WO2016201528A1 - Treatment of inflammation and/or cancer - Google Patents

Abstract

Provided are methods for preventing and/or treating inflammation and/or cancer in a subject by administration of an isolated EgKI-1 protein. Isolated EgKI-1 proteins, including nucleic acids encoding same, and an antibody raised against an EgKI-1 protein are also provided. Compositions including a wildtype or mutant EgKI-1 protein and/or the aforementioned antibody are further provided. Also provided are methods for preventing and/or treating a parasitic infection in a subject by administration of one or more of the aforementioned EgKI-1 proteins, antibodies, and/or compositions.

Description

TITLE

TREATMENT OF INFLAMMATION AND/OR CANCER

TECHNICAL FIELD THIS INVENTION relates to isolated proteins from the tapeworm Echinococcus granulosus and their use in methods for preventing and/or treating inflammatory disease and/or cancer.

BACKGROUND

The dog tapeworm Echinococcus granulosus is one of a group of medically important parasitic helminths of the family Taeniidae (Platyhelminthes; Cestoda; Cyclophyllidea). Its life cycle involves two mammals: an intermediate host, usually a domestic or wild ungulate, with humans being accidental hosts, and a canine definitive host such as the domestic dog. The larval metacestode stage causes cystic echinococcosis (CE) (hydatidosis; cystic hydatid disease), a chronic cyst-forming disease in the intermediate/ human host (1). Hermaphrodite adult worms of E. granulosus reside in the small intestine of canines and pass eggs containing embryos (oncospheres) in faeces. Following ingestion by a human or an intermediate host such as a sheep, the egg hatches in the intestine to release the oncosphere which penetrates through the gut wall and is carried in the blood system to various internal organs, mainly the liver or lungs, where it develops into a hydatid cyst. Dogs and other canines get infected by eating offal with fertile hydatid cysts containing larval protoscoleces. These larvae evaginate, attach to the gut, and develop into 3-6 mm long adult parasites which reach sexual maturity 4-5 weeks later (2). The molecular mechanisms whereby the adult worms are able to survive in the dog gut without being damaged from host intestinal proteases and how oncospheres evade host immune attack in the blood system remain largely unknown. However, the recent deciphering of the E. granulosus genome and transcriptome (3,4) provides insights as to how these processes might occur.

Kunitz type proteins, which belong to the 12 family of protease inhibitors, have been characterised from many organisms including sea anemone (5), cone snail (6), scorpion (7), spider (8), ticks and biting flies (9, 10), parasitic helminths (11, 12) and mammals (13). Bovine pancreatic trypsin inhibitor (BPTI) is the classic member of this family of proteins and was the first Kunitz-type protease inhibitor described (14). These proteins possess one or more Kunitz domains; the Kunitz-type motif consists of around 60 amino acids and has six conserved cysteine residues which connect with three disulphide bonds in a characteristic pattern (C1-C6, C2-C4, and C3-C5). The C1-C6 and C3-C5 bonds are required for the maintenance of native confirmation (16) whereas the C2-C4 bond stabilizes the folded structure (17). Position Pi (18) of the reactive site is the major determinant of the energetic and specificity of protease recognition by Kunitz inhibitors (19).

In invertebrates, Kunitz inhibitors are involved in various physiological processes such as blood coagulation, fibrinolysis, inflammation and ion channel blocking with or without protease inhibition (15). As powerful inhibitors of mammalian intestinal proteases, the Kunitz type protease inhibitors of E. granulosus (EgKIs) additionally may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. In light of these key functions, the EgKIs may represent novel agents in the treatment of inflammatory disease and cancer. To this end and notwithstanding the existence of a number of existing therapies for inflammatory diseases and cancer, there remains a need for new compounds for treating such diseases, especially given the fact that they are often poorly managed and/or do not respond to current treatments.

SUMMARY

The present invention is broadly directed to methods and compositions for preventing and/or treating inflammation and/or cancer.

In a first aspect, the invention provides an isolated protein comprising, consisting or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment, variant or derivative thereof.

In one embodiment, the variant is a mutant protein which has one or a plurality of amino acids normally present in a full-length or wild-type protein mutated or modified.

In one embodiment, the variant protein comprises an amino acid sequence at least 70% identical to that set forth in SEQ ID NO: 1 or SEQ ID NO:2.

Suitably, the isolated protein of the present aspect is capable of preventing and/or treating inflammation and/or cancer upon administration to a subject.

The isolated protein of this first aspect may be referred to herein as an "EgKI-1" protein, or a fragment, variant or derivative thereof.

In a second aspect, the invention provides a method for preventing and/or treating inflammation in a subject, the method including the step of administering to the subject a therapeutically effective amount of the EgKI-1 protein of the first aspect, to thereby prevent and/or treat the inflammation.

Suitably, the method of the present aspect further includes the step of administering to the subject an additional agent. Preferably, the additional agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), an aminosalicylate, a corticosteroid, an immunosuppressant, an anti-cytokine/cytokine receptor agent, an antibiotic, and combinations thereof.

In particular embodiments, the inflammation is associated with, or secondary to, an inflammatory disease disorder or condition in the subject. Suitably, the inflammatory disease, disorder or condition is an inflammatory disease, disorder or condition of the respiratory system. Preferably, the inflammatory disease, disorder or condition of the respiratory system is selected from the group consisting of asthma, emphysema, chronic bronchitis, and chronic obstructive pulmonary disease (COPD).

In a third aspect, the invention provides a method for preventing and/or treating cancer in a subject, the method including the step of administering to the subject a therapeutically effective amount of the EgKI-1 protein of the first aspect, to thereby prevent and/or treat the cancer.

In certain embodiments, administration of the isolated EgKI-1 protein prevents and/or inhibits metastasis of said cancer.

Suitably, the method of the present aspect further includes the step of administering to the subject an additional agent. Preferably, the additional agent is or comprises one or a plurality of anti-cancer treatments selected from the group consisting of an endocrine therapy, a chemotherapy, an immunotherapy, a molecularly targeted therapy and combinations thereof.

In particular embodiments, the cancer is selected from the group consisting of breast cancer, head and neck cancer, melanoma and cervical cancer.

In one embodiment, one or a plurality of cells of the cancer express or otherwise contain or comprise a neutrophil elastase.

In a fourth aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the EgKI-1 protein of the first aspect.

This aspect also includes fragments, variants and derivatives of said isolated nucleic acid. In a fifth aspect, the invention provides a genetic construct comprising: (i) the isolated nucleic acid of the fourth aspect operably linked or connected to one or more regulatory sequences in an expression vector.

In a sixth aspect, the invention provides a host cell comprising the isolated nucleic acid of the fourth aspect or the genetic construct of the fifth aspect.

In a seventh aspect, the invention provides a method of producing the EgKI-1 protein of the first aspect, including the steps of; (i) culturing the previously transformed host cell of the sixth aspect; and (ii) isolating said protein from said host cell cultured in step (i).

In an eighth aspect, the invention provides an antibody or antibody fragment which binds and/or is raised against the EgKI-1 protein of the first aspect.

In a ninth aspect, the invention provides a composition comprising:

(i) the isolated EgKI-1 protein of the first aspect;

and/or

(ii) the antibody or antibody fragment of the eighth aspect;

and one or more pharmaceutically acceptable carriers, diluents or excipients.

Suitably, the composition further comprises at least one additional agent. In particular embodiments, the at least one additional agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), an aminosalicylate, a corticosteroid, an immunosuppressant, an anti-cytokine/cytokine receptor agent, an antibiotic, and combinations thereof. In alternative embodiments, the at least one additional agent is or comprises one or a plurality of anti-cancer treatments selected from the group consisting of a endocrine therapy, chemotherapy, immunotherapy, a molecularly targeted therapy and combinations thereof.

In a tenth aspect, the invention provides a method of preventing and/or treating a parasitic infection in a subject, the method including the step of administering to the subject a therapeutically effective amount of:

(i) the EgKI-1 protein of the first aspect;

(ii) the antibody or antibody fragment of the eighth aspect; and/or

(iii) the composition of the ninth aspect;

to thereby prevent and/or treat the parasitic infection.

Suitably, the parasitic infection is caused by and/or associated with, at least in part, Echinococcus granulosus. Suitably, the subject referred to herein is a mammal. Preferably, the subject is a human.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further elements, components, integers or steps but may include one or more unstated further elements, components, integers or steps.

It will be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" cell includes one cell, one or more cells and a plurality of cells.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. A. Amino acid sequences of EgKI-1 (1) and EgKI-2 (2). Signal sequences (18 amino acids) are in red, the Kunitz domain of both proteins is boxed and the Kunitz family signature highlighted in black. The conserved cysteine residues are shown in orange; EgKI-1 has six whereas EgKI-2 has five with one position replaced by a glycine (blue). The Pi reactive sites of both proteins are highlighted in green. B. Schematic diagram of (i) EgKI-1 showing three disulphide bridges, and (ii) EgKI-2 presenting two disulphide bridges (general structure reproduced from that described by Chand, Schmidt et al. (52)).

Figure 2. Partial amino acid sequence comparison of EgKI-1 and EgKI-2 with other Kunitz type protease inhibitors from: Fasciola hepatica (FhKTM, AAB46830.1); BPTI (1510193A); Echinococcus granulosus (EgKU8, ACM79010.1); Ancylostoma ceylanicum (AceKI, AAD51334.1); Conus striatus (Conk-Sl, P0C1X2.1); the first domain of Ancylostoma caninum Kunitz inhibitor (Ac-KPI-1, AAN10061.1). The six conserved cysteine residues are marked by * and the pattern of disulphide bond formation is shown in brackets. The Pi reactive site is marked by the arrow head and the Kunitz family signature by the dashed double head arrow.

Figure 3. Cladogram phylogenetic analysis (53) of five Kunitz inhibitors (FhKTM, Fasciola hepatica; EgKU8, Echinococcus granulosus; BPTI, basic pancreatic trypsin inhibitor; AceKI, Ancylostoma caninum; and Conk-Sl,Conus striatus) and EgKI-1 and EgKI-2.

Figure 4. Clustal alignment of the EgKI proteins with other single domain putative Kunitz type protease inhibitors present in the E. granulosus genome (4). The Pi reactive sites are highlighted in black. Typical trypsin inhibitors have an arginine (R) at the Pi site whereas typical chymotrypsin inhibitors have a leucine (L).

Figure 5. Normalized expression levels of the EgKI genes in E. granulosus. The Y axis represents the number of copies after dividing the number of copies of either gene of interest (GOI) by the number of copies of the house keeping gene (HKG). PSC, protoscoleces; AW, adult worms; HCM, hydatid cyst membrane; ONC, oncospheres. Error bars represent the mean ± SEM.

Figure 6. Western blotting with mouse antisera raised against the rEgKI proteins. The purified EgKI proteins (Lane 1, EgKI-1; Lane 2, EgKI-2) and mite serpin protein as control (Lane c) were electrophoresed on (A) SDS-PAGE, stained with Coomassie Blue and immunoblotted with antisera (1 :2,000) against (B) recombinant EgKI-1 and (C) EgKI-2, confirming the specificity of the antibodies and the lack of cross reactivity with the unrelated E. coli expressed serpin.

Figure 7. Immunolocalisation of EgKI-2 in histological sections of an adult worm of E. granulosus (A) Bright field, (B) probed with pre-immune sera as negative control, (C) probed with EgKI-2 immunized mouse serum (1 :200). DAPI counterstained nuclei are stained blue and the positive green fluorescence marks the presence of EgKI-2 along the tegument (t). Scale Bar = 100 μπι.

Figure 8. (A) Predicted calcium binding site of EgKI-1, shown in blue (B) Calcium binding assay: SDS-PAGE gel (left) and corresponding western blot membrane of the calcium binding assay showing clear bands corresponding to the EgKI-1 protein: M- Marker; 1, 6 μ§ EgKI-1; 2, 2 μg EgKI-1; 3, 6 μg EgKI-2; 4, 6 μg BSA.

Figure 9. Inhibition of different serine proteases with increasing concentrations of the EgKI proteins. The relative activity (as a %) with: (A) nanomolar (nM) concentration range of EgKI-1 with trypsin, pancreatic elastase and cathepsin G, and EgKI-2 with trypsin (B) picomolar (pM) concentration range of EgKI-1 with neutrophil elastase and chymotrypsin. (C) Progress curves for trypsin inhibition with increasing concentrations of EgKI-2 (0 nM, 10 nM, 20 nM, 100 nM) with different substrate concentrations ([S]). Figure 10. Inhibition of neutrophil chemotaxis by EgKI-1 in the mouse air pouch model. The data represent the mean ± SEM values of groups of eight mice in two independent experiments and analysis by one-way ANOVA. * P value, 0.03; ** P value, 0.001; ns, not significant (p value > 0.05). Figure 11. The secondary structure of EgKI-1 showing its reactive sites. Kunitz inhibitors react with proteases mainly with the Pi- P₃' peptide bond, and the P₃ and Pis' sites also play roles in binding.

Figure 12. Graphs on cell viability % with Log concentrations of EgKI-1 in nM.

Figure 13. Graph on increase/ decrease of scratch area (mm²) with time (hours) in wells containing control cells (with buffer) and EgKI-1 (3750 nM) treated cells.

Figure 14. Growth of MDA-MB-231 cells from pre-treatment to 24 hours after scratch wound making compared to same time points of control well.

Figure 15. Cytokine response of PBMC against different EgKI-1 concentrations and controls

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 = peptide sequence EgKI-1 of Figure 1A; sequence includes 18 amino acid signal peptide (total = 79 amino acids)

SEQ ID NO: 2 = peptide sequence EgKI-1 (mature peptide) of Figure 1A and B; sequence excludes 18 amino acid signal peptide (total = 61 amino acids)

DETAILED DESCRIPTION

The present invention is predicated, at least in part, on the surprising discovery of a secreted single domain Kunitz type protease inhibitor (EgKI-1) in the E. granulosus genome that acts as a potent chymotrypsin and neutrophil elastase inhibitor. EgKI-1 may also bind calcium and reduce neutrophil infiltration associated with inflammation and thus has potential as a novel therapeutic targeting inflammatory diseases and cancer. In other embodiments, inhibition or suppression of EgKI- 1 may facilitate treatment of parasitic infections such as by E. granulosus.

In one aspect, the invention provides an isolated EgKI-1 protein comprising, consisting or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment, variant or derivative thereof.

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material includes material in native and recombinant form. The term "isolated" also encompasses terms such as "enriched", "purified" and/or "synthetic". Synthetic includes recombinant synthetic and chemical synthetic.

By "EgKI-l" is meant Kunitz type protease inhibitor-1, an excretory/secretory protein from Echinococcus granulosus. EgKI-l (Genbank: # EUB56407) is a 79 amino acid polypeptide. EgKI-l may also be referred to as Venom basic protease inhibitor 2.

In the context of the present invention, by "consisting essentially of" means that the isolated protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 together with 1, 2, 3, 4 or 5 additional amino acids at the N- and/or C-terminus.

By "protein" is meant an amino acid polymer. The amino acids may be natural or non- natural amino acids, D- or L-amino acids, as are well understood in the art.

A "peptide" is a protein having no more than sixty (60) amino acids.

A "polypeptide" is a protein having more than sixty (60) amino acids.

This aspect also includes fragments, variants and derivatives of said EgKI-l protein.

For the purposes of the present invention, a protein "fragment" includes an amino acid sequence that constitutes less than 100%, but at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, 92%, 94%, 96%, 98%, or 99% of said EgKI-l protein (e.g., SEQ ID NO: l and SEQ ID NO: 2).

In particular embodiments, a protein fragment may comprise, for example, at least 10, 15, 20, 25, 30 35, 40, 45, 50, 55, 60, 65, 70 and 75 contiguous amino acids of said EgKI-l protein (e.g., SEQ ID NO: l and SEQ ID NO: 2).

It will be appreciated that a peptide may be a protein fragment, for example comprising at least 6, 10, 12 preferably at least 15, 20, 25, 30, 35, 40, 45, and more preferably at least 50 contiguous amino acids.

Peptide fragments may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques, such as those described herein. Alternatively, peptides can be produced by digestion of an isolated protein of the invention with proteases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques as are well known in the art.

It will also be appreciated that larger peptides and isolated proteins comprising a plurality of the same or different fragments are contemplated. In one preferred embodiment, the EgKI-1 protein fragment comprises, consists or consists essentially of the Kunitz domain of EgKI-1 (e.g., amino acids 23 to 73 of SEQ ID NO: 1).

The invention also provides variants of the EgKI-1 proteins.

As used herein, a protein variant shares a definable nucleotide or amino acid sequence relationship with an isolated protein disclosed herein. Preferably, EgKI-1 protein variants share at least 70% or 75%, preferably at least 80% or 85% or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a wildtype EgKI-1 amino acid sequence, such as SEQ ID NO: 1 or SEQ ID NO: 2.

Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity " and "substantial identity" . Because respective nucleic acids/proteins may each comprise (1) only one or more portions of a complete nucleic acid/protein sequence that are shared by the nucleic acids/proteins, and (2) one or more portions which are divergent between the nucleic acids/proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

The term "sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "match percentage " calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA). Preferably, sequence identity is measured over the entire amino acid sequence of the EgKI-1 protein.

As used herein "variant" proteins disclosed herein have one or more amino acids deleted or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted or deleted without changing the activity of the isolated protein (conservative substitutions).

The term "variant" includes peptidomimetics and orthologs of an isolated protein comprising an amino acid sequence set forth in SEQ ID NOS: l-2. By "peptidomimetic" is meant a molecule containing non-peptidic structural elements that are capable of mimicking or antagonising the biological action(s) of a natural parent peptide. Examples of peptidomimetics include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see, e.g., James et al, Science 260: 1937-42, 1993) and "retro-inverso" peptides (see, e.g., US Pat. No. 4,522,752). The term also refers to a moiety, other than a naturally occurring amino acid, that conformationally and functionally serves as a substitute for a particular amino acid in a protein without adversely interfering to a significant extent with the function of the protein. Examples of amino acid mimetics include D-amino acids. Proteins substituted with one or more D-amino acids may be made using well known peptide synthesis procedures. Additional substitutions include amino acid analogs having variant side chains with functional groups, such as, for example, b-cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxyphenylalanine, 5- hydroxytryptophan, 1-methylhistidine, and 3-methylhistidine.

The term "variant" also includes isolated proteins or fragments thereof disclosed herein, produced from, or comprising amino acid sequences of, naturally occurring (e.g., allelic) variants, orthologs (e.g., from intestinal helminths, such as tapeworms, hookworms, whipworms and roundworms, other than Echinococcus granulosus) and synthetic variants, such as produced in vitro using mutagenesis techniques.

Variant proteins can be produced by a variety of standard, mutagenic procedures known to one of skill in the art, such as those described herein.

Derivatives of the EgKI-1 proteins are also provided.

As used herein, "derivative" proteins have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification {e.g., phosphorylation, acetylation and the like), modification of glycosylation {e.g., adding, removing or altering glycosylation) and/or inclusion of additional amino acid sequences as would be understood in the art.

Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein. Non-limiting examples include metal-binding {e.g., polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences {e.g., GFP), epitope tags such as myc, FLAG and haemagglutinin tags.

Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the isolated EgKI-1 proteins, fragments and variants of the invention.

As would be appreciated by the skilled, EgKI-1 fragments, variants or derivatives may be produced with the aim of improving, for example, their anti-inflammatory activity, anti-cancer activity, side effects or toxicity profile, pharmacodynamics and/or pharmacokinetic s .

Preferably, the EgKI-1 fragment, variant or derivative is a "biologically active" fragment, variant or derivative. In some embodiments, the biologically active fragment, variant or derivative has no less than 10%, preferably no less than 25%, more preferably no less than 50%, and even more preferably no less than 75%, 80%, 85%, 90%, or 95% of the anti-inflammatory activity and/or anti-cancer activity of the EgKI-1 protein (such as set forth in SEQ ID NO: l or SEQ ID NO:2). Such activity may be evaluated using standard testing methods and bioassays recognizable by the skilled artisan in the field as generally being useful for identifying such activity. In particular embodiments, the invention provides a mutant EgKI-1 protein which has one or a plurality of amino acids normally present in a full-length or wild-type EgKI-1 protein mutated or modified.

Suitably, the amino acid sequence of the full-length or wild-type EgKI-1 protein is that set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Accordingly, in particular embodiments, the mutant EgKI-1 protein comprises an amino acid sequence at least 70% or 75%, preferably at least 80% or 85% or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical with the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2.

By "mutant" is meant a form of an original, wildtype or equivalent sequence, inclusive of protein and nucleic acid sequences, which varies from said original, wildtype or equivalent sequence. Such changes or variations can arise either spontaneously or by manipulations by man, by chemical energy (e.g., X-ray), or by other forms of chemical mutagenesis, such as those provided herein, or by genetic engineering, or as a result of mating or other forms of exchange of genetic information. Mutations include, for example, substitutions, deletions, insertions, inversions, translocations, or duplications.

A mutation can involve the modification of the nucleotide sequence of a single gene, blocks of genes or a whole chromosome, with the subsequent production of one or more mutant proteins. Changes in single genes may be the consequence of point mutations, which involve the removal, addition or substitution of a single nucleotide base within a DNA sequence, or they may be the consequence of changes involving the insertion or deletion of large numbers of nucleotides.

Mutations occur following exposure to chemical or physical mutagens. Such mutation-inducing agents include ionizing radiation, ultraviolet light and a diverse array of chemical agents, such as alkylating agents and polycyclic aromatic hydrocarbons, all of which are capable of interacting either directly or indirectly (generally following some metabolic biotransformations) with nucleic acids. The DNA lesions induced by such environmental agents may lead to modifications of base sequence when the affected DNA is replicated or repaired and thus to a mutation, which can subsequently be reflected at the protein level. Mutation also can be site-directed through the use of particular targeting methods.

Mutagenic procedures of use in producing EgKI-1 proteins comprising one or more mutations include, but are not limited to, random mutagenesis (e.g., insertional mutagenesis based on the inactivation of a gene via insertion of a known DNA fragment, chemical mutagenesis, radiation mutagenesis, error prone PCR (Cadwell and Joyce, PCR Methods Appl. 2:28-33, 1992)) and site-directed mutagenesis (e.g., using specific oligonucleotide primer sequences that encode the DNA sequence of the desired mutation). Additional methods of site-directed mutagenesis are disclosed in U.S. Pat. Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377; and 5,789, 166.

Mutant forms of EgKI-1 proteins can display either increased or decreased antiinflammatory activity and/or either increased or decreased anti-cancer activity relative to a wildtype EgKI-1 protein, such as that set forth in SEQ ID NO: l or SEQ ID NO: 2. Suitably, the mutant EgKI-1 protein is capable of preventing and/or treating inflammation and/or cancer upon administration to a subject.

Specifically, mutant EgKI-1 proteins of the invention may be produced with the aim of improving their anti-inflammatory and/or anti-cancer activity without affecting or reducing their side effects and/or toxicity profile. Such mutant EgKI-1 proteins may therefore achieve similar or improved anti-inflammatory results with fewer treatments or a shorter course of treatments. Similarly, mutant EgKI-1 proteins of the invention may be produced with the aim of reducing their side effects and/or toxicity profile whilst maintaining their anti-inflammatory and/or anti-cancer activity.

In alternative embodiments, mutant EgKI-1 proteins have reduced or absent antiinflammatory and/or anti-cancer activity. Such mutant proteins may be useful in the prevention and/or treatment of a parasitic infection, such as that caused by and/or associated with Echinococcus granulosus. By way of example, the mutant EgKI-1 protein may possess an active site mutation inactivating EgKI-l 's enzymatic activity, such that the mutant EgKI-1 protein may still bind and/or interact with a substrate molecule, but without cleavage thereof, thereby protecting the substrate molecule from subsequent degradation and/or inactivation by wild-type EgKI- 1.

In particular embodiments, the mutant EgKI-1 protein of the present aspect is a recombinant protein.

In this regard, mutant EgKI-1 proteins may be obtained through the application of standard recombinant techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 18 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Coligan et al. Eds (John Wiley & Sons, 1995-2000). Suitably, the mutant EgKI-1 protein comprises a Kunitz domain (e.g., amino acids 23 to 73 of SEQ ID NO: 1) having one or a plurality of residues of said Kunitz domain mutated or modified.

Suitably, the isolated EgKI-1 protein of the present aspect is capable of preventing and/or treating inflammation and/or cancer, as are hereinafter described, upon administration to a subject.

Accordingly, in a related aspect, the invention provides a method for preventing and/or treating inflammation in a subject, the method including the step of administering to the subject a therapeutically effective amount of the isolated EgKI-1 protein hereinbefore described (e.g., an isolated EgKI-1 protein comprising, consisting or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment, variant or derivative thereof) to thereby prevent and/or treat the inflammation.

As used herein, "treating" (or "treat" or "treatment") refers to a therapeutic intervention that ameliorates a sign or symptom of a disease, disorder or condition after it has begun to develop. The term "ameliorating" , with reference to a disease, disorder or condition, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

As used herein, "preventing" (or "prevent" or "prevention") refers to a course of action (such as administering a therapeutically effective amount of one or more EgKI-1 proteins or a biologically active fragment, variant or derivative thereof) initiated prior to the onset of a symptom, aspect, or characteristic of a disease, disorder or condition, such as those associated with inflammation and/or cancer, so as to prevent or reduce the symptom, aspect, or characteristic. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease, disorder or condition or exhibits only early signs for the purpose of decreasing the risk of developing a symptom, aspect, or characteristic of a disease, disorder or condition.

By "administration" is meant the introduction of a composition (e.g., a composition comprising one or more isolated EgKI-1 proteins, or a biologically active fragment, variant or derivative thereof) into a subject by a chosen route.

The term "therapeutically effective amount" describes a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising one or more isolated EgKI- 1 proteins (or a biologically active fragment, variant or derivative thereof) necessary to treat and/or prevent inflammation and/or cancer in said subject. In some embodiments, a "therapeutically effective amount" is sufficient to reduce or eliminate a symptom of a disease, disorder or condition, such as inflammation or cancer. In other embodiments, a "therapeutically effective amounf is an amount sufficient to achieve a desired biological effect, for example: (a) an amount that is effective to decrease the degree of inflammation associated with an inflammatory disease, disorder or condition; or (b) an amount that is effective to decrease or prevent cancer growth and/or metastasis.

Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent, for example one or more EgKI-1 proteins (or a biologically active fragment, variant or derivative thereof), useful for treating and/or preventing inflammation and/or cancer will be dependent on the subject being treated, the type and severity of any associated disease, disorder and/or condition, and the manner of administration of the therapeutic composition.

A therapeutically effective amount of a one or more EgKI-1 proteins (or a biologically active fragment, variant or derivative thereof) and/or a composition provided herein may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the frequency of administration is dependent on the preparation applied, the subject being treated, the severity of disease, and the manner of administration of the therapy or composition.

Any safe route of administration may be employed for administering the isolated EgKI-1 protein and/or composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be achieved by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be achieved by using other polymer matrices, liposomes and/or microspheres.

The quantity of the therapeutic agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the therapeutic agent(s) required to be administered will depend on the judgement of the clinician. The total dose required for each treatment may be administered by multiple doses or in a single dose. In any event, suitable dosages of the therapeutic agents of the invention may be readily determined by those skilled in the art. Such dosages may be in the order of nanograms to milligrams of the therapeutic agents of the invention.

As used herein, "inflammation" refers to the well known localised response to various types of injury or infection, which is characterised by redness, heat, swelling, and pain, and often also including dysfunction or reduced mobility. Inflammation may represent an early defence mechanism to contain an infection and prevent its spread from the initial focus. Major events in inflammation include dilation of capillaries to increase blood flow, changes in the microvasculature structure, leading to escape of plasma and proteins and leukocytes from the circulation, and leukocyte emigration from the capillaries and accumulation at the site of injury or infection.

Inflammation is often associated with, or secondary to, a disease, disorder and/or condition in a subject, including an inflammatory and/or immunological disease, disorder and/or condition (such as an autoimmune disease, disorder and/or condition) and allergic reactions. Exemplary inflammatory and/or immunological diseases, disorders and/or conditions include, without limitation, Addison's disease, ankylosing spondylitis, celiac disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Crohn's disease, demyelinating neuropathies, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), insulin-dependent diabetes (type 1), juvenile arthritis, Kawasaki syndrome, multiple sclerosis, myasthenia gravis, postmyocardial infarction syndrome, primary biliary cirrhosis, psoriasis, idiopathic pulmonary fibrosis, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE), thrombocytopenic purpura (TTP), ulcerative colitis, vasculitis, vitiligo, and Wegener's granulomatosis. As will be understood by one of ordinary skill in the art, diseases of the respiratory system (e.g., asthma, emphysema, chronic bronchitis, and chronic obstructive pulmonary disease (COPD)) have an inflammatory component, and thus are particularly amenable to treatment using the disclosed methods.

Accordingly, in one embodiment the inflammatory disease, disorder or condition is an inflammatory disease, disorder or condition of the respiratory system, such as asthma, emphysema, chronic bronchitis, and chronic obstructive pulmonary disease (COPD).

Suitably, the method of the present aspect further includes the step of administering to the subject an additional agent. Preferably, the additional agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), an aminosalicylate, a corticosteroid, an immunosuppressant, an anti-cytokine/cytokine receptor agent, an antibiotic, and combinations thereof.

As is well known to one of skill in the art, nonsteroidal anti-inflammatory drugs (NSAIDs), also referred to as nonsteroidal anti-inflammatory agents (NSAIAs), are drugs with analgesic, antipyretic and anti-inflammatory effects, and include salicylates (e.g., aspirin) and propionic acid derivatives (e.g., ibuprofen and naproxen).

Aminosalicylates are well known in the art for use in the treatment of inflammatory bowel disease (particularly ulcerative colitis), and include, for example, balsalazide, mesalazine, olsalazine, and sulfasalazine.

As is well known to one of skill in the art, corticosteroids are drugs that closely resemble Cortisol, a hormone produced by the adrenal glands. Exemplary corticosteroids include, without limitation, cortisone, prednisone, prednisolone, and methylprednisolone.

Immunosuppressants are well known in the art for use in the treatment of inflammation associated with certain diseases or conditions, and include, for example, the drugs cyclosporine, azathioprine and mycophenolate.

As is well known to one of skill in the art, anti-cytokine/cytokine receptor agents (e.g., anti-T Fa agents, anti-IL-5 agents, anti-IL-13 agents, anti-IL-17 agents, and anti- IL-6R agents) include, without limitation, small molecule inhibitors and antibodies.

In one embodiment, an EgKI-1 protein, such as that comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 (or a biologically active fragment, variant or derivative thereof), is administered prior to the administration of the additional agent. In another embodiment, an EgKI-1 protein, such as that comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 (or a biologically active fragment, variant or derivative thereof), is administered after the administration of the additional agent. In still another embodiment, an EgKI-1 protein, such as that comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 (or a biologically active fragment, variant or derivative thereof), is administered simultaneously with the administration of the additional agent. In yet another embodiment, administration of an EgKI-1 protein, such as that comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 (or a biologically active fragment, variant or derivative thereof), and the administration of the additional agent (either sequentially or concurrently) results in treatment or prevention of inflammation that is greater than such treatment or prevention from administration of either the said protein or the additional agent in the absence of the other.

In a related aspect, the invention provides a method for preventing and/or treating cancer in a subject, the method including the step of administering to the subject a therapeutically effective amount of an EgKI-1 protein comprising, consisting or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment, variant or derivative thereof to thereby prevent and/or treat the cancer.

Cancer may include any aggressive or potentially aggressive cancers, tumours or other malignancies such as listed in the NCI Cancer Index at http://www.cancer.gov/cancertopics/alphalist, including all major cancer forms such as sarcomas, carcinomas, lymphomas, leukaemias and blastomas, although without limitation thereto. These may include breast cancer, lung cancer inclusive of lung adenocarcinoma, cancers of the reproductive system inclusive of ovarian cancer, cervical cancer, uterine cancer and prostate cancer, cancers of the brain and nervous system, head and neck cancers, gastrointestinal cancers inclusive of colon cancer, colorectal cancer and gastric cancer, liver cancer, kidney cancer, skin cancers such as melanoma and skin carcinomas, blood cell cancers inclusive of lymphoid cancers and myelomonocytic cancers, cancers of the endocrine system such as pancreatic cancer and pituitary cancers, musculoskeletal cancers inclusive of bone and soft tissue cancers, although without limitation thereto.

In particular embodiments, the cancer is selected from the group consisting of breast cancer, head and neck cancer, melanoma and cervical cancer, inclusive of primary and metastatic disease. Breast cancers include all aggressive breast cancers and cancer subtypes such as triple negative breast cancer, grade 2 breast cancer, grade 3 breast cancer, lymph node positive (LN⁺) breast cancer, HER2 positive (HER2⁺) breast cancer and ER positive (ER⁺) breast cancer, although without limitation thereto.

In one embodiment, the cancer is triple negative breast cancer. As used herein,

"triple negative breast cancer" (T BC) is an often aggressive breast cancer subtype lacking or having significantly reduced expression of estrogen receptor (ER) protein, progesterone receptor (PR) protein and HER2 protein. TNBC and other aggressive breast cancers are typically insensitive to some of the most effective therapies available for breast cancer treatment, including HER2-directed therapy, such as trastuzumab, and endocrine therapies, such as tamoxifen and aromatase inhibitors.

Suitably, the method of the present aspect further includes the step of administering to the subject an additional agent. Preferably, the additional agent is or comprises one or a plurality of anti-cancer treatments selected from the group consisting of an endocrine therapy, a chemotherapy, an immunotherapy, a molecularly targeted therapy and combinations thereof. The anti-cancer treatment may be any known in the art, including those hereinbefore described.

Endocrine therapy (also called hormone therapy) typically slows or stops the growth of hormone-sensitive tumours by blocking the body' s ability to produce hormones or by interfering with hormone action. For example, with breast cancer, the female hormones oestrogen and progesterone can promote the growth of some breast cancer cells. Therefore in some patients, hormone therapy is given in an attempt to block the body's naturally occurring oestrogen to slow or stop the cancer's growth. Non-limiting examples of endocrine therapy include aromatase inhibitors (e.g., anastrozole, letrozole, exemestane), gonadotropin-releasing hormone agonists (e.g., goserelin, leuprolide) and oestrogen receptor antagonists (e.g., tamoxifen, ralexifene, toremifene, fulvestrant).

Non-limiting examples of chemotherapy include a pyrimidine analogue (e.g., 5- fluorouracil, capecitabine), a taxane (e.g., paclitaxel), an anthracycline (e.g., doxorubicin, epirubicin), an anti-folate drug (e.g., the dihydrofolate reductase inhibitor methotrexate), an alkylating agent (e.g., cyclophosphamide) or any combination thereof. It would be appreciated that the chemotherapy may be administered as adjuvant, neoadjuvant and/or as standard therapy.

Insofar as they relate to cancer, immunotherapy or immunotherapeutic agents use or modify the immune mechanisms of a subject so as to promote or facilitate treatment of a cancer. In this regard, immunotherapy or immunotherapeutic agents used to treat cancer include cell-based therapies, antibody therapies (e.g., anti-PDl or anti-PDLl antibodies) and cytokine therapies. These therapies all exploit the phenomenon that cancer cells often have subtly different molecules termed cancer antigens on their surface that can be detected by the immune system of the cancer subject. Accordingly, immunotherapy is used to provoke the immune system of a cancer patient into attacking the cancer' s cells by using these cancer antigens as targets.

Non-limiting examples of immunotherapy or immunotherapeutic agents include adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, BMS-936559, brentuximab, certolizumab, cituximab, daclizumab, eculizumab, ibritumomab, infliximab, ipilimumab, lambrolkizumab, mepolizumab, MPDL3280A muromonab, natalizumab, nivolumab, ofatumumab, omalizumab, pembrolizumab, pexelizumab, pidilizumab, rituximab, tocilizumab, tositumomab, trastuzumab, ustekinumab, abatacept, alefacept and denileukin diftitox. In particular preferred embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor, such as an anti-PDl antibody (e.g., pidilizumab, nivolumab, lambrolkizumab, pembrolizumab), an anti-PDLl antibody (e.g., BMS- 936559, MPDL3280A) and/or an anti-CTLA4 antibody (e.g., ipilimumab).

Molecularly targeted therapies are agents that typically block the growth and spread of cancer by interfering with specific molecules ("molecular targets"), such as receptors, signalling molecules and kinases, that are involved in the growth, progression, and spread of cancer. In certain embodiments, the molecularly targeted therapy comprises an ALK inhibitor (e.g., TAE684), an Aurora kinase inhibitor (e.g., Alisertib, AMG-900, BI-847325, GSK-1070916A, ilorasertib, MK-8745, danusertib), a BCR-ABL inhibitor (e.g., Nilotinib, Dasatinib, Ponatinib), a HSP90 inhibitor (e.g., Tanespimycin (17-AAG), PF04291 13, AUY922, Luminespib, ganetespib, Debio-0932), an EGFR inhibitor (e.g., Afatinib, Erlotinib, Lapatinib, cetuximab), a PARP inhibitor (e.g., ABT-888, AZD-2281), retinoic acid (e.g., all-trans retinoic acid or ATRA), a Bcl2 inhibitor (e.g., ABT-263), a gluconeogenesis inhibitor (e.g., metformin), a p38 MAPK inhibitor (e.g., BIRB0796, LY2228820), a MEKl/2 inhibitor (e.g., trametinib, cobimetinib, binimetinib, selumetinib, pimasertib, refametinib, TAK-733), an ERK inhibitor (e.g., FR 180204), a mTOR inhibitor (e.g., BEZ235, JW-7-25-1), a PI3K inhibitor (e.g., Idelalisib, buparlisib/apelisib, copanlisib, GSK-2636771, pictilisib, AMG-319, AZD-8186), an IGF1R inhibitor (e.g., BMS-754807, dalotuzumab, ganitumab, linsitinib), a PLCy inhibitor (e.g., U73122), a INK inhibitor (e.g., SP600125), a PAK1 inhibitor (e.g., IP A3), a SYK inhibitor (e.g., BAY613606), a HDAC inhibitor (e.g., Vorinostat), an FGFR inhibitor (e.g., Dovitinib), a XIAP inhibitor (e.g., Embelin), a PLK1 inhibitor (e.g., Volasertib, P-937), an ERK5 inhibitor (e.g., XMD8-92), a MPS 1/TTK inhibitor (e.g., BAY-1 161909) and any combination thereof.

In particular embodiments, an EgKI-1 protein, such as that comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 (or a biologically active fragment, variant or derivative thereof), is administered (i) prior to; (ii) after; or (iii) simultaneously with, the administration of the additional agent. In one embodiment, administration of an EgKI-1 protein, such as comprising an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 (or a biologically active fragment, variant or derivative thereof), and the administration of the additional agent (either sequentially or concurrently) results in treatment or prevention of cancer that is greater than such treatment or prevention from administration of either the said protein (or a biologically active fragment, variant or derivative thereof) or the additional agent in the absence of the other.

It would be appreciated that the method of this aspect further includes within its scope the prevention and/or inhibition of metastasis by the cancer in question. Accordingly, in particular embodiments, administration of the isolated EgKI-1 protein prevents and/or inhibits metastasis of said cancer.

As used herein, "metastasis" or "metastatic", refers to the migration or transfer of malignant tumour cells, or neoplasms, via the circulatory or lymphatic systems or via natural body cavities, typically from the primary focus of tumour, cancer or a neoplasia to a distant site in the body, and the subsequent development of one or more secondary tumours or colonies thereof in the one or more new locations. "Metastases" refers to the secondary tumours or colonies formed as a result of metastasis and encompasses micro- metastases as well as regional and distant metastases.

In particular embodiments, one or a plurality of cells of the cancer express, overexpress , or otherwise contain or comprise a neutrophil elastase.

The skilled artisan would appreciated that neutrophil elastase is a serine protease of the chymotrypsin superfamily that is stored in the primary azurophilic granules of polymorphonuclear neutrophils (PMN). Neutrophil elastase is typically released from activated PMN's and has been implicated in the pathogenesis of cancer. To this end, some cancers lack endogenous neutrophil elastase expression, but may still express neutrophil elastase by uptake of this enzyme by one or more cancer cells of said cancer from one or more associated cells, such as tumor-associated neutrophils (TAN), stromal cells, cancer- associated fibroblasts (CAFs), pericytes and endothelial cells.

Accordingly, in one further embodiment, one or more cells associated with the cancer express, overexpress , or otherwise contain or comprise a neutrophil elastase.

The expression level of neutrophil elastase may be relative or absolute.

Additionally, the expression level of neutrophil elastase may be measured by any means known in the art, such as immunoblotting, immunohistochemistry, immunocytochemistry, immunoprecipitation, ELISA, flow cytometry, magnetic bead separation, biosensor-based detection systems, nucleic acid amplification techniques as described herein and in situ hybridization.

In some embodiments, the expression of neutrophil elastase in one or more cells of the cancer is increased (i.e., overexpression). By way of example, the expression of neutrophil elastase is increased if its level of expression, such as extracellular expression, is more than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%), 400%) or at least about 500% greater than the level of expression of the corresponding neutrophil elastase in a control sample or further biological sample from a subject.

The term "control sample" typically refers to a biological sample from a (healthy) non-diseased individual not having cancer. In one embodiment, the control sample may be from a subject known to be free of cancer. Alternatively, the control sample may be from a subject in remission from cancer. The control sample may be a pooled, average or an individual sample. An internal control is a marker from the same biological sample being tested.

In yet another aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the EgKI-1 protein described herein.

This aspect also includes fragments, variants and derivatives of said isolated nucleic acid.

The term "nucleic acid" as used herein designates single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.

Accordingly, in particular embodiments, the isolated nucleic acid is cDNA.

In further embodiments, the isolated nucleic acid is codon-optimised nucleic acid. A "polynucleotide " is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide " has less than eighty (80) contiguous nucleotides.

A "probe" may be a single or double- stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

A "primer^'" is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™ (see, for example, Table 1).

Another particular aspect of the invention provides a variant of an isolated nucleic acid that encodes an isolated protein of the invention.

In another embodiment, nucleic acid variants share at least 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more preferably at least 80%, 81%, 82%, 83%, 84%, or 85%, and even more preferably at least 90%, 91%, 92%), 93%), 94%), or 95% nucleotide sequence identity with an isolated nucleic acid of the invention. Percent sequence identity may be determined as previously described.

Also included are nucleic acid fragments. A "fragment" is a segment, domain, portion or region of a nucleic acid, which respectively constitutes less than 100% of the nucleotide sequence. A non-limilting example is an amplification product or a primer or probe. In particular embodiments, a nucleic acid fragment may comprise, for example, at least 10, 15, 20, 25, 30 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200 and 225 contiguous nucleotides of said nucleic acid.

Nucleic acids may also be isolated, detected and/or subjected to recombinant DNA technology using nucleic acid sequence amplification techniques.

Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-β replicase amplification and helicase-dependent amplification, although without limitation thereto. As used herein, an "amplification product" refers to a nucleic acid product generated by nucleic acid amplification.

Nucleic acid amplification techniques may include particular quantitative and semi-quantitative techniques such as qPCR, real-time PCR and competitive PCR, as are well known in the art.

In still a further aspect, the invention provides a genetic construct comprising: (i) the isolated nucleic acid provided herein operably linked or connected to one or more regulatory sequences in an expression vector.

Suitably, the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and/or expression of the nucleic acid or an encoded protein of the invention.

For the purposes of host cell expression, the genetic construct is an expression construct. Suitably, the expression construct comprises the nucleic acid of the invention operably linked to one or more additional sequences in an expression vector. An "expression vector" may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome. Non-limiting examples of expression constructs include adenovirus vectors, adeno-associated virus vectors, herpesviral vectors, retroviral vectors, lentiviral vectors, and the like. For example, adenovirus vectors can be first, second, third, and/or fourth generation adenoviral vectors or gutless adenoviral vectors. Adenovirus vectors can be generated to very high titers of infectious particles, infect a great variety of cells, efficiently transfer genes to cells that are not dividing, and are seldom integrated in the host genome, which avoids the risk of cellular transformation by insertional mutagenesis (Douglas and Curiel, Science and Medicine, March/ April 1997, pages 44-53; Zern and Kresinam, Hepatology 25:484-91, 1997). Representative adenoviral vectors are described by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626-30, 1992), Graham and Prevec (In Methods in Molecular Biology: Gene Transfer and Expression Protocols 7: 109-28, 1991) and Barr et al. (Gene Therapy, 2: 151-55, 1995).

Adeno-associated virus (AAV) vectors also are suitable for administration of the nucleic acids of the invention. Methods of generating AAV vectors, administration of AAV vectors and their uses are well known in the art (see, e.g., U.S. Patent No. 6,951,753; U.S. Patent Application Publication Nos. 2007/036757, 2006/205079, 2005/163756, 2005/002908; and PCT Publication Nos. WO 2005/116224 and WO 2006/119458).

By "operably linked" is meant that said additional nucleotide sequence(s) is/are positioned relative to the nucleic acid of the invention preferably to initiate, regulate or otherwise control transcription.

Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. Non-limiting examples of promoters include SV40, cytomegalovirus (CMV), and HIV-1 LTR promoters.

The expression construct may also include an additional nucleotide sequence encoding a fusion partner (typically provided by the expression vector) so that the recombinant isolated protein of the invention is expressed as a fusion protein, as hereinbefore described.

In still another aspect, the invention provides a host cell transformed with the isolated nucleic acid or the genetic construct described herein.

Suitable host cells for expression may be any as are well known in the art, including prokaryotic or eukaryotic host cells. For example, suitable host cells may be mammalian cells (e.g. HeLa, HEK293T, Jurkat cells), yeast cells (e.g. Saccharomyces cerevisiae), insect cells (e.g. Sf9, Trichoplusia ni) utilized with or without a baculovirus expression system, or bacterial cells, such as E. coli, or a Vaccinia virus host. Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 9 and 16.

In a related aspect, the invention provides a method of producing the EgKI-1 protein hereinbefore described, comprising; (i) culturing the previously transformed host cell of the previous aspect; and (ii) isolating said protein from said host cell cultured in step (i).

The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. 1995-2009), in particular Chapters 1, 5 and 6.

In still another aspect, the invention provides an antibody or antibody fragment which binds and/or is raised against the EgKI-1 protein described herein (e.g., an EgKI-1 protein comprising, consisting or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment, variant, derivative or mutant thereof).

Suitably, said antibody or antibody fragment specifically binds said isolated EgKI-

1 protein. In some embodiments, antibodies raised against the mutant EgKI-1 protein described herein may demonstrate reduced affinity or little or no affinity for the wildtype EgKI-1 protein (e.g., SEQ ID NO: 1 or 2).

In a particular embodiment, the antibody or antibody fragment is an inhibitory or antagonist antibody or antibody fragment. In some embodiments, the antibody may reduce, eliminate, inhibit or suppress the activity of EgKI-1 and/or may inhibit reduce, eliminate, inhibit or suppress binding of EgKI-1 to a substrate molecule.

Antibodies of the invention may be polyclonal or monoclonal, native or recombinant. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, which are both herein incorporated by reference.

Generally, antibodies of the invention bind to or conjugate with an isolated EgKI- 1 protein, fragment, variant, or derivative, such as that described herein. For example, the antibodies may be polyclonal antibodies. Such antibodies may be prepared for example by injecting an isolated EgKI-1 protein, fragment, variant or derivative hereinbefore described into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.

Monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler & Milstein, 1975, Nature 256, 495, which is herein incorporated by reference, or by more recent modifications thereof as for example, described in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the isolated EgKI-1 proteins, fragments, variants or derivatives, such as those provided herein.

The invention also includes within its scope antibody fragments, such as Fc, Fab or F(ab)2 fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349:293, which are incorporated herein by reference. The invention is also contemplated to include multivalent recombinant antibody fragments, so-called diabodies, triabodies and/or tetrabodies, comprising a plurality of scFvs, as well as dimerisation-activated demibodies (e.g., WO/2007/062466). By way of example, such antibodies may be prepared in accordance with the methods described in Holliger et al, 1993 Proc Natl Acad Sci USA 90:6444-6448; or in Kipriyanov, 2009 Methods Mol Biol 562: 177-93 and herein incorporated by reference in their entirety.

Antibodies and antibody fragments may be modified so as to be administrable to one species having being produced in, or originating from, another species without eliciting a deleterious immune response to the "foreign" antibody. In the context of humans, this is "humanization" of the antibody produced in, or originating from, another species. Such methods are well known in the art and generally involve recombinant "grafting" of non-human antibody complementarity determining regions (CDRs) onto a human antibody scaffold or backbone.

Antibodies and antibody fragments of the invention may be particularly suitable for affinity chromatography purification of the isolated EgKI-1 proteins described herein. For example reference may be made to affinity chromatographic procedures described in Chapter 9.5 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra. In another embodiment, antibodies or antibody fragments that bind or are raised against an isolated EgKI-1 protein provided herein may be used for passive immunization against and/or the treatment of a parasitic infection, such as that caused by and/or associated with Echinococcus granulosus inclusive of cystic echinococcosis and hydatid disease.

In particular embodiments, the antibody or antibody fragment is a recombinant antibody or antibody fragment.

In a further aspect, the invention provides a composition comprising:

(i) the isolated EgKI-1 protein hereinbefore described (e.g., an isolated EgKI-1 protein comprising, consisting or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment, variant or derivative thereof; and/or

(ii) the antibody or antibody fragment provided herein;

and one or more pharmaceutically acceptable carriers, diluents or excipients.

In further embodiments, the EgKI-1 protein, comprising, consisting or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 and/or the mutant EgKI-1 protein are recombinant proteins.

By "pharmaceutically -acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference.

Compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the therapeutic agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

It is contemplated that the composition may alternatively comprise (i) an isolated nucleic acid, for example, one or more encoding an EgKI-1 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) and/or a mutant EgKI-1 protein of the present invention, inclusive of variants, derivatives and fragments thereof; (ii) an expression construct encoding the isolated nucleic acid of (i); and/or (iii) a host cell comprising the expression construct of (ii).

In particular embodiments, the composition is suitable for treating and/or preventing inflammation and/or cancer in a subject. To this end, the composition may further comprise at least one additional agent.

In particular embodiments, the at least one additional agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), an aminosalicylate, a corticosteroid, an immunosuppressant, an anti-cytokine/cytokine receptor agent (e.g., anti- TNFa agents, anti-IL-5 agents, anti-IL-13 agents, anti-IL-17 agents, and anti-IL-6R agents), an antibiotic, and combinations thereof.

In alternative embodiments, the at least one additional agent is or comprises one or a plurality of anti-cancer treatments selected from the group consisting of a endocrine therapy, chemotherapy, immunotherapy, a molecularly targeted therapy and combinations thereof.

In further embodiments, the composition is suitable for treating and/or treating a parasitic infection in a subject, such as that caused by and/or associated with Echinococcus granulosus inclusive of cystic echinococcosis and hydatid disease.

Accordingly, in yet still a further aspect, the invention provides a method of preventing and/or treating a parasitic infection in a subject, the method including the step of administering to the subject a therapeutically effective amount of:

(i) the EgKI-1 protein provided herein;

(ii) the antibody or antibody fragment provided herein; and/or

(iii) the composition provided herein;

to thereby prevent and/or treat the parasitic infection. As would be appreciated by the skilled artisan, a "parasitic infection" is one caused by a plant or animal that at some stage of its existence obtains nourishment from another living organism typically called the host. Examples are helminths, fleas, ticks, mosquitoes and the like that prey on hosts such as mammals.

Suitably, the parasitic infection is a helminth infection, including, but not limited to that caused by and/or associated with tapeworms, flukes, hookworms, whipworms and roundworms. Preferably, the parasitic infection is caused by and/or associated with, at least in part, Echinococcus granulosus, such as cystic echinococcosis or hydatid disease.

The term "subject", as used herein, includes both human and veterinary subjects. For example, administration to a subject can include administration to a human subject or a veterinary subject. In particular embodiments, the term "subject" includes but is not limited to humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Preferably, the subject is a human.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting example.

EXAMPLE 1

A previous study described a multigene family of eight (EgKUl-EgKU8) secreted monodomain Kunitz proteins from E. granulosus protoscoleces preferentially expressed by pepsin/H (+)-treated worms (20). Only one (EgKU8) behaved as a potential protease inhibitor whereas structural modelling revealed EgKUl as a cation-channel blocker which suggested involvement of the majority of these Kunitz proteins in functions other than protease inhibition (20).

By interrogation of the available genome sequence data for E. granulosus (3,4) we identified two gene sequences (designated EgKI-1 and EgKI-2) encoding two polypeptides similar to single domain Kunitz proteins. The two cDNAs were expressed in Escherichia coli, and the recombinant proteins (rEgKI-1 and rEgKI-2) were purified and functionally characterized. EgKI-2 reacted as a typical trypsin inhibitor, whereas EgKI-1 a potent inhibitor of chymotrypsin and neutrophil elastase, was able to significantly reduce neutrophil infiltration in the carrageenan mouse air pouch model of local inflammation, and is the first Kunitz type serine protease inhibitor shown to bind calcium.

EXPERIMENTAL PROCEDURES

Identification and expression of EgKI-1 and EgKI-2 - Two nucleotide sequences (EgKI-1 and EgKI-2), encoding single domain Kunitz type protease inhibitors, were identified by interrogation of available E. granulosus genomic sequence for Kunitz domains. Searches for similar nucleotide sequences were performed using BLAST (bli -.//blast . ncbi . nim . mh . gov/B last) on the NCBI (National Centre for Biotechnology Information) web site. The presence of a signal sequence in both proteins was checked using signalP 4.1 (http ://www, cbs. dtu.dk/services/SignalP ) (21). Protein domains were identified by searching the PRO SITE database ( h ttp : //pro site, expasy . org/) (22) and multiple sequence alignment was generated with the Clustal Omega programme (htip://w vv.ebi.ac.uk/1^ ols/insa/ciustaio/) (23). Protein structure prediction was performed with Phyre2 online programme (www. sbg.bio. ic. ac.uk/phyre2/) (24) and binding site predictions were carried out with 3DLigandSite (http://www.sbg.bio.ic.ac.uk/3dligandsite/) (25). Other amino acid sequences of E. granulosus, homologous to Kunitz proteins, were searched by blast using the GeneDB online database (http://www.genedb.org/Homepage/Egranulosus).

PCR primers with an introduced N-terminal 6><His tag, were designed and produced by Sigma^® Aldrich (Supplementary Table). Both EgKI-1 and EgKI-2 were PCR-amplified using cDNA from adult worms and MyTaq™ DNA polymerase. Purified PCR products were digested with the restriction enzymes Ncol and EcoRl by incubating in a 37°C water bath for 3 h. Products were ligated into the pET28a expression vector and the recombinant plasmids were transformed in E. coli BL21 (DE3) cells. Positive recombinant clones were identified by PCR screening and a single colony was grown in 5 ml of LB (Luria-Bertani) medium containing 30 mg/ml Kanamycin as the starter culture. Recombinant protein production was induced with 1 mM isopropyl β-D-l- thiogalactopyranoside (IPTG) at mid-log phase (A₆oo -0.5-0.7) at 37°C and samples were collected after 4 h post-induction. Harvested induced cells were lysed with lysozyme (10 mg/ml) in Tris buffer (100 mM NaH₂P0₄, 10 mM Tris-Cl) and incubated for 1 h. Samples were homogenized using a Potter-Elvehjem homogenizer and sonicated for 45 s 3 times with 30 s intervals. Inclusion bodies, collected by centrifugation at 12,000 g for 20 min, washed three times with Tris buffer containing 0.5% (v/v) Triton-X 100 and solubilised in 6 M GuHCl, were allowed to bind to Ni charged resin (Novagen, Madison, USA) at 4°C. The flow through was collected and then the EgKI proteins bound to Ni resin were washed three times with 4 ml Tris buffer containing 40 mM, 50 mM and 70 mM imidazole, sequentially. Then, 50 ml of elution buffer (50 mM NaH₂P0₄, 300 mM NaCl), without imidazole, were allowed to pass through the column, the refolded proteins were eluted with the elution buffer containing 250 mM imidazole, their concentration determined using the Bradford assay and stored at -80°C. Aliquots of the recombinant EgKI proteins were electrophoresed on 15% (w/v) sodium dodecyl sulphate (SDS) polyacrylamide gels and stained with Coomassie Blue to determine their purity and relative molecular mass.

Real time PCR - Preparations of cDNA from adult worms (AW), protoscoleces

(PSC), hydatid cyst membrane (HCM) and oncospheres (ONC) were used for real time PCR. Primers used for quantitative PCR (qPCR) were designed using the online primer design software, Primer3 (http :// simgetie . com/Pr imer3 ) (Table 1). Each cDNA sample (25 ng per reaction) was tested in quadruplicate and all reactions were performed twice. E. granulosus eukaryotic translation initiation factor (Eg-eif) was used as housekeeping gene for normalization of data. The confidence threshold (CT) of the second results set was normalized to the first set before evaluation by importing the standard curve of the first set, to the second. The results were analyzed using Rotor-Gene 6000 software.

Production of polyclonal antibodies against EgKI-1 and EgKI-2 proteins - Purified recombinant EgKI-1 and EgKI-2 were dialyzed in PBS using 3500 MWCO Slide- A-Lyzer^® dialysis cassettes following the manufacturer's instructions. Antiserum production was undertaken using six Swiss mice per protein. For the first immunization each mouse was inoculated subcutaneously with 50 μg of protein (dissolved in 50 μΐ PBS) emulsified with an equal amount of Freund's complete adjuvant. Subcutaneous boosts of 50 μg of protein (dissolved in 50 μΐ PBS), emulsified with the same amount of Freund's incomplete adjuvant, were undertaken on two occasions at two weekly intervals. Mice were bled for serum one week after the third immunization and the presence of serum anti-EgKI antibodies was confirmed by Western blotting. Firstly, 0.5 μg of EgKI- lor EgKI-2 and an unrelated protein produced in E. coli were fractioned on a 15% (w/v) SDS- PAGE gel and transferred to Immun-Blot low fluorescence-PVDF membrane. Overnight blocking was performed with Odyssey buffer at 4°C. Then, the membrane was subjected to incubation with the mouse anti-EgKI-1 or -EgKI-2 anti-serum (1 :2,000 dilution in Odyssey buffer and 0.1% Tween-20) for 1 h followed by incubation with IRDye-labeled rabbit anti-mouse antibody (1 : 15,000 diluted in Odyssey buffer with 0.1% Tween-20 and 0.01%) SDS) for 1 h on a shaker in a dark chamber. After a final wash with distilled water, the membrane was allowed to dry and visualized using the Odyssey^® imaging system.

Western blotting and immunolocalization - Western blotting was carried out with both anti-EgKI-1 and anti-EgKI-2 antibodies using soluble extracts of AW, PSC, HCM and ONC as described (26). Paraffin blocks were made by embedding AW, HCM and PSC fixed in 10%> phosphate buffered formalin in wax- filled moulds. Sections (4 μπι) of these paraffin blocks were then adhered onto microscope slides. Following de- paraffinization and rehydration, antigen retrieval was done with RevealtA^® solution. Then the tissue sections were blocked with 1%> (v/v) bovine serum albumin in Tris buffered saline (TBS) for 1 h and incubated with mouse anti-EgKI-1 or -EgKI-2 anti-serum (1 :200) at 4°C overnight. After washing with TBS-T (TBS with 0.1% Tween20), the sections were incubated with Alexa Fluor^® 488 goat anti-mouse IgG (1 :500) (Invitrogen, Carlsbad, USA) at 37°C for 60 min. Nuclei in the tissue sections were counterstained with DAPIgold^® (Invitrogen, Carlsbad, USA) and observed under an EVOS^® fluorescence microscope.

Serine protease inhibition assays - The inhibitory activity of the recombinant EgKI proteins was tested using several commercially available mammalian proteases: bovine trypsin, bovine chymotrypsin, porcine pancreatic elastase, human neutrophil elastase, human cathepsin G and human proteinase3. Enzyme and EgKI protein mixture were first incubated together in 96 well plates at 37°C for 10 min. Subsequently a chromogenic or fluorogenic substrate was added at concentrations ranging from 100 mM to 5 μΜ and product release was measured using a plate reader every min for 30 min.

Bovine pancreatic trypsin, bovine pancreatic a-chymotrypsin and the fluorogenic substrates N_a-Benzoyl-L-arginine-7-amido-4-methylcoumarin hydrochloride and N- Succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin were purchased from Sigma Aldrich (St Louis, USA). Trypsin and chymotrypsin assays were performed in 200 mM Tris-HCl (pH 8.2) containing 20 mM CaCl₂ and 0.1% PEG 8000. The kinetic rate of substrate hydrolysis was measured at excitation/emission wavelengths of 370/460 nm with a fluorescence microplate reader. Inhibitory activity of porcine pancreatic elastase (PPE) was observed using the Enzcheck elastase assay kit (Life technologies, Carlsbad, USA) following the manufacturer's instructions. Fluorescence signals were measured at 505/515 nm.

Neutrophil elastase, Cathepsin G and Proteinase 3, with corresponding substrates N-Methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin, Suc-Ala-Ala-Pro-Phe- pNA and Boc-Ala-Ala-Nva-SBzl, respectively, were purchased from Enzolifesciences (NY, USA). The neutrophil elastase (NE) inhibition assay was carried out with buffer containing 100 mM HEPES, 300 mM NaCl and 0.05% Tween-20 (pH 8) with 2.5 nM enzyme and fluorescence signals were detected at 370/460 nm. Cathepsin G activity was determined in 100 mM Tris-HCl, 1.6 M NaCl buffer (pH 7.5) with 100 nM enzyme and release of Pro-Phe-pNA was measured at 405 nm. Buffer containing 100 mM HEPES pH 7.5, 500 mM NaCl, 10% DMSO was used to detect Proteinase 3 activity and substrate hydrolysis was detected at 412 nm following the addition of 170 μΜ 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB).

Results were expressed as a percentage of the relative activity of the EgKI proteins using the formula:

Percentage of relative activity = (ARFU of inhibitor/ ARFU of enzyme control) x 100% Δ Relative unit (RU) = R₂ - Ri, Readings Ri and R₂ were taken at ti and t₂ time points respectively, when the reaction was in the linear range. IC₅₀ values of EgKI- 1 and EgKI-2 for each protease were calculated using GraphPad Prism version 6.02 software.

Ki values were calculated using Graph Pad Prism version 6.02 software according to the formula; v = Vma_X [S]/ K_m (1+ [I]/Ki) + [S], where v is velocity, Vmax is maximum velocity, [S] is substrate concentration, Km is the Michaelis constant of the substrate.

Blood clotting assays - As the clotting of blood involves multiple serine proteases, two standard tests (activated partial thromboplastin time, APTT; and the prothrombin time, PT) were performed to determine whether the two recombinant EgKIs had any effect on the intrinsic and the extrinsic pathways of coagulation. Fresh healthy human blood (30 ml) was collected into sodium citrate vacutainers and the plasma was separated. Then, 800 μΐ plasma was mixed with 50 μΐ of each EgKI protein (final concentrations of 200 pM, 200 nM and 2 μΜ for EgKI-2 and 200 pM, 200 nM and 10 μΜ for EgKI-1) and incubated in a 37°C water bath for 10 min. After adding CaCl₂ to the mixture, the time taken for clot formation was measured by a Sta-R coagulometer (Diagnostica stago, Asnieres, France). The kits, TriniCLOT APTT HS (Trinity Biotech) and Thromborel S (Siemens) were used for the determination of the APTT and the PT, respectively. Aprotinin (Sigma Aldrich, St Louis, USA) and FVII negative plasma were used as positive controls for APTT and PT, respectively.

Calcium binding assay - As the 3DLigandsite predicted that EgKI-1 would bind calcium, a calcium binding assay was carried out using a published procedure (27) with minor modifications to confirm the prediction. Recombinant EgKI-1, recombinant EgKI-2 and bovine serum albumin (BSA), as positive control, were separated on 15% (w/v) SDS- PAGE gels and the electrophoresed proteins transferred to a PVDF membrane. The membrane was washed with wash buffer (10 mM imidazole, 60mM potassium chloride, 5 mM magnesium chloride, pH 6.8) for 1 h at 37°C with gentle shaking. After rinsing with distilled water, the blot was incubated with 1 mM CaCl₂ for 1 h. Following washing (3X) with 20%) (v/v) ethanol and a final wash with distilled water, the membrane was incubated with ImM Quin-2 (AM) (Sigma Aldrich, St Louis, USA) for 1 h prior to visualizing bound calcium using a UV transilluminator.

Carrageenan mouse air pouch model of local inflammation - The subcutaneous air pouch model, which is an in vivo model that can be used to study acute and chronic inflammation, was used according to a published protocol (28). Briefly, female BALB/c mice weighing <25g were anesthetized with isoflurane and a subcutaneous dorsal pouch was made in each animal by injecting 3 ml sterile air. The pouch was reinjected with 1.5 ml sterile air, after two days. On the sixth day, a 15 μΜ protein sample (rEgKI-1 or the anti-inflammatory drug Ulinastatin [Prospec-Tany TechnoGene Ltd, Ness Ziona, Israel] as positive control: 200 μΐ) in PBS or PBS alone as negative control was injected into the pouch. An LPS control (L2880; Sigma Aldrich, St Louis, USA) was also included and consisted of 12 μg of LPS, which was an amount equivalent to the calculated LPS contamination of the bacterially produced rEgKI-1 protein measured using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific Inc., IL, USA) with a sensitivity of 0.1 EU/ml (approximately 0.01 ng endotoxin per ml). After 30 mins, 300 μΐ of 1%) (w/v) lambda (λ) carrageenan (Sigma Aldrich, St Louis, USA) in sterile saline were injected into the air pouch. On the following day, mice were euthanized and the pouches were washed with 1 ml ice cold lavage solution (0.5%> EDTA in 0.9% saline). The collected lavage solution was immediately placed on ice and then centrifuged at 200 g for 10 min at 4°C. The resulting cell pellet was then resuspended in 500 μΐ lavage solution. Thin smears were made on microscope slides with the cell solution, stained with Diffquick stain and examined under xlOO magnification. Differential cell counts were performed with the stained thin smears by counting 300 cells in total with the percentage of neutrophils in each sample calculated. The total cell count in the lavage solution was determined using a hemocytometer. Then, the total neutrophil count per mouse in the lavage solution was calculated by multiplying the percentage of neutrophils by the total cell count. Statistical analysis was performed with one-way analysis of variance (ANOVA) using GraphPad prism 6. Statistical significance was established at P < 0.05 compared with the PBS control and the LPS control.

Ethics statement - The QIMR Berghofer (QEVIRB) Medical Research Institute Animal Ethics Committee approved the animal work performed in this study. Human venous blood was prepared from a healthy volunteer after he/she provided informed consent and following review by the QIMRB Medical Research Institute Human Ethics Committee.

RESULTS

Identification and analysis of Kunitz protein genes - EG 08721 (GenBank:

EUB56407.1), reported as being highly expressed in oncospheres and EG 07242 (GenBank: EUB57880.1), reportedly highly expressed in adult worms (4) were selected for further characterization and named EgKI-1 and EgKI-2 respectively. The full-length EgKI-l and EgKI-2 cDNAs have open reading frames of 240 and 252 nucleotides. Both translated peptides contain 18 amino acid signal sequences (Fig. 1A) and have molecular masses of 8.08 kDa (EgKI-1) and 8.3 kDa (EgKI-2). EgKI-1 has six conserved cysteine residues whereas EgKI-2 has only five, apparently lacking the second disulphide bond (Fig. IB). A Clustal alignment comparison of EgKI-1 and EgKI-2 with well characterized Kunitz inhibitors available in GenBank revealed that the Kunitz family signature is highly conserved among different species (Fig. 2). Phylogenetic analysis of the two EgKIs (Fig. 3) confirmed their relatedness with Kunitz protein sequences from other taxa. Further interrogation indicated the presence of several other putative Kunitz inhibitors in the E. granulosus genome and transcriptome (4), suggesting these proteins likely play an important role in the parasite's biology. Some of these Kunitz proteins contain a non inhibitory Pi amino acid whereas others contain a protease inhibitory amino acid. Also, several contain only Kunitz domains, whereas some associate with other domains such as Ig or spondin. Clustal alignment performed with the ten putative single domain Kunitz inhibitors of E. granulosus identified nine typical trypsin inhibitors and one typical chymotrypsin inhibitor (Fig. 4). Real time PCR - EgKI-1 was highly expressed in oncospheres, the infective stage of E. granulosus for humans and intermediate hosts, such as sheep, whereas EgKI-2 was more highly expressed in adult worms compared with EgKI-1 (Fig. 5).

Western blotting and immunolocalization - Purified yields of 1 mg/ L and 0.4 mg/ L, respectively, were obtained for recombinant EgKI-1 and EgKI-2 (Fig. 6 A). There was no cross immuno-reactivity between EgKI-1 and EgKI-2 as shown by western blotting of the recombinant proteins with antisera from mice immunized with the two proteins; moreover, no reactivity was observed with either antiserum to an E. coli produced non- related scabies (Sarcoptes) mite serpin, SMSB4 (29) (Fig. 6B and 6C). There was no positive reactivity with soluble antigen extracts from AW, PSC, HCM or ONC by either the anti-EgKI-1 or anti-EgKI-2 murine antisera in western blots (not shown). A positive reaction was evident with the EgKI-2 anti serum along the tegument of sections of adult worms (Fig. 7) but no positive reactivity was observed with sections from any of the life cycle stages probed with the EgKI-1 antiserum.

Calcium binding assay - Recombinant EgKI-1 bound calcium whereas EgKI-2 did not (Fig. 8B). According to the 3DLigandsite prediction, five calcium ions bind with the glutamine (Glu) residue at position 49 in EgKI-1 (Fig. 8 A). As EgKI-2 does not have a Glu at this position it was unable to bind calcium.

Protease inhibition and coagulation assays - In serine protease inhibition assays (Table 2, Fig. 9) EgKI-2 reacted as a typical trypsin inhibitor, having no significant inhibitor activity against the other tested proteases. EgKI-1 inhibited all tested proteases, except proteinase3, showing a high potency for inhibiting chymotrypsin and neutrophil elastase (Table 2, Fig. 9). Varying the pre-incubation time period of the EgKI proteins with the serine proteases did not affect their inhibitory capacity suggesting that they are not "slow binders" (30).

Neither recombinant EgKI-1 nor EgKI-2 interfered with blood coagulation when tested for APTT and PT. Neither protein prolonged the time taken for clot formation in either test indicating none of the proteases involved in the coagulation cascade were inhibited.

Mouse air pouch model - The results of the mouse air pouch model indicated that the infiltration of neutrophils to the inflammatory site was significantly reduced by around 50% (P value <0.05) in the presence of 15 μΜ EgKI-lor the positive control Ulinastatin compared with the PBS control; injection of the LPS control had no effect on the numbers of neutrophils infiltrating the pouch (Fig. 10). DISCUSSION

The host-parasite relationship is complex being mediated both by parasite virulence factors and exacerbated by host responses. The presence of Kunitz type proteins in numerous phylogenetically diverse species suggests that these molecules perform important biological functions even though their precise role in each organism is not yet fully understood. We focused on two nucleotide sequences (EgKI-1 and EgKI-2) encoding secreted single domain Kunitz type protease inhibitors (EgKI-1 and EgKI-2) from E. granulosus, that we had identified by interrogation of the available genomic sequence.

Real time PCR showed EgKI-1 is highly expressed in the oncosphere which is the stage infective to humans and ungulate hosts. The fact that EgKI-1 inhibited the activities of trypsin, chymotrypsin and PPE may be a feature that helps protect oncospheres from digestion by these enzymes in the small intestine of the mammalian host. In contrast EgKI-2 is highly expressed in adult worms and, as shown by immunofluorescence, the protein is localized to the tegument, suggesting a possible role in protecting the parasite from the constant trypsin exposure that it is subjected to in the small intestine of the canine definitive host. Due to the considerable risk of handling and the difficulties in obtaining material, immunolocalization of the two EgKIs in activated oncospheres was not possible. The fact that neither murine anti-EgKI-1 nor anti-EgKI-2 antisera showed any positive reactivity with any of the western blot-tested soluble antigen extracts from different life cycle stages of E. granulosus suggests that either both EgKI proteins are produced in low quantity or are only expressed following an external stimulus, such as when the worm comes in contact with host proteases. A recent transcriptomic study identified five protease inhibitors, including EgKU8 (31), in the excretory/secretory products of E. granulosus protoscoleces but more sensitive technologies may be required to identify other proteins expressed at lower levels of abundance as may be the case with EgKI-1 and EgKI-2.

The two Kunitz proteins were recombinantly expressed and purified by column refolding from induced lysates of Escherichia coli cells transformed with Z¾ 7/pET28a plasmids. Refolding of bacterially produced protein inclusion bodies immobilised by nickel chelating chromatography is a proven method for reconstituting the native properties of recombinant proteins and making them suitable for structural and functional analysis (32). As a result, we were able to show that both EgKI proteins are potent serine protease inhibitors. Nanomolar range inhibition of trypsin activity was disclosed for EgKI-2, while EgKI-1 inhibited chymotrypsin and neutrophil elastase in the picomolar range. The specificity of a protease inhibitor against a protease is mainly determined by the nature of the amino acid residue at position Pi of its active site. The results we obtained with the EgKI proteins are in agreement with previous findings of Kunitz inhibitors from other taxa, where typical trypsin inhibitors have Arg (R) or Lys (K) at Pi, and chymotrypsin inhibitors have Leu (L) or Met (M) (33); EgKI-1 and EgKI-2 have Leu and Arg residues at the Pi site, respectively. The EgKIs are likely to play an important role in E. granulosus survival within their mammalian hosts and thus represent new drug and/or vaccine targets as control interventions. EgKI-1 may prevent the oncosphere from being digested in the gut by inhibiting trypsin, chymotrypsin and pancreatic elastase before it penetrates the intestinal wall. Similarly, trypsin inhibition by EgKI-2 may be involved in providing protection to the adult worms while residing in the small intestine of the canine host.

Inflammatory responses occur after surgery, trauma and infection, and involve neutrophil activation and infiltration into the injured tissue. Neutrophil infiltration also occurs in the early stages of echinococcal infection (34). Activated neutrophils release proteases such as neutrophil elastase, cathepsin G and proteinase 3 which, if not appropriately controlled, can result in severe damage to healthy tissue. Uncontrolled proteolysis can lead to various diseases/disease syndromes including emphysema, idiopathic pulmonary fibrosis, respiratory distress syndrome, cystic fibrosis, rheumatoid arthritis and glomerulonephritis (35). Neutrophil elastase is the major protease responsible for extracellular proteolysis and it plays a pivotal role in the inflammatory response (36). By releasing neutrophil elastase in the presence of foreign material in blood, infiltrating neutrophils activate a signalling pathway which triggers macrophages to secrete cytokines as well as to attract more neutrophils (37).

The most potent, specific human neutrophil elastase inhibitor described to date is a protein engineered from the Kunitz domain of human inter a inhibitor (EPI-HNE-4), which was shown to have a Ki value of 5.45 x 10^"12 M (38). Excessive accumulation of neutrophil elastase in pulmonary fluids and tissues of patients with cystic fibrosis (CF) is thought to act on the lungs, compromising their structure and function, so that EPI-HNE-4 has been suggested as an anti-inflammatory compound for the treatment of CF (38). We show here that EgKI-1 is also a highly potent inhibitor (Ki = 6.42x10^"11 M) of neutrophil elastase and it thus warrants further investigation as a potentially effective therapeutic for treating acute and chronic inflammatory diseases. Cathepsin G has chymotrypsin-like catalytic activity and also has potent proinflammatory activity (36). Calcium mobilization is one of the earliest events that occurs with neutrophil activation and is a key factor for modulating numerous neutrophil biological responses (39). Once stimulated, the intracellular Ca⁺⁺ concentration within neutrophils rises rapidly due to mobilization of ions from intracellular pools and influxes from the extracellular medium (40). Being a secretory protein, EgKI-1, as well as playing a role in preventing proteolytic damage to oncospheres by neutrophil elastase and cathepsin G once they enter the blood circulation, it may also act to suppress further neutrophil activation by binding calcium ions (39) in the extracellular medium, thus making them unavailable to neutrophils.

Neutrophil chemotaxis plays an important role in the inflammatory response and, when excessive or persistent, may augment tissue damage. The fact that neutrophils have a short life span of around 6-8 hours after purification from whole blood is a limitation for performing assays with primary neutrophils (41). Resting neutrophils become primed and then mobilized to the site of infection which involves receptor activation and secretion of cytokines, chemokines and other components (35). Because of this, the molecular properties of primed neutrophils are very different to their resting state. The regulatory functions of macrophages are also shared by primed neutrophils. Hence, in vitro experiments with freshly isolated neutrophils can often fail to recognize their full functional activity (35). For a more complete understanding of the functional activities of EgKI-1, we undertook in vivo experiments using the carrageenan induced mouse air pouch model. Subcutaneous injections of air over several days cause morphological changes in the cellular lining of the pouch and resemble a synovial cavity (42). As an irritant, λ-carrageenan induces localized inflammation characterized by the infiltration of cells and a marked increase in the production of biochemical mediators. Thus, this model has been proven for use in pre-clinical studies of anti-inflammatory drugs (28). EgKI-1 significantly reduced neutrophil infiltration to the inflammatory site, most likely as a result of its inhibition of neutrophil elastase.

There is much current interest in developing novel potent drugs as treatments for inflammatory-related diseases to inhibit excessive and uncontrolled neutrophil serine protease activity (43,44). Clinical therapies that utilize protease inhibitors in controlling sepsis are currently restricted to the use of urinary trypsin inhibitor (UTI), mainly in Japan (45). UTI, also referred to as ulinastatin or bikunin is a multivalent Kunitz type serine protease inhibitor found in human urine and produces several anti-inflammatory effects (46). Proteases also play important roles beyond their involvement in inflammation. In different types of cancers, the secretion of various proteases correlates with the aggressiveness of the tumour. Kunitz type inhibitors have been shown to exhibit promising anti-cancer properties which may be used in their development as novel cancer therapies (47). Bikunin (48), TFPI-2 (49) and SPINT2 (50) are Kunitz proteins with anticancer effects, and as potent protease inhibitors, the EgKI proteins may also exhibit similar properties which can be exploited in cancer therapy.

In summary, this study further broadens knowledge of E. granulosus biology and emphasises the potential importance of the EgKI Kunitz proteins in protecting the worm by inhibiting or inactivating host digestive enzymes in the gut. As such they represent novel intervention targets for the control of cystic echinococcosis. Being a small molecule, a potent neutrophil elastase inhibitor and an inhibitor of neutrophil chemotaxis, EgKI-1 warrants further study as a potential therapeutic agent against inflammatory diseases (51). As well, the potential anti-cancer effects of the EgKIs and their possible interactions with cytokines should be investigated.

Table 1: Forward and reverse primers used for amplification of EgKI-1 and EgKI-2 by PCR and real-time PCR

Ncol and EcoRI restriction sites are underlined and 6><His tags are highlighted in grey

Table 2: IC₅₀ values for EgKI-1 and EgKI-2

NI - No inhibition

REFERENCES

1. McManus, D. P., Gray, D. J., Zhang, W., and Yang, Y. (2012) Diagnosis, treatment, and management of echinococcosis. BM J 344

2. Eckert, J., and Deplazes, P. (2004) Biological, Epidemiological, and Clinical Aspects of Echinococcosis, a Zoonosis of Increasing Concern. Clinical Microbiology Reviews 17,

107-135

3. Tsai, I. J., Zarowiecki, M., Holroyd, N., Garciarrubio, A., Sanchez-Flores, A., Brooks, K.

L., Tracey, A., Bobes, R. J., Fragoso, G., Sciutto, E., Aslett, M., Beasley, H., Bennett, H. M., Cai, J., Camicia, F., Clark, R., Cucher, M., De Silva, N., Day, T. A., Deplazes, P., Estrada, K., Fernandez, C, Holland, P. W. H., Hou, J., Hu, S., Huckvale, T., Hung, S. S.,

Kamenetzky, L., Keane, J. A., Kiss, F., Koziol, U., Lambert, O., Liu, K., Luo, X., Luo, Y., Macchiaroli, N., Nichol, S., Paps, J., Parkinson, J., Pouchkina-Stantcheva, N., Riddiford, N., Rosenzvit, M., Salinas, G., Wasmuth, J. D., Zamanian, M., Zheng, Y., Cai, X., Soberon, X., Olson, P. D., Laclette, J. P., Brehm, K., and Berriman, M. (2013) The genomes of four tapeworm species reveal adaptations to parasitism. Nature 496, 57-63

4. Zheng, H., Zhang, W., Zhang, L., Zhang, Z., Li, J., Lu, G., Zhu, Y., Wang, Y., Huang, Y., Liu, J., Kang, H., Chen, J., Wang, L., Chen, A., Yu, S., Gao, Z., Jin, L., Gu, W., Wang, Z., Zhao, L., Shi, B., Wen, H., Lin, R., Jones, M. K., Brejova, B., Vinar, T., Zhao, G., McManus, D. P., Chen, Z., Zhou, Y., and Wang, S. (2013) The genome of the hydatid tapeworm Echinococcus granulosus. Nature genetics 45, 1168-1175

5. Peigneur, S., Billen, B., Derua, R., Waelkens, E., Debaveye, S., Beress, L., and Tytgat, J.

(2011) A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties. Biochem Pharmacol 82, 81-90

6. Dy, C. Y., Buczek, P., Imperial, J. S., Bulaj, G., and Horvath, M. P. (2006) Structure of conkunitzin-Sl, a neurotoxin and Kunitz-fold disulfide variant from cone snail. Acta

Crystallogr D Biol Crystallogr 62, 980-990

7. Chen, Z. Y., Hu, Y. T., Yang, W. S., He, Y. W., Feng, J., Wang, B., Zhao, R. M., Ding, J.

P., Cao, Z. J., Li, W. X., and Wu, Y. L. (2012) Hgl, novel peptide inhibitor specific for Kvl .3 channels from first scorpion Kunitz-type potassium channel toxin family. / Biol Chem 287, 13813-13821

8. Wan, H., Lee, K. S., Kim, B. Y., Zou, F. M., Yoon, H. J., Je, Y. H., Li, J., and Jin, B. R.

(2013) A spider-derived Kunitz-type serine protease inhibitor that acts as a plasmin inhibitor and an elastase inhibitor. PLoS One 8, e53343

9. Chudzinski-Tavassi, A. M., De-Sa-Junior, P. L., Simons, S. M., Maria, D. A., de Souza Ventura, J., de Fatima Correia Batista, I., Faria, F., Duraes, E., Reis, E. M., and Demasi,

M. (2010) A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system. Toxicon 56, 1145-1154

10. Tsujimoto, H., Kotsyfakis, M., Francischetti, I. M., Eum, J. H., Strand, M. R., and Champagne, D. E. (2012) Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses. PLoS One 7, e29964

11. Kooyman, F. N., van Balkom, B. W., de Vries, E., and van Putten, J. P. (2009) Identification of a thrombospondin-like immunodominant and phosphorylcholine- containing glycoprotein (GP300) in Dictyocaulus viviparus and related nematodes. Mol Biochem Parasitol 163, 85-94

12. Hawdon, J. M., Datu, B., and Crowell, M. (2003) Molecular Cloning of a Novel Multidomain Kunitz-Type Proteinase Inhibitor From the Hookworm Ancylostoma caninum. Journal of Parasitology 89, 402-407

13. MacLean, J. A., Chakrabarty, A., Xie, S., Bixby, J. A., Roberts, R. M., and Green, J. A.

(2003) Family of Kunitz proteins from trophoblast: Expression of the trophoblast Kunitz domain proteins (TKDP) in cattle and sheep. Molecular Reproduction and Development 65, 30-40

14. Kunitz, M., and Northrop, J. H. (1936) Isolation from Beef Pancreas of Crystalline Trypsinogen, Trypsin, a Trypsin Inhibitor, and an Inhibitor-Trypsin Compound. / Gen Physiol 19, 991-1007

15. Ranasinghe, S., and McManus, D. P. (2013) Structure and function of invertebrate Kunitz serine protease inhibitors. Dev Comp Immunol 39, 219-227

16. Creighton, T. E. (1975) The two-disulphide intermediates and the folding pathway of reduced pancreatic trypsin inhibitor. Journal of molecular biology 95, 167-199

17. Laskowski, M., Jr., and Kato, I. (1980) Protein inhibitors of proteinases. Annual review of biochemistry 49, 593-626

18. Schechter, I., and Berger, A. (1967) On the size of the active site in proteases. I. Papain.

Biochem Biophys Res Commun 27, 157-162

19. Krowarsch, D., Dadlez, M., Buczek, O., Krokoszynska, I., Smalas, A. O., and Otlewski, J.

(1999) Interscaffolding additivity: binding of PI variants of bovine pancreatic trypsin inhibitor to four serine proteases. J Mol Biol 289, 175-186

0. Gonzalez, S., Flo, M., Margenat, M., Duran, R., Gonzalez-Sapienza, G., Grana, M., Parkinson, J., Maize Is, R. M., Salinas, G., Alvarez, B., and Fernandez, C. (2009) A family of diverse Kunitz inhibitors from Echinococcus granulosus potentially involved in host- parasite cross-talk. PLoS One 4, e7009

1. Petersen, T. N., Brunak, S., von Heijne, G., and Nielsen, H. (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature methods 8, 785-786 22. Sigrist, C. J., de Castro, E., Cerutti, L., Cuche, B. A., Hulo, N., Bridge, A., Bougueleret, L., and Xenarios, I. (2013) New and continuing developments at PROSITE. Nucleic Acids Res 41, D344-347

23. Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Soding, J., Thompson, J. D., and Higgins, D. G. (2011)

Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7

24. Kelley, L. A., and Sternberg, M. J. (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4, 363-371

25. Wass, M. N., Kelley, L. A., and Sternberg, M. J. (2010) 3DLigandSite: predicting ligand- binding sites using similar structures. Nucleic acids research 38, W469-473

26. Zhang, W., Zhang, Z., Shi, B., Li, J., You, H., Tulson, G., Dang, X., Song, Y., Yimiti, T., Wang, J., Jones, M. K., and McManus, D. P. (2006) Vaccination of Dogs against Echinococcus granulosus the Cause of Cystic Hydatid Disease in Humans. / Infect Dis 194, 966-974

27. Kurien, B. T., and Bachmann, M. (2009) Detection of calcium binding by Ro 60 multiple antigenic peptides on nitrocellulose membrane using Quin-2. Methods in molecular biology (Clifton, N.J.) 536, 483

28. Duarte, D. B., Vasko, M. R., and Fehrenbacher, J. C. (2012) Models of inflammation: carrageenan air pouch. Current protocols in pharmacology / editorial board, S.J. Enna

(editor-in-chief) ... [et al.] U6 Chapter 5, Unit5.6

29. Mika, A., Reynolds, S. L., Mohlin, F. C, Willis, C, Swe, P. M., Pickering, D. A., Halilovic, V., Wijeyewickrema, L. C, Pike, R. N., Blom, A. M., Kemp, D. J., and Fischer, K. (2012) Novel scabies mite serpins inhibit the three pathways of the human complement system. PLoS One 7, e40489

30. Morrison, J. F., and Walsh, C. T. (1988) The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 61, 201-301

31. Pan, W., Shen, Y., Han, X., Wang, Y., Liu, H., Jiang, Y., Zhang, Y., Wang, Y., Xu, Y., and Cao, J. (2014) Transcriptome profiles of the protoscoleces of Echinococcus granulosus reveal that excretory-secretory products are essential to metabolic adaptation.

PLoS neglected tropical diseases 8, e3392

32. Rogl, H., Kosemund, K., Kuhlbrandt, W., and Collinson, I. (1998) Refolding of Escherichia coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography. FEBS letters 432, 21-26

33. Grzesiak, A., Helland, R., Smalas, A. O., Krowarsch, D., Dadlez, M., and Otlewski, J.

(2000) Substitutions at the Ρ Γ position in BPTI strongly affect the association energy with serine proteinases. Journal of Molecular Biology 301, 205-217 34. Zhang, W., Wen, H., Li, J., Lin, R., and McManus, D. P. (2012) Immunology and Immunodiagnosis of Cystic Echinococcosis: An Update. Clinical and Developmental Immunology 2012, 10

35. Wright, H. L., Moots, R. J., Bucknall, R. C, and Edwards, S. W. (2010) Neutrophil function in inflammation and inflammatory diseases. Rheumatology (Oxford) 49, 1618-

1631

36. Owen, C. A., and Campbell, E. J. (1999) The cell biology of leukocyte-mediated proteolysis. Journal of leukocyte biology 65, 137

37. Soehnlein, O., Kenne, E., Rotzius, P., Eriksson, E. E., and Lindbom, L. (2008) Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages. Clinical

& Experimental Immunology 151, 139-145

38. Delacourt, C, Herigault, S., Delclaux, C, Poncin, A., Levame, M., Harf, A., Saudubray, F., and Lafuma, C. (2002) Protection against acute lung injury by intravenous or intratracheal pretreatment with EPI-HNE-4, a new potent neutrophil elastase inhibitor. Am / Respir Cell Mol Biol 26, 290-297

39. Pettit, E. J., Davies, E. V., and Hallett, M. B. (1997) The microanatomy of calcium stores in human neutrophils: relationship of structure to function. Histol Histopathol 12, 479-490

40. Malinin, V. S., Kazarinov, K. D., and Putvinsky, A. V. (1999) Calcium Dependent Activation of Human Blood Neutrophils by Electric Field Pulses, in Electricity and Magnetism in Biology and Medicine (Bersani, F. ed.), Springer US. pp 569-572

41. Nuzzi, P. A., Lokuta, M. A., and Huttenlocher, A. (2007) Analysis of neutrophil chemotaxis. Methods in molecular biology ( Clifton, N.J.) 370, 23

42. Edwards, J. C, Sedgwick, A. D., and Willoughby, D. A. (1981) The formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system. The Journal of pathology 134, 147-156

43. Korkmaz, B., Horwitz, M. S., Jenne, D. E., and Gauthier, F. (2010) Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacological reviews 62, 726-759

44. Lucas, S. D., Costa, E., Guedes, R. C, and Moreira, R. (2013) Targeting COPD: advances on low- molecular-weight inhibitors of human neutrophil elastase. Medicinal Research

Reviews 33, E73-E101

45. Inoue, K.-i., and Takano, H. (2010) Urinary trypsin inhibitor as a therapeutic option for endotoxin-related inflammatory disorders. Expert Opinion on Investigational Drugs 19, 513-513

46. Lee, S.-H., Kim, H.-J., Han, H.-J., Li, M., Kwak, S.-H., and Park, S. (2012) Urinary trypsin inhibitor attenuates lipopolysaccharide-induced neutrophil activation. Korean journal of anesthesiology 63, 540 Kafarski, K. G. a. P. (2011) Inhibitors of Proteinases as Potential Anti-Cancer Agents, in Drug Development - A Case Study Based Insight into Modern Strategies (Rundfeldt, D. C. ed.

Kobayashi, H., Suzuki, M., Tanaka, Y., Kanayama, N., and Terao, T. (2003) A Kunitz- type Protease Inhibitor, Bikunin, Inhibits Ovarian Cancer Cell Invasion by Blocking the Calcium-dependent Transforming Growth Factor-βΐ Signaling Cascade. Journal of Biological Chemistry 278, 7790-7799

Sierko, E., Wojtukiewicz, M. Z., and Kisiel, W. (2007) The role of tissue factor pathway inhibitor-2 in cancer biology. Semin Thromb Hemost 33, 653-659

Kongkham, P. N., Northcott, P. A., Ra, Y. S., Nakahara, Y., Mainprize, T. G., Croul, S. E., Smith, C. A., Taylor, M. D., and Rutka, J. T. (2008) An epigenetic genome-wide screen identifies SPINT2 as a novel tumor suppressor gene in pediatric medulloblastoma. Cancer Res 68, 9945-9953

Groutas, W. C, Dou, D., and Alliston, K. R. (2011) Neutrophil elastase inhibitors. Expert Opin Ther Pat 21, 339-354

Chand, H. S., Schmidt, A. E., Bajaj, S. P., and Kisiel, W. (2004) Structure-Function Analysis of the Reactive Site in the First Kunitz-type Domain of Human Tissue Factor Pathway Inhibitor-2. Journal of Biological Chemistry 279, 17500-17507

Dereeper, A., Lescot, M., Claverie, J. M., Gascuel, O., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J. F., Guindon, S., and Lefort, V. (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research 36, W465- W469

EXAMPLE 2

Recent studies have shown that neutrophils in the tumor micro environment act in the detriment of the host by playing critical roles in tumorigenesis [3]. Breast and most of the lung cancer cell lines tend to produce neutrophil elastase on their own to facilitate invasion into adjacent host tissues [4, 5]. Therefore low molecular weight potent neutrophil elastase inhibitors hold great interest as potential anti-cancer therapeutics.

Materials and Methods

SRB Assay. Cells (5000/well) from cancer cell lines NFF, MCF-7, T47D, SCC15, FaDu, MDA-MB231 and CJM were plated and treated with different concentrations of EgKI-1/ buffer control after 24 hours. After confluence of control well becomes 100%, usually in 2 days, Sulforhodamine B (SRB) colorimetric assay was carried out to determine the cell density in each well. An IC₅₀ value of EgKI-1 for each cancer cell line was calculated using GraphPad Prism software.

Scratch wound assay to analyse effect of EgKI-1 on cancer cell migration. MDA-

MB-231 cells (50,000/ well) were plated and treated with 3750 nM EgKI-1/ buffer after 24 hours incubation. The plate was then put in the incucyte to take images every 3 hours. After 24 hours scratch wound was made using a scratch wound maker. Plate was washed and topped with similar amount of EgKI-1/ buffer and continued to be imaged in the incucyte. A graph was generated by analysing the scratch wound area (mm²) in both treated and control wells over time using IncucyteZOOM software.

Cytokine expression analysis in human peripheral blood mononuclear cells (PBMC). PBMC were cultured in the presence of different concentrations of EgKI-1, buffer control and LPS (bacterial endotoxin) and PMA as positive controls. Culture supernatants were then analysed with flow cytometry to determine cytokine expression.

Results

Recombinant EgKI-1 inhibits the proliferation of a range of human cancer cells in a dose dependent manner (Table 3 and Figure 12); Oestrogen dependent breast cancer lines (MCF-7 and T47D), triple negative breast cancer (MDA-MB-231), head and neck cancer lines (FaDu, SCC15 and CAL27), melanoma (CJM) and cervical adenocarcinoma (HeLa) cell lines. However, EgKI-1 does not affect the growth of normal cells as shown using Neonatal Foreskin Fibroblast (NFF) cell culture assay. Compared to Sivelestat which is also a neutrophil elastase inhibitor and already marketed as an anti-inflammatory drug, EgKI-1 demonstrated significantly lower IC₅₀ values.

Table 1: IC₅₀ values of EgKI-1 against different cancer cell lines

In referring to the results for the scratch wound assay, EgKI-1 (3 μg in 5 μΐ/ well) effectively inhibited the migration of MDA-MB-231 cancer cells across the scratch wound compared to the control well containing a similar volume of buffer (Tris 20 mM NaCl 150 mM) (Figure 13 and 14).

For the cytokine expression studies, EgKI-1 increased the expression of a range of cytokines which are related to inflammation and cancer from PBMC (Figure 15):

MIP (macrophage inflammatory protein) - Release by macrophages and crucial for immune responses towards infection and inflammation

T F (tumor necrosis factor) - Monocyte-derived cytotoxin which affects in tumor regression

IL-6 - stimulate immune response

IL-8 - Pro-inflammatory cytokine and promotes angeogenic responses in endothelial cells IL-1 - Mainly released by macrophages and induces production of IL-2. IL-2 is produced by T cells and responds to antigenic or mitogenic stimulation

IL-10 - Anti-inflammatory cytokine

Conclusions:

- EgKI-1 inhibit the growth of multiple different cancer cell lines possibly by inhibiting specific target molecule/s highly express in cancer cells.

- Cancer cell culture assays are free of neutrophils or neutrophil elastase indicating one or more other anti-cancer mechanisms of EgKI-1.

Cancer cell growth inhibition by EgKI-1 without affecting the normal cell growth is a promising step towards therapeutic development. Cancer cell migration towards the blood stream is the first step in metastasis [6]. Therefore, EgKI-1 may also inhibit tumour metastasis.

In 24 hours post treatment cancer cells are already dying as can be seen in Figure 14 (i.e., less cell density compared to the control).

After making the scratch wound, cancer cells start to migrate over time into the control well but not into the treated well.

EgKI-1 modulates the expression of a number of cytokines having roles in inflammation and cancer

REFERENCES Ranasinghe, S.L., et al, Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus. PLoS Negl Trop Dis, 2015. 9(12): e0004268.

Ranasinghe, S. and D.P. McManus, Structure and function of invertebrate Kunitz serine protease inhibitors. Dev Comp Immunol, 2013. 39(3): 219-227.

Gregory, A.D. and A.M. Houghton, Tumor-associated neutrophils: new targets or cancer therapy. Cancer Res, 2011. 71(7): 2411-6.

Sato, T., et al, Neutrophil elastase and cancer. Surg Oncol, 2006. 15(4): 217-22. Yamashita, J., et al, Local increase in polymorphonuclear leukocyte elastase is associated with tumor invasiveness in non-small cell lung cancer. Chest, 1996. 109(5): 1328-34.

Clark, A. G., and Vignjevic, D. M. (2015) Modes of cancer cell invasion and the role of the microenvironment. Curt Opin Cell Biol 36, 13-22.

Claims

1. An isolated EgKI-1 protein comprising, consisting or consisting essentially of an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment, variant or derivative thereof.

2. The isolated EgKI-1 protein of Claim 1, wherein said variant is a mutant EgKI-1 protein which has one or a plurality of amino acids normally present in a full-length or wild-type EgKI-1 protein mutated or modified.

3. The isolated EgKI-1 protein of Claim 1 or Claim 2, wherein the variant EgKI-1 protein comprises an amino acid sequence at least 70% identical to that set forth in SEQ ID NO: l or SEQ ID NO:2.

4. The isolated EgKI-1 protein of any one of the preceding claims, wherein the isolated EgKI-1 protein is capable of preventing and/or treating inflammation and/or cancer upon administration to a subject.

5. A method for preventing and/or treating inflammation in a subject, the method including the step of administering to the subject a therapeutically effective amount of the isolated EgKI-1 protein of any one of Claims 1 to 4, to thereby prevent and/or treat the inflammation.

6. The method of Claim 5, further including the step of administering to the subject an additional agent.

7. The method of Claim 6, wherein the additional agent is selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), an aminosalicylate, a corticosteroid, an immunosuppressant, an anti-cytokine/cytokine receptor agent, an antibiotic, and combinations thereof.

8. The method of any one of Claims 5 to 7, wherein the inflammation is associated with, or secondary to, an inflammatory disease disorder or condition in the subject

9. The method of Claim 8, wherein the inflammatory disease, disorder or condition is an inflammatory disease, disorder or condition of the respiratory system.

10. The method of Claim 9, wherein the inflammatory disease, disorder or condition is selected from the group consisting of asthma, emphysema, chronic bronchitis, and chronic obstructive pulmonary disease (COPD).

11. A method for preventing and/or treating cancer in a subject, the method including the step of administering to the subject a therapeutically effective amount of the isolated EgKI-1 protein of any one of Claims 1 to 4, to thereby prevent and/or treat the cancer.

12. The method of Claim 11, wherein administration of the isolated EgKI-1 protein prevents and/or inhibits metastasis of said cancer.

13. The method of Claim 11 or Claim 12, further including the step of administering to the subject an additional agent.

14. The method of Claim 13, wherein the additional agent is or comprises one or a plurality of anti-cancer treatments selected from the group consisting of an endocrine therapy, a chemotherapy, an immunotherapy, a molecularly targeted therapy and combinations thereof.

15. The method of any one of Claims 11 to 14, wherein the cancer is selected from the group consisting of breast cancer, head and neck cancer, melanoma and cervical cancer.

16. The method of any one of Claims 11 to 15, wherein one or a plurality of cells of the cancer express or otherwise contain or comprise a neutrophil elastase.

17. An isolated nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the EgKI-1 protein of any one of Claims 1 to 4.

18. A fragment, variant or derivative of the isolated nucleic acid of Claim 17.

19. A genetic construct comprising: (i) the isolated nucleic acid of Claim 17; or (ii) the fragment or variant of Claim 18; (iii) a nucleotide sequence complementary thereto; operably linked or connected to one or more regulatory sequences in an expression vector.

20. A host cell transformed with an isolated nucleic acid molecule according to Claim 17 or 18 or the genetic construct of Claim 19.

21. A method of producing the EgKI-1 protein of any one of Claims 1 to 4, comprising; (i) culturing the previously transformed host cell of Claim 20; and (ii) isolating said protein from said host cell cultured in step (i).

22. An antibody or antibody fragment which binds and/or is raised against the isolated EgKI-1 protein, fragment, variant or derivative according to any one of Claims 1 to 4..

23. A composition comprising:

(i) the isolated EgKI-1 protein of any one of Claims 1 to 4; and/or

(ii) the antibody or antibody fragment of Claim 22;

and one or more pharmaceutically acceptable carriers, diluents or excipients.

24. The composition of Claim 23, further comprising at least one additional agent.

25. The composition of Claim 24, wherein the at least one additional agent is selected from the group consisting of nonsteroidal anti-inflammatory drugs (NSAIDs), aminosalicylates, corticosteroids, immunosuppressants, anti-cytokine/cytokine receptor agents, antibiotics, and combinations thereof.

26. The composition of Claim 24, wherein the at least one additional agent is or comprises one or a plurality of anti-cancer treatments selected from the group consisting of an endocrine therapy, chemotherapy, immunotherapy, a molecularly targeted therapy and combinations thereof.

27. A method of preventing and/or treating a parasitic infection in a subject, the method including the step of administering to the subject a therapeutically effective amount of: (i) the EgKI-1 protein of any one of Claims 1 to 4;

(ii) the antibody or antibody fragment of Claim 22; and/or

(iii) the composition of any one of Claims 23 to 26;

to thereby prevent and/or treat the parasitic infection.

28. The method of Claim 27, wherein the parasitic infection is caused by and/or associated with, at least in part, Echinococcus granulosus.