

1.6 Data representation and statistical analysis

The results of the NIH / 3T3 migration test carried out are disclosed in the tables and bar graphs shown in Figure and Table 7.1 to Figure and Table 7.9.

Data are represented in bar columns as MEDIUM ± 95% confidence interval (CI).

The one-way ANOVA, followed by Dunnett's post-test (control column data: 1 nM HMGB1 sample + HMGB1 Box A sample WT), is the statistical analysis carried out.

When evaluating the results data, the data of the HMGB1 Box A variants that have a post-test p-value <0.05 are considered significantly different from the wild type of the HMGB1 Box A. If the average of the polypeptide variant of Box A is greater than that of the wild type of Box A, the column is colored red in the graph of the experiment shown in Figures 7.1 to 7.9. These red columns represent polypeptide variants of the HMGB1 Box A that show less activity than the wild type when it comes to inhibiting HMGB1-induced cell migration.

If the average of the polypeptide variant is lower than that of the wild-type Box A, then the column is colored light blue in the graph of the experiment shown in Figures 7.1 to 7.9. These variants represent variants of the HMGB1 Box A that show an activity greater than that of the wild type of HMGB1 Box A in inhibiting HMGB1-induced cell migration.

The data of the HMGB1 Box A variants that have a post-test p-value> 0.05 are not considered significantly different from the wild type of the HMGB1 Box A. The columns of bars of these variants are colored in green. These variants represent variants of the HMGB1 Box A that show the same activity as the wild type when it comes to inhibiting HMGB1-induced cell migration.

1.7 Results

The activity of the polypeptide variants of the high affinity binding domain Box A of human HMGB1 of SEQ ID NOS: 2 to 116 was evaluated in comparison with the wild type of Box A of human HMGB1 of SEQ ID NO: 1 as inhibition of HMGB1 induced cell migration, in order to determine the preferred polypeptide variants useful as a remainder of the HMGB1 Box A of the preferred polymer conjugate of the present invention.

The results of the chemotaxis assays revealed (Figures 7.1 to 7.9) that, for 26 polypeptide variants, the

5 mutation according to the present invention could lead to increased activity in the inhibition of cell migration compared to the activity of the wild-type human HMGB1 Box A. In particular, greater activity was shown in the inhibition of cell migration for the polypeptide variants of SEQ ID NOS: 30-32, 35, 38, 4041, 43, 48, 51, 57, 63-64, 69, 70, 94, 95, 100, 103-104, 106-107, 109-111 and 113.

In addition, the results of chemotaxis assays revealed (Figures 7.1 to 7.9) that 41 polypeptide variants

10 showed no changes in their activity in inhibiting HMGB1-induced cell migration compared to the activity of the wild-type polypeptide of Box A. In particular, this is the case for the polypeptide variants of SEQ ID NOS: 2 , 7, 9, 11, 15, 18, 22, 26-28, 33, 37, 39, 42, 44-47, 49, 52, 55, 59, 61, 62, 66-67, 71, 80-84 , 86-87, 97-99, 101-102, 112 and 114.

All these polypeptide variants of Box A that show an activity similar or greater than that of the wild type

15 of Box A were tested for protease resistance in vitro, in order to choose the most resistant ones that are at least as active as the wild type of Box A (see the protease resistance test in Example 2) .

2. In vitro protease resistance test

The purpose of this study was to evaluate the in vitro protease resistance of the HMGB1 Box A 20 variants shown in Example 1, and compare it with that of the wild type HMGB1 Box A of SEQ ID NO: 1 in order of identifying variants with enhanced protease resistance with respect to the wild type polypeptide.

2.1 Materials  Wild type of Box A marked with HMGB1 His and selected variants (Nautilus biotech) Trypsin (Sigma; catalog number T8658; lot number 045K5113)

25 -chymotrypsin (Sigma; catalog number C6423; lot number 109H74858),  Endoproteinase Asp-N (Sigma; catalog number P3303; lot number 046K1049)  Endoproteinase Glu-C (Sigma; catalog number P6181; lot number 075K5100)  EDTA free protease inhibitor cocktail, Complete Mini (Roche; catalog number 11836170 001)  Trizma base (Sigma; catalog number T6066)

30  40% acrylamide / bis solution in water (Sigma; catalog number 01709)  SDS (Sigma; catalog number 71729)  Glycerol 99% (Sigma; catalog number G9012)  Temed (Sigma; catalog 87689)  APS (Sigma; catalog number A 3678)

35  Molecular weight standards for polypeptide SDS-PAGE (Bio-Rad; catalog number 161-0326)  Tris / tricine / SDS 10x premixed buffer (Bio-Rad; catalog number 161-0744) -Mercaptoethanol ( Sigma; catalog number M7154)  Methanol (VWR; catalog number 20864.320)  Acetic acid (VWR; catalog number 20104.323)

40  Bright Blue R (Sigma; catalog number B0149)  Bromophenol blue (Sigma; catalog number B0126)

 Hydrochloric acid (Merck; catalog number 1.00319.2511)

 3X sample loading buffer for Tricine gels (composition: 150 mM Tris-HCl, pH 6.8; 12% SDS; 36% glycerol; 6% -mercaptoethanol; 0.04% blue bromophenol)

2.2 Preparation of the protease mixture

A mixture of proteases containing trypsin, -chymotrypsin, Asp-N endoproteinase and endoproteinase was used Glu-C Table 1 gives the specificity of each of the proteases used in this study.

Table 1: Specificity of proteases.

Protease

 Specificity

Trypsin

Term C of K, R (not if P is at the C term of the cutting site; slower digestion if the acidic residue on either side of the cutting site)

-chymotrypsin

Term C of T, P, W, L (secondary hydrolysis: term C of M, I, S, T, V, H, G, A)

Asp-N endoproteinase

Term N of D, C

Endoproteinase Glu

Term C of E, D (not if P is in term C of the cutting site)

10 Each lyophilized protease was dissolved according to the manufacturer's recommendations to obtain a stock solution that was divided into aliquots and stored at -80 ° C.

100 µg of trypsin was dissolved in 100 µl of dH2O to obtain a stock solution of 1 µg / µl. 25 µg of -chymotrypsin was dissolved in 50 µl of a solution of 1 mM HCl, 2 mM CaCl2 to obtain a 0.5 µg / madrel stock solution. 2 µg of Asp-N endoproteinase was dissolved in 50 µl of dH2O to obtain a stock solution

15 0.04 g / l. 25 µg of Asp-N endoproteinase was dissolved in 50 µl of dH2O to obtain a stock solution of 0.5 µg / µl.

Before carrying out the experiment, an aliquot of each protease stock solution was allowed to thaw on ice.

Aliquots of the trypsin and Glu-C endoproteinase stock solution were diluted in dH2O to obtain a

20 final working solution of 0.1 /g / l. The mother aliquot of -chymotrypsin was diluted in a 1 mM solution of HCl, 2 mM CaCl2 to obtain a final working solution of 0.1 g / l. The aliquot of endoproteinase Asp-N was used without dilution.

Just before carrying out the experiment, a mixture of proteases containing 1% (by weight / weight of the total Box A contained in the sample) of each protease was recently prepared, and added immediately to Box A

25 of HMGB1 to digest.

2.3 Digestion with wild type proteases of Box A of HMGB1 and variants

In each experiment 18 g were digested in total from each Box A of HMGB1 (wild type or variants).

The HMGB1 Box A to be tested was allowed to thaw on ice, and the volume corresponding to 18 g was taken. The volume of this solution was then brought with dH2O to a final volume of 90 µl in order to obtain the same

30 final volume for each Box A of HMGB1 to be tested. 10 µl of this solution (corresponding to 2 µg of Box A of HMGB1) is taken before adding the protease mixture. This sample corresponds to an undigested sample of "time 0".

The remaining sample (16 g of HMGB1 Box A) with 8.8 l (corresponding to 0.16 g of each protease of the freshly prepared mixture is added; see 2.2) of the protease mixture for digestion.

35 Protease digestion is carried out at 25 ° C, and a volume corresponding to 2 g of Box A of HMGB1 (originally present in the mixture) is sampled at different time points. Digestion is stopped by adding 4 µl of a protease inhibitor cocktail solution free of EDTA Complete Mini (a tablet dissolved in 10 ml of dH2O). The time points for sampling are: 0.5 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours and 4 hours.

Very shortly after protease inhibition, the samples are added with the appropriate amount of 3X sample loading buffer and incubated at 95 ° C for about 3 minutes.

2.4 Wild-type SDS-PAGE trichine and digested HMGB1 Box A variants

After digestion with proteases and sample preparation, samples from the time points of each HMGB1 Box A are loaded onto SDS-PAGE tricine gel (see for references: Schägger and von Jagow, “Tricinesodium dodecyl sulphate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa ”, Anal. Biochem. 166, 368-379, 1987). For reference in each gel, 5 µl of polypeptide molecular weight standards were loaded for SDS-PAGE (Bio-Rad).

Each well of the gel is loaded with 10 µl of sample (volume corresponding to 1 µg of Box A of HMGB1 before digestion).

Electrophoresis is carried out at 30 V until bromophenol blue has entered the separating portion of the gel, and then carried out at 120 V (Mini Protean 3 System; Bio-Rad) until the end of the experiment.

The gels are stained by soaking them in Coomassie Brilliant Blue R staining solution (0.1% w / v in 50% methanol, 10% acetic acid) for 1 hour, and they were faded overnight in fader solution (methanol at 30%, 10% acetic acid).

The gel images are acquired with an image forming system Gel Document 2000 (Bio-Rad).

2.5 Results

Under the test conditions disclosed above, the wild-type HGMB1 protein resisted approximately 30 minutes until complete digestion with proteases. In Figure 8, the band corresponding to the full length fragment of 84 wild-type amino acids of the human HMGB1 Box A of the protein of SEQ ID NO: 1 is visible up to 30 minutes of digestion with proteases.

The 21 Box A polypeptide variants tested showed increased protease resistance (Figure 10). Under the test conditions disclosed, these variants resist from 1 hour to 2 hours to digestion with proteases. The polypeptide variants of SEQ ID NOS: 33, 35, 37, 38, 39, 42, 43, 44, 47, 48, 57, 62, 69 and 104 showed a 1 hour resistance to digestion with proteases. Figures 9.14, 9.15, 9.16, 9.17, 9.18, 9.21, 9.22, 9.23, 9.26, 9.27, 9.32, 9.35, 9.40 and 9.59 show a band corresponding to the unlabeled protein with His of 84 amino acids, which is visible up to 1 hour of digestion with proteases.

The polypeptide variants of SEQ ID NOS: 45, 49, 52, 55 and 67 showed a 1.5 hour resistance to digestion with proteases. Figures 9.24, 9.28, 9.30, 9.31 and 9.39 show a band corresponding to the 84-amino acid His unlabeled protein that is clearly visible 1 and a half hours after digestion with proteases. The polypeptide variants of SEQ ID NOS: 59 and 64 even show a resistance of up to 2 hours to digestion with proteases. Figures 9.33 and 9.37 show a band of the 84-amino acid His unlabeled protein that is clearly visible up to 2 hours after digestion with proteases.

3. Pharmacokinetic study of Box A (wild type and variants) and PEGylated Box A (wild type and variants) after a single subcutaneous administration in mice,

3.1 Object of the study

The purpose of the study was to evaluate and compare the pharmacokinetic profile of Box A (wild type and variants) and PEGylated Box A (wild type and variants) after a single subcutaneous administration of the compounds in mice.

3.2 Materials and methods

 Test articles: Wild type and variants of Box A and the corresponding PEGylated molecules. The

PEGylated molecules were obtained by reacting linear mPEG-aldehyde (40 kDa) with the N terminus of

the respective Box A molecule by reductive amination.

3.3 In vivo experiment

 Animals: mice (Balb / c mice, males, 7-9 weeks, supplied by Charles River Laboratories Italy

SpA, Calco, one week before the experiment) with an average weight of 22.2-22.4 g at the time

of the experiment

 Animal farm: the animals were caged in a ventilated thermostatic container, adjusted to maintain

temperature and relative humidity at 22ºC ± 2ºC and 55 ± 15%, respectively, with a 12-hour cycle of

light darkness. The animals were caged in an amount of up to 10 in a cage, in transparent polycarbonate cages (Techniplast, Buguggiate, Italy); Water to drink was supplied at will via bottles of water, and a commercially available laboratory rodent diet (4RF21, Mucedola s.r.I., Settimo Milanese, Italy).

 Experimental groups: 4 (four test articles), 20 animals / group, randomly grouped.

 Dose administered: the wild type and variants of Box A were supplied subcutaneously at 1 mg / kg.

The wild type and variants of PEGylated Box A were administered subcutaneously at 5 mg / kg. These

doses ensured the equimolarity of the test compounds.

 Administration of test articles: test articles were administered subcutaneously using

an insulin syringe with a 0.45 x 12 mm needle (26G x ½ ”) at a volume of 0.25 ml / mouse (10 ml / kg

of body weight).

 Formulation of the test article: phosphate buffered saline solution.

 Animal sacrifice and blood collection:

Blood samples were collected at the following time points after treatment:

 wild type and variants of Box A: 5, 20 and 40 minutes, 1.5 and 2.5 hours after administration of test compounds;

 wild type and variants of PEGylated Box A: 5, 40 minutes, 1.5, 5 and 10 hours after administration of test compounds.

Different time points for the collection of blood samples between the test compounds were decided based on the highest expected permanence of the PEGylated molecules in the bloodstream.

At each sampling time, approximately 0.4 ml of blood samples were collected from the ventral aorta of each animal using an insulin syringe, under deep ether anesthesia, and transferred to Eppendorf polyethylene tubes containing 5  l of heparin (5000 IU / ml) to prevent blood clotting. Blood samples were kept on ice until centrifugation at 1400 g for 5 minutes in a refrigerated centrifuge (2-4 ° C). Plasma samples from each tube were then recovered, placed in new Eppendorf tubes, and frozen at -80 ° C until analysis.

3.4 Analytical determination

Plasma concentrations of Box A (wild type and variants) and PEGylated Box A (wild type and variants) were determined in mouse plasma by an ELISA method.

Briefly, a coating solution was prepared by diluting a monoclonal antibody against the N-terminal of Box A up to 10 ng / ml in 100 mM carbonate-bicarbonate coating buffer. 100 µl in aliquots were applied to each well of a Nunc Maxisorp ELISA plate, which was incubated overnight at 4 ° C. The plate was washed with 0.05% Tween PBS 6 times, and 300 µl of 5% milk in 1% Tween PBS was added to each well to block the remaining binding sites on the plate. The plate was incubated for 1 hour at room temperature at 300 rpm. The samples were diluted 1:20 in 1% Tween PBS.

The plate was washed 6 times, and 100 µl of standards and diluted samples were transferred to the designated wells of the coated plate, and incubated at room temperature for 1 hour at 300 rpm. The plate was washed 6 times again before the addition of the secondary antibody against the C-terminal of Box A (1: 200). After 1 hour of incubation and 6 washes, the 1: 20000 goat anti-rabbit biotin-rabbit conjugate solution was added to each well (100 µl / well). After 1 hour of incubation (at room temperature, 300 rpm) and six washes, the plate was incubated with 100 µl / well of the streptavidin-HRP 1: 100,000 solution for 25 minutes at 300 rpm. The plate was washed 6 times, and 100 µl of preheated TMB substrate was added to each well. The signal was developed at room temperature on the worktable, and, after 30 minutes, 100 µl / well of Stop Solution was added, and the plate was immediately read at 450 nm.

3.5 Results

The mean plasma concentrations of Box A (wild type and variants) and PEGylated Box A (wild type and variants) were calculated for each of the PK samples described above, and the pharmacokinetic profile was determined. The results are shown in Fig. 11 and 12.

As an example, the PK profiles of the wild type and of the 64th variant (M511) of the Box A, and of the PEGilated Box A and of the 64th variant of the PEGilated Box A (M511) are disclosed below . The results are given as mean values ± error (SEM) (4 mice for each time point, duplicate analysis).

In the following table, the last calculated AUC (Area Under the Curve, calculated at the last experimental point) for the wild type curves of Box A, of variant number 64 of Box A, of the wild type of PEGilated Box A and the 64th variant of PEGilada Box A.

Table 2. Last AUC data calculated for the wild type curves of Box A (WT), variant number 64 of Box A (64), wild type of PEGilated Box A (PEG-WT) and of variant number 64 of the PEGylated Box A (PEG-64).

Last

(M * min)

Wt

 8,899

64

 14.97

PEG-WT

 275.5

PEG-64

 332.5

3.6 Discussion

The relative gain in AUClast conferred on WT by the mutation is 1.68X (WT vs. 64), most likely

10 due to the increased resistance to proteases in the sub acute compartment. The relative gain in AUClast conferred on WT by PEGylation is 31X (WT vs. PEG-WT), mainly due to an altered renal filtration of the PEG conjugate, but also to protection against the action of proteases. Putting the mutation and PEGylation together gives a relative gain in AUCultimate of 37X (WT vs. PEG-64). Unexpectedly, the two modifications together have a positive effect on the animal's exposure to protein.

15 which is greater than the sum of the contributions of the individual modifications (ie 37X> 1.68X + 31X). Thus, single point mutation and PEGylation have a synergistic cooperative effect on the pharmacokinetic profile of the native protein. A possible explanation for this phenomenon could be that PEGylated proteins are not completely protected from proteolysis in the sub-acute compartment. The introduction of a single point mutation gives a resistance boost in this compartment, allowing them to enter the

20 blood circulation higher amounts of protein and then be protected from renal filtration by the bulky PEG chain.

Claims (21)

one.

 Polymer conjugate of the high affinity binding domain Box A of HMGB1 wild-type human and / or non-human (Box A of HMGB1) or a biologically active fragment of Box A of HMGB1.

2.

 Polymer conjugate of a polypeptide variant of the high affinity binding domain Box A of human and / or non-human HMGB1 (Box A of HMGB1) or of a biologically active fragment of Box A of HMGB1, whereby the amino acid sequence of said polypeptide variant differs from the amino acid sequence of the wild type HMGB1 Box A by the mutation of one or more individual amino acids.

3.

 The polymer conjugate of claim 1 or 2, wherein the polymer is a linear or branched polymer moiety of the group consisting of polyalkylene glycol, poly (alkylene oxide), poly (acrylic acid), polyacrylate, polyacrylamide or their N- derivatives alkyl, poly (methacrylic acid), polymethacrylate, poly (ethylacrylic acid), polyethylacrylate, polyvinyl pyrrolidone, poly (vinyl alcohol), poly (glycolic acid), poly (lactic acid), poly (lactic-co-glycolic acid), dextran, chitosan opoliamino acid.

Four.

 The polymer conjugate of claim 1 or 2, wherein the polymer is a linear or branched polymer moiety selected from polyethylene glycol (PEG) or methoxy polyethylene glycol (m-PEG), preferably with an average molecular weight of 20,000 or a molecular weight average of 40,000 Da.

5.

The polymer conjugate of any one of claims 1 to 4, wherein the polymer moiety is covalently linked to the HMGB1 Box A polypeptide, to the HMGB1 Box A polypeptide variant or to a biologically active fragment thereof.

6.

 The polymer conjugate of any one of claims 1 to 5, wherein the conjugation site in the polypeptide, polypeptide variant or biologically active fragment thereof from Box A of HMGB1 is selected from a lysine, cysteine, histidine moiety. arginine, tyrosine, serine, threonine, aspartate, and glutamate, or the N-terminal amino group.

7.

 The polymer conjugate of claims 1 to 6, wherein the polypeptide variant differs from the wild-type HMGB1 Box A sequence in the mutation of 1 to 10 individual amino acids, preferably in only a single amino acid.

8.

 The polymer conjugate of claim 7, wherein the mutation is a substitution, a deletion or an addition of individual amino acids.

9.

 The polymer conjugate of claim 8, wherein the substitution is a conservative substitution or a non-conservative substitution obtained by a different genetically encoded amino acid or by non-genetically encoded amino acids.

10.

 The polymer conjugate of any one of claims 1 to 9, wherein the polypeptide variant of Box A of human HMGB1 is selected from the group consisting of the amino acid sequences as defined in any of SEQ ID NO: 2 to 116 .

eleven.

 The polymer conjugate of any one of claims 1 to 9, wherein the biologically active fragments of the human wild-type HMGB1 Box A is a fragment of at least 77 or at least 54 amino acids respectively, and comprise the amino acid sequences as they are defined in SEQ ID NO: 117 or 223 respectively, and wherein the polypeptide variant of said biologically active fragments of human HMGB1 Box A is selected from the group consisting of amino acid sequences as defined in any of SEQs. ID NO: 118 to 222 or 224 to 300.

12.

 The polymer conjugate of any one of claims 1 to 9, wherein the non-human HMGB1 Box A is the Anopheles gambia HMGB1 Box A (SEQ ID NO: 301), and wherein the non-human polypeptide variant of said Box A of HMGB1 of Anopheles gambia is selected from the group consisting of amino acid sequences as defined in any of SEQ ID NO: 302-418.

13.

 The polymer conjugate of any one of claims 1 to 9 and 12, wherein the biologically active fragments of the Anopheles gambia wild type HMGB1 Box is a fragment of at least 77 or at least 54 amino acids respectively and comprises the sequences of amino acids as defined in SEQ ID NO: 419 or 529 respectively, and wherein the polypeptide variant of said biologically active fragments of Anopheles gambia HMGB1 Box A is selected from the group consisting of amino acid sequences as defined in any of SEQ ID NO: 420 to 528 or 530 to 610.

14.

 A pharmaceutical composition comprising an effective amount of at least one polymer conjugate of any of the preceding claims as an active agent, and optionally together with pharmaceutically acceptable carriers, adjuvants, diluents or / and additives.

fifteen.

 The composition of claim 14 for diagnostic applications or for therapeutic applications.

16.

 Use of a polymer conjugate of any one of claims 1 to 13 for the manufacture of a medicament for the prevention, relief or treatment of pathologies associated with HMGB1 or pathologies associated with HMGB1 homologous proteins, particularly selected from pathological conditions mediated by activation of the cascade of inflammatory cytokines.

17.

 The use of claim 16, wherein the pathological conditions are selected from the group consisting of inflammatory disease, autoimmune disease, systemic inflammatory response syndrome, reperfusion injury after organ transplantation, cardiovascular conditions, obstetric and gynecological disease, disease infectious (viral and bacterial), allergic and atopic disease, pathologies of solid and liquid tumors, transplant rejection diseases, congenital diseases, dermatological diseases, neurological diseases, cachexia, kidney diseases, iatrogenic intoxication conditions, metabolic and idiopathic diseases, and ophthalmic diseases

18.

 Use of a polymer conjugate of any one of claims 1 to 13 for the manufacture of a medicament for the prevention, relief or treatment of pathologies related to RAGE, in particular type I and / or type II diabetes.

19.

 The use of any one of claims 16 to 18 in combination with an additional agent capable of inhibiting an early mediator of the inflammatory cytokine cascade, wherein the additional agent is particularly selected from

(i)

 an antagonist or inhibitor of a cytokine selected from the group consisting of TNF, IL-1, IL-1, IL-Ra, IL-6, IL-8, IL-10, IL-13, IL-18, IFN -, MIP-1, MIF-1, MIP-2, MIF and PAF;

(ii)

 an antibody against RAGE, a nucleic acid or a nucleic acid analog capable of inhibiting the expression of RAGE, for example an antisense molecule, a ribozyme or an RNA interference molecule, or a small synthetic molecule antagonizing the interaction of HMGB1 with RAGE or soluble RAGE (sRAGE);

(iii) an inhibitor of the interaction of a Toll-like receptor (TLR), in particular of TLR2, TLR4, TLR7, TLR8 or / and TLR9, with HMGB1, preferably a monoclonal or polyclonal antibody, a nucleic acid or an analogue of nucleic acid capable of inhibiting the expression of TLR, for example an antisense molecule, a ribozyme or an RNA interference molecule, or a synthetic molecule having a size smaller than 1000 Daltons.

twenty.

 The composition of claim 14, wherein the at least one polymer conjugate is in combination with the at least one additional agent as defined in claim 19.

twenty-one.

 Medical device reversibly coated with at least one polymer conjugate of any one of claims 1 to 13.