WO2020053426A1 - Use of beta-antithrombin in the treatment of bacterial infections - Google Patents

Abstract

The present invention relates to beta- antithrombin for use in treating infections. The invention also relates to identification of novel interactions between antithrombin and certain receptors and binding protein and the use of beta- antithrombin in regulating Toll- Like Receptor pathways.

Description

USE OF BETA-ANTITHROMBIN IN THE TREATMENT OF BACTERIAL

INFECTIONS

Field of the Invention

The present invention relates to beta- antithrombin for use in treating bacterial infections. The invention also relates to identification of novel interactions between antithrombin and certain receptors and binding protein and the use of beta-antithrombin in regulating Toll-Like Receptor pathways.

Background to the Invention

Antithrombin III (AT III) is the most important physiological anticoagulant in human blood. It circulates in the plasma with a concentration of 150-300 pg/ml (2.5-5 mM) as a single-chain glycoprotein. AT III is composed of 432 amino acids and has a molecular weight of 58 kDa. The protein, encoded by SERPIN1C, constitutes a major inhibitor of thrombin but it also has inhibitory effect on other activated serine proteases of the coagulation system in various strength including factor Vila, IXa, Xa, XIa and Xlla. When bound to its target, AT III forms an equimolar complex resulting in an irreversible inhibition of the proteinase. The formed thrombin- antithrombin (TAT) complex is removed from the blood circulation by serpin receptor 1 on hepatocytes with a half-life of 5 min. In the circulation AT III can bind to heparan sulphate proteoglycans (HSPG), expressed on the surface of endothelial cells and thus its affinity to proteases is increased. Once the protease is captured, the complex dissociates from the vessel surface and is cleared by the liver. This high-affinity state is reached by sulfated 9 glycosaminoglycans (GAGs) inducing a conformational change in AT III. These GAGs include therapeutic heparin and physiological HSPG. Hence, the inhibitory potential of AT III as a serine protease inhibitor (serpin) is 1000-fold enhanced in the presence of heparin.

Pentasaccharide sequences of heparin are described to be responsible for the binding to AT III. In contrast to the inhibition of factor Xa, only a full-length heparin with several pentasaccharide chains promotes the AT Ill-dependent inactivation of thrombin. This is rather explained by the heparin bridging mechanism, the simultaneous binding of heparin to both AT III and thrombin, than by the conformational change. AT III is found in healthy individuals in two most abundant isoforms, aAT and bAT which differ in their amount of glycosylation. The major form aAT representing 90- 95 % of the inhibitor in plasma, is fully N-glycosylated (at Asn96, Asnl35, Asnl55 and Asnl92), whereas the minor glycoform bAT lacks the carbohydrate chain at Asnl35. The b-isoform, which constitutes 5-10 % of plasma has a higher heparin affinity than aAT.

This is due to the incomplete glycosylation resulting from the presence of serine instead of threonine in the recognition sequence for glycan attachment. The difference in

glycosylation can be explained since Asnl35 can interfere with the heparin-induced conformational change triggered by binding of the positively charged helix D of AT III to the negatively charged heparin molecule. Hence, bAT is suggested to be mainly responsible for the inhibitory effect of AT III in vivo.

Founder et al. published in 1992 that AT III plasma levels in patient with severe sepsis and especially in DIC patients are significantly reduced and they concluded that AT III can be used as a predictor of death. Because of its anti-coagulative activity, application of AT III has been considered as treatment of DIC and sepsis. Several clinical studies have been performed and it was found that administration of concentrated human AT III in patients with DIC positive sepsis shortened the duration of DIC, but failed in improvement of the overall survival. In 2001, in a randomized, prospective, placebo-controlled phase III multicenter clinical trial (KyberSept) including patients with severe sepsis (total of 2,314) was conducted. Patients were treated with high-dose of 30,000 IU AT III over four days. No significant reduction in 28-day mortality was found when patients were

prophylactically treated with heparin. However, AT III treatment of patients with no concomitant heparin achieved after 28 days a reduction in mortality, which became significant after 90-day. Regarding sepsis patients with DIC, an absolute decrease in 28- day mortality of 15 % was shown upon AT Ill-treatment without heparin. These clinical findings are in line with animal and in vitro studies demonstrating anti-inflammatory properties of AT III. Early studies explained the anti-inflammatory activity of AT III by the induction of endothelial prostaglandin I₂ production in rodents models of hepatic ischemia/reperfusion lesions and endotoxin-induced multiple organ dysfunction. Using in vitro studies it has been found that AT III has anti-inflammatory activity as it inhibits NF- KB activation and leads to a reduction of IL-6, TNFa and TF production after stimulation of monocytes with LPS. However, this mechanism is still not completely understood. The inhibitory potential of AT III is supposed to be associated with the binding to heparan sulphate proteoglycans, confirmed by the finding that b-isoform prevent the activation of NF-kB more effective than aAT.

Summary of the Invention

The present inventors have identified a direct antimicrobial effect of bAT, allowing it to be useful in treating infections in individuals. Compared to short peptides, administering a full length bAT (or variant or fragment thereof) has the advantage of having a higher specificity, a lower chance of being easily degraded, a lower sensitivity to proteases and therefore a higher half-life.

The inventors have also identified novel interactions between ATIII and certain proteins, such as Cathepsin G, CD300f/CLMl/CMRF35-like molecules 1, Aminopeptidase N/CD13, LRP1/CD91, protein EVI2B/CD361, MEGF9 and Heparin-binding protein (also known as azurocidin or CAP37), thus suggesting potential use of bAT in regulating TLR pathways and diseases or conditions associated with TLR-associated inflammation.

The invention provides an antimicrobial for use in a method of treating a bacterial infection, wherein the antimicrobial is administered to an individual in need, and wherein the antimicrobial is beta- antithrombin (bAT).

The invention also provides the use of an antimicrobial as described herein in the manufacture of a medicament for the treatment of a bacterial infection, wherein said infection is as described herein.

The invention also provides a method of treating a bacterial infection in an individual in need thereof, wherein the method comprises administering an antimicrobial as described herein, wherein said infection is as described herein.

Brief Description of the Sequences

SEQ ID NO: 1 is the amino acid sequence of human AT without any signal sequences. Glycosylation sites are found at positions 96, 135, 155 and 192 based on the numbering in this sequence. SEQ ID NO: 2 is the amino acid sequence of human antithrombin (AT) containing signal sequences from positions 1 to 32. Glycosylation sites are found at positions 128, 167, 187 and 224 based on the numbering in this sequence.

SEQ ID NO: 3 is the amino acid sequence of

KTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKSLTFNE which corresponds to positions 114 to 156 in SEQ ID NO: 1.

Brief Description of the Figure

Figure 1: Preliminary results of AT concentration and activity in plasma of healthy, intensive care unit (ICU) and infected patients. AT concentration was analysed by total AT EFISA. Activity assay was performed by using Stachrom AT III Kit (Diagnostica Stago, Asnieres sur Seine, France). By performing the activity assay in presence of 1.1 M NaCI, bAT is exclusively activated. Activity of aAT was calculated as the difference between total AT activity and bAT activity. Means ± SEM of 15 patients per group are shown. Significance is determined using one-way ANOVA with a Kruskal- Wallis multiple comparison test (p-value: 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****)).

Figure 2: Validation of activity of dialysed and resuspended aAT and bAT fractions.

Thrombin time (TT) in human citrate plasma was measured to examine the activity of the isolated AT isoforms. TT of 4 batches was tested in human citrate plasma of different donors. N=4, mean ± SEM is presented. Values are significant (p-value: 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****)) using a one-way ANOVA with Tukey’s multiple comparisons test.

Figure 3: Binding of AT to Gram-negative bacteria. (A) E. coli and P. aeruginosa were incubated with citrate plasma for 4-16 hours at 37°C. Bacterial cell pellets were collected and bound proteins were detached by acid buffer. Citrate plasma (c.p.) and 2 pg aAT were subjected to the SDS-PAGE as control. (B) E. coli was incubated with either 3 pg aAT or bAT for 8 hours at 37°C. Unbound AT was detected in supernatant (S) and bound protein in bacterial pellet (P). AT was detected by western blot using AT specific antibodies. Figure 4: Binding of LPS to aAT and bAT. Biacore graph shows the association and dissociation curve for LPS binding to immobilised AT isoforms. LPS were injected in diluted concentrations (1.875-30 x 10⁵ EU/ml) over the coated surfaces (at 10 mΐ/min in running buffer). EU, Endotoxin Unit.

Figure 5: Binding of aAT and bAT visualized by negative electron microscopy. aAT and bAT were incubated with LPS for 30 mins at room temperature. The binding and structural conformational changes of proteins were studied using negative staining electron microscopy.

Figure 6: Electron microscopy pictures of interaction between AT isoforms and E. coli. (A) Bacteria were incubated with either 10 mM aAT (left panels) or bAT (right panels) for 2 h at 37°C. Interaction was visualized by TEM and using gold-labelled monoclonal antibodies against AT (black spots). Insert shows a higher magnification. (B) E. coli bacteria were incubated for one hour with or without AT

variants and antimicrobial activity was visualised in electron microscopy. Scale Bar: 5 pm.

Figure 7: Antimicrobial activity of aAT and bAT in the presence or absence of plasma. Antimicrobial activity of 10 mM aAT and bAT against various Gram- negative (. E . coli, P. aeruginosa) and Gram-positive ( S . aureus, S. pyogenes) bacteria in 10 mM Tris buffer supplemented with 150 mM NaCI was measured. Results are means ± SEM of 3 independent experiments. Values are significant (p-value: 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****)) different as analysed using a one-way ANOVA with Tukey’s multiple comparisons test ns, non-significant.

Figure 8: Phagocytosis of bAT-opsonized bacteria is increased. (A) RAW 264.7 cells were pre-incubated with 5 mM aAT or bAT for 1 hour, the supernatant was removed and cells were incubated with E. coli particles for 2 hours. (B) E. coli particles were pre- incubated with 5 pM aAT or bAT in 20% human citrate plasma for 2 hours, followed by 2 hours-incubation with RAW 264.7 cells. Fluorescence was measured at 485 nm excitation and 535 nm emission. The percentage indicates the phagocytosis activity of AT in comparison to untreated cells served as control (ctr). Results are means ± SEM of two independent experiments with 4 values. Values are significant (p-value: 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****)) different from the control as analysed using a one-way ANOVA with Tukey’s multiple comparisons test.

Figure 9: AT Ill-binding to cells in human blood. Neutrophils and monocytes isolated from human blood were incubated with aAT or bAT and binding was recorded by FACs analysis. The results show both cell types bind more bAT on their surfaces than aAT.

Figure 10: Analysis of released inflammatory mediators (pro-, anti-inflammatory cytokines, chemokines, growth factors) after whole blood stimulation. Whole blood (50%) was incubated for 16 h at 37°C with 2.5 ng/ml LPS ± 10 mM a/bAT. Incubation with a/bAT alone and untreated samples served as controls. Additionally, LPS and AT isoforms were pre-incubated (pre-inc.) for 1.5 h at room temperature and then added to blood samples. Supernatants were analysed by multiplex immunoassay. Results are means ± SEM of 8 donors. Values are significantly (p-value: 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****) different from LPS as analysed using a one-way ANOVA with a Dunnett’s multiple comparisons test.

Figure 11: Survival studies using an LPS model. B ALB/c mice received an

intraperitoneal LPS injection (10 mg/kg) and were intravenously treated with 0.5 mg aAT or bAT 1 h and 5 h post-infection. Survival was monitored up to 48 h. (n=6/group, means ± SEM are indicated, Log-rank test, p-value (**) = 0.0039).

Figure 12: Modulation of inflammatory reactions by bAT in a murine E. coli infection model. BALB/c mice were intraperitoneally infected with 8-8.5 x 10⁷ cfu E. coli and intravenously treated with 0.5 mg aAT or bAT 1 h and 5 h post-infection. Citrate blood was analysed by multiplex immunoassay (n=l0/group of two independent experiments; means ± SEM are indicated). Values are significantly (p-value: 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****)) different from LPS as analysed using a one-way ANOVA with a Dunnett’s multiple comparisons test ns, non-significant. Figure 13: Reactive oxygen species (ROS) production and lung section of B ALB/c mice. BALB/c mice were intraperitoneally infected with 8-8.5 x 10⁷ cfu E. coli and intravenously treated with 0.5 mg aAT or bAT 1 h and 5 h post-infection. In vivo image of mice injected subcutaneously with ROS probe 8 hours after infection. (A) ROS production was quantified by radiance. Results are mean ± SEM of two independent experiments with 5-7 mice per group. A representative image (B) of 13 mice per group at 8 h after infection with E. coli. (C) Lungs of healthy and E. coli infected mice ± aAT/bAT administration were analysed by scanning electron microscopy 8 h post-infection. One representative lung section per group (n=3) is shown. Indicated scale bar corresponds to 100 pm.

Figure 14: Antimicrobial effects of bAT in a murine E. coli infection model. BALB/c mice were intraperitoneally infected with 8-8.5 x 10⁷ cfu E. coli and intravenously treated with 0.5 mg aAT or bAT 1 h and 5 h post-infection. Cfu of kidney, liver, lobe of lung and spleen (n=l2/group, means ± SEM are indicated). Values are significantly (p-value:

0.0332 (*), 0.0021 (**)) different as analysed using a one-way ANOVA with a Kruskal- Wallis multiple comparison test.

Figure 15: Survival studies using an E. coli infection model. BALB/c mice were intraperitoneally infected with 8-8.5 x 10⁷ cfu E. coli and intravenously treated with 0.5 mg aAT or bAT 1 h and 5 h post-infection. (A) Survival and (B) weight loss of mice was monitored up to 7 days. (n=6/group, means ± SEM are indicated, Log-rank test, p-value (**) = 0.0039).

Figure 16: Biacore analysis. Surface plasmon resonance (SPR) sensorgrams illustrating interactions between aAT and bAT (analytes) and immobilized CatG or CD300f or CD13/AMPN or HBP (ligands). The curves were obtained after injection of different concentrations of aAT and bAT at indicated concentrations and analysis shows binding incidence with association and dissociation curves between analytes and ligands. Figure 17: Inhibition of cathepsin G (CatG) activity by aAT and bAT. Cathepsin G activity in THP-l cell lysates were determined in presence of aAT and bAT. 1 x 10⁶ cells were lysed with 300 mΐ of the Cathepsin G Activity Assay Kit (abeam) lysis buffer.

Cathepsin G activity (abeam Cathepsin G Activity Assay Kit) in the cell pellets were then determined following manufacturer's instructions.

Figure 18: Treatment of gene modified mice with LPS. Gene modified mice were generated using the crispr-cas9 technology. The mouse AT III gene was replaced with human AT III or human bAT. Heterozygous mice were used for studying the cytokine response upon treatment with 4 mg/kg E. coli LPS. (mAT n=6; hAT n= 6 and bAT n=4).

Figure 19: Survival of bacteria in blood from gene modified mice. Blood from wild- type mice (mAT), heterozygous (native human AT III hAT (tAT)), aAT, and bAT, respectively; n=5) mice were infected with E. coli bacteria and bacterial survival was monitored.

Figure 20: Schematic cartoon of the signalling pathways involving bAT. The figure shows that (i) bAT permeabilises bacteria; (ii) bAT neutralises LPS by binding; (iii) bAT binding to CD300f leads to activation of SHP-l, which further inhibits MyD88 and TRIF pathways; (iv) bAT binding to CD 13 leads inhibition of TRIF pathway.

Figure 21: Treatment of transgenic mice expressing hAT or hbAT with LPS.

Modulation of inflammatory responses in. (A) Experimental design for the employed animal models. (B-C) Transgenic mice were i.p. injected with LPS (10 mg/kg) followed by monitoring survival and weight loss for up to 7 days on a daily basis (mAT n=5, hAT n=6 and HbAT n=7). (D) Mouse and human hAT gene expression levels in wild type and genetically modified mice were determined in liver (Healthy: mAT n=8, hAT n=9 and HbAT n=9; LPS: mAT h=10, haAT n=6 and IibAT n=8). (E) Determination of the concentration of hAT in murine blood of healthy and LPS-challenged mice (Healthy: mAT h=10, hAT n=5 and IibAT n=5; LPS: mAT n=6, hAT n=7 and IibAT n=7). (F) E. coli bacteria were grown in mouse blood AT and the colony forming units (CFU) were determined after 4h incubation (n=5/group). (G) Mice were infected with E. coli bacteria and CFUs were determined in kidney, liver, lung and spleen (n=6/group).

Figure 22: Generation of a transgenic mice replacing mouse AT gene with human AT gene. Mice were genetically modified by using CRISPR/Cas-mediated genome engineering technology to replace murine AT (mAT) gene with the gene of either both human AT glycosylation isoforms (hAT) or beta isoform (ΉbAT). (A) Comparison of mAT, hAT, and IibAT in gene and protein level. (B) Strategy for creating human AT transgenic mouse. The arrows indicate the primers used to check for targeted event by genomic PCR. (C-D) Genomic PCR demonstrating targeted DNA insertion in EC cells (C) and heterozygous mice tails (D).

Detailed Description of the Invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singular forms“a”,“an”, and“the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to“a polypeptide” includes“polypeptides”, and the like.

Structural features of beta-antithrombin

This section sets out the structural features of antithrombin (AT) and beta- antithrombin (b-AT or bAT) for use in the invention. “Antithrombin” can be used interchangeably with“AT” and“AT III”. Human AT has a molecular weight of 58kDa and is composed of 432 amino acids. AT is found in healthy individuals in two most abundant isoforms, alpha- antithrombin (a-AT or aAT) and bAT which differ in their amount of glycosylation. The major form aAT representing 90-95 % of the inhibitor in plasma, is fully N-glycosylated (at Asn96, Asnl35, Asnl55 and Asnl92, based on SEQ ID

NO: 1, and at Asnl28, Asnl67, Asnl87 and Asn224, based on SEQ ID NO: 2), whereas the minor glycoform bAT lacks the carbohydrate chain at Asnl35. The full amino acid sequence of human AT excluding signal sequences is shown in SEQ ID NO: 1. The full amino acid sequence of human AT including the signal sequences is shown in SEQ ID NO: 2. The term“bAT”, as used herein, denotes a polypeptide that may comprise, consist essentially, or consist of the sequence of SEQ ID NO: 1, where position 135 lacks a carbohydrate chain but where positions 96, 155 and 192 may be N-glycosylated

(numbering based on SEQ ID NO: 1). References to the position numbering in the amino acid sequence herein will be based on SEQ ID NO: 1, though the skilled person would understand how to work out the corresponding positions in a sequence of SEQ ID NO: 2. “bAT” is also understood to encompass variants and fragments thereof as described herein.

The polypeptide of the invention may also comprise, consist essentially, or consist of a variant of the amino acid sequence of SEQ ID NO: 1 which is at least 50%, at least 60%, at least 70%, at least 80%, at least, 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence of SEQ ID NO: 1, where the bAT lacks a carbohydrate chain at position 135, optionally where it is N-glycosylated at positions 96, 155 and/or 192. The identity level is preferably at least 85% or higher. Identity relative to the sequence of SEQ ID NO: 1 can be measured over a region of at least 100, at least 200, at least 300, at least 350, or at least 400 or more contiguous amino acids of the sequence shown in SEQ ID NO: 1, or more preferably over the full length of SEQ ID NO: 1. A variant is typically of a length which is no more than 50 amino acids longer or shorter than the reference sequence, and is preferably of approximately (or exactly) the same length as the reference sequence.

Amino acid identity may be calculated using any suitable algorithm. For example the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300;

Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. Lor example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG Package provides the BESTLIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, 387-395).

The sequence of a polypeptide of the invention may comprise a variant of the amino acid sequence of SEQ ID NO: 1 in which modifications, such as amino acid additions, deletions or substitutions are made relative to the sequence of SEQ ID NO: 1. Unless otherwise specified, the modifications are preferably conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the

conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table Al below. Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains in Table A2. A sequence of a polypeptide of the invention may comprise a variant of the amino acid sequence of SEQ ID NO: 1 in which up to 10, 20, 30, 40, 50 or 60 conservative substitutions are made.

Table Al - Chemical properties of amino acids

Table A2 - Hydropathy scale

Side Chain Hydropathy

lie 4.5

Val 4.2

Leu 3.8

Phe 2.8

Cys 2.5

Met 1.9

Ala 1.8

Gly 0.4

Thr 0.7

Ser 0.8

Trp 0.9

Tyr 1.3

Pro 1.6

His 3.2

Glu 3.5

Gin 3.5

Asp 3.5

Asn 3.5

Lys 3.9

Arg 4.5

The amino acid sequence of a polypeptide of the invention may comprise a variant of the amino acid sequence of SEQ ID NO: 1 as described above. However, certain residues in the amino acid sequence of SEQ ID NO: 1 are preferably retained within the said variant sequence such that the polypeptide retains its antimicrobial activity. For example, the said variant sequence typically retains a stretch of amino acids, KTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKSLTFNE (SEQ ID NO: 3).

The stretch of amino acids may be present in the polypeptide at positions corresponding to positions 114 to 156 with reference to SEQ ID NO: 1. A polypeptide of the invention may comprise a variant of the amino acid sequence of SEQ ID NO: 1 which does not comprise a residue capable of being N-glycosylated, thus not allowing a carbohydrate chain to be attached at position 135 of SEQ ID NO: 1 as with a wild-type amino acid sequence of AT, SEQ ID NO: 1.

Alternatively, a polypeptide of the invention may comprise, consist essentially, or consist of a shorter fragment of SEQ ID NO: 1 or of a variant thereof as described above. The fragments may be described as a truncated form of SEQ ID NO: 1 which retains its antimicrobial activity. Such fragments are shorter than SEQ ID NO: 1 and are typically at least 350, 360, 370, 380, 390, 400, 410 or 420 amino acids in length. The fragments typically comprise the sequence,

KTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKSLTFNE (SEQ ID NO: 3). General polypeptide features

A“polypeptide” is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term “peptide” thus includes short peptide sequences and also longer polypeptides and proteins. The terms“protein”,“peptide”,“polypeptide” and“glycoprotein” may be used

interchangeably. A glycoprotein is a protein that contains oligosaccharide chains (glycans) or carbohydrates attached to amino acid side chains. The carbohydrate may be attached to the polypeptide in a cotranslational or posttranslational modification, known as

glycosylation. The two most common types of glycosylation are N-glycosylation and O- glycosylation. In N-glycosylation, the carbohydrates are typically attached to a nitrogen that may be found on an amide side chain of asparagine (Asn). In O-glycosylation, the carbohydrates are typically attached to an oxygen on serine (Ser), threonine (Thr) or tyrosine (Tyr). As used herein, the term“amino acid” and“amino acid residue” may be used interchangeably, and refers to either natural and/or unnatural or synthetic amino acids, including both D or L optical isomers, and amino acid analogs and peptidomimetics.

A polypeptide e.g. bAT, may be purified from plasma using known techniques in the art. Different isoforms of a polypeptide may be separated and purified using affinity chromatography techniques known in the art. For instance, in the context of bAT and aAT, the different heparin affinity can be used in order to separate both isoforms via by heparin sepharose affinity chromatography, in which aAT is eluted from the column at 1 M NaCl and bAT at a concentration of more than 1.4 M NaCl, for instance. Purified bAT has less than 25%, 20%, 15%, 10%, 5% or 1% of the AT in alpha form. Preferably, less than 5% of the AT is in alpha form.

A polypeptide may also be produced by any suitable method, including

recombinant or synthetic methods, or semi- synthetic methods for example by combining a recombinant and synthetic production. For example, the polypeptide may be synthesised directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry or by solution phase peptide synthesis. A polypeptide may also be synthesised using in vitro translation of mRNA. Suitable cell-free expression systems include rabbit reticulocyte lysate, wheat germ extract, canine pancreatic microsomal membranes, E. coli S30 extract, and coupled transcription/translation systems such as the TNT-system (Promega). These systems allow the expression of recombinant peptides or proteins upon the addition of cloning vectors, DNA fragments, or RNA sequences containing coding regions and appropriate promoter elements. Alternatively, a peptide may be produced by transforming a cell with a nucleic acid molecule or vector which encodes said peptide. A large number of suitable methods exist in the art to produce peptides in appropriate hosts under appropriate culture conditions, such as in a mammalian cell, yeast cell, plants cell, bacteria or insect cell. The produced protein is harvested from the culture medium, lysates of the cultured cells or from isolated (biological) membranes by established techniques. For example, nucleic acid sequences of the peptide can be synthesised by PCR and inserted into an expression vector. Subsequently a suitable host cell may be transfected or transformed with the expression vector. The host cell is then cultured to produce the desired peptide, which is isolated and purified. Subsequent in vitro glycosylation of recombinant proteins may be carried out as described in the art.

The terms“nucleic acid molecule” and“polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either

deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide encodes a polypeptide for use in the invention and may be provided in isolated or substantially isolated form. By substantially isolated, it is meant that there may be substantial, but not total, isolation of the polypeptide from any surrounding medium. The polynucleotides may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. A nucleic acid sequence which“encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences, for example in an expression vector. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the purposes of the invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

Polynucleotides can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the peptide in vivo. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors). Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide may be administered to a host subject. Preferably the polynucleotide is prepared and/or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide for use in the invention.

A polypeptide may be derivatised or modified to assist with their production, isolation or purification. For example, the polypeptide may be derivatised or modified by addition of a ligand which is capable of binding directly and specifically to a separation means. Alternatively, the peptide may be derivatised or modified by addition of one member of a binding pair and the separation means comprises a reagent that is derivatised or modified by addition of the other member of a binding pair. Any suitable binding pair can be used. For example, where the polypeptide for use in the invention is derivatised or modified by addition of one member of a binding pair, the peptide may be histidine-tagged or biotin-tagged. Typically the amino acid coding sequence of the histidine or biotin tag is included at the gene level and the peptide is expressed recombinantly. The histidine or biotin tag is typically present at either end of the peptide. It may be joined directly to the peptide or joined indirectly by any suitable linker sequence, such as 3, 4 or 5 glycine residues, or a mixture of glycine and serine residues. The histidine tag typically consists of six histidine residues, although it can be longer than this, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2 or 1 amino acids.

The polypeptide for use in the invention or a variant thereof may be fused to a further polypeptide, such as a tag, signal peptide or an antigenic determinant that is known in the art. Such additional sequences may aid with expression and/or purification, increase the solubility of the peptide or be used to target the peptide of interest to an organ or tissue wherein the cells express certain antigens to which the tag bind. For example, the tag may be a histidine tag, human influenza hemagglutinin (HA) tag, FLAG-tag or biotin tag. The tag may be linked to the N or C terminus by a linker. A linker may be used to connect or fuse the peptides. The linker may physically separate the polypeptides to ensure that neither polypeptide is limited in their function due to the close vicinity to the other.

Depending on what the further polypeptide is, the linker can be a peptide bond, an amino acid, a peptide of appropriate length, or a different molecule providing the desired features, or any appropriate linker known to the skilled person. For example, peptide linkers can be chosen from the LIP (Loops in Proteins) database (Michalsky et al (2003) Protein Eng Des Sel, (12): 979-985). A linker may be attached to the N- or the C-terminus of the polypeptide. The linker is preferably located at the N-terminus. In a preferred

embodiment, the linker is a lysine, glycine, serine, an ether, ester or a disulphide. Signal peptides are short amino acid sequences capable of directing the peptide or protein to which they are attached to different cellular compartments or to the extracellular space. Antigenic determinants allow for the purification of the fusion peptides via antibody affinity columns.

The N-and C-terminus of the polypeptide may be derivatized using conventional chemical synthetic methods. The polypeptides may contain an acyl group, such as an acetyl group. Methods for acylating, and specifically for acetylating the free amino group at the N-terminus are well known in the art. For the C-terminus, the carboxyl group may be modified by esterification with alcohols or amidated to form -CONFb or CONHR. Methods of esterification and amidation are well known in the art.

A polypeptide may be provided in a substantially isolated or purified form. That is, isolated from the majority of the other components present in a cellular extract from a cell in which the polypeptide was expressed. By substantially purified, it will be understood that the polypeptide is purified to at least 50%, 60%, 70%, 80% or preferably at least 90% homogeneity. Purity level may be assessed by any suitable means, but typically involves SDS-PAGE analysis of a sample, followed by Coomassie Blue detection. A polypeptide may be mixed with carriers, diluents or preservatives which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated or purified.

Where a polypeptide is provided in a composition with an additional active component, such as another polypeptide, each said polypeptide will individually be purified to a high level of homogeneity prior to mixing in an appropriate ratio for the intended purpose of each. For example, two polypeptides may be each be purified to at least 90% homogeneity prior to combining in a 1: 1 ratio. In any such composition, less than 25%, 20%, 15%, 10%, 5% or 1% of the AT is in alpha form. Preferably, less than 5% of the AT is in alpha form.

A polypeptide (or mixture thereof) may be provided in lyophilised form, suitable for reconstitution in aqueous solution prior to use. The lyophilised composition has improved stability enabling longer storage of the polypeptide. A method of preparing a polypeptide (or mixture thereof) in lyophilised form, comprising freeze-drying said polypeptide (or mixture) in a suitable buffer, such as Tris-buffered saline (TBS), is provided herein. A polypeptide is typically substantially purified prior to freeze-drying. The resulting polypeptide (or mixture) in lyophilised form is also provided. A method of preparing a solution of a polypeptide (or mixture), comprising providing the polypeptide (or mixture) in lyophilised form and reconstituting with a suitable carrier or diluent, such as water, is also provided.

Methods of Treatment/Medical Use The present invention relates to an antimicrobial, bAT as described above, for use in a method of treating an infection, wherein the method comprises administering to the individual an effective amount of the bAT as described herein.

An infection is the invasion of body tissues by disease-causing (infectious) agents or microbes. Such infectious agents or microbes include for example viruses, bacteria, fungi, parasites, viroids, prions, nematodes, arthropods and helminths. An“infection” may be used interchangeably with“infectious disease”. The infectious agents or microbes multiply and the host tissue react to them, such as involving inflammation. Preferably, the infection is a bacterial infection.

bAT may be used to treat infections caused by bacteria such as Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma (e.g. Anaplasma phagocytophilum), Azorhizobium caulinodans, Azotobacter vinelandii, viridans streptococci, Bacillus (e.g. Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis), Bacillus Thuringiensis, Bacteroides (e.g. Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus (now known as Prevotella melaninogenica )), Bartonella (e.g. Bartonella henselae, Bartonella quintana), Bordetella (e.g. Bordetella

bronchiseptica, Bordetella pertussis), Borrelia burgdorferi, Brucella (e.g. Brucella abortus, Brucella melitensis, Brucella suis), Burkholderia (e.g. Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia), Calymmatobacterium granulomatis, Campylobacter (e.g. Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylon), Chlamydia (e.g. Chlamydia trachomatis), Chlamydophila (e.g. Chlamydophila pneumoniae (previously called Chlamydia pneumoniae), Chlamydophila psittaci (previously called Chlamydia psittaci)), Clostridium (e.g. Clostridium botulinum, Clostridium difficile, Clostridium perfringens (previously called Clostridium welchii), Clostridium tetani), Corynebacterium (e.g. Corynebacterium diphtheriae,

Corynebacterium fusiforme), Coxiella burnetii, Ehrlichia chajfeensis, Enterobacter cloacae, Enterococcus (e.g. Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus), Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus (e.g. Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis), Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus (e.g. Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis), Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium (e.g. Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis), Mycoplasma (e.g. Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria (e.g. Neisseria gonorrhoeae, Neisseria meningitidis), Pasteurella (e.g.

Pasteurella multocida, Pasteurella tularensis), Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenica (previously called Bacteroides melaninogenicus), Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia (e.g. Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae), Rochalimaea (e.g. Rochalimaea henselae, Rochalimaea quintana), Rothia dentocariosa, Salmonella (e.g. Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium), Serratia marcescens, Shigella dysenteriae, Spirillum volutans, Staphylococcus (e.g.

Staphylococcus aureus, Staphylococcus epidermidis), Stenotrophomonas maltophilia, Streptococcus (e.g. Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae,

Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius. Streptococcus sanguis, Streptococcus sobrinus), Treponema (e.g. Treponema pallidum, Treponema denticola), Vibrio (e.g. Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus), Wolbachia, Yersinia (e.g. Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis). The bAT for use of the invention may be used to treat infections caused by either Gram-positive or Gram-negative bacteria. The bAT is preferably used to treat bacterial infections caused by Gram-negative bacteria, such as E. coli or P.

aeruginosa. For instance, the bAT may be used to treat bacteraemia, infections which lead to sepsis, lung infections, skin infections or systemic inflammatory complications. bAT may also be used to treat infections caused by viruses such as adenovirus, Herpes simplex type 1, Herpes simplex type 2, Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Human herpesvirus type 8, Human papillomavirus, BK virus, JC virus, Smallpox, Hepatitis B virus, Parvovirus B19, Human astrovirus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, Severe acute respiratory syndrome virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, Rubella virus, Hepatitis E virus, Human immunodeficiency virus (HIV), Influenza virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Rabies virus, Hepatitis D, Rotavirus, Orbivirus, Coltivirus, Banna virus. The treatment may allow the dampening of a pathologic inflammatory response.

bAT may also be used to treat infections caused by fungi such as Candida (e.g. Candida albicans, Candida tropicalis, Candida kruser), Aspergillus (e.g. Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus), Cryptococcus (e.g. Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii),

Histoplasma (Histoplasma capsulatum), Pneumocytis (e.g. Pneumocystis jirovecii (or Pneumocystis carinii), Stachybotrys (e.g. Stachybotrys chartarum). The treatment may lead to dampening of a pathologic inflammatory response and may exhibit anti-fungal activity.

The infection may be in any part of the body, including but not limited to the blood, lungs, liver, abdomen, urinary tract, pelvis, skin, sinuses, respiratory tract, eye, stomach, genitals, brain, lymph, or some or all of the above.

An individual to be treated by the administration of the peptide may be a human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is preferred.

The method of the invention may be for treating an infection. In the case of treating, the patient typically has an infection, i.e. has been diagnosed as having an infection, or is suspected as having an infection, i.e. shows the symptoms of an infection. As used herein, the term“treating” includes any of following: the prevention of an infection or of one or more symptoms associated with an infection; a reduction or prevention of the development or progression of an infection or symptoms; and the reduction or elimination of the existing infection or symptoms. In some embodiments the beta- antithrombin is not for use in a method of treating sepsis.

The method may be for preventing an infection. In this embodiment, the individual may be asymptomatic. As used herein, the term“preventing” includes the prevention of acquiring an infection or of one or more symptoms associated with an infection.

The method may be for ameliorating the symptoms associated with an infection.

As used herein, the term“ameliorating” includes the reduction or elimination of the existing infection or symptoms.

Therapy and prevention includes, but is not limited to preventing, alleviating, reducing, curing or at least partially arresting symptoms and/or complications resulting from or associated with an infection. When provided therapeutically, the therapy is typically provided at or shortly after the onset of a symptom of an infection. Such therapeutic administration is typically to prevent or ameliorate the progression of, or a symptom of an infection or to reduce the severity of such a symptom or infection. When provided prophylactic ally, the treatment is typically provided before the acquisition of an infection or of a symptom of an infection. Such prophylatic administration is typically to prevent the onset of symptoms of an infection.

Methods of diagnosing an infection include e.g. microbial culture methods, microscopy techniques, biochemical tests, PCR etc. which are known in the art.

Antimicrobial activity of a substance e.g. bAT, can also be determined using these techniques, as well as those shown in the Examples. For instance, electron microscopy may be used to observe pore formations on the bacterial surface and release of bacterial exudates which represents good antimicrobial activity, standard viable count assays and survival studies may also be employed.

Combination therapies

An antimicrobial agent e.g. bAT may be used in combination with one or more other therapies or agents intended to prevent and/or to treat infections, or to ameliorate the symptoms associated with an infection in the same individual. The therapies or agents may be administered simultaneously, in a combined or separate form, to an individual.

The therapies or agents may be administered separately or sequentially to an individual as part of the same therapeutic regimen. The other therapy or administration of an agent may be a general therapy aimed at treating or improving the condition of an individual with an infection. The other therapy or administration of an agent may be a specific treatment directed at an infection or directed at a particular symptom of an infection.

For example, treatment may include administering agents such as antibacterials or antibiotics, antivirals, antifungals or antiparasitics, antiprotozoals, antihelminthics etc that are commonly used. As bAT leads to a permeable cell membrane, it can be used in combination of antibiotics such as gyrase inhibitors or other transcriptional and

translational bacterial inhibitors. A combination may lead to better efficiency of the intracellularly acting drug.

Effects of bAT administration

bAT may have a direct antimicrobial effect on the infectious agents, such as E. coli or P. aeruginosa. For instance, bAT may lead to pore formations on a bacterial surface and cause release of bacterial exudates, which may be observed through an electron microscope. bAT may bind to the infectious agent, e.g. bacteria. Binding can be assessed using SDS-PAGE and Western blot techniques and surface plasmon resonance techniques. bAT may also lead to enhanced phagocytosis of e.g. bacteria, by macrophages observed through carrying out a phagocytosis assay known in the art.

bAT may prolong the clotting time of blood, as measure using a thrombin time test known in the art.

bAT may modulate the inflammatory response. The measurement of inflammation parameters associated with infection may be carried out by analysing blood samples.

At least one of the above effects are observed. Alternatively, all of these effects are observed.

Administration routes, formulations and dosages

Specific routes, dosages and methods of administration of the bAT for use in the invention may be routinely determined by the medical practitioner. Typically, a therapeutically effective or a prophylactically effective amount of the bAT is administered to the patient. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of an infection. A therapeutically effective amount of the compound is an amount effective to ameliorate one or more symptoms of an infection. A therapeutically or prophylactically effective amount of the bAT is administered. The dose may be determined according to various parameters, especially according to the compound used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.

The bAT can be administered to the patient by any suitable means. The polypeptide can be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraosseous, intraperitoneal, intraarticular, topical or other appropriate administration routes. The polypeptide is preferably administered intravenously, intranasally or topically. For instance, when used to treat an infection leading to sepsis, the polypeptide is preferably administered intravenously. When used to treat lung infections, the polypeptide is preferably administered intranasally. When used to treat skin infections, the polypeptide is preferably administered topically.

The bAT may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intraosseously, intrastemally, transdermally or by infusion techniques. The polypeptide may also be administered as a suppository. A physician will be able to determine the required route of administration for each particular patient.

The polypeptide can be formulated for use with a pharmaceutically acceptable carrier or diluent and this may be carried out using routine methods in the pharmaceutical art. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes. Liquid dispersions for oral administration may be syrups, emulsions and

suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.

The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.

Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit“S”, Eudragit“L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.

Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermally, may also be used.

A daily dosage for administration to a subject such as a human may range from about 25mg/kg to about 50 mg/kg, from about 25 mg/kg to about 75 mg/kg, from 50 mg/kg to 100 mg/kg, from about 75 mg/kg to about 150 mg/kg, from about 100 mg/kg to about 250 mg/kg, from about 150 mg/kg to about 500 mg/kg, from about 200 mg/kg to about 750 mg/kg, from about 500 mg/kg to about 1500 mg/kg. Administration may be in single or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations.

Alternatively, doses can be via a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the bAT in the patient and the duration of treatment desired.

The skilled person and particularly an appropriate physician will be able to identify an appropriate dosage, for instance taking factors such as age, sex, weight, conditions of the patient to be treated, the severity of the disease and the frequency and route of administration and so on into account.

Novel AT interactions with receptors/binding proteins/ligands

ATIII is a natural anticoagulant protein and tissue factor pathway inhibitor. It also has potent anti-inflammatory properties. bAT in particular is primarily responsible for the anticoagulant effect of AT due to the lack of glycosylation at Asnl35. However, the molecular mechanisms of the anti-inflammatory properties of ATIII are not well understood. The present inventors have identified novel interactions between ATIII (both the alpha and beta forms) and certain proteins that play a role in inflammatory signaling pathways, which suggest ways in which ATIII is able to modulate inflammatory responses.

In particular, interactions between ATIII and the following proteins have been identified: CD300f/CLMl/CMRF35-like molecules 1, LRP1/CD91, Cathepsin G, aminopeptidase N/CD13, prolow-density lipoprotein receptor-related protein

(LRP1/CD91), protein EVI2B/CD361, multiple epidermal growth factor like domains protein 9 (MEGF9) and Heparin-binding protein (also known as azurocidin or CAP37).

ATIII levels are depleted in certain diseases, e.g. sepsis. During sepsis for instance, inflammatory cytokines and endotoxin are released and are involved in the inhibition of fibrinolysis and activation of the coagulation cascade by inducing cell surface receptor tissue factor. Upon stimulation with cytokines or bacterial components like LPS, macrophages express tissue factor. The tissue factor triggers the extrinsic coagulation pathway and mediates systemic inflammation-induced coagulation. Some of the activated serine proteases e.g. thrombin, can trigger the complement cascade. The impaired balance of coagulation can result in disseminated intravascular coagulation and ultimately lead to multiple organ failure in patients with severe sepsis due to the consumption of anticoagulants. Thus, ATIII, particularly bAT, is administered to restore and replenish the levels of ATIII to a“normal” level as that in a healthy individual.

The administration of bAT therefore provides a dual benefit. On the one hand, in certain disease conditions, such as sepsis, bAT is able to inhibit thrombin and other clotting factors (clotting-dependent mechanisms). On the other hand, bAT can exert coagulation-independent effects through interaction with the proteins identified herein.

The present invention provides bAT for use to regulate a Toll-Like Receptor (TLR) pathway in an individual in need thereof. The individual may have a condition related to TLR- associated inflammation. For instance, the bAT may be used to treat diseases or conditions such as a coagulation disorder caused by a consumption or deficiency of ATIII or bAT. The bAT may also be used to treat a complication caused by depleted, overexpressed, or malfunctioning cathepsin G, heparin-binding protein, CMRF35-like molecule 1, aminopeptidase N, prolow-density lipoprotein receptor-related protein 1, EVI2B, multiple epidermal growth factor like domains protein 9. The bAT may also be used to treat a complication caused by infection, trauma, or a malfunctioning immune response.

The amount or the change in level (depletion/consumption/deficiency or overexpression) of the proteins described herein, including ATIII and bAT can be measured by methods well known in the art, including immunological methods such as ELISA. Mass spectrometric methods may also be used. Chromogenic substrates may be employed. Malfunctioning of the proteins means that the proteins fail to carry out their normal function and may lead to their depletion or overexpression.

The invention also provides use of bAT in the manufacture of a medicament for the treatment of such a disease or condition. Also provided is a method of treating such a disease or condition by administering bAT to an individual.

The invention is illustrated by the following Examples:

Examples

MATERIALS AND METHODS

Isolation and Purification of Antithrombin Glycosylation Isoforms by FPLC In order to separate and purify aAT and bAT affinity chromatography was used by utilizing the different heparin affinity of the glycosylation isoforms using a fast protein liquid chromatography (FPLC) system (AKTA purifier, GE Healthcare). 40 g hAT (III Baxalta 1000 IE, Baxalta Innovations GmbH, Vienna, Austria) diluted in 0.1 M Tris, 0.01 M citric acid, 0.225 M NaCl, pH 7.4 (Buffer A) was applied to a HiTrap 5 x 5 ml Heparin HP column (GE Healthcare, Uppsala, Sweden) equilibrated with 55 % buffer A / 45 % buffer B (0.1 M Tris, 0.01 M citric acid, 2 M NaCl, pH 7.4). The elution of the isoforms was performed by using buffers according to the GE Healthcare protocol for isolation of bovine AT III. After injection, samples were eluted with two elution steps: haAT was eluted with 60 ml of 55% buffer A/45 % buffer B (~ 1.2 M NaCl) and IibAT with 31 ml 100 % buffer B (2 M NaCl) at a flow of lml/min. Fractions of each peak were collected manually and analyzed by measuring absorbance at 280 nm. For storage at 4 °C the column was washed with degased water and stored in 20 % ethanol. The isolated glycosylation isoforms were dialyzed in 12-14 kDa molecular weight cut off dialysis tubes (Spectra/Por® Dialysis Tubing 12-14 kD MWCO, Spectrum Laboratories, Rancho Dominguenz, USA) against 0.01 M Tris, pH 7.4 to reduce the salt concentration. Dialysis was performed in 5 liter buffer for 2 days with changing the buffer twice a day. The protein solution was frozen at -80 °C and concentrated by freeze drying (Freezone Plus 6, Labconco, Kansas City, USA). Then, pellet was dissolved in water to desired

concentration. The protein concentration was measured at 280 nm using the NanoDrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, Waltham, USA) based on the Bradford Protein Assay (A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248- 54 (1976)). Aliquots of the AT III isoform samples were stored at -20°C.

Coagulation Assay

Activities of purified haAT and hbAT were tested by analyzing thrombin induced fibrin-network formation (thrombin time, TT) using a coagulometer (Me 10 Plus merlin medical, Lemgo, Germany). Clotting time was measured by incubating 50 mΐ citrated human plasma supplemented with 5 mM purified AT III for 1 min at 37°C in cuvettes with metal ball. Clotting was initiated by the addition of 50 mΐ thrombin reagent (Technoclone, Vienna, Austria). TT is detected when plasma starts clotting. Human AT Assay

The concentration of the total AT III in a bottle of Antithrombin III Baxalta 1000 IE dissolved in 15 ml buffer A was measured by using human Antithrombin III Total Antigen ELISA Kit (Molecular Innovations, Novi, USA) according to manufacturer’s protocols. Absorbance was measured at 450 nm in iMark™ Microplate Reader (Bio-Rad, Hercules, USA).

Cell Culture

The murine macrophages cell line RAW 264.7 (ATCC® TIB-71™) was purchased from American Type Culture Collection (Manassas, USA) and cultured in DMEM/high modified without phenol red (GE Healthcare Life Science, HyClone Laboratories, Logan, USA) supplemented with 10% (v/v) heat-inactivated LBS (Invitrogen, Carlsbad, USA) and 1% (v/v) antibiotic-antimycotic solution (Invitrogen, Carlsbad, USA) at 37 °C in a 5 %

C0₂ incubator. Sub-confluent (70-80%) RAW cells were split by scraping and maintained until passage 17.

Viable Count Analysis

The viable count analysis was performed to determine the antimicrobial effect of haAT und IibAT on different types of bacteria. E. coli ATCC 25922, Pseudomonas aeruginosa PA01, Staphylococcus aureus ATCC 29213, Streptococcus pyogenes AP1, Candida albicans ATCC 90028 and Candida parapsilosis ATCC 90018 were grown to mid-log phase in Todd-Hewitt broth. Bacteria/fungi were washed twice with lOmM Tris, pH 7.4. Bacteria/fungi were diluted either in 10 mM Tris, pH 7.4, with or without 150 mM NaCl, or with 20 % human citrate plasma. 2 x 10⁶ cfu/ml bacteria were incubated in 50 mΐ, at 37 °C for 2 h with haAT and IibAT in the concentration of 5 mM and 10 mM or without effector as control. To determine the antimicrobial effect of bAT within whole citrate blood of hAT- and hbAT-mice, E. coli (1 x 10⁸ cfu/ml) bacteria were incubated in 100 mΐ of citrate whole blood (n=5 mice/group) at 37 °C for 1 h. Wild-type whole citrate blood served as control. Serial dilutions of the incubation mixture were plated on Todd-Hewitt agar, followed by incubation at 37 °C overnight and cfu determination. Reactions were performed in triplicates.

Pull Down Assay To investigate hAT III binding to bacteria surface, a pull down assay was performed by incubating 450 mΐ human citrate plasma with either 50 mΐ of 2 x 10⁹ cfu/ml E. coli or P. aeruginosa diluted in 10 mM Tris, 150 mM NaCl, pH 7.4 in 1.5 ml-reaction tubes for 4 h, 8 h, 16 h or overnight at 37 °C in a 5 % C0₂ incubator. (Culturing of bacteria see VCA). Additionally, 50 mΐ of the same 2 x 10⁹ cfu/ml E. coli suspension was incubated with 3 pg haAT or IibAT for 8 h under equal conditions. After incubation time bacteria were spun down at 5800 g for 5 min. The supernatant was collected and frozen at -20 °C after adding 100 mΐ of 100 % trichloroacetic acid. The bacteria pellet was washed twice with 1 ml 10 mM Tris. The pellet was resuspended in 360 mΐ of 0.1 M glycine-HCl, pH 2.0, vigorously vortexed and incubated for 5 to 10 min at room temperature to elute bound proteins. Followed by centrifugation at 1500 g for 5 min, the supernatant containing the bound proteins fraction was collected in a separate tube. 20 mΐ of 1 M Tris was added to raise pH to 7.4. Precipitation of proteins was achieved by adding 100 mΐ of 100 % trichloroacetic acid to the solution and vortexing. After keeping for at least 10 min at -20 °C, the solution was centrifuged at 15,000 g for 15 min at 4 °C, supernatant was discarded and the pellet was washed with 200 mΐ of 100 % acetone. After centrifugation at 15,000 g for 5 min the supernatant was discarded and the pellet was air dried. The precipitated material was dissolved in 4x reducing SDS sample buffer (50 mM Tris, 5 % (w/v) SDS, 20 % (v/v) glycerol, 0.02 mg/ml bromophenol blue, 5 % (v/v) b-mercaptoethanol), denatured at 95 °C for 10 min and subjected to a SDS-gel electrophoresis followed by

immunoblotting .

SDS-Gel Electrophoresis

Denaturated samples were separated on 4-20 % Mini PROTEAN® TGX™ or 10 % Criterion™ precast gels in lx Tris-Glycine-SDS running buffer for 60 min at 100 V. Gels, buffers and equipment (Mini PROTEAN® Tetra Cell) were derived from Bio-Rad

(Miinchen, Germany). Gels were stained with Simplyblue™ SafeStain (Invitrogen, Carlsbad, CA) and pattern were documented using a GelDoc™ EZ Imager (Bio-Rad, Hercules, USA). PageRuler™ prestained protein ladder, 10 to 180 kDa (Thermo Fisher Scientific, Waltham, USA) was used to evaluate the separation of the SDS-PAGE.

Western Blot After SDS-gel electrophoresis, gels were equilibrated in transfer buffer and PVFD- membrane (Immobilon®-P Membrane, EMD Millipore Corporation, Billerica, USA) was activated by methanol for 10 s, rinsed with water followed by transfer buffer (0.025 M Tris, 0.19 M glycine, 20 % (v/v) methanol, 10 % (w/v) SDS) for 10 min. The transfer cassette was assembled, placed in a Citerion™ Blotter (Bio-Rad, Hercules, USA) and performed at 100 V for 60 min. Subsequently, the membrane was blocked with 3 % non fat dry milk (Blotting-Grade Blocker, Bio-Rad, Hercules, USA) in PBS containing 0.05 % Tween-20 for 60 min at RT while shaking and incubated with monoclonal (EP5372) rabbit anti-human AT III antibodies (abl26598, Abeam, Cambridge, UK) diluted 1:1000 in blocking solution for 60 min at RT. Next, the membrane was washed thrice for 5 min in PBS-Tween (0.05 %) and incubated with goat anti-rabbit IgG-HRP conjugate (Bio-Rad, Hercules, USA) diluted 1:1000 in blocking solution for 45 min at RT. The membrane was washed thrice for 5 min in PBS-Tween (0.05 %), developed with Super Signal® West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, USA) for 3 min and visualized using ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, USA).

Surface Plasmon Resonance/BiaCore

The affinity between hAT and LPS, CatG, CD13 and CD300f was analysed by using surface plasmon resonance (SPR) analysis. Analyses were performed with a BIAcore XI 00 instrument (GE Healthcare, Uppsala, Sweden) using Sensor Chip CM5 technology at 25°C in a degassed HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Surfactant P20, pH 7.4) with a flow rate of 10 mΐ/min. Both isoforms of AT III were immobilized via amine coupling to flow cell 1 (aAT) or flow cell 2 (bAT). For amine coupling, the dextran surface was activated by injecting a 1:1 mixture of 0.4 M l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide and 0.1 M N-hydroxysuccinimide for 6-10 min. Then the ligand was immobilized by an injection of 50 pg/ml of haAT or IibAT diluted in 10 mM sodium acetate (pH 5.5) for 5-10 min. Next, 1 M ethanolamine-HCl, pH 8.5 was injected for 6-7 min to deactivate excess reactive groups of the Sensor Chip surface. One Flow cell was subjected without protein and used as a control in this experiment. LPS, CD13, CD300f, or Heparin-binding protein were added as analyte in indicated

concentrations to the coated surface at 10 mΐ/inin in HBS-EP buffer. The Sensor Chip surfaces were regenerated by an injection of 30 mΐ 50 mM NaOH. All buffers, reagents and equipment were purchased from GE Healthcare (Uppsala, Sweden).

Sensorgrams were analyzed using the BIA Evaluation 4.1 software (GE

Healthcare). After X/Y normalization of data, blank curves from the vehicle flow cell were subtracted, and the association (k_a) and dissociation (k_d) rate constants were determined using a Langmuir model in the evaluation program.

Transmission Electron microscopy

For visualization of the binding of haAT or IibAT to the bacterial surface, transmission electron microscopy (TEM) was performed as described in detail by

Abdillahi et al. (Abdillahi el ah, J Innate Immun, 7, 506-17. (2015)). Briefly, monoclonal (EP5372) rabbit anti-human AT III antibodies (ab 126598, Abeam, Cambridge, UK) were labeled with 20 nm gold colloid (BBIntemational, Cardiff, UK). E. coli ATCC 25922 (1 x 10⁵ cfu/50 mΐ) were incubated with 10 mM of haAT or IibAT in 10 mM Tris, 150 mM NaCl, pH 7.4 for 2 h at 37 °C. Bacteria were spun down (3000 x g) and then incubated with gold-labeled antibodies for 30 min at room temperature. For negative staining samples were adsorbed onto 400-mesh carbon-coated copper grids (Agar scientific, Stansted, UK) for 2 min, washed briefly by two drops of water, and stained by two drops of 0.75% uranyl formate. All samples were examined with a JEOL JEM 1230

transmission electron microscope (JEOL, Peabody, Mass., USA) at 60 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 charge-coupled device camera using DigitalMicrograph™ software.

Scanning Electron Microscopy

For scanning electron microscopy (SEM), murine lungs were fixed overnight at room temperature with 2.5% glutaraldehyde in 0.15 M cacodylate buffer, pH 7.4. They were then washed with cacodylate buffer four times for 10 min and dehydrated with an ascending ethanol series from 50% (v/v) to absolute ethanol according the following protocol: 2 x 15 min 50 % ethanol, 2 x 15 min 70 % ethanol, 2 x 15 min 95 % ethanol, 15 min absolute ethanol, 30 min absolute ethanol, followed by 1 h in absolute ethanol. Next, samples were subjected to critical point drying with carbon dioxide (3 x 10 min) using critical point dryer CPD 030 (Bal-Tec, Los Angeles, USA). The tissue samples were mounted on aluminum holders (32 x 5 mm) with 25 nm carbon tabs and silver paint and sputtered with 20 nm palladium/gold by using vacuum coater Leica EM ACE200 (Leica Microsystems A/S, Ballerup, Denmark). Samples were examined in a PHENOM PROX scanning electron microscope. Reagents and equipment were purchased from Agar scientific (Stansted, UK).

Phagocytosis Assay

The phagocytosis activity of haAT and IibAT was analyzed either by pre incubating macrophages or E. coli BioParticles with hAT III using the Vybrant™

Phagocytosis Assay Kit (Molecular Probes, Eugene, USA). RAW 264.7 cells (1 x 10⁶ cells/ml) were pre-incubated with 5 mM haAT or IibAT for 1 hour at 37 °C in 100 mΐ of cell culture medium (depending on cell type) performed in a 96-well plate according to the manufacturer’s protocol. Briefly, after incubation the supernatant was replaced with 100 mΐ of Fluorescein-labelled Escherichia coli K-12 BioParticles dissolved in Hanks’ balanced salt solution (HBSS) and incubated for 2 hours at 37 °C in a 5% C0₂ incubator. The supernatant was discarded and cells were treated with 50 mΐ of 0.25 mg/ml trypan blue for 1 min. To quench the fluorescence of adherent bacteria, the supernatant was replaced with trypan blue for 1 min before the acquisition. After removing the trypan blue, fluorescence was measured in the fluorescence plate reader Wallac 1420 Victor3™ Multilabel Counter (Perkin Elmer Life and Analytical Science, Wallac Oy, Turku, Finland) using 485 nm excitation and 535 nm emission. For pre-incubation of BioParticles, 100 mΐ BioParticles were incubated with 5 mM haAT or hbAT in 20 % citrate human plasma for 2 hours at 37 °C followed by centrifugation at 2000 x g for 10 min. Pellet was diluted in 100 mΐ of serum-free cell culture medium and replaced with supernatant of the cells. Following steps were performed as before described. Wells without cells served as negative control and cells without treatment of phagocytosis effector served as positive control. The test was performed in 5 replicates for each experimental condition. The net phagocytosis was calculated by subtracting the average fluorescence intensity of the negative control wells from all positive control and experimental wells. The percentage of phagocytosis effect is the fraction of the net positive control phagocytosis: net experimental phagocytosis x 100 %

% phagocytosis effect net positive control

Isolation of Neutrophils and Monocytes To investigate the effect of aAT and bAT on induced NETs, human neutrophils were isolated from healthy donors by density centrifugation. 15 ml of PolymorphPrep™ (Axis-Shield, Oslo, Norway) were overlaid with 15 ml heparinized blood (Safety- Multifly® Kaniile 23G; S-Monovette Li-Heparin LH / 9 ml, Niimbrecht, Germany) and centrifuged at 370 x g for 30 min without brake. Layer of Plasma and peripheral blood mononuclear cells (PBMCs) was removed. Polymorph nuclear cells (PMNs) including neutrophils was collected and mixed with PBS to a total volume of 50 ml followed by centrifugation at 370 x g for 10 min. Lysis of erythrocytes was performed twice by adding 5 ml water to the cell pellet, incubation for 15 s, filling up to a volume of 50 ml with PBS and centrifugation at 370 x g for 10 min. Then, neutrophils were resuspended in Hank's Balanced Salt Solution without CaCl₂, MgCl₂ and MgS0₄ (HBSS, Thermo Lisher Scientific, Waltham, USA). CDl4⁺ monocytes were isolated from leukocyte concentrate (CD 14 microbeads from Miltenyi Biotech). Cells were counted using a Biirker counting chamber (Paul Marienfeld GmbH & Co. KG, Lauda- Konigshofen, Germany).

Whole Blood Stimulation

Whole blood stimulation was used to investigate if hAT III can avoid the cellular responsiveness to LPS. Peripheral blood was collected from healthy volunteers into a 2.7 ml vacutainer containing citrate (0.3 ml 0.109 M sodium citrate, BD Vacutainer® system including Plus Blood Collection Tubes, Blood Collection Set Safety Lok, Luer Lok™ Access Device, New Jersey, USA). Whole blood samples were diluted two-fold with RPMI 1640 Medium + GlutaMAX™ (GE Healthcare Life Science, Logan, Australia), stimulated with 2.5 ng/ml of LPS (Sigma- Aldrich GmbH, Steinheim, Germany) and incubated in the presence or absence of 10 mM haAT or IibAT in a final volume of 100 mΐ in 1.5 ml reaction tubes. Additionally, 5 ng/ml LPS was pre-incubated with 20 mM haAT or hbAT for 1.5 hours, followed by mixing with the same part of whole blood in a total volume of 100 mΐ and a final concentration of 10 mM hAT III. Whole blood diluted with RPMI 1640 + GlutaMAX™ served as control. Samples were incubated overnight (-16 hours) at 37°C in a 5 % C0₂ atmosphere incubator and then centrifuged at 400 x g for 10 min. Supernatants were removed and frozen at -20°C for cytokine analysis. Pellets were used for LACS analysis.

Cytokine Assay The levels of 45 cytokines/ chemokines/growth factors were measured in supernatant of whole blood stimulation using the multiplex Immunoassay ProcartaPlex® (affymetrix eBioscience, Bender MedSystems GmbH, Vienna, Austria) according to the manufacturer’s instructions.

Murine Cytokine Assay

Murine cytokines and chemokines were measured in plasma of infected mice as described above. Multiplex Immunoassay ProcartaPlex® mCytokine/Chemokine Panel 1A 36plex (affymetrix eBioscience, Bender MedSystems GmbH, Vienna, Austria) was performed according to the manufacturer’s instructions. Multiplex assay is designed in a capture sandwich format. Briefly, Color-coded beads coated with analyte specific antibodies were incubated with 25 mΐ of plasma sample. After several washing steps, beads were mixed with 25 mΐ biotinylated detection antibodies specific for a different epitope on the analyte molecule. By adding of 50 mΐ of Streptavidin- Phycoerythrin (PE) the reaction mix was labelled, followed by analysis in a flow-based Bio-Plex™ 200 system (Bio-Rad, Hercules, USA). Cytokines were identified based on bead color and quantified by the PE-fluorescence signal.

E. coli Infection Model

Animal experiments were performed according to a protocol approved by the Local Ethics Committee at Lund University. Animals were housed under standard conditions of light and temperature and had free access to chow and water. In order to reduce the imaging background, animal were fed with special chow (AIN-93 purified diet, Envigo, Cambridgeshire, UK) for at least 4 days before start of the infection experiment. For experiments E. coli ATCC 25922 was grown to mid exponential phase (OD620nm ~0.4), harvested, washed in PBS twice and diluted in the same buffer to 8-8.5 x l0⁸cfu/ml.

BALB/c mice (female, 8-9 weeks; Janvier Labs, Le Genest-Saint-Isle, France) were injected intraperitoneally (i. p.) with 100 pL of the bacterial suspension (n= 14 mice per group). One and five hours after bacterial injection, 0.5 mg (100 mΐ) of hAT III isoforms or PBS alone was administrated intravenous (i. v.). PBS, haAT and IibAT injection alone were used as a control (n=6/group). 8 hours post- infection, mice were anesthetized with 3.5 % Isoflurane (Baxter, Deerfield, USA) in the induction chamber (XGI-8 Gas

Anesthesia System, Xenogen, Alameda, CA), followed by subcutaneous (s. c.) injection of 25 mg/kg body weight of the chemiluminescence probe L-012 (Wako Chemicals, Neuss, Germany) was diluted in water to a total volume of 100 mΐ. After 10 min the presence of reactive oxygen species in the mice was measured using an IVIS® Spectrum with Life Image® version 4.4 (Caliper Life Science, Hopkinton, USA). Results are expressed as radiance (photons per s). After imaging, mice were sacrificed to evaluate cfu in kidney, spleen, lungs and liver as well as for SEM of lungs. Blood was immediately collected in 0.1 M sodium citrate (1:10 citrate:blood) by cardiac puncture. Blood was centrifuged at 1000 x g for 10 min and supernatant was used for analysis of inflammatory mediator cytokine as described above. In an independent experiment with the same treatments, mice were monitored (animal status, daily weight) for up to 7 days to obtain survival data. Mice showing predefined endpoint-criteria (e. g. immobilization) were sacrificed and counted as non- survivors.

For transgenic mice experiments, 100 mΐ of E. coli ATCC 25922 bacteria

(3 - 4 x 10⁷ cfu/ml) were injected i. p. to wild-type, hAT- and hpAT-mice and sacrificed 8 h post-infection. In addition to liver collection for qPCR and lung for SEM, liver, kidney and spleen were collected for cfu determination.

LPS inflammation mouse model

All mAT-, hAT- and hpAT-mice were male and 6-9 weeks old. Mice were i. p. injected with 4 mg/kg of E. coli 011 LB4 LPS (Sigma). After indicated time points (Fig.

5 A) mice were anaesthetised with isoflurane (Baxter) and blood was taken by cardiac puncture, collected in 0.1 M sodium citrate (1:10 citrate:blood), and centrifuged for 10 min at 3500 rpm. Citrate blood was used for coagulation assays, total cell count analysis and flow cytometry. Citrate plasma was carefully removed, aliquoted and stored at - 80°C for further studies. Livers were collected in RNAlater™ (QIAGEN) for expression analysis and lungs were collected in SEM-Fix (2.5 % glutaraldehyde in 0.15 M cacodylate buffer, pH 7.4) for scanning electron microscopy. For survival studies 5 mg/kg of LPS were injected i. p . to BALB/c and 10 mg/kg of LPS were injected i. p. into transgenic mice. Survival status and weight loss was measured for 7 days.

Mice ( Mus musculus) were housed under standard conditions of light and

temperature and had free access to chow and water. An ethical approval number (Ml 85- 14) permits inbreeding and performance of infection/inflammation experiments. Mice were checked every day to ensure health and provide care for the developing young mice. At the age of 3-4 weeks, females and males were weaned from their parents, tagged at their ears and tails were cut to isolate DNA for genotyping PCR. For genotyping and determination, whether the humanized mice are wild-type (WT), homozygote (HO) or heterozygote (HT), collected tails were boiled in 150 mΐ 50 mM NaOH at 99 °C and 350 rpm for 1 h and the digested tails were neutralized by adding 1 M TrisHCl pH 7.5. 1 mΐ of tail lysate served as template.

Human ethical statement

The study of patient samples was approved by the local ethics committee of Lund University (nr 2010/205) and generic approval of EC for patients with coagulopathies (approval number 123, approval date 2/01/2014). Patients are a part of a previously published study (Linder, A., Christensson, B., Herwald, H., Bjorck, L. & Akesson, P. Heparin-binding protein: an early marker of circulatory failure in sepsis. Clin Infect Dis 49, 1044-50, doi: 10.1086/605563 (2009). Patients were enrolled with suspicion of infection and at least 1 SIRS criteria were enrolled at the Emergency department at Lund University Hospital in Lund, Sweden.

Establishment of humanized mouse model

Genetically modified mice have been made using CRISPR/Cas-mediated genome editing. A single guide RNA targeting mouse 5’ UTR was designed and cloned into pSpCas9 BB-2A-GLP (pX458; Addgene). cDNAs for the human AT glycosylation isoforms (hAT, hbeta AT) were flanked by homologous arms identical to the genomic mouse sequences of 790 and 842 bp (gBlocks, Integrated DNA tech) and cloned into a pUCl9 vector. Cas9 and sgRNA expressing plasmid and the targeting construct were then transfected into TCL2.2 ES cells (hybrid L1-129S2; C57BL/B6N line, established at the Transgenic Core Lacility, University of Copenhagen) in serum-free, feeder-free 2i + LIE culture conditions. ES cell clones were picked and genomic DNA was analyzed for correct replacement of murine AT- gene with human AT- gene (hAT or IibAT, Figure 22) using the indicated primers. Transgenic Fl mice (Primer Table) were backcrossed for 3 generations to C57BL/6J.

Isolation and purification of AT from humanized mouse plasma Heterozygous 6-9 weeks old hAT- and hpAT-mice as well as wildtype mice were anaesthetised by inhalation of isoflurane (JD Medical), blood was taken by cardio vascular puncture and collected in tubes containing 0.1 M citrate (1:10 citrate:blood). Citrate blood was centrifuged for 10 min at 3500 rpm and citrate plasma was carefully removed. Five millilitres of mAT-, hAT- and hpAT-mice citrate plasma were diluted in 5.0 ml buffer containing 0.1 M Tris, 0.01 M citric acid, 0.225 M NaCl, pH 7.4. According to the GE Healthcare Protocol for the isolation of bovine AT (GE Healthcare Bio-Sciences AB, Data file 18-1134-77 AE), two different buffers were used: Buffer A contained 0.1 M Tris,

0.01 M citric acid, 0.225 M NaCl, pH 7.4 and buffer B 0.1 M Tris, 0.01 M citric acid, 2 M NaCl, pH 7.4. 5 ml of mAT-, hAT- and hpAT-mice citrate plasma were diluted in 5.0 ml of buffer A and loaded on an 1 ml Heparin HP column equilibrated with 87.5 % buffer A and 12.5 % buffer B with a flow rate of 1 ml/min. Chromatography was performed by following elution steps: 18 ml 12.5 % buffer B; 5 ml, 25 % buffer B; 8 ml 100 % buffer B and monitored by measuring the absorbance at 280 nm. Protein peak was collected, dialysed against wlO mM Tris pH 7.4, freeze dried and the pellet was dissolved in water. The concentrations of purified proteins were measured by Bradford Protein Assay¹. 2 pg of purified AT from mice plasma were dissolved in 2 x Laemmli Sample Buffer (BIO-RAD) containing 5 % b-mercaptoethanol, denatured at 95 °C for 10 min and subjected to a SDS- PAGE gel followed by Coomassie staining (GelCode™ Blue Safe Protein Stain, Thermo Scientific) and followed immunoblotting using hAT specific monoclonal antibodies (EPR5371) mouse anti-human AT antibodies (abl24808, Abeam, Cambridge, UK).

Antithrombin RNA expression analysis

For analysing AT gene expression in liver of healthy, LPS injected and E. coli infected humanized mice, RNA was isolated followed by DNase digestion, cDNA synthesis and quantitative PCR analysis.

RNA isolation and DNase digestion

For analysing relative AT RNA expression by quantitative polymerase chain reaction (qPCR), livers were transferred from R/VAlater™ into a 2.0 ml SC Micro Tube

PCR-PT containing 600 mΐ of RLT Plus buffer and 1 % of b-mercaptoethanol and 10 to 15

1.4 mm ceramic beads (QIAGEN). Livers were homogenized in this solution for 30 sec at

7000 rpm using a MagNA Lyser Instrument Version 4.0 (ROCHE). RNA was isolated following the protocol of RNeasy® Plus Mini Kit (QIAGEN) and eluted with 50 mΐ of nuclease-free water (Ambion®). Furthermore, a NanoDrop® Spectrophotometer ND-1000 was used to determine RNA concentration and purity. To exclude DNA contamination, further DNase digestion by using Deoxyribonuclease I, Amplification Grade (18068015, Invitrogen by Thermo Fisher Scientific) according to manufacturer’s protocol was performed. Reaction mixture served as template for further complementary DNA (cDNA) synthesis.

Complementary DNA Synthesis

One 1 mΐ of recent DNase digestion containing 1 mg of isolated RNA from liver was used for transcription into cDNA by using iScript™ cDNA Synthesis Kit (1708891, BIO-RAD, Sweden) performed according to manufacturer’s protocol.

Quantitative-PCR

The Applied Biosystems™ TaqMan™ Pre-Developed Assay Reagents from Thermo Fisher were used for real-time relative quantitative evaluation of human AT gene expression in wild-type-, hAT- and hpAT-mice and was performed according

manufacturer’s protocol. Briefly, anti-human AT probe (Hs00l66654_ml), anti-mouse AT probe (Mm00446573_ml) and anti-mouse GAPDH probe (Glycerinaldehyd-3-phosphat- Dehydrogenase; 4352932E) purchased from Applied Biosystems™ (by Fife Technology) were diluted 1:20 and used to detect murine and human AT gene segments. GAPDH expression serves as reference. All samples were run in duplicates and Quantstudio-7 from applied Biosystems (Thermo Fisher) and appropriate software was used.

ELISA

hAT concentration was measured by using human Antithrombin Total Antigen EFISA Kit (HATIIIKT-TOT, Molecular Innovations) according to manufacturer’s protocols. Absorbance was measured at 450 nm in iMark™ Microplate Reader (Bio-Rad). Based on the EFISA quantification of the total amount of AT in one bottle of Baxalta AT, the isolation by FPFC yielded 6 mg bAT (photometric quantified) out of 116 mg total AT (A 5.2 %). Alternatively, the concentration of hAT in mouse citrate plasma was measured using Human AT Simple Step EFISA Kit ab222507 purchased from Abeam and absorbance was measured at 450 nm. Human thrombin and antithrombin TAT complexes in plasma were measured by using human TAT ELISA Kit (ADG833, Sckisui Diagnostics GmbH) and absorbance was measured at 450 nm.

Flow cytometry

hAT binding to white blood cells was investigated by immunofluorescence and flow cytometry using BD Accuri™ C6 (B&D). 100 mΐ of citrate blood were used and red blood cells were lysed with Dako Uti-Lyse™ S3325. After lysis, Fc-Block 553142

(0.5 mg/ml; purified rat anti-mouse CD16/CD32 Clone 2.4G2, BD Bioscience) were added (1: 12.5) to the cell suspension and incubated 10 min at 4 °C in the dark. Alternatively, purified 5 x 10⁶ cells/ml neutrophils and monocytes were incubated with 10 mM haAT or IibAT for 30 min at 37 °C. Prior adding primary antibodies samples were incubated with mouse Fc-Block for 10 min at 4 °C. For each sample, one reaction with IgG control (2.0 mg/ml, normal rabbit IgG, AB-L05-C, RD Systems), diluted 1: 10, and one with primary anti-mouse/human AT antibody (0.5 mg/ml; SERPINC1 polyclonal, PA5-13974, Thermo Fisher), diluted 1: 10, were added to the cell suspension for 30 min at 4 °C. After washing twice with 1 % BSA in PBS, secondary goat anti-rabbit antibody labelled with Alexa Fluor® 488 (2 mg/ml; A11034, Life Technology) was added (1:50) and incubated for 30 min at 4 °C in the dark. Samples were washed once with 1 % BSA in PBS and the cell pellet was resuspended in 100 mΐ of washing buffer. FL-l was used to detect the fluorescence signal of Alexa Fluor® 488 (filter 533/30). The percentage of antithrombin binding to cells was calibrated with control cells that are treated with primary and secondary antibody. The results are presented as mean values ± SEM.

Flow cytometry was performed using BD FACS Verse in combination with

CellQuest Pro software.

Cathepsin G Activity Assay hAT inhibition activity of cathepsin G in THP-l cells was determined by Cathepsin G Activity Assay Kit according to manufacturer's instructions (Abeam). THP-l cells were lysed with assay lysis buffer and further incubated with 10 mM haAT or IibAT for 30 min at 37°C. After incubation, substrate solution was added to each sample and incubated 1 h at 37°C and absorbance was measured at 450 nm.

Statistical analysis Student t test was used for comparisons using GraphPad Prism 7 (GraphPad Software, San Diego, CA). P-values were considered statistically significant at p>0.05 (p>0.05; * p<0.05; ** p<0.0l; *** p<0.00l and **** p<0.000l).

E. coli infection and LPS inflammation studies in vivo were performed at least in two separate experiments. In vitro experiments were done in duplicates at three

independent trials. Data were analysed with Microsoft Excel 2016 (Microsoft, Redmond, WA) and GraphPad Prism 7.0 (GraphPad Software, San Diego, CA). All results are presented as mean values ± SEM with the number of independent experiments and mice per group indicated in the figure legends. Comparison of data was performed by one-way or two-way ANOVA and Turkey’s multiple comparison test. Values were significantly when p-value ranging from 0.05 to 0.0332 (*), to 0.0021 (**), to 0.0002 (***) or < 0.0001 ****)

RESULTS

Example 1

Preliminary results of AT concentration and activity in plasma of healthy, intensive care unit (ICU) and infected patients

Fig 1 shows that the concentration of AT III as measured by ELISA is decreased in patients with an infection (left panel). Lowest levels were found in ICU patients. Similar findings were recorded when the activity of AT III was determined by the Stachrom AT III Kit (Diagnostica Stago, Asnieres sur Seine, France) (Fig 1, middle panel left). This applies also when measuring the activity of aAT (Fig 1, middle panel right), while bAT concentrations dropped to background levels in both patient groups (Fig 1, right panel). Because bAT has a better protease inhibitory activity compared to aAT, it is earlier depleted in plasma samples from infected patients.

Validation of activity of dialysed and resuspended aAT and bAT fractions

Fig 2 depicts that bAT is more potent than aAT or the control to prolong the clotting time (TT) (>995 s). Because of the increased proteinase activity bAT is also more potent than aAT in blocking normal clotting.

Binding of AT to Gram-negative bacteria The binding of AT to E. coli and P. aeruginosa was investigated by pull down assays and followed by western blot analyses. As shown in Fig 3A, both AT III forms bind to the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, respectively. An increased binding of AT in human citrate plasma was observed over the time (4-16 h). The bound amount of AT to P. aeruginosa was more enhanced compared to E. coli (Figure 3A).

E. coli was then incubated with isolated aAT or bAT instead of human plasma. Unbound AT was detected in supernatant (S) and bound protein in bacterial pellet (P). As shown in Fig 3B, the binding of both isoforms to E. coli was detected, whereas the band of bAT was more intensive compared to aAT, suggesting a stronger interaction with the bacterial surface (Figure 3B).

Interaction of aAT and bAT proteins with bacterial endotoxin LPS

(lipopolysaccharide)

Surface plasmon resonance experiments were conducted by the immobilisation of aAT or bAT on the sensor chip and a flow of LPS (range 1.875 -30 x 10⁵ EU/ml) was applied. The highest net response was achieved by 30 x 10⁵ EU/ml, as shown in Figure 4. By stopping LPS injection and washing with running buffer the dissociation phase was initiated. Subsequent analysis of the biacore curve revealed that LPS avidly binds to both AT isoforms, followed by a quick dissociation. Similar curves were obtained for both aAT and bAT, indicating comparable LPS binding capacity. Sensor chip surface without immobilised protein showed no signal by LPS flow. Thus, unspecific binding of LPS to the chip surface can be excluded.

Thus, as shown in Fig 4, both AT III isoforms bind to LPS with the same affinity.

The binding and structural conformational changes of proteins were then studied using negative staining electron microscopy. As shown in Fig 5, while LPS seems to make monomeric complexes with aAT (upper panel), its interaction with bAT led to the formation of larger complexes.

Electron microscopic analysis of AT III binding to bacteria

The interaction between AT and E. coli was examined by electron microscopy, as illustrated in Fig 6 A (TEM). bAT exerted its antimicrobial effect by formation of pores on the bacterial surface (lower right), resulting in a massive release of bacterial exudates (top right). In contrast to the detection of labelled bAT on the bacterial surface, less binding of aAT was observed (lower left) and consequently weak disruption of the bacterial cell wall was obtained (top left). Thus, it appears that only bAT but not aAT has antimicrobial activity.

Antimicrobial activity of aAT and bAT

Concentrations of aAT and bAT at 1 OmM showed sufficient killing of Gram negative bacteria (Fig 7). Both isoforms had significant antibacterial activity against E. coli in the viable count assay performed in physiological buffer (10 mM Tris, 150 mM NaCl, Fig 7), whereby bAT could achieve almost a complete eradication of E. coli (top).

In comparison to E. coli, the killing of P. aeruginosa by bAT was significantly less effective in physiological buffer. AT isoforms yielded insufficient (for S. pyogenes in Tris buffer) or no killing of the Gram-positive bacteria S. aureus and S. pyogenes. These results display a significant bAT-mediated killing of Gram-negative bacteria, which is dependent on the salt concentration and presence of plasma components. bAT has some, but reduced, activity against S. pyogenes in Tris buffer.

Phagocytosis of bAT-opsonized bacteria is increased

To determine the phagocytic effect of AT isoforms on murine macrophages, a phagocytosis assay using Vybrant™ Phagocytosis Assay Kit with fluorescence labelled E. coli particles was performed. In order to compare the phagocytosis activity by the binding of AT to particles or cells, either cells or particles were pre-incubated with 5 mM aAT or bAT. As shown in Fig 8, a significant increase of phagocytosis for both the experimental setups with bAT (131 % ± 5% for pre-incubation of RAW 264.7 cells/macrophages; 140 % ± 13% for pre-incubation of E. coli particles) was observed, while the phagocytic activity could not be enhanced upon aAT treatment.

Thus, in agreement with EM results, it appears that bAT can bind directly to E. coli particles as well as to RAW 264.7 cells to enhance the phagocytosis activity of

macrophages. Based on these findings we propose a model that bAT can opsonize E. coli particles which functions as an“eat me” signal for macrophages. Furthermore, bAT seems to be a better pattern recognition molecule than aAT. AT Ill-binding to cells in human blood

Neutrophils and monocytes isolated from human blood were incubated with aAT or bAT and binding was recorded by FACs analysis. The results show both neutrophils and monocytes bind more bAT on their surfaces than aAT (Fig 9). The differences in glycosylation provide bAT with an increased affinity to bind to neutrophils and

monocytes. aAT and bAT can modulate the inflammatory response

To study the immune-modulating effect of AT isoforms ex vivo, stimulation of whole blood from 8 donors, followed by the quantification of cytokine/chemokine response were performed. Due to the confirmed binding of AT to LPS, the stimulation was accomplished by LPS in a concentration of 2.5 ng/ml and an incubation time of 16 hours. This time point and the concentration of LPS were chosen based on previous optimisation experiments by detecting IL-6 levels. Blood samples untreated, treated with LPS and AT isoforms alone served as controls. Immune responses were assessed by a multiplexed magnetic beads immunoassay.

Fig 10 shows the results of 22 out of 45 inflammatory mediators with detectable differences between LPS and its co-incubation with AT isoforms. No general pattern of down- or up-regulated proinflammatory cytokines (IL-l, 2, 5, 6, 7, 8, 22, 23, TNFa, Interferon (IFN)y) could be observed upon the simultaneous incubation with LPS and AT.

The production of the proinflammatory cytokines TNFa was considerably augmented upon AT isoforms combined with LPS in comparison to LPS alone.

Furthermore, combination of LPS and bAT increased the production of IL-7significantly. Interestingly, bAT alone also elevated this cytokine level compared to the control and LPS, whereas the effect of aAT was similar to LPS alone.

For proinflammatory cytokines IL-2 (protects abscess formation), IL-5 (protects sepsis by regulating PMNs) and IL-22 (involved in host defence against infections), an upward trend was observed by co-incubation of bAT and LPS, while the combined effect with aAT was less or similar to incubation with LPS alone. Levels of the proinflammatory cytokines IL-la, IL- 1 b, IL-6, IL-8, IL-23 and IFNy were significantly reduced upon co-incubation of LPS and both AT isoforms. Except from IL-23, the inhibitory effect of bAT is stronger than aAT.

Regarding the measured levels of anti-inflammatory cytokines, both AT isoforms, together with LPS, decreased the production of IL-10 and IL-l receptor antagonist (IL- lRa) significantly in comparison to LPS stimulation, whereby aAT had less of an effect than bAT. In the same way, the levels of the chemokines INRg-induced protein 10 (IP-10), macrophage inflammatory protein (MlP)-la and MIR-Ib, stromal cell derived factor (SDL)- la, Regulated upon Activation Normally T Cell Expressed and Secreted (RANTES) and growth related oncogene (GRO)-a were significantly reduced by both AT isoforms in combination LPS. Moreover, levels of growth factors were also affected upon AT treatment. aAT and bAT decreased the LPS-stimulated production of hepatocyte growth factor (HGL) in the same way. Vascular endothelial growth factor A (VEGL-A) was decreased by aAT, but increased by bAT concomitant with LPS. Taken together, bAT blocked chemokine production and maintained the balance between LPS-triggered pro- and anti-inflammatory responses. Interestingly, production of GM-CSL was increased by bAT but not aAT compared to control, as well as being further boosted in combination with LPS. AT III treatment increased the production of GM-CSL around 5 -fold.

Assuming that the different effects of aAT and bAT on LPS-stimulated blood cells resulted from time limited or weaker binding of aAT to LPS, the same inflammatory mediators in the same blood samples were analysed after pre-incubation of LPS with AT isoforms for 1.5 h. Under these conditions, the same effect of aAT and bAT was observed as for the simultaneous incubation of blood with AT and LPS for all mediators (Pig 10).

Survival studies using an LPS model

Mice challenged with LPS died after 24 h. Treatment with bAT significantly prolonged survival, while treatment with aAT was similar to control (Pig 11).

Modulation of inflammatory reactions by bAT in a murine E. coli infection model aAT and bAT are able to modulate inflammatory responses in vivo. Despite obvious improvement of survival and bacterial killing by bAT, no difference in

cytokine/chemokine production was observed between both AT isoforms, as shown in Figure 12. Proinflammatory cytokines IFNy, IL- 1 b and IL-17A were significantly decreased upon aAT and bAT treatment. In the same way, AT treatment reduced levels of the proinflammatory cytokines TNFa, IL-6 and 1L-22 as well as the anti-inflammatory cytokine IL-10 (without determined significance), whereas a downward trend for IL-22 and IL-10 was observed upon bAT treatment. Moreover, chemokine levels of monocyte chemoattractant protein- 1 (MCP-l), MIP-2 and IP- 10 dropped similarly in response to aAT and bAT administration compared to control. Regarding GM-CSF, a slight reduction of its plasma level was detected. Mice administrated with aAT or bAT alone showed no detectable or no significant different levels of cytokines/chemokines to healthy control animals (data not shown). bAT-mediated prevention of bacteria-caused complication in mice

The progress of inflammation and infection was evaluated by the analysis of ROS production using luminescence labelling and in vivo imaging (Fig 13 A, 13B). The radiance signal induced by E. coli infection was less reduced by aAT, but was effectively decreased by bAT treatment. The highest signal was obtained in the centre of the mouse body close to the site of infection.

Scanning electron microscopy analyses of the lungs from infected mice and controls were performed, as shown in Fig 13C. Supporting the reduced bacterial numbers in lungs from bAT-administrated mice, decreased pulmonary leakage of protein and formation of thrombi as well as big alveolar spaces were observed upon bAT treatment after E. coli infection, similar to healthy animals. These improvements could not be seen by aAT-treatment. It was noted that infected lungs showed dramatic damage, involving reduced pulmonary volume, inflamed thick walls, cell infiltration and protein deposition. Neither aAT nor bAT alone initiated detectable effects on lung tissue, compared to controls.

Thus, bAT and to some extent also aAT down-regulated signs of inflammation (ROS production), bacteria spreading and lung damage (Fig 13). bAT prevents bacterial spreading into other organs

To explore the bacterial burden upon AT treatment, mice were sacrificed 8 h after infection and organs were homogenised and plated. In accordance with survival results, considerable amounts of bacteria were reduced in liver, lung and spleen for bAT-treated mice compared to control and aAT (Figure 14). No significant decrease of bacteria was obtained in kidneys, but a small downward trend was seen in response to bAT treatment.

Survival studies using an E.coli model As shown in Fig 15, bAT has a significant impact on survival in an animal model of severe infection. When mice were infected with a lethal E. coli bacteria dose, treatment with IibAT, given one hour after infection, improved survival rates froml00% mortality to more than 70% survival (Fig. 15C). As seen with the LPS challenge, treatment with haAT had no impact on survival (Fig. 15C). Additionally, the weight loss was monitored for 7 days (Fig 15B), displaying a dramatic drop of the weight in all groups, followed by slight regeneration of bAT-administered mice. Infected mice treated with IibAT regained their normal weight after one week, demonstrating that they were fully recovered.

Example 2 - Identification of AT receptors and binding proteins

In order to identify bAT ligands LRC-TriCEPS ligand-receptor capture proteomics analysis was performed {Dualsystems). Samples were analysed on a Thermo LTQ

Orbitrap XL spectrometer fitted with an electro spray ion source.

Using this technology, a number of proteins were enriched > 2 fold compared to the control sample: Cathepsin G (CATG # P08311), CMRF35-like molecule 1 (CLM1 # Q8TDQ1) and Prolow-density lipoprotein receptor (LRP1 #Q07954). Notably, these proteins may interfere with the regulation of inflammatory pathway.

Table 1

Binding of CLMl/Cd300f and AMPN/CD13 negatively regulates TLR4 signaling. Regulation by LRP1 affects cytokine secretion, phagocytosis and migration of cells of the immune system. CATG and HBP are suggested as a biomarker and their inhibition may improve the survival. Functions of EVI2B and MEGF9 are unknown.

Biacore analysis

As shown in Fig 16, the BiaCore analysis confirmed the LRC-TriCEPS showing that aAT and bAT bind with the same affinity to CATG (cathepsin G), while bAT had a higher affinity to CLM1 (CD300Fc) and AMPN (rhAminopeptidase) than aAT. Cathepsin G is a neutrophil-derived serine proteinase that shares high homology with heparin-binding protein (HBP). HBP is serine proteinase-like protein that lacks a proteinase activity.

When the interaction of aAT and bAT with HBP was tested a binding affinity in the nano molar range was determined. Notably, HBP is a sepsis biomarker that can induce vascular leakage, one of hallmarks in the pathology of sepsis.

As cathepsin G and HBP belong the family of serine and thus it seems plausible that aAT and bAT bind to these proteins with a similar affinity. The release of these two proteins from neutrophils has been shown to trigger inflammatory reactions and their blockage by aAT and bAT may help to regulate the host response. CLM1 and AMPN are two receptors involved in the induction of inflammatory signaling pathways, such as interferon-g. Both receptors show a higher affinity to bAT, which also explains why bAT can modulate the inflammatory response better than aAT.

Inhibition of cathepsin G activity by aAT and bAT When employing cathepsin G (CATG) substrates, it was found that bAT, but not aAT, was able to block the enzymatic activity of the proteinase, as illustrated in Fig 17.

Like thrombin, cathepsin G belongs to the family of serine proteinases. The activity of the enzyme can be blocked by fast- acting serpins such as alpha l-anti- chymotrypsin. As mentioned before AT III is a specific thrombin inhibitor, but it can also block other serine proteinases with bAT being the more potent inhibitor compared to aAT. Therefore bAT can partially reduce the activity of cathepsin G, while aAT lacks an inhibitory activity.

Example 3 - Generation of gene modified mice

Treatment of gene modified mice with LPS

Using the crispr-cas9 technology, the mouse AT III gene was replaced with human AT III or human bAT. While male homozygotes carrying the gene for hAT or IibAT survived up to 28 (hAT) and 49 (ΉbAT) days, all hAT and IibAT female homozygote mice died 4 weeks after birth because of massive internal hemorrhage. As heterozygote mice did not show any obvious phenotype, experiments were therefore performed with 4- to 6-week old male heterozygote animals. Mice were challenged with LPS or infected with E. coli bacteria as outlined in Figure 21A. Choosing a LPS dose of 10 mg/kg only 40% of all wild-type mice (mAT) survived, whereas in hAT and IibAT mice survival rates were as high as 60% and 80% (Fig 21B). Likewise the weight loss of LPS-challenged of hAT and IibAT mice was less compared to mAT mice, though only the difference in weight loss of mAT mice and IibAT mice reached statistical significance (Fig. 21C). As these

experiments were performed with heterozygote mice, the gene modified animals expressed in addition to hAT or IibAT also mAT. Thus, the expression profiles of the three proteins in healthy and LPS-challenged mice were compared. Figure 21D (left panel) illustrates that under healthy conditions the mRNA levels in the liver of mAT were higher in wild-type mice compared to that from CRISPR-Cas9 genome edited mice carrying the genes hAT or IibAT. However, when challenged with LPS mice in all three groups had similar levels of mAT mRNA which were about twice as high as seen in healthy wild-type mice. This was in contrast to the mRNA expression levels of hAT or IibAT. Here we found that LPS treatment did not induce a significant increase (Fig. 21D, right panel). However, we noted that the protein concentrations of hAT and IibAT in murine blood samples from LPS- challenged mice were more than 50% and 80% reduced compared to unchallenged mice (Fig. 21E).

To test the antimicrobial activity of the different AT glycosylation isoforms under ex vivo conditions, blood from wild-type and CRISPR-Cas9 hAT and IibAT mice were incubated with E. coli bacteria for 4 hours followed by determination of bacterial survival. Figure 21L shows that the lowest number of bacteria were found in murine blood from mice expressing hbAT, while bacterial counts were higher in blood samples from hAT expressing mice compared to wild-type mice. An explanation for the latter finding is that under non- stimulating conditions, expression levels of hAT in the CRISPR-Cas9 genetically modified mice are very low when compared to the mAT levels in wild-type mice (Fig. 21D). As hAT mice express mAT upon stimulation, the killing activity under the chosen experimental condition (4 hours) is entirely dependent on human homologue which has a concentration in blood that is approximately more than 10 times below the concentration of mAT in wild- type mice. This in turn implies that the antimicrobial activity in the blood from hAT expressing mice is lower compared to blood samples derived from wild-type animals, which then is responsible for the better bacterial survival. It should be noted that also HbAT is expressed at low level in the genetically modified mice. As bacterial growth is decreased in blood from these mice, our findings further confirm our previous findings (Fig. 7) that the protein is a potent antimicrobial agent. Finally, we infected transgenic mice with E. coli bacteria for 8 hours. Subsequent analyses of the bacterial counts in the infected organs revealed lower number of colonies measured in livers and spleens from animals expressing hAT or HbAT compared to wild- type mice (Fig. 21M).

Survival of bacteria in blood from gene modified mice

Blood from normal and heterozygous (human AT III, aAT, and bAT, respectively) mice were incubated with E. coli bacteria and bacterial survival was monitored (Fig 19). The results show that bAT can restore the antimicrobial activity in plasma depleted of murine AT III. Thus, bAT is an important antimicrobial factor in the circulation. Sequence

SEQ ID NO: 1 - full length sequence of human ATI I I including signal sequences (underlined)

MYSNVIGTVTSGKRKVYLLSLLLIGFWDCVTCHGSPVDICTAKPRDIPMNPMCIYRSPEK KATEDEGSEQKIPEATNRRVWELSKANSRFATTFYQHLADSKNDNDNIFLSPLSISTAFA MTKLGACNDTLQQLMEVFKFDTISEKTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFG DKSLTFNETYQDISELVYGAKLQPLDFKENAEQSRAAINKWVSNKTEGRITDVIPSEAIN ELTVLVLVNTIYFKGLWKSKFSPENTRKELFYKADGESCSASMMYQEGKFRYRRVAEGTQ VLELPFKGDDITMVLILPKPEKSLAKVEKELTPEVLQEWLDELEEMMLWHMPRFRIEDG FSLKEQLQDMGLVDLFSPEKSKLPGIVAEGRDDLYVSDAFHKAFLEVNEEGSEAAASTAV VIAGRSLNPNRVTFKANRPFLVFIREVPLNTI IFMGRVANPCVK SEQ ID NO: 2 - full length sequence of human ATI I I without signal sequences

HGSPVDICTAKPRDIPMNPMCIYRSPEKKATEDEGSEQKIPEATNRRVWELSKANSRFATTFYQHLADSKNDN DNIFLSPLSISTAFAMTKLGACNDTLQQLMEVFKFDTISEKTSDQIHFFFAKLNCRLYRKANKSSKLVSANRL FGDKSLTFNETYQDISELVYGAKLQPLDFKENAEQSRAAINKWVSNKTEGRITDVIPSEAINELTVLVLVNTI YFKGLWKSKFSPENTRKELFYKADGESCSASMMYQEGKFRYRRVAEGTQVLELPFKGDDITMVLILPKPEKSL AKVEKELTPEVLQEWLDELEEMMLWHMPRFRIEDGFSLKEQLQDMGLVDLFSPEKSKLPGIVAEGRDDLYVS DAFHKAFLEVNEEGSEAAASTAWIAGRSLNPNRVTFKANRPFLVFIREVPLNTI IFMGRVANPCVK SEQ ID NO: 3 - stretch of amino acids contributing to antimicrobial effect, corresponding to positions 114 to 156 of SEQ ID NO: 1

KTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKSLTFNE

Claims

1. An antimicrobial for use in a method of treating a bacterial infection, wherein the antimicrobial is administered to an individual in need, and wherein the

antimicrobial is beta- antithrombin (bAT).

2. The antimicrobial for use according to claim 1, wherein the bAT comprises:

(a) an amino acid sequence of SEQ ID NO: 1, wherein the bAT lacks a

carbohydrate chain at position 135;

(b) an amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 1, wherein the bAT lacks a carbohydrate chain at position 135; or

(c) an amino acid sequence which is a fragment of the sequence of SEQ ID NO: 1 or a fragment of an amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 1, wherein the bAT lacks a carbohydrate chain at position 135.

3. The antimicrobial for use according to claim 2, wherein the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 of (b), or the fragment thereof of (c), comprises the amino acid sequence of

KTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKSLTFNE (SEQ ID NO: 3), optionally wherein said stretch of amino acids is present in said

antimicrobial at positions corresponding to positions 114 to 156 of SEQ ID NO: 1.

4. The antimicrobial for use according to any one of claims 1 to 3, wherein the

bacteria is a Gram-negative bacteria.

5. The antimicrobial for use according to claim 4, wherein the Gram-negative bacteria is Escherichia coli or Pseudomonas aeruginosa.

6. Use of an antimicrobial as defined in any one of claims 1 to 3, in the manufacture of a medicament for the treatment of a bacterial infection, optionally wherein said infection is as defined in claim 4 or 5.

7. A method of treating a bacterial infection in an individual in need thereof, wherein the method comprises administering an antimicrobial, wherein the antimicrobial is beta- antithrombin (bAT).

8. The method according to claim 7, wherein the bAT comprises:

(a) an amino acid sequence of SEQ ID NO: 1, wherein the bAT lacks a

carbohydrate chain at position 135;

(b) an amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 1, wherein the bAT lacks a carbohydrate chain at position 135; or

(c) an amino acid sequence which is a fragment of the sequence of SEQ ID

NO: 1 or a fragment of an amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 1, wherein the bAT lacks a carbohydrate chain at position 135.

9. The method according to claim 8, wherein the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 of (b), or the fragment thereof of (c), comprises the amino acid sequence of

KTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKSLTFNE (SEQ ID NO: 3), optionally wherein said stretch of amino acids is present in said antimicrobial at positions corresponding to positions 114 to 156 of SEQ ID NO: 1.

10. The method according to any one of claims 7 to 9, wherein the bacteria is a Gram negative bacteria.

11. The method according to claim 10, wherein the Gram-negative bacteria is

Escherichia coli or Pseudomonas aeruginosa.