GB2500184A - Polypeptide derived from helix A of heparin cofactor II - Google Patents

Abstract

The present invention provides polypeptides comprising or consisting of an amino acid sequence derived from helix A of heparin cofactor II, or a biologically active fragment or variant thereof, together with isolated nucleic acid molecules, vectors and host cells for making the same. In particular, the invention provides polypeptides comprising or consisting of an amino acid sequence of SEQ ID NO: 1, or a biologically active fragment or variant thereof, for use in the treatment and prevention of infection, inflammation and/or excessive coagulation. Additionally provided are pharmaceutical compositions comprising a polypeptide of the invention, as well as methods of use of the same in the treatment and/or prevention of inflammation and/or excessive coagulation. Exemplified are compositions consisting of the amino acid sequence of SEQ ID No 1: GKSRIQRLNILNAKFAFNLYRVLKDQ

Description

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POLYPEPTIDES AND USES THEREOF

Field of the invention

10 The present invention relates to novel polypeptides derived from helix A of heparin cofactor II (HCII), and their use in medicine. In particular, the invention provides polypeptides comprising or consisting of an amino acid sequence of SEQ ID NO: 1, or a biologically active fragment or variant thereof, for use in the treatment and prevention of microbial infection, inflammation and/or excessive coagulation.

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Introduction

Infectious and inflammatory diseases account for millions of deaths worldwide each 20 year and incur tremendous health care costs. The disease spectrum is broad and includes acute disease, such as erysipelas, sepsis, pneumonia and numerous other infections, having a direct association to a given pathogen, as well as chronic diseases, where microbes often cause a long-standing inflammatory state. Sepsis is an infection-induced syndrome characterized by a generalized inflammatory state 25 and represents a frequent complication in the surgical patient, in immunocompromized patients, or in relation to burns. Severe sepsis is a common, expensive and frequently fatal condition, having a documented worldwide incidence of 1.8 million each year, but this number is confounded by a low diagnostic rate and difficulties in tracking sepsis in many countries. It is estimated that with an incidence 30 of 3 in 1000 the true number of cases each year reaches 18 million, and with a mortality rate of almost 30% it becomes a leading cause of death worldwide. Sepsis costs on average US$22 000 per patient, and its treatment therefore has a great impact on hospitals' financial resources, with US$16.7 billion each year being spent in the USA alone. The cost of treating an ICU patient with sepsis is six times greater 35 than that of treating a patient without sepsis. In other settings, harmful inflammatory cascades are initiated by other mechanisms than bacterial, such as during trauma, surgery, extracorporeal circulation, ischemia, burns, drug reactions, hemorrhagic

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shock, toxic epidermal necrolysis, and transfusion reactions, leading to ARDS or SIRS. Chronic obstructive pulmonary disorder (COPD) refers to a range of chronic disorders in the airways characterized by irreversible and progressing decline in airflow to the lung capillaries. Although several factors contribute to the development 5 of COPD, smoking and recurring infections are the most important causes. COPD predominantly develops in long-term smokers from their late-30s and progressively develops in an irreversible fashion. According to 2007 estimates by WHO, there are currently 210 million patients with COPD, and 3 million people died of COPD in 2005. WHO also predicts that COPD will become the fourth leading cause of death 10 worldwide by 2030. Several factors are expected to contribute to this increase, including increased diagnosis rates, lack of treatments that reverse the inflammatory disease progression, and a globally ageing population burden. Microbes cause, and/or aggravate, a spectrum of diseases including bacterial conjunctivitis and keratitis, otitis, postoperative and burn wound infections, chronic leg ulcers, 15 pneumonia, and cystic fibrosis.

New agents addressing infection are therefore needed, and there is significant interest in the potential use of AMPs as novel treatment modalities (Marr, A. K., W. J. Gooderham, et al. (2006). Curr Opin Pharmacol 6(5): 468-472). Considering the 20 increasing resistance problems against conventional antibiotics, antimicrobial peptides have recently emerged as potential therapeutic candidates. AMPs provides a first line of defense against invading microbes in almost all organisms (Tossi, A., L. Sandri, et al. (2000). Biopolvmers 55(1): 4-30; Lehrer, R. I. and T. Ganz (2002). Curr Opin Hematol 9(1): 18-22; Zasloff, M. (2002). Nature 415(6870): 389-95; Yount, N. 25 Y., A. S. Bayer, et al. (2006). Biopolvmers 84: 435-458; Harder, J., R. Glaser, et al. (2007). J Endotoxin Res 13(6): 317-38). Ideally, AMP should display high bactericidal potency, but low toxicity against (human) eukaryotic cells. Various strategies, such as use of combinational library approaches (Blondelle, S. E. and K. Lohner (2000). Biopolvmers 55(1): 74-87), stereoisomers composed of D-amino acids (Sajjan, U. S., 30 L. T. Tran, et al. (2001). Antimicrob Agents Chemother 45(12): 3437-44) or cyclic D,L-a-peptides ( Fernandez-Lopez, S., H. S. Kim, et al. (2001). Nature 412(6845): 452-5), high-throughput based screening assays (Hilpert, K., R. Volkmer-Engert, et al. (2005). Nat Biotechnol 23(8): 1008-12; Taboureau, O., O. H. Olsen, et al. (2006). Chem Biol Drug Pes 68(1): 48-57), quantitative structure-activity relationship (QSAR) 35 approaches (Hilpert, K., R. Volkmer-Engert, et al. (2005). Nat Biotechnol 23(8): 1008-12; Marr, A. K., W. J. Gooderham, et al. (2006). Curr Opin Pharmacol 6(5): 468-472; Jenssen, H., T. Lejon, et al. (2007). Chem Biol Drug Pes 70(2): 134-42; Pasupuleti,

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M., B. Walse, et al. (2008). Biochemistry 47(35): 9057-70), and identification of endogenous peptides ( Papareddy, P., V. Rydengard, et al. PLoS Pathoq 6(4): e1000857; Nordahl, E. A., V. Rydengard, et al. (2005). J Biol Chem 280(41): 34832-9; Malmsten, M., M. Davoudi, et al. (2006). Matrix Biol 25(5): 294-300; Malmsten, M., 5 M. Davoudi, et al. (2007). Growth Factors 25(1): 60-70; Pasupuleti, M., B. Walse, et al. (2007). J Biol Chem 282(4): 2520-8) are currently employed for identifying selective and therapeutically interesting AMPs (Hancock, R. E. and H. G. Sahl (2006). Nat Biotechnol 24(12): 1551-7; Marr, A. K., W. J. Gooderham, et al. (2006). Curr Opin Pharmacol 6(5): 468-472). Despite the potential of these approaches, 10 naturally occuring peptide epitopes may show advantages in a therapeutic setting considering low immunogenecity as well as inherent additional biological functions.

The coagulation cascade also represents a fundamental system activated in response to injury and infection (Davie, E.W. and J.D. Kulman, Semin Thromb 15 Hemost, 2006. 32 SuppI 1: p. 3-15; Bode, W., Semin Thromb Hemost, 2006. 32 SuppI 1: p. 16-31). Through a series of cascade-like proteinase activation steps, thrombin is formed, leading to fibrinogen degradation and clot formation. The coagulation cascade is controlled by various regulatory proteins, such as heparin cofactor II (HCII), antithrombin III (ATIII) (two serine proteinase inhibitors, serpins), 20 protein C inhibitor, and tissue factor proteinase inhibitor (TFPI) Furthermore, histidine-rich glycoprotein may modulate coagulation by interacting with fibrinogen as well as plasminogen.

Heparin cofactor II is a 66.5 kDa, 480 amino acid glycoprotein present in plasma at 25 ~80 ug/ml. However, although HCII blocks free and clot-associated thrombin, its exact physiological role is not fully understood. Similar to antithrombin III, the inhibition of thrombin by HCII is accelerated by glycosaminoglycans, such as heparin (Tollefsen, 1995 Thromb Haemost. 74(5): 1209-14.). While ATIII deficiency is clearly linked to thrombosis, HCII homozygous deficient mice do not suffer from 30 thrombophilia under normal conditions. Plasma concentrations of HCII are significantly decreased during inflammation and infection (Noda et al. (2002), Clin. Appl. Thromb. Hemost.. 8(3): 265-271). Indeed, recent evidence suggest that the primary physiological function of HCII is to inhibit thrombin's non-hemostatic roles such as in the development of atherosclerosis ( Rau, J. C., L. M. Beaulieu, et al. 35 (2007). J Thromb Haemost 5 SuppI 1: 102-15). It has also been shown that HCII could function as an extravascular thrombin inhibitor and may play a role in the regulation of wound healing (Hoffman, Loh et al. 2003), and furthermore,

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chemotactic products have been described upon proteolysis of HCII ( Hoffman, M., C. W. Pratt, et al. (1990). J Leukoc Biol 48(2): 156-62), further illustrating the potential latent biological activities of this antiproteinase. Structural studies on HCII have revealed that the molecule undergoes an unusual conformational change, 5 termed the Stressed to Relaxed (S to R) transition. The inventors have made the unexpected discovery that a series of peptides derived from HCII have antiinflammatory and anticoagulative functions, thus representing a previously unknown property of HCII derived peptides.

10 The present invention seeks to provide new polypeptide agents, derived from heparin cofactor II, for use in the treatment or prevention of microbial infection, inflammation and/or excessive coagulation of the blood.

15 Summary of the invention

The invention derives from the unexpected discovery by the inventors that heparin cofactor II (HCII) comprises "cryptic peptides" within its internal regions capable of exhibiting anti-bacterial and anti-inflammatory activity. It is believed that such 20 peptides represent an active epitope which is conformationally activated during proteolysis.

Thus, a first aspect of the invention provides a polypeptide comprising or consisting of an amino acid sequence derived from helix A of heparin cofactor II, or a 25 biologically active fragment or variant thereof.

By an amino acid sequence "derived from helix A" of heparin cofactor II, we mean that the amino acid sequence is found within (i.e. shares 100% sequence identity with) the amino acid sequence of helix A of heparin cofactor II.

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By "helix A" of heparin cofactor II, we include the amino acid sequence corresponding to amino acids S121 to D143 (Swiss Port Accession No. P05546) or S102 to D124 (numbering without signal peptide) of human heparin cofactor II.

35 It will be appreciated by persons skilled in the art that the polypeptide may be isolated, i.e. provided in a form in which it is not found in nature. For example, the

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polypeptide may be substantially pure, provided in a cell-free preparation, dissolved in solution and/or otherwise provided in a pharmaceutical formulation.

In addition to polypeptides which comprise or consist of the an amino acid sequence 5 derived from helix A of heparin cofactor II, the invention also extends to biologically active fragments and variants of such amino acid sequences.

By "biologically active" in this context we mean that the fragment or variant retains (at least in part) at least one biological activity of the polypeptide of SEQ ID NO: 1 below. 10 In particular, the fragment or variant may retain (at least in part) an anti-bacterial and/or anti-inflammatory and/or anti-coagulant activity of the polypeptide of SEQ ID NO: 1.

By "anti-bacterial" activity we mean an ability to attenuate the growth and/or to kill 15 bacterial cells (Gram positive bacteria and/or Gram negative bacteria). Such antibacterial activity of polypeptides may be determined using methods well known in the art (for example as described in Examples A and B below). In particular, we include the ability to attenuate the growth and/or to kill bacteria of the species Pseudomonas aeruginosa.

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By "anti-inflammatory activity" we mean an ability to reduce or prevent one or more biological processes associated with inflammatory events. Such anti-inflammatory activity of polypeptides may be determined using methods well known in the art, for example by measuring LPS-induced release of pro-inflammatory cytokines from 25 macrophages (e.g. TNFa, IL-6, IF-y), or neutrophils (see Examples below). Other relevant assays comprise effects of lipoteichoic acid, zymosan, DNA, RNA, flagellin or peptidoglycan in the above systems as well as determination of regulation at the transcriptional level (e.g. Gene-array, qPCR etc). Furthermore, dendritic cell activation or activation of thrombocytes may also be used as a measure of anti-30 inflammatory activity.

By "anti-coagulant activity" we mean an ability to increase the prothrombin time (PT), the thrombin clotting time (TCT) and/or the activated partial thromboplastin time (aPTT). Alternatively, peripheral blood mononuclear cells (PBMNC)s can be 35 stimulated by E. coli LPS with or without the peptide and tissue factor and clot formation followed after addition of human plasma, or clotting times for whole blood can be measured.

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In one embodiment of the polypeptides of the invention, the polypeptide is not a naturally occurring protein, e.g. a holoprotein (although it will, of course, be appreciated that the polypeptide may constitute an incomplete portion or fragment of 5 a naturally occurring protein).

It will be appreciated by persons skilled in the art that the heparin cofactor II may be from a human or non-human source. For example, the heparin cofactor II may be derived (directly or indirectly) from a non-human mammal, such as an ape 10 (e.g. chimpanzee, bonobo, gorilla, gibbon and orangutan), monkey (e.g. macaque, baboon and colobus), rodent (e.g. mouse, rat) or ungulates (e.g. pig, horse and cow).

In a preferred embodiment, however, the heparin cofactor II is human heparin cofactor II (for example, see Swiss Port Accession No. P05546).

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In a further embodiment of the polypeptides of the invention, the polypeptide comprises a heparin-binding region. By "heparin-binding region" we mean an amino acid sequence within the polypeptide which is capable of binding heparin under physiological conditions. Such sequences often comprise XBBXB and XBBBXXB 20 (where B = basic residue and X = hydropathic or uncharged residue), or clusters of basic amino acids (XBX, XBBX, XBBBX). Spacing of such clusters with non-basic residues (BXB, BXXB) may also occur. Additionally, a distance of approximately 20 A between basic amino acids constitutes a prerequisite for heparin-binding.

25 In a related embodiment of the polypeptides of the invention, the polypeptide is capable of binding lipopolysaccharide (LPS).

In one preferred embodiment of the first aspect of the invention, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:1:

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"GKS2&': GKSRIQRLNILNAKFAFNLYRVLKDQ [SEQ ID NO:1]

or a biologically active fragment or variant thereof.

35 It will be appreciated by persons skilled in the art that the term 'amino acid', as used herein, includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the'd' form (as compared to the natural 'l' form),

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omega-amino acids other naturally-occurring amino acids, unconventional amino acids (e.g., a,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids (see below).

5 When an amino acid is being specifically enumerated, such as 'alanine' or 'Ala' or 'A', the term refers to both l-alanine and d-alanine unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where 10 appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.

In one embodiment, the polypeptides of the invention comprise or consist of l-amino acids.

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Where the polypeptide comprises an amino acid sequence according to a reference sequence (i.e. SEQ ID NO: 1), it may comprise additional amino acids at its N- and/or C- terminus beyond those of the reference sequence, for example, the polypeptide may comprise additional amino acids at its N-terminus. Likewise, where the 20 polypeptide comprises a fragment or variant of an amino acid sequence according to a reference sequence, it may comprise additional amino acids at its N- and/or C-terminus.

It will be appreciated by persons skilled in the art that the polypeptides of the 25 invention may be of various lengths. In one embodiment, the polypeptide is between 10 and 200 amino acids in length, for example between 10 and 150, 15 and 100, 15 and 50, 15 and 30, 20 and 30, or 25 and 27 amino acids in length. For example, the polypeptide may be at least 20 amino acids in length.

30 In one embodiment, the polypeptide comprises or consists of a fragment of the amino acid sequence of SEQ ID NO: 1. Thus, the polypeptide may comprise or consist of at least 5 contiguous amino acid of the reference sequence, for example at least 5 contiguous amino acids of SEQ ID NO: 1, for example at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acids of SEQ 35 ID NO: 1.

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In one embodiment the polypeptide fragment commences at an amino acid residue selected from amino acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of SEQ ID NO:1. Alternatively/additionally, the polypeptide fragment may terminate at an amino acid residue selected from amino 5 acid residues 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 of SEQ IDNO:1.

For example, the polypeptide fragment may comprise or consist of amino acids 3 to 25 of SEQ ID NO: 1.

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Persons skilled in the art will further appreciate that the polypeptide of the invention may comprise or consist of a variant of the amino acid sequence of SEQ ID NO: 1, or fragment of said variant. Such a variant may be non-naturally occurring.

15 By 'variants' of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. For example, conservative substitution refers to the substitution of an amino acid within the same general class (e.g. an acidic amino acid, a basic amino acid, a non-polar amino acid, a polar amino acid or an aromatic amino acid) by another amino acid within the same class. Thus, the 20 meaning of a conservative amino acid substitution and non-conservative amino acid substitution is well known in the art. In particular we include variants of the polypeptide which exhibit an anti-bacterial and/or anti-inflammatory activity.

In a further embodiment the variant has an amino acid sequence which has at least 25 50% identity with the amino acid sequence of SEQ ID NO: 1, or with a fragment thereof, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

The percent sequence identity between two polypeptides may be determined using 30 suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (as 35 described in Thompson et al., 1994, Nuc. Acid Res. 22:4673-4680, which is incorporated herein by reference).

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The parameters used may be as follows:

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

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

Scoring matrix: BLOSUM.

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

In one embodiment of the polypeptides of the invention, one or more amino acids 15 from the above reference sequence may be mutated in order to reduce proteolytic degradation of the polypeptide, for example by l,F to W modifications (see Stromstedt et al, Antimicrobial Agents Chemother 2009, 53, 593).

Variants may be made using the methods of protein engineering and site-directed 20 mutagenesis well known in the art using the recombinant polynucleotides (see example, see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2000, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference).

25 In one embodiment, the polypeptide comprises or consists of an amino acid which is a species homologue of SEQ ID NO: 1. By "species homologue" we include that the polypeptide corresponds to the same amino acid sequence within an equivalent protein from a non-human species, i.e. which polypeptide exhibits the maximum sequence identity with SEQ ID NO: 1 (for example, as measured by a GAP or BLAST 30 sequence comparison). Typically, the species homologue polypeptide will be the same length as the human reference sequence (i.e. SEQ ID NO: 1).

In a still further embodiment, the polypeptide comprises or consists of a fusion protein.

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By 'fusion' of a polypeptide we include an amino acid sequence corresponding to SEQ ID NO: 1, or a fragment or variant thereof, fused to any other polypeptide.

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For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art.

5 Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. In addition, fusions comprising a hydrophobic oligopeptide end-tag may be used. Fusions to any variant or derivative of said polypeptide are also included in the scope of the invention. It will be appreciated that fusions (or variants or derivatives thereof) 10 which retain desirable properties, such as an anti-inflammatory activity, are preferred.

The fusion may comprise a further portion which confers a desirable feature on the said polypeptide of the invention; for example, the portion may be useful in detecting or isolating the polypeptide, or promoting cellular uptake of the polypeptide. The 15 portion may be, for example, a biotin moiety, a streptavidin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art. The moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting 20 cellular uptake of the polypeptide, as known to those skilled in the art.

In one embodiment, the polypeptide of the invention is a fusion polypeptide comprising a polypeptide derived from helix D of heparin cofactor II.

25 For example, the polypeptide derived from helix D of heparin cofactor II may comprise or consist of the amino acid sequence of any one of SEQ ID NOS: 2 to 4:

or a fragment or variant thereof which retains a biological activity of polypeptide of any one of SEQ ID NOS: 2 to 4.

35 It will be appreciated by persons skilled in the art that the polypeptide of the invention may comprise one or more amino acids that are modified or derivatised, for example by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

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KYE28'\ KYEITTIHNLFRKLTHRLFRRNFGYTLR KYE21": KYEITTIHNLFRKLTHRLFRR NLF20": NLFRKLTHRLFRRNFGYTLR

[SEQ ID NO:2] [SEQ ID NO:3] [SEQ ID NO:4]

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As appreciated in the art, Pegylated proteins may exhibit a decreased renal clearance and proteolysis, reduced toxicity, reduced immunogenicity and an increased solubility [Veronese, F.M. and J.M. Harris, Adv Drug Deliv Rev, 2002.

5 54(4): p. 453-6., Chapman, A.P., Adv Drug Deliv Rev, 2002. 54(4): p. 531-45.]. Pegylation has been employed for several protein-based drugs including the first pegylated molecules asparaginase and adenosine deaminase [Veronese, F.M. and J.M. Harris, Adv Drug Deliv Rev, 2002. 54(4): p. 453-6., Veronese, F.M. and G. Pasut, Drug Discov Today, 2005.10(21): p. 1451-8.].

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In order to obtain a successfully Pegylated protein, with a maximally increased half-life and retained biological activity, several parameters that may affect the outcome are of importance and should be taken into consideration. The PEG molecules may differ, and PEG variants that have been used for Pegylation of proteins include PEG 15 and monomethoxy-PEG. In addition, they can be either linear or branched [Wang, Y.S., et al., Adv Drug Deliv Rev, 2002. 54(4): p. 547-70]. The size of the PEG molecules used may vary and PEG moieties ranging in size between 1 and 40 kDa have been linked to proteins [Wang, Y.S., et al., Adv Drug Deliv Rev, 2002. 54(4): p. 547-70., Sato, H., Adv Drug Deliv Rev, 2002. 54(4): p. 487-504, Bowen, S., et al., 20 Exp Hematol, 1999. 27(3): p. 425-32, Chapman, A.P., et al., Nat Biotechnol, 1999. 17(8): p. 780-3], In addition, the number of PEG moieties attached to the protein may vary, and examples of between one and six PEG units being attached to proteins have been reported [Wang, Y.S., et al., Adv Drug Deliv Rev, 2002. 54(4): p. 547-70., Bowen, S., et al., Exp Hematol, 1999. 27(3): p. 425-32]. Furthermore, the presence 25 or absence of a linker between PEG as well as various reactive groups for conjugation have been utilised. Thus, PEG may be linked to N-terminal amino groups, or to amino acid residues with reactive amino or hydroxyl groups (Lys, His, Ser, Thr and Tyr) directly or by using y-amino butyric acid as a linker. In addition, PEG may be coupled to carboxyl (Asp, Glu, C-terminal) or sulfhydryl (Cys) groups. 30 Finally, Gin residues may be specifically Pegylated using the enzyme transglutaminase and alkylamine derivatives of PEG has been described [Sato, H., Adv Drug Deliv Rev, 2002. 54(4): p. 487-504].

It has been shown that increasing the extent of Pegylation results in an increased in 35 vivo half-life. However, it will be appreciated by persons skilled in the art that the Pegylation process will need to be optimised for a particular protein on an individual basis.

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PEG may be coupled at naturally occurring disulphide bonds as described in WO 2005/007197. Disulfide bonds can be stabilised through the addition of a chemical bridge which does not compromise the tertiary structure of the protein. This 5 allows the conjugating thiol selectivity of the two sulphurs comprising a disulfide bond to be utilised to create a bridge for the site-specific attachment of PEG. Thereby, the need to engineer residues into a peptide for attachment of to target molecules is circumvented.

10 A variety of alternative block copolymers may also be covalently conjugated as described in WO 2003/059973. Therapeutic polymeric conjugates can exhibit improved thermal properties, crystallisation, adhesion, swelling, coating, pH dependent conformation and biodistribution. Furthermore, they can achieve prolonged circulation, release of the bioactive in the proteolytic and acidic 15 environment of the secondary lysosome after cellular uptake of the conjugate by pinocytosis and more favourable physicochemical properties due to the characteristics of large molecules (e.g. increased drug solubility in biological fluids). Block copolymers comprising hydrophilic and hydrophobic blocks form polymeric micelles in solution. Upon micelle disassociation, the individual block copolymer 20 molecules are safely excreted.

Chemical derivatives of one or more amino acids may also be achieved by reaction with a functional side group. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine 25 hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain 30 naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite 35 activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications.

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It will be further appreciated by persons skilled in the art that peptidomimetic compounds may also be useful. Thus, by 'polypeptide' we include peptidomimetic compounds which have an anti-inflammatory activity. The term 'peptidomimetic' 5 refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent.

For example, the polypeptides of the invention include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in 10 which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al. (1997) J. Immunol. 159, 3230-3237, which is incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse 15 peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Alternatively, the polypeptide of the invention may be a peptidomimetic compound wherein one or more of the amino acid residues are linked by a -y(CH2NH)- bond in place of the conventional amide linkage.

20 In a further alternative, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it may be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond.

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It will be appreciated that the polypeptide may conveniently be blocked at its N- or C-terminal region so as to help reduce susceptibility to exoproteolytic digestion.

A variety of uncoded or modified amino acids such as d-amino acids and N-methyl 30 amino acids have also been used to modify mammalian peptides. In addition, a presumed bioactive conformation may be stabilised by a covalent modification, such as cyclisation or by incorporation of lactam or other types of bridges, for example see Veber et al., 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thursell et al., 1983, Biochem. Biophys. Res. Comm. 111:166, which are incorporated herein by 35 reference.

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A common theme among many of the synthetic strategies has been the introduction of some cyclic moiety into a peptide-based framework. The cyclic moiety restricts the conformational space of the peptide structure and this frequently results in an increased specificity of the peptide for a particular biological receptor. An added 5 advantage of this strategy is that the introduction of a cyclic moiety into a peptide may also result in the peptide having a diminished sensitivity to cellular peptidases.

Thus, exemplary polypeptides of the invention comprise terminal cysteine amino acids. Such a polypeptide may exist in a heterodetic cyclic form by disulphide bond 10 formation of the mercaptide groups in the terminal cysteine amino acids or in a homodetic form by amide peptide bond formation between the terminal amino acids. As indicated above, cyclising small peptides through disulphide or amide bonds between the N- and C-terminal region cysteines may circumvent problems of specificity and half-life sometime observed with linear peptides, by decreasing 15 proteolysis and also increasing the rigidity of the structure, which may yield higher specificity compounds. Polypeptides cyclised by disulphide bonds have free amino and carboxy-termini which still may be susceptible to proteolytic degradation, while peptides cyclised by formation of an amide bond between the N-terminal amine and C-terminal carboxyl and hence no longer contain free amino or carboxy termini. 20 Thus, the peptides of the present invention can be linked either by a C-N linkage or a disulphide linkage.

The present invention is not limited in any way by the method of cyclisation of peptides, but encompasses peptides whose cyclic structure may be achieved by any 25 suitable method of synthesis. Thus, heterodetic linkages may include, but are not limited to formation via disulphide, alkylene or sulphide bridges. Methods of synthesis of cyclic homodetic peptides and cyclic heterodetic peptides, including disulphide, sulphide and alkylene bridges, are disclosed in US 5,643,872, which is incorporated herein by reference. Other examples of cyclisation methods includes 30 cyclization through click chemistry, epoxides, aldehyde-amine reactions, as well as and the methods disclosed in US 6,008,058, which is incorporated herein by reference.

A further approach to the synthesis of cyclic stabilised peptidomimetic compounds is 35 ring-closing metathesis (RCM). This method involves steps of synthesising a peptide precursor and contacting it with an RCM catalyst to yield a conformational^ restricted peptide. Suitable peptide precursors may contain two or more unsaturated C-C

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bonds. The method may be carried out using solid-phase-peptide-synthesis techniques. In this embodiment, the precursor, which is anchored to a solid support, is contacted with a RCM catalyst and the product is then cleaved from the solid support to yield a conformationally restricted peptide.

5

Another approach, disclosed by D. H. Rich in Protease Inhibitors, Barrett and Selveson, eds., Elsevier (1986), which is incorporated herein by reference, has been to design peptide mimics through the application of the transition state analogue concept in enzyme inhibitor design. For example, it is known that the secondary 10 alcohol of staline mimics the tetrahedral transition state of the scissile amide bond of the pepsin substrate.

In summary, terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion and therefore to prolong the half-life of the 15 peptides in solutions, particularly in biological fluids where proteases may be present. Polypeptide cyclisation is also a useful modification because of the stable structures formed by cyclisation and in view of the biological activities observed for cyclic peptides.

20 Thus, in one embodiment the polypeptide of the first aspect of the invention is cyclic. However, in a preferred embodiment, the polypeptide is linear.

As stated at the outset, the polypeptides of the invention exhibit one or more of the biological activities of helix A of heparin cofactor II, most notably anti-microbial 25 activity, anti-inflammatory activity and/or anti-coagulant activity.

Thus, the polypeptides may be capable of inhibiting the growth and/or number of microorganisms, such as bacteria, mycoplasmas, yeasts, fungi and/or viruses.

30 In one embodiment, the polypeptide is capable of inhibiting the growth of bacteria, for example Pseudomonas aeruginosa (see Examples), Escherichia coii, Staphylococcus aureus, or Group A streptococci.

In a further embodiment, the polypeptides are capable of inhibiting the release of one 35 or more pro-inflammatory cytokines from human monocyte-derived macrophages, such as monocyte-derived macrophages, including macrophage inhibitory factor,

15

TNF-alpha, HMGB1, C5a, IL-17, IL-8, MCP-1, IFN-gamma, II-6, IL-1b, IL-12. Antiinflammatory IL-10 may be unaffected or transiently increased.

Other markers may also be affected: These include tissue factor on monocytes and 5 endothelial cells, procalcitonin, CRP, reactive oxygen species, bradykinin, nitric oxide, PGE1, platelet activating factor, arachidonic acid metabolites, MAP kinase activation.

In particular, the polypeptide may exhibit anti-inflammatory activity in one or more of 10 the following models:

(i) in vitro macrophage models using microbial stimulants such as LPS, LTA, zymosan, flagellin, viral or bacterial DNA or RNA, or peptidoglycan as well as endogenous damage associate molecular patterns (DAMPs), such as 15 DNA, HMGB1, or S100 proteins;

(ii) in vivo mouse models of endotoxin shock; and/or

(iii) in vivo infection models, either in combination with antimicrobial therapy, 20 or given alone.

In a further embodiment of the invention, the polypeptide exhibits anticoagulant activity.

25 By "anti-coagulant activity" we mean an ability to reduce or prevent coagulation {i.e. the clotting of blood) or an associated signal or effect. Such activity may be determined by methods well known in the art, for example using the activated partial thromboplastin time (aPTT) test, prothrombin time (PT) test or the thrombin clotting time (TCT) test. Furthermore, specific measurements of prekallikrein activation or the 30 activity of Factor X and other coagulation factors may be performed. In a preferred embodiment, the polypeptide inhibits (at least in part) the intrinsic coagulation pathway.

In a still further embodiment of the invention, the polypeptide exhibits Toll-like 35 receptor (TLR) blocking activity. Such receptor blocking activity can be measured using methods well known in the art, for example by analysis of suitable down-stream effectors, such as iNOS, nuclear factor kappa B and cytokines.

16

The present invention includes pharmaceutical^ acceptable acid or base addition salts of the above described polypeptides. The acids which are used to prepare the pharmaceutical^ acceptable acid addition salts of the aforementioned base 5 compounds useful in this invention are those which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulphonate, ethanesulphonate, 10 benzenesulphonate, p-toluenesulphonate and pamoate [i.e. 1,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others.

Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutical^ acceptable salt forms of the polypeptides. The chemical bases that 15 may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations (e.g. calcium and 20 magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.

It will be appreciated that the polypeptides of the invention may be lyophilised for 25 storage and reconstituted in a suitable carrier prior to use, e.g. through freeze drying, spray drying, spray cooling, or through use of particle formation (precipitation) from supercritical carbon dioxide. Any suitable lyophilisation method (e.g. freeze-drying, spray drying, cake drying) and/or reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can 30 lead to varying degrees of activity loss and that use levels may have to be adjusted upward to compensate. Preferably, the lyophilised (freeze dried) polypeptide loses no more than about 1% of its activity (prior to lyophilisation) when rehydrated, or no more than about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or no more than about 50% of its activity (prior to lyophilisation) when rehydrated.

35

Methods for the production of polypeptides of the invention are well known in the art.

17

Conveniently, the polypeptide is or comprises a recombinant polypeptide. Suitable methods for the production of such recombinant polypeptides are well known in the art, such as expression in prokaryotic or eukaryotic hosts cells (for example, see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, 5 Cold Spring Harbor, New York, the relevant disclosures in which document are hereby incorporated by reference).

Polypeptides of the invention can also be produced using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheatgerm lysate 10 (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system may be coupled to a transcription system, such as the TNT transcription-translation system (Promega). This system has the advantage of producing suitable mRNA transcript from an encoding DNA polynucleotide in the same reaction as the translation.

15

It will be appreciated by persons skilled in the art that polypeptides of the invention may alternatively be synthesised artificially, for example using well-known liquid-phase or solid phase synthesis techniques (such as £-Boc or Fmoc solid-phase peptide synthesis).

20

Thus, included within the scope of the present invention are the following related aspects:

(a) a second aspect of the invention provides an isolated nucleic acid molecule 25 which encodes a polypeptide according to the first aspect of the invention;

(b) a third aspect of the invention provides a vector (such as an expression vector) comprising a nucleic acid molecule according to the second aspect of the invention;

30

(c) a fourth aspect of the invention provides a host cell comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention; and

35 (d) a fifth aspect of the invention provides a method of making a polypeptide according to the second aspect of the invention comprising culturing a population of host cells according to the fourth aspect of the invention under

18

conditions in which said polypeptide is expressed, and isolating the polypeptide therefrom.

A sixth aspect of the invention provides a pharmaceutical composition comprising a 5 polypeptide according to the first aspect of the invention together with a pharmaceutically acceptable excipient, diluent or carrier.

As used herein, 'pharmaceutical composition' means a therapeutically effective formulation for use in the treatment or prevention of disorders and conditions 10 associated with microbial infection, inflammation and/or excessive coagulation of the blood.

The pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals. 15 The pharmaceutical compositions may be lyophilised, e.g. through freeze-drying, spray-drying, spray cooling, or through use of particle formation from supercritical particle formation.

By "pharmaceutically acceptable" we mean a non-toxic material that does not 20 decrease the effectiveness of the biological activity of the active ingredients, i.e. the anti-inflammatory polypeptide(s). Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed ., Pharmaceutical Press (2000).

25

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, 30 AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The term "diluent" is intended to mean an aqueous or non-aqueous solution with the 35 purpose of diluting the peptide in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

19

The term "adjuvant" is intended to mean any compound added to the formulation to increase the biological effect of the peptide. The adjuvant may be one or more of colloidal silver, or zinc, copper or silver salts with different anions, for example, but 5 not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. The adjuvant may also be cationic polymers such as PHMB, cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as polyvinyl 10 imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrines, 15 which are added to the composition, e.g., for facilitating lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose, carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, 20 polyethylenglycol/polyethylene oxide, polyethyleneoxide/ polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, poly(lactic acid), poly(glycolic acid) or copolymers thereof with various composition, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g. for viscosity control, for achieving bioadhesion, or for protecting the 25 active ingredient from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc 30 oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

The pharmaceutical composition may also contain one or more mono- or di-sacharides such as xylitol, sorbitol, mannitol, lactose, lactitiol, isomalt, maltitol or 35 xylosides, and/or monoacylglycerols, such as monolaurin. The characteristics of the carrier are dependent on the route of administration. One route of administration is topical administration. For example, for topical administrations, a preferred carrier is

20

an emulsified cream comprising the active peptide, but other common carriers such as certain petrolatum/mineral-based and vegetable-based ointments can be used, as well as polymer gels, liquid crystalline phases and microemulsions.

5 Additional compounds may also be included in the pharmaceutical compositions, such as chelating agents (for example EDTA, citrate, EGTA or glutathione).

It will be appreciated that the pharmaceutical compositions may comprise one or more polypeptides of the invention, for example one, two, three or four different 10 peptides. By using a combination of different peptides the therapeutic benefits to the patient may be increased.

As discussed above, the polypeptide may be provided as a salt, for example an acid adduct with inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, 15 hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, boric acid etc. or with organic acid such as formic acid, acetic acid, haloacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, sulfanilic acid etc. Inorganic salts such as monovalent 20 sodium, potassium or divalent zinc, magnesium, copper calcium, all with a corresponding anion, may be added to improve the biological activity of the antimicrobial composition.

The pharmaceutical compositions of the invention may also be in the form of a 25 liposome, in which the polypeptide is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. 30 Suitable lipids also include the lipids above modified by poly(ethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations is can be found in for example US4,235,871.

The pharmaceutical compositions of the invention may also be in the form of 35 biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PI_A and PGA (PLGA) or poly(carprolactone) (PCL), and polyanhydrides have been widely used as

21

biodegradable polymers in the production of microshperes. Preparations of such microspheres can be found in US 5,851,451 and in EP 213 303.

The pharmaceutical compositions of the invention may also be formulated with 5 micellar systems formed by surfactants and block copolymers, preferably those containing poly(ethylene oxide) moieties for prolonging bloodstream circulation time.

The pharmaceutical compositions of the invention may also be in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose, 10 carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates, chitosan, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylenglycol/polyethylene oxide, polyethylene-oxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinyl-15 acetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the peptide. The polymers may also comprise gelatin or collagen.

Alternatively, the polypeptides of the invention may be dissolved in saline, water, 20 polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers.

The pharmaceutical composition may also include ions and a defined pH for potentiation of therapeutic action of polypeptides of the invention.

25

The compositions of the invention may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc., e.g., as disclosed elsewhere herein.

30

It will be appreciated by persons skilled in the art that the pharmaceutical compositions of the invention may be administered locally or systemically. Routes of administration include topical, ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral, vaginal and rectal. Also 35 administration from implants is possible. Suitable preparation forms are, for example granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, microemulsions, defined as optically isotropic thermodynamically stable

22

systems consisting of water, oil and surfactant, liquid crystalline phases, defined as systems characterised by long-range order but short-range disorder (examples include lamellar, hexagonal and cubic phases, either water- or oil continuous), or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, 5 aerosols, droplets or injectable solution in ampoule form and also preparations with protracted release of active compounds, in whose preparation excipients, diluents, adjuvants or carriers are customarily used as described above. The pharmaceutical composition may also be provided in bandages, plasters or in sutures or the like.

10 In preferred embodiments, the pharmaceutical composition is suitable for parenteral administration or topical administration.

In alternative preferred embodiments, the pharmaceutical composition is suitable for pulmonary administration or nasal administration.

15

For example, the pharmaceutical compositions of the invention can be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, 20 trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution 25 or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a polypeptide of the invention and a suitable powder base such as lactose or starch.

30

Aerosol or dry powder formulations are preferably arranged so that each metered dose or 'puff' contains at least 0.1 mg of a polypeptide of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in 35 divided doses throughout the day.

The pharmaceutical compositions will be administered to a patient in a

23

pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose is dependent on the, activity of the compound, manner of administration, nature and severity of the disorder, age and 5 body weight of the patient different doses may be needed. The administration of the dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.

10 The pharmaceutical compositions of the invention may be administered alone or in combination with other therapeutic agents, such as anti-inflammatory, immunosuppressive, vasoactive and/or antiseptic agents (such as anti-bacterial agents, anti-fungicides, anti-viral agents, and anti-parasitic agents). Examples of suitable antibiotic agents include penicillins, cephalosporins, carbacephems,

15 cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide. Likewise, the pharmaceutical compositions may also contain additional anti-inflammatory drugs, such as steroids

20 and macrolactam derivatives.

In one embodiment, the pharmaceutical compositions of the invention are administered in combination with a steroid, for example a glucocorticoid (such as dexamethasone).

25

It will be appreciated by persons skilled in the art that the additional therapeutic agents may be incorporated as part of the same pharmaceutical composition or may be administered separately.

30 In one embodiment of the seventh aspect of the invention, the pharmaceutical composition is associated with a device or material to be used in medicine (either externally or internally). By 'associated with' we include a device or material which is coated, impregnated, covalently bound to or otherwise admixed with a pharmaceutical composition of the invention (or polypeptide thereof).

35

For example, the composition may be coated to a surface of a device that comes into contact with the human body or component thereof (e.g. blood), such as a device

24

used in by-pass surgery, extracorporeal circulation, wound care and/or dialysis. Thus, the composition may be coated, painted, sprayed or otherwise applied to or admixed with a suture, prosthesis, implant, wound dressing, catheter, lens, skin graft, skin substitute, fibrin glue or bandage, etc. In so doing, the composition may impart 5 improved anti-microbial, anti-inflammatory and/or anti-coagulant properties to the device or material.

Preferably, the device or material is coated with the pharmaceutical composition of the invention (or the polypeptide component thereof). By 'coated' we mean that the 10 pharmaceutical composition is applied to the surface of the device or material. Thus, the device or material may be painted or sprayed with a solution comprising a pharmaceutical composition of the invention (or polypeptide thereof). Alternatively, the device or material may be dipped in a reservoir of a solution comprising a polypeptide of the invention.

15

Advantageously, the device or material is impregnated with a pharmaceutical composition of the invention (or polypeptide thereof). By 'impregnated' we mean that the pharmaceutical composition is incorporated or otherwise mixed with the device or material such that it is distributed throughout.

20

For example, the device or material may be incubated overnight at 4°C in a solution comprising a polypeptide of the invention. Alternatively, a pharmaceutical composition of the invention (or polypeptide thereof) may be immobilised on the device or material surface by evaporation or by incubation at room temperature.

25

In an alternative embodiment, a polypeptide of the invention is covalently linked to the device or material, e.g. at the external surface of the device or material. Thus, a covalent bond is formed between an appropriate functional group on the polypeptide and a functional group on the device or material. For example, methods for covalent 30 bonding of polypeptides to polymer supports include covalent linking via a diazonium intermediate, by formation of peptide links, by alkylation of phenolic, amine and sulphydryl groups on the binding protein, by using a poly functional intermediate e.g. glutardialdehyde, and other miscellaneous methods e.g. using silylated glass or quartz where the reaction of di- and trialkoxysilanes permits derivatisation of the 35 glass surface with many different functional groups. For details, see Enzyme immobilisation by Griffin, M., Hammonds, E.J. and Leach, C.K. (1993) In Technological Applications of Biocatalysts (BIOTOL SERIES), pp. 75-118,

25

Butterworth-Heinemann. See also the review article entitled 'Biomaterials in Tissue Engineering' by Hubbell, J.A. (1995) Science 13:565-576.

In a preferred embodiment, the device or material comprise or consists of a polymer. 5 The polymer may be selected from the group consisting of polyesters (e.g. polylactic acid, polyglycolic acid or poly lactic acid-glycolic acid copolymers of various composition), polyorthoesters, polyacetals, polyureas, polycarbonates, polyurethanes, polyamides) and polysaccharide materials (e.g. cross-linked alginates, hyaluronic acid, carageenans, gelatines, starch, cellulose derivatives).

10

Alternatively, or in addition, the device or material may comprise or consists of metals (e.g. titanium, stainless steel, gold, titanium), metal oxides (silicon oxide, titanium oxide) and/or ceramics (apatite, hydroxyapatite).

15 Such materials may be in the form of macroscopic solids/monoliths, as chemically or physicochemically cross-linked gels, as porous materials, or as particles.

Thus, the present invention additionally provides devices and materials to be used in medicine, to which have been applied a polypeptide of the invention or 20 pharmaceutical composition comprising the same.

Such devices and materials may be made using methods well known in the art.

A seventh aspect of the invention provides a polypeptide according to the first aspect 25 of the invention or a pharmaceutical composition according to the sixth aspect of the invention for use in medicine.

In particular, the polypeptides and pharmaceutical compositions of the invention are for use:

30

(a) the treatment and/prevention of microbial infection (e.g. bacterial infection);

(b) the treatment and/prevention of acute and/or chronic inflammation;

(c) the modulation of blood coagulation; and/or

(d) the treatment of wounds.

35

Thus, in one embodiment, the polypeptides and pharmaceutical compositions of the invention the treatment and/prevention of microbial infection.

26

By "treatment and/prevention" of microbial infection we mean that the polypeptide of the invention is capable of attenuating the growth (at least in part) and/or killing microbial organisms.

5

It will be appreciated by persons skilled in the art that attenuating the growth of microbial organisms may be in whole or in part. In a preferred embodiment, the polypeptide is capable of attenuating the growth by 20% or more compared to microbial organisms which have not been exposed to the polypeptide, for example by 10 at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

Methods for determining such antimicrobial properties are well known in the art (see Examples).

15 Advantageously, the polypeptides of the invention are capable of attenuating the growth and/or killing microbial organisms selectively. By 'selectively', in this context, we mean that the polypeptide attenuates the growth and/or kills microbial organisms to a greater extent than it attenuates the growth and/or kills of non-microbial (e.g. mammalian) cells.

20

It will be appreciated that the polypeptides and pharmaceutical compositions may be used for killing a number of types of microorganism, including bacteria, mycoplasmas, yeasts, fungi and/or viruses. It will be further appreciated that the polypeptides and pharmaceutical compositions may be used to prevent and/or treat 25 infection with such microorganisms, i.e. the medicaments are suitable for prophylactic and/or therapeutic treatment. For example, the polypeptides and pharmaceutical compositions may be used to prevent or reduce the spread or transfer of a pathogen to other subjects, e.g. patients, healthcare workers, etc.

30 Preferably, the polypeptides and pharmaceutical compositions are for use in the curative and/or prophylactic treatment of bacterial infections such as Gram positive cocci (e.g. Streptococcus), Gram negative cocci (e.g. Neisseria), Gram positive bacilli (e.g. Corynebacterium species), Gram negative bacilli (e.g. Escherichia coli), acid-fast bacilli (e.g. a typical Mycobacterium) and including infections causing abscesses, 35 cysts, blood infection (bacteraemia), dermatological infections, wound infections, arthritis, urinary tract infections, pancreatitis, pelvic inflammatory disease, peritonitis, prostatitis, infections of the vagina, oral cavity (including dental infections), eye

27

and/or ear, ulcers and other localised infections; actinomyces infections; fungal infections such as Candida albicans, Aspergillus and Blastomyces; viral infections such as HIV, encephalitis, gastro-enteritis, haemorrhagic fever, hantavirus, viral hepatitis, herpesvirus (e.g. cytomegalovirus, Epstein-Barr, herpesvirus simiae, 5 herpes simplex and varicella-zoster); protozoal infections such as amoebiasis, babesiosis, coccidiosis, cryptosporidiosis, giardiasis, Leishmaniasis, Trichomoniasis, toxoplasmosis and malaria; helminthic infections such as caused by nematodes, cestodes and trematodes, e.g. ascariasis, hookworm, lymphatic filariasis, onchocerciasis, schistosomiasis and toxocariasis; prion diseases; and inflammatory 10 diseases such as soft-tissue rheumatism, osteoarthritis, rheumatoid arthritis and spondyloarthropathies.

More preferably, the polypeptides and pharmaceutical compositions are for use in the curative and/or prophylactic treatment of infections by Gram positive bacteria and/or 15 Gram negative bacteria.

Most preferably, the polypeptides and pharmaceutical compositions are for use in the curative and/or prophylactic treatment of infections by Gram negative bacteria, such as Pseudomonas aeruginosa.

20

The polypeptides and pharmaceutical compositions of the invention are also suitable for killing bacteria which have developed resistance to conventional antibiotic treatments, such as methicillin-resistant Staphylococcus aureus (MRSA).

25 In a further embodiment, the polypeptides of the first aspect of the invention are intended for use in the treatment or prevention of inflammation.

By "treatment or prevention of inflammation" we mean that the polypeptide of the invention is capable of preventing or inhibiting (at least in part) one or more symptom, 30 signal or effect constituting or associated with inflammation.

It will be appreciated by persons skilled in the art that inhibition of inflammation may be in whole or in part. In a preferred embodiment, the polypeptide is capable of inhibiting one or more markers of inflammation by 20% or more compared to cells or 35 individuals which have not been exposed to the polypeptide, for example by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

28

Advantageously, the polypeptides of the invention are capable of treating or preventing inflammation selectively.

By 'selectively' we mean that the polypeptide inhibits or prevents inflammation to a 5 greater extent than it modulates other biological functions. Preferably, the polypeptide or fragment, variant, fusion or derivative thereof inhibits or prevents inflammation only.

In a still further embodiment, the polypeptide also (or alternatively) inhibits or 10 prevents coagulation of the blood. As above, it will be appreciated by persons skilled in the art that inhibition of coagulation may be in whole or in part. In a preferred embodiment, the polypeptide is capable of inhibiting one or more measures and/or markers of coagulation by 20% or more compared to cells or individuals which have not been exposed to the polypeptide, for example by at least 30%, 40%, 50%, 60%, 15 70%, 80%, 90% or more.

In preferred but non-limiting embodiments of the invention, the polypeptides are for use in the treatment or prevention of a disease, condition or indication selected from the following:

20

i) Acute systemic inflammatory disease, with or without an infective component, such as systemic inflammatory response syndrome (SIRS), ARDS, sepsis, severe sepsis, and septic shock. Other generalized or localized invasive infective and inflammatory disease, 25 including erysipelas, meningitis, arthritis, toxic shock syndrome,

diverticulitis, appendicitis, pancreatitis, cholecystitis, colitis, cellulitis, burn wound infections, pneumonia, urinary tract infections, postoperative infections, and peritonitis.

30 ii) Chronic inflammatory and or infective diseases, including cystic fibrosis, COPD and other pulmonary diseases, gastrointestinal disease including chronic skin and stomach ulcerations, other epithelial inflammatory and or infective disease such as atopic dermatitis, oral ulcerations (aphtous ulcers), genital ulcerations and 35 inflammatory changes, parodontitis, eye inflammations including conjunctivitis and keratitis, external otitis, mediaotitis, genitourinary inflammations.

29

iii) Postoperative inflammation. Inflammatory and coagulative disorders including thrombosis, DIC, postoperative coagulation disorders, and coagulative disorders related to contact with foreign material, including

5 extracorporeal circulation, and use of biomaterials. Furthermore,

vasculitis related inflammatory disease, as well as allergy, including allergic rhinitis and asthma..

iv) Excessive contact activation and/or coagulation in relation to, but not 10 limited to, stroke.

v) Excessive inflammation in combination with antimicrobial treatment. The antimicrobial agents used may be administred by various routes; intravenous (iv), intraarterial, intravitreal, subcutaneous (sc),

15 intramuscular (im), intraperitoneal (ip), intravesical, intratechal,

epidural, enteral (including oral, rectal, gastric, and other enteral routes), or topically, (including dermal, nasal application, application in the eye or ear, eg by drops, and pulmonary inhalation). Examples of agents are penicillins, cephalosporins, carbacephems, cephamycins, 20 carbapenems, monobactams, aminoglycosides, glycopeptides,

quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide.

25 For example, the polypeptides may be for use in the treatment or prevention of an acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, wounds, asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis, thrombosis and/or disseminated intravascular coagulation (DIC).

30

In one embodiment, the polypeptide exhibits anti-microbial and anti-inflammatory and anti-coagulant activity and may be used in the concomitant treatment or prevention of infection, inflammation and coagulation. Such polypeptides may be particularly suited to the treatment and prevention of conditions where the combined inhibition of 35 both bacterial growth, as well as inflammatory and coagulant processes, is desirable, namely sepsis, chronic obstructive pulmonary disorder (COPD), thrombosis, DIC and acute respiratory distress syndrome (ARDS).

30

Furthermore, other diseases associated with excessive inflammation and coagulation changes may benefit from treatment by the polypeptides, such as cystic fibrosis, asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis.

5

In a further embodiment, the polypeptides of the invention are for use in combination with one or more additional therapeutic agent. For example, the polypeptides of the invention may be administered in combination with antibiotic agents, antiinflammatory agents, immunosuppressive agents and/or antiseptic agents, as well as 10 vasoactive agents and/or receptor-blockers or receptor agonists. The antimicrobial agents used may be applied iv, sc, im, intratechal, per os, or topically. Examples of agents are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, 15 clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide. For example, the peptides of the invention may serve as adjuvants to antiseptic treatments, for example silver/PHMB treatment of wounds to quench LPS effects.

20 Thus, the peptides of the invention may serve as adjuvants (for blocking inflammation) to complement antibiotic, antiseptic and/or antifungal treatments of internal and external infections (such as erysipelas, lung infections including fungal infections, sepsis, COPD, wounds, and other epithelial infections). Likewise, the peptides of the invention may serve as adjuvants to antiseptic treatments, for 25 example silver/PHMB treatment of wounds to quench LPS effects.

In one embodiment, the polypeptides of the invention are for use in combination with an anti-bacterial agent, such as a cephalosporin antibiotic agent {e.g. ceftazidime). Such combination therapies may provide a synergistic effect in the treatment or 30 prevention of bacterial infection (e.g. sepsis induced by P. aeruginosa; see Examples below).

In a further preferred embodiment, the polypeptides of the invention are for use in combination with penetration enhancing agents, such as poly(ethyleneimine), or 35 antibiotic agents which exhibit such penetration-enhancing capability (e.g. polymyxin or colistin).

31

Dosages of the polypeptides the invention to be used will depend on several factors; including the particular polypeptide used, the formulation, route of administration and the indication for which the polypeptide is used. Typically, however, dosages will range from 0.01 to 25 mg of polypeptide per kilogram of body weight, preferably from 5 0.1 to 15 mg/kg, for example from 1 to 10 mg/kg of body weight.

A related eighth aspect of the invention provides the use of a polypeptide according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention in the preparation of a medicament for the treatment or 10 prevention of microbial infection and/or inflammation and/or excessive coagulation (as described above).

A ninth aspect of the invention provides a method for treating or preventing microbial infection and/or inflammation and/or excessive coagulation in a patient, the method 15 comprising administering to the patient a therapeutically-effective amount of a polypeptide according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention (as described above).

In preferred but non-limiting embodiments, the method is for the treatment or 20 prevention of an acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis, thrombosis and/or disseminated intravascular coagulation (DIC).

25 Persons skilled in the art will further appreciate that the polypeptides of the present invention have utility in both the medical and veterinary fields. Thus, the polypeptides may be used in the treatment of both human and non-human animals (such as horses, dogs and cats). Advantageously, however, the patient is human.

30

Preferred aspects of the invention are described in the following non-limiting examples, with reference to the following figures:

Figure 1: HCII suppresses bacterial growth ex vivo and in vivo.

35 a, P. aeruginosa was grown in mouse blood from HCII+/+, HCII"'" and HCII"'" supplemented with 100 pg/ml HCII (HCII"/'(+)). Cfu evaluation after 6 h (HCII+/+ n=19, HCII"'" n=18, HCII"/"(+) n=13). b-e, HCII+/+ and HCII"'" mice were infected

32

intraperitoneally with P. aeruginosa and analysis was performed after 12 h. (b) Bacterial counts (HCII+/+ n=17, HCII"'" n=21), (c) cytokines in plasma (HCII+/+ n=8, HCII"'" n=8), and (d) platelet counts (HCII+/+ n=17, HCir'" n=18) in comparison to non-infected mice (HCII+/+ n=9, HCII"'" n=8). e, Determination of aPTT and PT 5 coagulation times (HCII+/+ n=7, HCII"'" n=8). f, Bacterial counts in organs of HCII+/+ and HCII"'" mice subcutaneously infected with P. aeruginosa (HCII+/+ n=7, HCII"'" n=6). g, Cytokine levels in mouse plasma 20 h after injection of E. coli LPS (HCII+/+ n=14, HCH"/"n=13). (*p<0.05, **p<0.01 and ***p<0.001, ns = not significant)

10 Figure 2: HCII is decreased and fragmented during infection and inflammation.

a, b, HCII+/+ mice were injected intraperitoneally with P. aeruginosa bacteria or E. coli LPS. (a) HCII detection in mouse plasma by immunoblotting (Con; plasma from non-infected mice), (b) HCII concentration in plasma 12 or 20 h after P. aeruginosa infection or LPS injection (n=10). c, Detection of HCII in mouse tissues. B-actin levels 15 are shown for comparison (representative blot out of 3 experiments is shown), d, HCII gene expression levels in mouse liver (n=6). e, Six acute wound fluid samples (AWF, left), one AWF sample incubated for 1 hour with increasing amounts of HLE (jjg) (middle) and six chronic wound fluid samples (CWF, right) were analysed by SDS-PAGE and western blot (CP; human citrate plasma).

20

Figure 3: Fragmented HCII binds to bacteria and is antimicrobial.

a, SDS-PAGE analysis of HCII subjected to HLE. b, Comparison of HCII (black) and HLE digested HCII (blue) using heparin affinity chromatography (FPLC). c, Binding of HCII, HCIIa (HLE+HCII) and HLE to 125l-labelled LPS. d, e, Binding of native or HLE-25 cleaved HCII to E. coli bacteria, (d) Binding was detected using flow cytometry. The graph represents a typical experiment of three (Control: bacteria only), (e) Evaluation of HCII binding using pull down assays. A representative western blot is shown (S; supernatant, P; pellet), f, Evaluation of antimicrobial activity of HLE, HCII or HCIIa against E. coli bacteria using a radial diffusion assay. Inhibition zones (as shown) 30 were measured and presented as means with standard deviation (n=3). g, Antimicrobial effect of HCII preparations against E. coli in viable count assays. Means with s.e.m. are presented (n=3). h, i, HCIIa binds bacterial surfaces as determined by electron microscopy, h, Analysis of E. coli treated with HCII or HLE-cleaved HCII in 10 mM Tris, pH 7.4. i, Acute wound fluid incubated with buffer or HLE 35 was added to P. aeruginosa bacteria. HCII was visualised using gold-labelled antibodies (-). (CWS) Representative scanning electron micrograph of fibrin slough from chronic wounds stained for HCII with gold-labelled antibodies.

33

Figure 4: Structural and functional analyses of HCII and HCII-derived peptides.

a, Electron micrographs of native HCII or HLE-cleaved HCII (HCIIa) followed by incubation with LPS or LPS with Heparin. Arrows: white; native HCII, blue; HCIIa and 5 yellow; LPS. b, Illustration of HCII structure. Exposed helical peptide regions are indicated: Helix A; GKS26 (green), Helix D; KYE28 (orange), SGM22 (magenta), SDP18 (cyan) and LKS23 (yellow), c, LPS binding of HCII-derived peptides to 125l-labelled LPS. d, Antimicrobial activity of HCII peptides against £ coli in 10 mM Tris, 0.15 M NaCI with or without 20 % citrate plasma (n=3; mean±SD). e, RAW 264.7 10 cells stimulated with E. coli LPS and peptides. Nitrite levels were determined after 20 h (n=3). f, Cytokines in human blood 20 hours after stimulation with £ coli LPS and peptides (n=3).

Figure 5: Analyses of coagulation parameters. a,b The activated partial thrombin 15 time (aPTT) and prothrombin time (PT) were determined in fresh citrate plasma (a) derived from mouse blood ex vivo infected with P. aeruginosa for 6 h (HCII+/+ n=7-9; HCII"'" n=8-9). (b) Clotting times were measured 20h after injection of mice with £ coli LPS (HCII+/+ n=6-13; HCII"'" n= 9-12). (Broken lines indicate normal values.)

20 Figure 6: Sequence analysis of HCII. a, Sequence of HCII. Arrows indicating the cleavage sites of HLE. b, HLE digested HCII was transferred to a PVDF membrane, fragments were visualized by coomassie staining and indicated bands were characterized by N-terminal sequencing, c, Summary of detected fragments and their net-charge.

25

Figure 7: HPLC of intact and degraded HCII, and analyses of HCIIa. Intact or degraded HCII was analysed by HPLC. The indicated fractions of cleaved HCII (arrow) were pooled, and analysed. Results of RDA (left) against E.coli, SDS-PAGE (middle) as well 125l-labelled LPS binding data (right) are shown as inserts within the 30 HPLC graph.

Figure 8: Control experiments to Figure 3H. Electron micrographs are displaying non-treated E.coli bacteria (upper panel) and E.coli treated with HCII, HLE and Heparin (lower panel).

34

Figure 9: Control experiments to Figure 3i. Visualization of HCII (gold-labelled) at P. aeruginosa surfaces in patient-derived acute wound fluid treated with buffer (upper panel) or HLE and heparin (lower panel).

5 Figure 10: In vitro properties of GKS26 (A) Antibacterial effects of GKS26 against £ coli ATCC 25922 in viable count assays. 2 x 106 cfu/ml of bacteria were incubated with increasing peptide concentrations in 0.15 m NaCI, 10 mM Tris, pH 7.4 or 0.15 m NaCI, 10 mM Tris containing 20% human citrate plasma (CP) (n=3). Bacteria were determined after 18 hours. (B) The effect of increasing concentrations of GKS26 on 10 liposomes was assessed in 10 mM Tris using DOPE/DOPG liposomes. (C) RAW 264.7 macrophages were stimulated with 10 ng/ml E. coli LPS in combination with indicated concentrations of GKS26. Nitric oxide production was determined in the celi supernatants using the Griess reaction (n=3).

15 Figure 11: Effects of GKS26 in vivo (A-F) Septic shock in C57BL6 mice was induced by intraperitoneal (ip) injection of £. coli LPS (18 mg/kg) followed by ip injection of 0.2 or 0.5 mg of GKS26 or buffer. (A) Scoring of mice status 20 hours after LPS injection. (1; healthy, 4; septic shock). (B) Measurement of total white blood cell counts 20 hours after LPS injection in blood. (C) Lungs of non-treated 20 (Control), LPS treated and LPS and GKS26 treated mice were analysed 20 hours after LPS injection. Representative light microscopy images stained with haematoxylin-eosin (magnification 4x) are shown. (C) Number of platelets was determined in mice, 20 hours after injection of LPS and treatment with GKS26 (D) Activated partial thromboplastin time (aPTT) and prothrombin time (PT) were 25 measured using citrate plasma from mice 20 hours after injection of the indicated substances. (Data are from 1 experiment, n=5).

Figure 12: Treatment of Pseudomonas aeruginosa induced sepsis by GKS26

(A) C57BI6 mice were infected with 2x109 cfu/ml P. aeruginosa (ip). GKS26 (0.5mg) 30 alone, ceftazidime alone (AB) (300mg/kg) or a combination of both was subcutaneously administered 1.5 hour and 4.5 hours after injection of bacteria. The survival was monitored for 7 days. (Control n= 11, GKS26 only; n=5, ceftazidime only; n=14, ceftazidime with GKS26; n= 15).

35

35

EXAMPLE A

Proteolytic activation transforms heparin cofactor II into a host defence molecule

5

Abstract

The abundant serine proteinase inhibitor (serpin) heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin and to modulate neointimal smooth 10 muscle cell proliferation and atherosclerosis1. However, the exact physiological role of this plasma protein remained enigmatic. Here, we demonstrate a previously unknown role for HCII in host defence. Animals deficient in HCII showed increased susceptibility to lipopolysaccharide (LPS) induced shock and invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. 15 Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with LPS or bacteria. The host defence action of HCII was dependent on proteolytic activation of the molecule leading to a conformational change, conferring upon the molecule anti-endotoxic and antimicrobial properties. The protease induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host 20 defence factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.

Methods

25 Proteins, peptides, patient materials

Human HCII was obtained from Enzyme Research Laboratories. The following peptides were synthesized by Biopeptide Co., San Diego, USA (>95% purity):

Human acute and chronic wound fluids and chronic wound sloughs (CWS) were collected as previously described16. The research project was approved by the Ethics

30

KYE28: KYEITTIHNLFRKLTHRLFRRNFGYTLR

GKS26: GKSRIQRLNILNAKFAFNLYRVLKDQ)

LKG23: LKGETHEQVHSILHFKDFVNASS)

SDP18: SDPAFISKTNNHIMKLTK)

and

SGM22: SGMKTLEAQLTPRWERWQKSM)

[SEQ ID NO:2] [SEQ ID NO:1] [SEQ ID NO:5] [SEQ ID NO:6]

[SEQ ID NO:7]

35

36

committee at Lund University Hospital.

Microorganisms and cells

Bacterial isolates of Escherichia coli ATCC 25922 and Pseudomonas aeruginosa 5 15159 (Department of Bacteriology, Lund University Hospital) were cultured in Tod-Hewitt broth. Mouse macrophages RAW 264.7 (ATCC, Rockville, MD) were cultured in Dulbecco's modified Eagle medium (PAA laboratories) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen) and 1% (v/v) Antibiotic-Antimycotic solution (Invitrogen).

10

Animal models

All animal studies were approved by the Laboratory Animal Ethics Committee of Malmo/Lund, Sweden. Wild type C57BL/6 (HCII+/+) and HCII knockout (HCII"'") mice4 were bred in the local facilities. Age and sex matched mice were used between 2 and 15 3 months of age. i) Infection models. P. aeruginosa bacteria were grown to logarithmic phase (OD62o~0.5) washed twice and diluted in PBS. The bacterial suspension was injected either intraperitoneally (2 x 108 cfu /mouse) or subcutaneous (0.4-1 x 109 cfu/mouse). To study bacterial dissemination spleen, liver and kidney were harvested, placed on ice, homogenized, and colony-forming units (cfu) 20 were determined, ii) LPS model. Mice were intraperitoneally injected with 12 mg/kg E. coli 0111:B4 LPS (Sigma).

Platelet analysis

Mouse blood (anti-coagulated with EDTA) was taken by cardiac puncture and the 25 number of platelets was determined with the VetScan HM5 System (TRIOLAB).

Cytokine analyses

Cytokines were measured in mouse and human plasma using the Mouse Inflammation Kit (Becton Dickinson AB) and the BioSource CytoSet™ (Invitrogen).

30

Coagulation assays

Activated partial thromboplastin time (aPTT) was measured by incubating 50 pi citrated plasma for 1 minute followed by the addition of 50 pi Dapttin (Technoclone) for 200 seconds at 37°C. Clotting was initiated by the addition of 50 pi of CaCI2 35 (30 mM). For the prothrombin time (PT) clotting was initiated by addition of 50 pi TriniClot-PT Excel (TrinityBiotech).

37

Degradation of HCII

Heparin cofactor II (13.5 |jg, 0.6 pg/ml) was incubated at 37°C with human leukocyte elastase (HLE) (0.3 |jg, 20 units/pg) (Sigma) in a total volume of 45 pi in 10 mM Tris, 5 pH 7.4 for different lengths of time. The reaction was stopped by boiling at 95°C for 3 min before samples were analysed. For antimicrobial assays, the material was directly used. HCII degradation in wound fluid was assessed by incubation of wound fluid with different concentrations of HLE at 37°C for 1 h. The material was then subjected to SDS-PAGE under reducing conditions followed by immunoblotting.

10

SDS-PAGE and western blot

For sample separation with SDS-PAGE, 16.5% Tris-tricine gels (Bio-Rad) were used, followed by staining with Coomassie brilliant blue. For identification of HCII fragments in chronic leg ulcer wound fluids, 1.5 fil of sample were analysed by SDS-PAGE 15 under reducing conditions. Proteins from mouse tissues were isolated using a protein purification kit (Total Protein Extraction Kit, Chemicon). Subsequent to separation by SDS-PAGE proteins and peptides were transferred to nitrocellulose membranes (Hybond-C). Membranes were blocked by 3% (w/v) skimmed milk, washed, and incubated with human (1:1000) (Innovative Research) or mouse polyclonal HCII-20 antibodies (1:1000) (R&D Systems). After washing HRP-conjugated secondary antibodies (1:2000) (Dako) were applied and HCII bands were visualized by the Supersignal West Pico Chemiluminesent substrate developing system (Thermo Scientific). In pull down assays 50 pi of E. coli bacteria (1-2 x 109 cfu) were incubated with native or HLE-cleaved HCII for 2 h at 37°C. Heparin (10 mg/ml) was 25 added for binding competition. After centrifugation the bacterial pellet was washed with PBS, and bound proteins were eluted with 0.1 M glycine-HCI, pH 2.0. The pH of the eluted material was raised to 7.5 with 1 M Tris. Eluted proteins were precipitated by addition of TCA to the sample (1:4), followed by incubation for 30 min on ice and centrifugation at 15,000 x g (4 °C for 30 min). Precipitated material and supernatants 30 were dissolved in SDS sample buffer and analyzed under reducing conditions followed by western blot.

Expression of HCII

Total RNA was isolated from mouse liver using trizol reagent (Invitrogen). cDNA was 35 synthesized using iScript cDNA synthesis kit (Bio-Rad). HCII and B-actin real-time quantitative PCR was performed using the iQ SYBR Green Supermix (Bio-Rad) on

38

an iCycler real-time PCR detection system (Bio-Rad). The primer sequences used were: HCII forward, 5'-CCA GCG TCT TAA TAT CCT CAA TGC-3' [SEQ ID NO: 8] and reverse, 5'-TGC CAA CAG GTG CTA TGA AGA GG-3' [SEQ ID NO: 9]; 0-actin forward, 5'-TGA CGA GGC CCA GAG CAA GAG-3' [SEQ ID NO: 10] and reverse, 5 5'-CGC AGC TCA TTG TAG AAG GTG TG-3' [SEQ ID NO: 11], Real-time PCR was run in duplicates from six different cDNAs and AACt method was used to determine relative expression of mRNAs.

FPLC

10 Fifteen pg of native or digested HCII were subjected to FPLC (GE Healthcare AKTA purifier) using a HiTrap™ 1 ml Heparin HP columns (Amersham Biosciences). After injection, samples were eluted with a linear gradient of 0-0.8 M NaCI in 10 mM Tris, pH, 7.4.

15 Slot-blot assay

Peptides (1, 2 and 5 ^g) or proteins (2 and 5 |jg) were bound to nitrocellulose membranes (Hybond-C, GE Healthcare Biosciences), pre-soaked in PBS. Membranes were blocked by 2 wt% BSA in PBS, pH 7.4, for 1 h at RT, and subsequently incubated 1 h with 125l-labelled LPS (40 ng/ml; 0.13x10® cpm/|jg) with 20 or without heparin (10 mg/ml).. After incubation, membranes were washed in PBS and radioactivity was visualised by the Bas 2000 radioimaging system (Fuji).

Flow cytometry analysis

E. coli bacteria (1-2 x 109 cfu) were preincubated with buffer, native or cleaved HCII 25 with or without heparin for 30 min at 37°C. The samples were then divided into two equal parts, centrifuged, washed resuspended in 100 pi PBS with or without rabbit polyclonal anti-human HCII antibodies. After incubation for 1 h at RT, bacteria were pelleted, washed and incubated in 100 pi PBS containing goat anti rabbit IgG FITC-labelled antibodies (1:500, Sigma) for 30 min at RT. Flow cytometry analysis was 30 performed using a FACS Calibur (Becton-Dickinson).

Antimicrobial assays

The antimicrobial activity against E. coli was assessed by radial diffusion and viable count assays. Methods were performed as described previously12,19. For 35 determination of cfu in citrated mouse blood, samples were infected with P. aeruginosa grown overnight (10:1 ratio), and incubated under rotation for 2-6 h at

39

37°C. Every second hour aliquots were taken and cfu were determined. Survival (%) was normalised to survival of non-treated bacteria under the same conditions.

Electron microscopy

5 For transmission electron microscopy and visualisation of HCII effects on bacteria, 5 |j| of E. coli or P. aeruginosa (1-2 x 109 cfu/sample) were incubated for 2 h at 37°C in the absence or presence of heparin (10 mg/ml) and with i) HCII (8 pg) incubated with buffer or HLE (0.3 pg) for 30 min, ii) human acute wound fluid (5 pi) incubated alone or supplemented with HLE (2.5 pg) for 1 h. HCII was visualised using a polyclonal 10 goat anti-human HCII AB which was gold labelled with EM-Rabbit anti-Goat IgG (H&L); 20 nm Gold (BB International). For the negative staining suspensions were adsorbed onto 400 mesh carbon-coated copper grids and stained with 0.75% (w/v) uranyl formate as described previously12. Fibrin slough from patients with chronic venous ulcers (CWS) was processed as recently described12. The labelling of HCII 15 was performed as for the negative staining. All samples were observed in a FEI Tecnai BioTWIN (North America NanoPort Hillsbro, Oregon USA) transmission electron microscope operated at 60kV accelerating voltage. Images were recorded with an Eagle™ 2k CCD camera.

20 LPS assay in vitro

3.5x10s RAW 264.7 cells were stimulated with 10 ng/ml E. coli LPS (0111:B4) with and without peptides. NO levels in supernatants were determined after 20 h using the Griess reaction20 or whole blood was stimulated 20 hours with 100ng/m E. coli LPS and peptides.

25

High performance liquid chromatography (HPLC)

Peptide/protein fragments of HCII, digested by HLE, were separated by HPLC (Perkin Elmer Series 200) on a reversed phase column (Perkin Elmer®, BROWNLEE BIO C18 5 pM 250x4.6 mm). After injection, samples were eluted with a gradient of 30 acetonitrile in 0.1% aqueous trifluroacetic acid at 1 ml per minute. Fractions were collected and stored at -80°C. Samples were freeze-dried, dissolved in water, and analysed by RDA, SDS-PAGE, or for binding to 125l-labelled LPS.

Statistics

35 Cfu data from several ex vivo or in vivo experiments were pooled and the median is indicated. In other experiments data are presented as mean from 2-3 experiments,

40

error bars indicate s.e.m. Statistically significance was evaluated using the Mann-Whitney U-test with *p<0.05, **p<0.01 and ***p<0.001.

Results and Discussion

5

The serpins have evolved into a family of structurally related molecules with diverse functions in human biology. Being the most abundant proteinase inhibitors in humans, serpins regulate multiple proteolytic pathways in blood and tissues2. Antithrombin III (ATIII) and heparin cofactor II (HCII), which are present in human 10 blood, inhibit proteases of the coagulation cascade2,3. Although current data indicate a pivotal role for ATIII in coagulation control, much less is known about the exact function of HCII. For example, while inherited deficiency of ATIII is clearly associated with thrombotic disorders, neither humans nor mice deficient in HCII present any evidence for thrombophilia under normal conditions3"5. However, both molecules 15 interact with heparin and other glycosaminoglycans in a similar manner, undergoing significant conformational changes that facilitate protease inhibition by the common serpin mechanism2. In contrast to ATIII, HCII exploits its conformational mobility in a unique way. HCII has an unusual N-terminal acidic region, which upon binding to glycosaminoglycans is expelled from HCII's cationic site, followed by proteolytic 20 scission of the flexible N-terminal tail6. Proteolysis of HCII by leukocyte elastase releases chemoattractant peptides derived from the N-terminal tail7. Thus, although HCII shares similar overall conformational characteristics with other serpins, mounting evidence suggests that HCII may have evolved novel biological functions2. For instance, while ATIII levels are reduced in almost all conditions associated with 25 disseminated intravascular coagulation, reduced plasma levels of HCII are only detected during infections8'10 Increasing evidence pointing to a close link between haemostasis, proteolysis, and host defence11"14 raised the hypothesis that HCII could play a role in host defence against infections.

30 Initial experiments were conducted to investigate whether a lack of HCII affects bacterial growth ex vivo. P. aeruginosa bacteria showed an enhanced growth in whole blood from HCII knockout (HCII"'") mice when compared to blood from wild type C57BL/6 mice (HCH+/+). The capacity to kill bacteria was restored by addition of physiological amounts of HCII to blood from HCII"'" mice (Fig. 1a). Coagulation 35 assays showed a similar prolongation of the activated partial thromboplastin time (aPTT) and prothrombin times (PT) in both groups, indicating a similar activation of the coagulation system in plasma derived from HCII+/+ and HCII"7" mice during

41

infection ex vivo (Fig. 5a). In order to investigate a potential anti-infective role for HCII in vivo, HCII+/+ and HCII"'" mice were infected intraperitoneally with P. aeruginosa bacteria, and bacterial dissemination was recorded in the spleen, kidney, and liver of the animals (Fig. 1b). Analogously to the ex vivo experiments, significantly higher 5 bacterial levels were observed in HCII"'" mice when compared with control mice. This was accompanied by significantly elevated levels of cytokines, particularly IL-6 and interferon-y (Fig. 1c). A significant reduction in platelet counts (Fig. 1d), but no alterations in PT and aPTT were observed in HCII"'" animals vis-a-vis controls (Fig. 1e). In an infection model using subcutaneous administration of bacteria, HCII"'" mice 10 also displayed higher bacterial counts in the analyzed organs compared to wild type animals (Fig. 1f).

Since the increased cytokine levels detected in the infection model above could be related to higher bacterial levels in HCII"'" mice, we set out to determine possible 15 immunomodulatory effects of HCII per se, using a (bacteria-free) model of LPS-induced shock. Both HCM+/+ and HCII"'" mice showed increased concentrations of pro-inflammatory cytokines; however, IL-6, TNF-a and interferon-y were significantly higher in HCII"'" mice (Fig. 1g). Again, no significant difference in prolongation of the aPTT or PT between HCII+/+ and HCIl ' mice was detected (Fig. 5b). Taken together, 20 the results from the infection and inflammation models above indicate that the major role of HCII in vivo is related to bacterial clearance and modulation of the endotoxin response, and not coagulation control.

We hypothesised that HCII interacts directly with bacteria and, as a consequence, 25 the level of HCII in plasma would be reduced during bacterial infection. In order to test this, plasma samples taken at different time points from C57BL/6 mice challenged with LPS, or infected with P. aeruginosa, were analysed for HCII. Indeed, a reduction in plasma levels of HCII, as detected by western blot (Fig. 2a) and ELISA (Fig. 2b) was observed over time. Similar to the findings in plasma, reduced levels of 30 HCII, particularly in kidney and liver, were observed during P. aeruginosa infection and LPS-induced shock (Fig. 2c). Analyses of expression of HCII in the liver of wild-type animals showed a diminished HCII expression in response to bacterial infection and endotoxin shock (Fig. 2d).

35 We next investigated whether intact or proteolytically cleaved forms of HCII could be detected under inflammatory conditions, such as wounding. Intact HCII was found in wound fluids collected from patients with acute surgical wounds (Fig. 2e, left panel).

42

Notably, addition of increasing amounts of HLE (human leukocyte elastase) to these wound fluids, thus simulating conditions with excessive inflammation, yielded fragments similar to the HLE-cleaved HCII forms described previously and designated HCIIa below (Fig. 2d, middle panel)15. Chronic wounds represent a 5 clinical situation characterized by a high influx of neutrophils, and excessive and uncontrolled levels of HLE16,17. Given this, wound fluids from these patients were investigated for HCII, and as shown in Fig. 2d (right panel), these fluids contained mostly forms similar to HCIIa, indicative of a compartmentalization of HCIIa generation to specific microenvironments characterized by high inflammation. In 10 summary, the data obtained from the mouse models indicate that the observed decrease, particularly of HCII plasma levels, can be explained by both diminished HCII expression combined with an extravascular consumption of HCII during infection or LPS-induced shock. The results with human wound fluids show that HCII, while present during wounding, is fragmented particularly in response to excessive 15 levels of HLE, conditions found in wounds characterized by high neutrophil influx and activation.

In light of the above observations, we investigated the properties of the residual, proteolytically cleaved form of HCII further. Similarly to the results with human wound 20 fluid above, digestion of pure HCII with HLE yielded one major truncated HCII form with a molecular weight -50 kDa, which was promptly generated and appeared to be stable during the course of the digestion. Along with this, minor fragments of lower molecular weights, were identified (Fig. 3a, Fig. 6). HCIIa exhibited a significantly higher affinity for heparin in comparison with the intact molecule (Fig. 3b, Fig. 7), data 25 concurrent with a previous report15,18. Considering the increase in affinity for heparin, a highly anionic glycosaminoglycan, we reasoned that this feature would lead to enhanced interactions with other anionic components, such as LPS. Consistent with this idea, we found that in contrast to HCII, HCIIa bound LPS and the interaction was completely blocked by an excess of heparin (Fig. 3c). Since LPS is an integral 30 component of Gram-negative bacterial surfaces, we decided to investigate interactions between the two HCII forms and bacteria. Similar to the observations with LPS above, FACS analyses demonstrated that only HCIIa bound to bacteria, and heparin inhibited the binding (Fig. 3d). Considering the presence of multiple minor fragments, including the possible release of the N-terminal tail of HCII7, we 35 wanted to define the major HCII form bound to bacteria. Thus, by again exploiting the affinity to bacteria, this time using a pull down assay, we demonstrated that whereas HCII showed no affinity to bacterial cells, the major form bound was identified as the

43

-50 kDa, heparin-releasable, HCIIa form (Fig. 3e). Whether binding to bacterial surfaces is leading to direct bacterial killing was addressed by using the Gram-negative Escherichia coli for detection of possible antibacterial effects. As judged by radial diffusion assay, digestion of HCII with HLE yielded antimicrobial activity after 5 5 min digestion with the enzyme, and the killing effect was retained after an extended incubation with the enzyme (Fig. 3f). Likewise, using viable counts, a direct antimicrobial activity was detected for cleaved HCII, but not for the intact form, at HCII concentrations close to physiological levels (Fig. 3g). Analysis by HPLC demonstrated that the -50 kDa HCIIa molecule was of relatively high hydrophobicity, 10 bound LPS, and was antimicrobial (Fig. 7), further linking the above antibacterial activities to HCIIa. At the ultrastructural level, as demonstrated by electron microscopy, only HCIIa caused disruption of bacteria, leading to disintegration and expulsion of cytoplasmic components (Fig. 3h). Bacteria incubated with HCIIa and heparin did not show any membrane damage and were similar to controls (Fig. 8). In 15 order to investigate the impact of the generation of HCIIa on bacteria under physiological conditions, acute wound fluid was incubated with P. aeruginosa bacteria with or without HLE. Electron microscopy analysis utilizing gold-labelled antibodies directed against HCII demonstrated that elastase activation of HCII significantly increased binding of the molecule to bacteria (Fig. 3i, Fig. 9). Taken 20 together, these results demonstrate that the generation by HLE of HCIIa is a prerequisite for its binding to LPS and bacteria, bacterial permeabilization and ultimately, bacterial killing.

The observations of a dramatic functional change of HCII upon cleavage by HLE, 25 adding new LPS-binding and antibacterial capabilities to the molecule, and the fact that addition of heparin seemed to completely inhibit these effects, indicated that release of the N-terminal acidic tail of HCII facilitates a conformational change, uncovering the heparin binding region of HCII which subsequently mediates the host defence activities of HCIIa. EM studies of single HCII molecules demonstrated that 30 intact HCII presented a "horse-shoe" like structure, this in contrast to the cleaved form, which assumed an almost stretched, linear shape (Fig. 4a). A significant co-localization between LPS (thin fibrillar structures) and the HCIIa forms was observed. Compatible with the above antibacterial experiments, the addition of heparin completely abolished the binding of LPS to HCIIa (Fig. 4a). Thus, these data further 35 indicate that a conformational change induced by proteolysis of HCII exposes a heparin- and LPS-binding region in the molecule. HCII has two heparin binding

44

regions, located to helices A and D6. In order to further define the epitopes responsible for the LPS-binding and antibacterial actions of intact HCIIa, peptides comprising those two helices, but also the other exposed helical regions of HCII (marked by different colours in Fig. 4b), were synthesised and utilised in subsequent 5 studies. Initial experiments using a slot-binding assay demonstrated that peptides from helix A and to a higher extent, helix D, bound LPS (Fig. 4c). The binding could be blocked by excess heparin (Fig. 4c), further verifying the overlap between heparin and LPS-binding activities of HCII. These results corresponded to the LPS binding data for HCIIa (Fig. 3c and Fig. 4a). Only peptides derived from helix A and D were 10 antibacterial (Fig. 4d), and it was notable that the doses required were compatible with those found to be effective for HCIIa mediated killing. The two peptides from helix A and D also blocked LPS-induced responses of RAW 264.7 macrophages and whole blood (Fig. 4e and f). Taken together, these data demonstrate that proteolytic activation results in a conformational change, which exposes a LPS-binding region in 15 HCIIa. The peptide epitopes were mapped to helix A and D of this region, and representative peptides covering these regions exerted antimicrobial and antiinflammatory effects.

In conclusion, the unique capacity of HCII to respond swiftly to proteases in 20 inflammatory milieus, acquiring a conformation critical for interactions with bacteria and endotoxins, represents to the best of our knowledge a conceptually novel mechanism in the increasing repertoire of host defence mechanisms and molecules characterising innate immunity. From a biological perspective, it is believed that the protease induced initial release of its chemotactic N-terminal peptide, paired with 25 shape-shifting of the residual main molecule, "tunes" HCII from an extracellular thrombin inhibitor to an activated host defence factor, thus facilitating a relevant and directed control of proteolytic as well as antibacterial activity in environments of localized and high inflammation. The observed decreased levels of the molecule during infections in patients, paralleled by similar findings in animal models of 30 endotoxic shock and severe P. aeruginosa infection, demonstrate novel treatment possibilities for severe infections based on supplementation with HCII or its functional correlates.

45

Tollefsen, D. M. Vascular dermatan sulfate and heparin cofactor II. Prog Mol Biol Trans! Sci 93, 351-372, doi: 10.1016/S1877-1173(10)93015-9 (2010). Huntington, J. A. Shape-shifting serpins-advantages of a mobile mechanism. Trends Biochem Sci 31, 427-435, doi:10.1016/j.tibs.2006.06.005 (2006). Rau, J. C., Beaulieu, L. M., Huntington, J. A. & Church, F. C. Serpins in thrombosis, hemostasis and fibrinolysis. J Thromb Haemost 5 SuppI 1, 102-115, doi: 10.1111/j. 1538-7836.2007.02516.x (2007).

He, L., Vicente, C. P., Westrick, R. J., Eitzman, D. T. & Tollefsen, D. M. Heparin cofactor II inhibits arterial thrombosis after endothelial injury. J Clin Invest 109, 213-219, doi: 10.1172/JC113432 (2002).

Villa, P. et al. Hereditary homozygous heparin cofactor II deficiency and the risk of developing venous thrombosis. Thromb Haemost 82, 1011-1014 (1999).

Baglin, T. P., Carrell, R. W., Church, F. C., Esmon, C. T. & Huntington, J. A. Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism. Proc Natl Acad Sci U SA 99, 11079-11084, doi: 10.1073/pnas. 162232399 (2002).

Hoffman, M., Pratt, C. W., Brown, R. L. & Church, F. C. Heparin cofactor II-proteinase reaction products exhibit neutrophil chemoattractant activity. Blood 73, 1682-1685(1989).

Noda, A. et al. Plasma levels of heparin cofactor II (HCII) and thrombin-HCII complex in patients with disseminated intravascular coagulation. Clin Appl Thromb Hemost 8, 265-271 (2002).

Kario, K., Matsuo, T., Kodama, K., Katayama, S. & Kobayashi, H. Preferential consumption of heparin cofactor II in disseminated intravascular coagulation associated with acute promyelocytic leukemia. Thromb Res 66, 435-444 (1992).

Rossi, E. B., Duboscq, C. L. & Kordich, L. C. [Heparin cofactor II, a thrombin inhibitor with a still not clarified physiologic role]. Medicina (B Aires) 59, 95-104(1999).

Papareddy, P. et al. Proteolysis of human thrombin generates novel host defense peptides. PLoS Pathog 6, e1000857, doi: 10.1371/journal.ppat. 1000857 (2010).

Papareddy, P. et al. C-terminal peptides of tissue factor pathway inhibitor are novel host defense molecules. J Biol Chem 285, 28387-28398, doi: 10.1074/jbc. M110.127019 (2010).

Nordahl, E. A., Rydengard, V., Morgelin, M. & Schmidtchen, A. Domain 5 of high molecular weight kininogen is antibacterial. J Biol Chem 280, 34832-34839, doi: 10.1074/jbc.M507249200 (2005).

Frick, I. M. et al. The contact system-a novel branch of innate immunity generating antibacterial peptides. Embo J 25, 5569-5578, doi: 10.1038/sj.emboj.7601422 (2006).

Maekawa, H., Sato, H. & Tollefsen, D. M. Thrombin inhibition by HCII in the presence of elastase-cleaved HCII and thrombin-HCII complex. Thromb Res 100, 443-451, doi:S0049-3848(00)00350-9 [pii] (2000).

Lundqvist, K., Herwald, H., Sonesson, A. & Schmidtchen, A. Heparin binding protein is increased in chronic leg ulcer fluid and released from granulocytes by secreted products of Pseudomonas aeruginosa. Thromb Haemost 92, 281-287, doi: 10.1267/THR004080281 (2004).

Schmidtchen, A. Degradation of antiproteinases, complement and fibronectin in chronic leg ulcers. Acta Derm Venereol 80, 179-184 (2000).

46

18 Pratt, C. W., Tobin, R. B. & Church, F. C. Interaction of heparin cofactor II with neutrophil elastase and cathepsin G. J Biol Chem 265, 6092-6097 (1990).

19 Lehrer, R. I., Rosenman, M., Harwig, S. S., Jackson, R. & Eisenhauer, P. 5 Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol

Methods 137, 167-173 (1991).

20 Pollock, J. S. et al. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci U SA 88, 10480-10484 (1991).

47

EXAMPLE B

A helix A-derived peptide of heparin cofactor II ameliorates septic shock and Pseudomonas aeruginosa induced sepsis

5

Introduction

Sepsis is an infection-induced syndrome characterized by a generalized inflammatory state and represents a frequent complication in the surgical patient, in 10 immunocompromized patients, or in relation to burns. Severe sepsis is a common, expensive and frequently fatal condition, having a documented worldwide incidence of 1.8 million each year. It is estimated that with an incidence of 3 in 1000 the true number of cases each year reaches 18 million, and with a mortality rate of almost 30% it becomes a leading cause of death worldwide. Sepsis is a major disease for 15 which efficient treatments are lacking, and for which hospital-associated society economic burden is large and growing. Considering this, novel therapies are urgently needed.

The abundant serine proteinase inhibitor (serpin) heparin cofactor II (HCII) has been 20 proposed to regulate coagulation by inhibiting thrombin within the extravascular areas and to modulate neointimal smooth muscle cell proliferation and atherosclerosis. However, the exact physiological role of this plasma protein has been enigmatic. Example A above describes a novel host defence function of HCII. Thus, in contrast to normal mice, HCII"'" animals were more susceptible to 25 lipopolysaccharide (LPS) induced shock and invasive infection by Pseudomonas aeruginosa. Concomitantly, a significantly increased cytokine response was observed in HCII"'" mice, and in parallel, HCII levels were reduced in normal mice. The LPS-binding and antimicrobial capacity of HCIIa was dependent on a conformational change, and the peptide regions responsible were mapped to helix A 30 and D of the molecule. Correspondingly, peptides representing these helices were antimicrobial, and exerted anti-endotoxic effects in macrophage models in vitro as well as in blood ex vivo. We here describe the activities of a helix A-derived peptide of HCII, GKS26. The peptide exerted potent antibacterial and anti-inflammatory activities, and showed efficiency in models of endotoxin-induces shock and sepsis in 35 vivo. The results demonstrate that functional epitopes of HCII may be utilized in the development of novel anti-infective and anti-inflammatory therapies.

48

Methods Peptides

The peptide GKS26 (NH2-GKSRIQRLNILNAKFAFNLYRVLKDQ-COOH [SEQ ID 5 NO:1] was synthesized by Biopeptide Co., San Diego, USA. The purity (>95%) was confirmed by mass spectral analysis (MALDI-ToF Voyager).

Viable-count analysis

E. coli ATCC 25922 bacteria were grown to mid-logarithmic phase in Todd-Hewitt 10 (TH) medium (Becton and Dickinson, Maryland, USA). The bacteria were then washed and diluted in 10 mM Tris, pH 7.4 containing 5 mM glucose. Following this, bacteria (50 pi; 2 x 106 cfu/ml) were incubated, at 37°C for 2 hours, with GKS26 (at 0.3, 0.6, 3, 6, 30, 60 |jM) in 10 mM Tris, 0.15 M NaCI, with or without 20% human citrate-plasma. To quantify the bactericidal activity, serial dilutions of the incubation 15 mixtures were plated on TH agar, followed by incubation at 37°C overnight and the number of colony-forming units was determined. 100% survival was defined as total survival of bacteria in the same buffer and under the same condition in the absence of peptide.

20 Nitrite assay

RAW 264.7 cells (3.5x106 /ml) in phenol red-free DMEM supplemented with 10% (v/v) FBS and 1% (v/v) AAS were seeded in 96-wells tissue culture plates (Nunc). Following 20 h of incubation to allow adherence, cells were washed and stimulated with 10 ng/ml E. coli (0111:B4) (Sigma-Aldrich, approximate 500.000 endotoxin 25 units/mg) with or without various concentrations of GKS26. The level of nitrite oxide (NO) in culture supernatants was determined after 20 h incubation at 37°C and 5% C02 using the Griess reaction as described previously and is presented as Nitrite (MM).

30 Liposome preparation and leakage assay

The liposomes investigated were anionic (DOPE/DOPG 75/25 mol/mol). DOPG (1,2-Dioleoyl-sn-Glycero-3-Phosphoglycerol, monosodium salt), DOPC (1,2-dioleoyl-sn-Glycero-3-phoshocholine), and DOPE (1,2-dioleoyl-sn-Glycero-3-phoshoetanolamine) were all from Avanti Polar Lipids (Alabaster, USA) and of >99% 35 purity). The lipid mixtures were dissolved in chloroform, after which solvent was removed by evaporation under vacuum overnight. Subsequently, 10 mM Tris buffer, pH 7.4, was added together with 0.1 M carboxyfluorescein (CF) (Sigma, St. Louis,

49

USA). After hydration, the lipid mixture was subjected to eight freeze-thaw cycles consisting of freezing in liquid nitrogen and heating to 60°C. Unilamellar liposomes of about 0140 nm were generated by multiple extrusions through polycarbonate filters (pore size 100 nm) mounted in a LipoFast miniextruder (Avestin, Ottawa, Canada) at 5 22°C. Untrapped CF was removed by two subsequent gel filtrations (Sephadex G-50, GE Healthcare, Uppsala, Sweden) at 22°C, with Tris buffer as eluent. CF release from the liposomes was determined by monitoring the emitted fluorescence at 520 nm from liposome dispersion (10 mM lipid in 10 mM Tris, pH 7.4). An absolute leakage scale was obtained by disrupting the liposomes at the end of each 10 experiment through addition of 0.8 mM Triton X-100 (Sigma-Aldrich, St. Louis, USA). A SPEX-fluorolog 1650 0.22-m double spectrometer (SPEX Industries, Edison, USA) was used for the liposome leakage assay in Tris buffer in the absence and presence of liposomes under conditions described above. Measurements were performed in triplicate at 37 °C.

15

LPS animal model

Male C57BL/6 mice (8 weeks, 21 +/- 5 g), were injected intraperitoneally (ip) with 18 mg E. coli 0111 :B4 LPS (Sigma-Aldrich, approximate 500.000 endotoxin units/mg) per kg of body weight. Thirty minutes after LPS injection 0.2 mg or 0.5 GKS26 in 10 20 mM Tris, pH 7.4 or buffer alone (control) were injected ip into the mice. The mice were sacrificed after 20 hours and blood was taken out for further analyses.

Determination of blood cell counts

The number of white blood cells, neutrophils lymphocytes, monocytes and platelets 25 in mouse blood (anti-coagulated with EDTA) taken by cardiac puncture was determined and analysed using the VetScan HM5 System (TRIOLAB)

Histochemistry

Organs were collected 20 h after LPS injection and were immediately fixed in 4% 30 formaldehyde prior to paraffin embedding and sectioning. Sections were stained with Mayers Haematoxylin (Histolab AB) and Eosin (Merck).

Clotting assay

All clotting times were analyzed using a coagulometer (Amelung, Lemgo, Germany). 35 For determination of prothrombin time (PT) a thromboplastin reagent (Trinity Biotech) was used, respectively. Fifty pi of fresh mouse plasma were pre-warmed for 60 sec

50

at 37°C before clot formation was initiated by adding 50 (jl of clotting reagent. To record the activated partial thromboplastin time (aPTT), 50 (jl of a kaolin-containing solution (Technoclone) was added to the plasma and incubated for 200 sec before clot formation was initiated by adding 50 (jl of 30 mM fresh CaCI2 solution.

5

Animal infection model

P. aeruginosa 15159 bacteria were grown to mid-exponential phase (OD620~0-5). harvested, washed twice in PBS, and diluted in PBS to 2-5 x 109 cfu/ml. Hundred microliter of this bacterial suspension was injected intraperitoneally (ip) into male 10 C57BL6 mice. After 1.5 and 4.5 h, 0.5 mg GKS26 or 300 mg/kg ceftazidime alone (AB) or a combination of both, was injected subcutaneously into the mice. For analysis of survival rate mice were followed up to 7 days.

Results

15

The helix A-derived peptide GKS26 was previously shown to bind LPS and was found to represent an antibacterial epitope of HCII. In order to explore the peptides effects in physiological environments, of importance for further in vivo studies, peptide effects were analysed in viable count assays in presence of salt as well as 20 plasma. (Figure 10A). The results showed that the peptide efficiently killed the bacteria in physiological salt as well in the presence of human plasma. Notably, the killing in plasma was increased when compared to the results in buffer only. Experiments with anionic liposomes showed that the peptide had significant permeabilising activity at or above concentrations of 0.1-0.2 |jM (Figure 10B).

25

Considering the LPS-binding activity of GKS26 we next explored the effects on LPS-stimulated macrophages. Mouse macrophages were stimulated with E. coli LPS and the resulting nitric oxide (NO) was measured in the cell supernatant. The NO concentration was reduced by GKS26 and completely blocked with a concentration 30 of 20 |jM (Figure 10C). Taken together these data illustrate the antimicrobial and antiinflammatory properties of GKS26 in vitro.

To determine a potential therapeutic effect of GKS26 in vivo, the peptide was used utilized in an animal model of LPS-induced septic shock in C57BI6 mice. Treatment 35 with GKS26, thirty minutes after LPS injection reduced septic symptoms as evidenced by a reduction in severity score in treated mice when compared to LPS

51

challenged control mice (Figure 11 A). This scoring was substantiated by analysing the different cell types. LPS challenge induced a reduction in the level of white blood cells (Figure 11B), most likely due to apoptosis of lymphocytes. This effect was partially reversed after treatment with GKS26.

5

Vascular leakage compromising lung function is one major hallmark symptom during sepsis. Therefore, lung sections from LPS challenged mice with or without GKS26 treatment were analyzed. In line with the clinical scoring and results from blood counts, less vascular leakage and infiltration of inflammatory cells was observed in 10 animals treated with the peptide. Interestingly, the status of the mice (Figure 11A) did not differ after treatment with either 0.2 or 0.5 mg of GKS26, whereas analyses of the lung sections showed that 0.5 mg of GKS26 was more effective in preventing the abovementioned LPS-induced effects on leakage and inflammation (Figure 11C).

15 Platelets are an important parameter in evaluation of septic shock, and during severe infections, the levels are significantly reduced. It is therefore notable that peptide-treatment reduced the consumption of platelets, a finding in line with the above observations on both clinical symptoms and lung status, with the improved status of the mice (Figure 11D). Finally, excessive coagulation is a compromising factor in 20 sepsis, characterized by consumption of coagulation factors, excessive fibrinolysis, leading to prolonged coagulation times in patients. Compatible to the results on platelet levels, mice challenged with LPS mice showed prolonged clotting times. Mice treated with GKS26 showed normalization of both the activated partial thromboplastin time (aPTT), as well as prothrombin time (PT), (Figure 11E). Taken together, the 25 data from these in vivo experiments demonstrate the treatment with GKS26 yielded reduction of many sepsis associated pathologies, including vascular leakage, excessive coagulation as well as platelet consumption.

In the last set of in vivo experiments C57BI56 mice were infected with a lethal dose of 30 Pseudomonas aeruginosa and the survival rate of mice treated twice with 0.5 mg of GKS26 alone or in combination with ceftazidime was monitored. The peptide alone did not prolong the survival time. Treatment with ceftazidime, a common antibiotic used in sepsis patients, increased the survival rate up to 43 percent. However, when ceftazime was administered in combination with GKS26, the survival rate was 35 increased to 53 percent (Figure 12).

52

53

SEQUENCE LISTING <110> Xlmmune AB

<120> POLYPEPTIDES AND USES THEREOF

<130> XIMBA/P50687GB

<160> 14

<170> SeqWin99

<210> 1 <211> 26 <212> PRT

<213> Artificial Sequence <220>

<223> GKS26 <400> 1

Gly Lys Ser Arg lie Gin Arg Leu Asn lie Leu Asn Ala Lys Phe Al 15 10 15

Phe Asn Leu Tyr Arg Val Leu Lys Asp Gin 20 25

<210> 2 <211> 28 <212> PRT

<213> Artificial Sequence <220>

<223> KYE28 <400> 2

Lys Tyr Glu lie Thr Thr lie His Asn Leu Phe Arg Lys Leu Thr Hi 15 10 15

Arg Leu Phe Arg Arg Asn Phe Gly Tyr Thr Leu Arg 20 25

<210> 3 <211> 21 <212> PRT

<213> Artificial Sequence <220>

<223> KYE21 <400> 3

Lys Tyr Glu lie Thr Thr lie His Asn Leu Phe Arg Lys Leu Thr Hi 15 10 15

Arg Leu Phe Arg Arg 20

54

<210> 4 <211> 20 <212> PRT

<213> Artificial Sequence <220>

<223> NLF20 <400> 4

Asn Leu Phe Arg Lys Leu Thr His Arg Leu Phe Arg Arg Asn Phe Gly 15 10 15

Tyr Thr Leu Arg 20

<210> 5 <211> 23 <212> PRT

<213> Artificial Sequence <220>

<223> LKG23 <400> 5

Leu Lys Gly Glu Thr His Glu Gin Val His Ser lie Leu His Phe Lys 15 10 15

Asp Phe Val Asn Ala Ser Ser 20

<210> 6 <211> 18 <212> PRT

<213> Artificial Sequence <220>

<223> SDP18 <400> 6

Ser Asp Pro Ala Phe lie Ser Lys Thr Asn Asn His lie Met Lys Leu 15 10 15

Thr Lys

<210> 7 <211> 22 <212> PRT

<213> Artificial Sequence <220>

<223> SGM22 <400> 7

Ser Gly Met Lys Thr Leu Glu Ala Gin Leu Thr Pro Arg Val Val Glu 15 10 15

55

Arg Trp Gin Lys Ser Met 20

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence <220>

<223> HCII forward primer

<400> 8

ccagcgtctt aatatcctca atgc

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence <220>

<223> HCII reverse primer

<400> 9

tgccaacagg tgctatgaag agg

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence <220>

<223> B-actin forward primer

<400> 10

tgacgaggcc cagagcaaga g

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence <220>

<223> B-actin reverse primer

<400> 11

cgcagctcat tgtagaaggt gtg

<210> 12

<211> 499

<212> PRT

<213> Unknown

<220>

<223> Heparin cofactor II (HCII)

23

21

23

sequence

<400> 12

Met Lys His Ser Leu Asn Ala Leu Leu lie Phe Leu lie lie Thr Ser 15 10 15

56

Ala Trp

Glu Thr

Asn Leu 50

Val Thr 65

Asp Leu

Asp Ser lie Leu

Leu Asn 130

Val Asn 145

Ala Met

Val His

Tyr Glu

Leu Phe 210

Tyr lie 225

Arg Glu

Ala Phe

Leu lie lie Leu 290

Val Glu 305

Gly Gly 20

Ala Gin 35

Ser Met

Asn Asp

Glu Lys

Leu Ser 100

Gin Leu 115

Ala Lys

Thr Phe

Gly Met

Ser lie 180

lie Thr 195

Arg Arg

Gin Lys

Tyr Tyr lie Ser 260

Lys Asp 275

Asn Cys Met Thr

Ser Lys

Ser Ala

Pro Leu

Trp lie 70

lie Phe 85

Val Ser

Phe His

Phe Ala

Asp Asn 150

lie Ser 165

Leu His

Thr lie

Asn Phe

Gin Phe 230

Phe Ala 245

Lys Thr Ala Leu lie Tyr

His Asn 310

Gly Pro

Asp Pro 40

Leu Pro 55

Pro Glu

Ser Glu

Pro Thr

Gly Lys 120

Phe Asn 135

lie Phe

Leu Gly

Phe Lys

His Asn 200

Gly Tyr 215

Pro lie

Glu Ala

Asn Asn

Glu Asn 280

Phe Lys 295

His Asn

Leu Asp 25

Gin Trp

Ala Asp

Gly Glu

Asp Asp 90

Asp Ser 105

Ser Arg

Leu Tyr lie Ala

Leu Lys 170

Asp Phe 185

Leu Phe

Thr Leu

Leu Leu

Gin lie 250

His lie 265

lie Asp Gly Ser Phe Arg

Gin Leu

Glu Gin

Phe His 60

Glu Asp 75

Asp Tyr

Asp Val lie Gin

Arg Val 140

Pro Val 155

Gly Glu

Val Asn

Arg Lys

Arg Ser 220

Asp Phe 235

Ala Asp

Met Lys

Pro Ala

Trp Val 300

Leu Asn 315

Glu Lys 30

Leu Asn 45

Lys Glu

Asp Asp lie Asp

Ser Ala 110

Arg Leu 125

Leu Lys

Gly lie

Thr His

Ala Ser 190

Leu Thr 205

Val Asn Lys Thr

Phe Ser

Leu Thr 270

Thr Gin 285

Asn Lys

Glu Arg

Gly Gly

Asn Lys

Asn Thr

Tyr Leu 80

lie Val 95

Gly Asn

Asn lie

Asp Gin

Ser Thr 160

Glu Gin 175

Ser Lys

His Arg

Asp Leu

Lys Val 240

Asp Pro 255

Lys Gly

Met Met

Phe Pro

Glu Val 320

57

Val Lys Val Ser Met Met Gin Thr Lys Gly Asn Phe Leu Ala Ala Asn 325 330 335

Asp Gin Glu Leu Asp Cys Asp lie Leu Gin Leu Glu Tyr Val Gly Gly 340 345 350

lie Ser Met Leu lie Val Val Pro His Lys Met Ser Gly Met Lys Thr 355 360 365

Leu Glu Ala Gin Leu Thr Pro Arg Val Val Glu Arg Trp Gin Lys Ser 370 375 380

Met Thr Asn Arg Thr Arg Glu Val Leu Leu Pro Lys Phe Lys Leu Glu 385 390 395 400

Lys Asn Tyr Asn Leu Val Glu Ser Leu Lys Leu Met Gly lie Arg Met 405 410 415

Leu Phe Asp Lys Asn Gly Asn Met Ala Gly lie Ser Asp Gin Arg lie 420 425 430

Ala lie Asp Leu Phe Lys His Gin Gly Thr lie Thr Val Asn Glu Glu 435 440 445

Gly Thr Gin Ala Thr Thr Val Thr Thr Val Gly Phe Met Pro Leu Ser 450 455 460

Thr Gin Val Arg Phe Thr Val Asp Arg Pro Phe Leu Phe Leu lie Tyr 465 470 475 480

Glu His Arg Thr Ser Cys Leu Leu Phe Met Gly Arg Val Ala Asn Pro 485 490 495

Ser Arg Ser

<210> 13

<211> 19

<212> PRT

<213> Unknown

<220>

<223> HCII signal peptide <400> 13

Met Lys His Ser Leu Asn Ala Leu Leu lie Phe Leu lie lie Thr Ser 15 10 15

Ala Trp Gly

<210> 14

<211> 12

<212> PRT

<213> Unknown

<220>

<223> HCII Chemotactic region

58

<400> 14

Asp Trp lie Pro Glu Gly Glu Glu Asp Asp Asp Tyr 15 10

59

Claims (1)

1. A polypeptide comprising or consisting of an amino acid sequence derived from helix A of heparin cofactor II, or a biologically active fragment or variant thereof.

2. A polypeptide according to Claim 1 wherein the polypeptide is isolated.

3. A polypeptide according to Claim 1 or 2 wherein the polypeptide is not a naturally occurring protein.

4. A polypeptide according to any one of the preceding claims wherein the heparin cofactor II is human heparin cofactor II

5. A polypeptide according to Claim 4 wherein the heparin cofactor II is Swiss Port Accession No. P05546.

6. A polypeptide according to any one of the preceding claims wherein the polypeptide comprises a heparin binding region.

7. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of binding LPS.

8. A polypeptide according to any one of the preceding claims comprising or consisting of the amino acid sequence of SEQ ID NO:1:

"GKS26GKSRIQRLNILNAKFAFNLYRVLKDQ [SEQ ID NO:1]

or a biologically active fragment or variant thereof.

9. A polypeptide according to Claim 8 comprising or consisting of the amino acid sequence of SEQ ID NO:1.

10. A polypeptide according to any one of the preceding claims wherein the polypeptide, or fragment or variant thereof, comprises or consists of L-amino acids.

60

11. A polypeptide according to any one of the preceding claims wherein the polypeptide, or fragment or variant thereof, comprises one or more amino acids that are modified or derivatised.

5 12. A polypeptide according to Claim 11 wherein the one or more amino acids are modified or derivatised by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

13. A polypeptide according to any one of the preceding claims wherein the

10 polypeptide comprises or consists of a fragment of the amino acid sequence of SEQ ID NO: 1.

14. A polypeptide according to Claim 13 wherein the fragment comprises or consists of at least 5 contiguous amino acids of SEQ ID NO: 1, for example at

15 least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25

contiguous amino acids.

15. A polypeptide according to any one of the preceding claims wherein the polypeptide comprises or consists of a variant of the amino acid sequence of

20 SEQ ID NO: 1.

16. A polypeptide according to Claim 15 wherein the variant has at least 50% identity with the amino acid sequence amino acid sequence of any one of SEQ ID NOS: 1 to 3, for example at least 55%, 60%, 65%, 70%, 75%, 80%,

25 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

17. A polypeptide according to any one of the preceding claims wherein the polypeptide is a fusion polypeptide.

30 18. A polypeptide according to Claim 17 wherein the fusion polypeptide comprises a polypeptide derived from helix D of heparin cofactor II.

19. A polypeptide according to Claim 18 wherein the polypeptide derived from helix D of heparin cofactor II comprises or consists of the amino acid

35 sequence of any one of SEQ ID NOS: 2 to 4:

KYE28T-. KYEITTIHNLFRKLTHRLFRRNFGYTLR [SEQ ID NO:2]

61

"KYE21KYEITTIHNLFRKLTHRLFRR [SEQ ID N0:3]

"NLF2CT\ NLFRKLTHRLFRRNFGYTLR [SEQ ID N0:4]

or a fragment or variant thereof which retains a biological activity of 5 polypeptide of any one of SEQ ID NOS: 2 to 4.

20. A polypeptide according to any one of the preceding claims wherein the polypeptide is between 10 and 200 amino acids in length, for example between 10 and 150, 15 and 100, 15 and 50, 15 and 30, 20 and 30, or 25 and

10 27 amino acids in length.

21. A polypeptide according to Claim 20 wherein the polypeptide is at least 20 amino acids in length.

15 22. A polypeptide according to any one of the preceding claims wherein the polypeptide is linear.

23. A polypeptide according to any one of the preceding claims wherein the polypeptide is a recombinant polypeptide.

20

24. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of inhibiting the growth of microorganisms.

25. A polypeptide according to Claim 24 wherein the microorganisms are selected

25 from the group consisting of bacteria, mycoplasmas, yeasts, fungi and viruses.

26. A polypeptide according to Claim 24 or 25 wherein the polypeptide is capable of inhibiting the growth of bacteria.

30 27. A polypeptide according to Claim 26 wherein the bacteria are of the species Pseudomonas aeruginosa.

28. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of inhibiting the release of one or more pro-

35 inflammatory cytokines from human monocyte-derived macrophages.

62

29. A polypeptide according to Claim 28 wherein the pro-inflammatory cytokines are selected from the group consisting of macrophage inhibitory factor, TNF-alpha, HMGB1, C5a, IL-17, IL-8, MCP-1, IFN-gamma, II-6, IL-1b, IL-12.

5 30. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of blocking platelet activation.

31. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of interfering with Toll-like receptor (TLR)-signalling in

10 leukocytes, epithelial cells (including keratinocytes) and/or mesenchymal cells

(including fibroblasts).

32. A polypeptide according to any one of the preceding claims wherein the polypeptide exhibits anti-inflammatory activity in one or more of the following

15 models:

(a) in vitro macrophage models using microbial stimulants such as LPS, LTA, zymosan, flagellin, viral or bacterial DNA or RNA, or peptidoglycan as well as endogenous damage associate molecular patterns (DAMPs), such as

20 DNA, HMGB1, or S100 proteins.

(b) in vivo mouse models of endotoxin shock;

(c) in vivo infection models, either in combination with antimicrobial therapy, 25 or given alone.

33. A polypeptide according to any one of the preceding claims wherein the polypeptide exhibits anticoagulant activity.

30 34. A polypeptide according to any one of the preceding claims wherein the polypeptide exhibits Toll-like receptor (TLR) blocking activity.

35. An isolated nucleic acid molecule which encodes a polypeptide according to any one of the preceding claims.

35

36.

A vector comprising a nucleic acid molecule according to Claim 35.

63

37. A vector according to Claim 36 wherein the vector is an expression vector.

38. A host cell comprising a nucleic acid molecule according to Claim 35 or a vector according to Claim 36 or 37.

5

39. A method of making a polypeptide according to any one of Claims 1 to 34 comprising culturing a population of host cells according to Claim 38 under conditions in which said polypeptide is expressed, and isolating the polypeptide therefrom.

10

40. A method of making a polypeptide according to any one of Claims 1 to 34 comprising liquid-phase or solid-phase synthesis of the polypeptide.

41. A pharmaceutical composition comprising a polypeptide according to any one

15 of Claims 1 to 34 together with a pharmaceutically acceptable excipient,

diluent, carrier, buffer or adjuvant.

42. A pharmaceutical composition according to Claim 41 suitable for administration via a route selected from the group consisting of topical, ocular,

20 nasal, pulmonar, buccal, parenteral (intravenous, subcutaneous, intratechal and intramuscular), oral, vaginal and rectal.

43. A pharmaceutical composition according to Claim 41 or 42 suitable for administration via an implant.

25

44. A pharmaceutical composition according to any one of Claims 41 to 43 wherein the pharmaceutical composition is associated with a device or material to be used in medicine.

30 45. A pharmaceutical composition according to Claim 44 wherein the device or material is for use in by-pass surgery, extracorporeal circulation, wound care and/or dialysis.

46. A pharmaceutical composition according to any one of Claims 41 to 5 wherein

35 the device or material comprise or consists of a polymer, metal, metal oxide and/or ceramic.

64

47. A pharmaceutical composition according to any one of Claims 41 to 46 wherein the pharmaceutical composition is coated, painted, sprayed or otherwise applied to a suture, prosthesis, implant, wound dressing, catheter, lens, skin graft, skin substitute, fibrin glue or bandage.

5

48. A polypeptide according to any one of Claim 1 to 34 for use in medicine

49. A polypeptide according to Claim 48 for use in the treatment or prevention of infection by microorganisms.

10

50. A polypeptide according to Claim 49 wherein the microorganisms are selected from the group consisting of bacteria, mycoplasmas, yeasts, fungi and viruses.

51. A polypeptide according to Claim 49 or 50 for use in the treatment or

15 prevention of bacterial infection.

52. A polypeptide according to Claim 51 wherein the bacteria are of the species Pseudomonas aeruginosa.

20 53. A polypeptide according to any one of Claims 48 to 52 for use in the treatment or prevention of inflammation.

54. A polypeptide according to Claim 53 for use in the treatment or prevention of inflammation associated with an infection.

25

55. A polypeptide according to Claim 52 or 54 for use in the concomitant treatment or prevention of inflammation and coagulation.

56. A polypeptide according to any one of Claims 48 to 55 for use in the treatment

30 or prevention of excessive coagulation of the blood.

57. A polypeptide according to any one of Claims 48 to 56 for use in the treatment or prevention of a disease, condition or indication selected from the following:

35

i)

Acute systemic inflammatory disease, with or without an infective component, such as systemic inflammatory response syndrome (SIRS), ARDS, sepsis, severe sepsis, and septic shock. Other

65

generalized or localized invasive infective and inflammatory disease, including erysipelas, meningitis, arthritis, toxic shock syndrome, diverticulitis, appendicitis, pancreatitis, cholecystitis, colitis, cellulitis, burn wound infections, pneumonia, urinary tract infections, postoperative infections, and peritonitis.

Chronic inflammatory and or infective diseases, including cystic fibrosis, COPD and other pulmonary diseases, gastrointestinal disease including chronic skin and stomach ulcerations, other epithelial inflammatory and or infective disease such as atopic dermatitis, oral ulcerations (aphtous ulcers), genital ulcerations and inflammatory changes, parodontitis, eye inflammations including conjunctivitis and keratitis, external otitis, mediaotitis, genitourinary inflammations.

Postoperative inflammation. Inflammatory and coagulative disorders including thrombosis, DIC, postoperative coagulation disorders, and coagulative disorders related to contact with foreign material, including extracorporeal circulation, and use of biomaterials. Furthermore, vasculitis related inflammatory disease, as well as allergy, including allergic rhinitis and asthma..

Excessive contact activation and/or coagulation in relation to, but not limited to, stroke.

Excessive inflammation in combination with antimicrobial treatment. The antimicrobial agents used may be administred by various routes; intravenous (iv), intraarterial, intravitreal, subcutaneous (sc), intramuscular (im), intraperitoneal (ip), intravesical, intratechal, epidural, enteral (including oral, rectal, gastric, and other enteral routes), or topically, (including dermal, nasal application, application in the eye or ear, eg by drops, and pulmonary inhalation). Examples of agents are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide.

66

58. A polypeptide according to any one of Claims 48 to 57 for use in the treatment or prevention of acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic 5 fibrosis, asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis, thrombosis and disseminated intravascular coagulation (DIC).

59. A polypeptide according to Claim 58 for use in the treatment or prevention of sepsis.

10

60. A polypeptide according to Claim 58 for use in the treatment or prevention of chronic obstructive pulmonary disease (COPD).

61. A polypeptide according to any one of Claims 48 to 60 for use in combination

15 with one or more additional therapeutic agent(s).

62. A polypeptide according to Claim 61 wherein the additional therapeutic agent is selected from the group consisting of antibiotic agents, anti-fungal agents, antiseptic agents, anti-inflammatory agents, immunosuppressive agents,

20 vasoactive agents, receptor blockers, receptor agonists and antiseptic agents.

63. A polypeptide according to Claim 62 wherein the antibiotic agents are selected from the groups consisting of anti-bacterial agents, anti-fungicides, anti-viral agents and anti-parasitic agents.

25

64. A polypeptide according to Claim 63 wherein the antibiotic agent is an antibacterial agent.

65. A polypeptide according to Claim 64 wherein the antibiotic agent is a 30 cephalosporin antibiotic agent.

66. A polypeptide according to Claim 65 wherein the antibiotic agent is ceftazidime.

35

67.

Use of a polypeptide according to any one of Claims 1 to 34 in the preparation of a medicament for the treatment or prevention of infection by microorganisms.

67

68. A use according to Claim 67 wherein the microorganisms are selected from the group consisting of bacteria, mycoplasmas, yeasts, fungi and viruses.

5 69. A use according to Claim 67 or 68 wherein the medicament is for the treatment or prevention of bacterial infection.

70. A use according to Claim 69 wherein the bacteria are of the species Pseudomonas aeruginosa.

10

71. Use of a polypeptide according to any one of Claims 1 to 34 in the preparation of a medicament for the treatment or prevention of inflammation.

72. A use according to Claim 71 wherein the inflammation is associated with an 15 infection.

73. A use according to Claim 71 or 72 wherein the medicament is for the concomitant treatment or prevention of inflammation and coagulation.

20 74. Use of a polypeptide according to any one of Claims 1 to 34 in the preparation of a medicament for the treatment or prevention of excessive coagulation of the blood.

75. A use according to any one of Claims 67 to 74 wherein the medicament is for 25 the treatment or prevention of a disease, condition or indication selected from the following:

vi) Acute systemic inflammatory disease, with or without an infective component, such as systemic inflammatory response syndrome 30 (SIRS), ARDS, sepsis, severe sepsis, and septic shock. Other generalized or localized invasive infective and inflammatory disease, including erysipelas, meningitis, arthritis, toxic shock syndrome, diverticulitis, appendicitis, pancreatitis, cholecystitis, colitis, cellulitis, burn wound infections, pneumonia, urinary tract infections, 35 postoperative infections, and peritonitis.

68

vii) Chronic inflammatory and or infective diseases, including cystic fibrosis, COPD and other pulmonary diseases, gastrointestinal disease including chronic skin and stomach ulcerations, other epithelial inflammatory and or infective disease such as atopic dermatitis, oral ulcerations (aphtous ulcers), genital ulcerations and inflammatory changes, parodontics, eye inflammations including conjunctivitis and keratitis, external otitis, mediaotitis, genitourinary inflammations.

viii) Postoperative inflammation. Inflammatory and coagulative disorders including thrombosis, DIC, postoperative coagulation disorders, and coagulative disorders related to contact with foreign material, including extracorporeal circulation, and use of biomaterials. Furthermore, vasculitis related inflammatory disease, as well as allergy, including allergic rhinitis and asthma..

ix) Excessive contact activation and/or coagulation in relation to, but not limited to, stroke.

x) Excessive inflammation in combination with antimicrobial treatment. The antimicrobial agents used may be administred by various routes; intravenous (iv), intraarterial, intravitreal, subcutaneous (sc), intramuscular (im), intraperitoneal (ip), intravesical, intratechal, epidural, enteral (including oral, rectal, gastric, and other enteral routes), or topically, (including dermal, nasal application, application in the eye or ear, eg by drops, and pulmonary inhalation). Examples of agents are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and other biguanides, chitosan, acetic acid, and hydrogen peroxide.

A use according to any one of Claims 67 to 75 wherein the medicament is for the treatment or prevention of acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, allergic and other types of rhinitis, cutaneous and

69

systemic vasculitis, thrombosis and disseminated intravascular coagulation (DIC).

77. A use according to Claim 76 wherein the medicament is for the treatment or 5 prevention of sepsis.

78. A polypeptide according to Claim 76 wherein the medicament is for the treatment or prevention of chronic obstructive pulmonary disease (COPD).

10 79. A use according to any one of Claims 67 to 78 wherein the medicament is for use in combination with one or more additional therapeutic agent(s).

80. A use according to Claim 79 wherein the additional therapeutic agent is selected from the group consisting of antibiotic agents, anti-fungal agents,

15 antiseptic agents, anti-inflammatory agents, immunosuppressive agents,

vasoactive agents, receptor blockers, receptor agonists and antiseptic agents.

81. A use according to Claim 80 wherein the antibiotic agents are selected from the groups consisting of anti-bacterial agents, anti-fungicides, anti-viral agents

20 and anti-parasitic agents.

82. A use according to Claim 81 wherein the antibiotic agent is an anti-bacterial agent.

25 83. A use according to Claim 82 wherein the antibiotic agent is a cephalosporin antibiotic agent.

84. A use according to Claim 83 wherein the antibiotic agent is ceftazidime.

30 85. A method for treating or preventing infection by microorganisms in a patient, the method comprising administering to the patient a therapeutically-effective amount of a polypeptide according to any one of Claims 1 to 34.

35

86.

A method for treating or preventing inflammation in a patient, the method comprising administering to the patient a therapeutically-effective amount of a polypeptide according to any one of Claims 1 to 34.

70

A method for treating or preventing excessive coagulation in a patient, the method comprising administering to the patient a therapeutically-effective amount of a polypeptide according to any one of Claims 1 to 34.

A method according to any one of Claims 85 to 87 for the treatment or prevention of a disease, condition or indication selected from the following:

i) Acute systemic inflammatory disease, with or without an infective component, such as systemic inflammatory response syndrome (SIRS), ARDS, sepsis, severe sepsis, and septic shock. Other generalized or localized invasive infective and inflammatory disease, including erysipelas, meningitis, arthritis, toxic shock syndrome, diverticulitis, appendicitis, pancreatitis, cholecystitis, colitis, cellulitis, burn wound infections, pneumonia, urinary tract infections, postoperative infections, and peritonitis.

ii) Chronic inflammatory and or infective diseases, including cystic fibrosis, COPD and other pulmonary diseases, gastrointestinal disease including chronic skin and stomach ulcerations, other epithelial inflammatory and or infective disease such as atopic dermatitis, oral ulcerations (aphtous ulcers), genital ulcerations and inflammatory changes, parodontitis, eye inflammations including conjunctivitis and keratitis, external otitis, mediaotitis, genitourinary inflammations.

iii) Postoperative inflammation. Inflammatory and coagulative disorders including thrombosis, DIC, postoperative coagulation disorders, and coagulative disorders related to contact with foreign material, including extracorporeal circulation, and use of biomaterials. Furthermore, vasculitis related inflammatory disease, as well as allergy, including allergic rhinitis and asthma..

iv) Excessive contact activation and/or coagulation in relation to, but not limited to, stroke.

v) Excessive inflammation in combination with antimicrobial treatment. The antimicrobial agents used may be administred by various routes;

71

intravenous (iv), intraarterial, intravitreal, subcutaneous (sc), intramuscular (im), intraperitoneal (ip), intravesical, intratechal, epidural, enteral (including oral, rectal, gastric, and other enteral routes), or topically, (including dermal, nasal application, application in 5 the eye or ear, eg by drops, and pulmonary inhalation). Examples of agents are penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents include iodine, silver, copper, clorhexidine, polyhexanide and 10 other biguanides, chitosan, acetic acid, and hydrogen peroxide.

89. A method according to any one of Claims 85 to 88 for the treatment or prevention of acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, 15 asthma, allergic and other types of rhinitis, cutaneous and systemic vasculitis,

thrombosis and disseminated intravascular coagulation (DIC).

90. A method according to Claim 89 wherein the medicament is for use in the treatment or prevention of sepsis.

20

91. A method according to Claim 89 wherein the medicament is for use in the treatment or prevention of chronic obstructive pulmonary disease (COPD).

92. A method according to any one of Claims 85 to 91 further comprising

25 administering to the patient one or more additional therapeutic agent(s).

93. A method according to Claim 92 wherein the additional therapeutic agent is selected from the group consisting of antibiotic agents, anti-fungal agents, anti-inflammatory agents, immunosuppressive agents, vasoactive agents and

30 antiseptic agents.

94. A polypeptide substantially as described herein with reference to the description and figures.

35 95. A nucleic acid molecule substantially as described herein with reference to the description and figures.

72

96. A pharmaceutical composition substantially as described herein with reference to the description and figures.

5 97. Use of a polypeptide substantially as described herein with reference to the description and figures.

98. A method of treatment/prevention substantially as described herein with reference to the description and figures.

10