Interleukin 18 variants

Abstract

There is provided an interleukin-18 (IL-18) variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1, and wherein in comparison to SEQ ID NO: 1, the IL-18 variant comprises an amino acid modification at the following amino acid positions: i) 48; and ii) 51 and / or 60.

Description

[0001] INTERLEUKIN 18 VARIANTS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to interleukin-18 (IL-18) molecules and variants thereof.

[0004] BACKGROUND

[0005] Microbial immunotherapy has been shown to be effective as post-surgery treatment to reduce the incidence of tumour recurrence, with BCG treatment being the current standard of care for certain cancer types, in particular for NMIBC patients (Alhunaidi, 2019). BCG therapy has limitations including the efficacy range (40-60% of patients will experience tumour recurrence within 5 years of surgery), adverse effects and safety concerns, complex manufacturing requirements, and a long treatment regimen (patients undergo 27 intra-vesical, transurethral instillations over 36 months). To improve efficacy, microbial immunotherapy together with co-immunostimulatory molecules has been proposed.

[0006] The evidence supporting the use of immuno-stimulatory molecules such as cytokines in the treatment of cancer has grown significantly in recent years. Interleukin 18 (IL-18) molecules are considered as promising immunotherapeutics because of their role in CDS T cells and NK activation and proliferation (Atallah- Yunes, 2022). However, one challenge associated with the use of IL-18 as an immuno-stimulatory anti-cancer treatment is the upregulation of a high-affinity decoy receptor, IL-18 binding protein (IL-18BP) in cancers which blocks downstream immuno-stimulatory signalling pathways.

[0007] Another challenge associated with IL-18, especially in the context of microbial living cures, is its expression in bacteria. There are many examples in the prior art of cytokines expressed and purified from E. coli, but a common problem with the use of bacterial systems to express eukaryotic cytokines is the formation of inclusion bodies. Bacterial expression of cytokines generally results in the formation of non-functional inclusion bodies. The depositing of cytokines in bacteria therefore imposes additional and laborious requirements of solubilising and refolding the proteins from inclusion bodies, which often leads to low yields of protein which is unsuitable for living therapeutics (Ferrer-Miralles, 2009; de Marco, 2009). A high rate of protein expression or unfavourable reducing conditions in the cytoplasm may impair the folding of nascent peptide resulting in formation of insoluble protein aggregates. Namely, the highly reducing environment of the bacterial cytoplasm hinders disulphide linkages forming between cysteine residues during protein folding - this can be problematic for the expression of cytokines (including IL-18) where there are disulphide bonds in the protein structure.

[0008] Attempts to improve cytokine expression levels and solubility in bacteria such as E. coli have included lowering the cultivation temperature, co-expression of chaperones, in vitro re-solubilisation and refolding protocols, use of weak promoters, fusion to solubility enhancer tags, adding signal peptides for periplasmic localisation or N-terminal fusion to carrier for extracellular export (Devi 2016, Nausch 2013).

[0009] The same limitations for cytokine expression efficiency observed in E. coli have also been observed in strains of Salmonella. A further challenge presented by protein expression in Gram-negative strains of bacteria such as E. coli and Salmonella is that, even following the successful expression of cytokines, the proteins must be correctly folded and then transported across two cellular membranes, in between which is the bacterial periplasm, in order to be secreted from the cell.

[0010] As such, it can be seen that there is a need to develop cytokines which can be optimally expressed in, and efficiently secreted by bacteria, which are also able to bind their receptor to allow activation of immuno-stimulatory signalling. SUMMARY OF INVENTION

[0011] The present invention provides IL-18 variants with optimised solubility, preserved bioactivity, and IL-18BP decoy-resistance. The inventors of the present invention have surprisingly identified certain combinations of mutations in IL-18 that result in IL-18 variants which are soluble, functional, and preferentially bind the IL-18 receptor (IL-18R) over the IL-18 binding protein (IL-18BP). The variants provided herein are therefore ideal candidates for expression in, and secretion from, efficient expression systems such as bacteria, for example Gram-negative bacteria and / or Gram-positive bacteria.

[0012] Accordingly, in a first aspect of the invention, there is provided an interleukin-18 (IL-18) variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at the following amino acid positions: i) 48; and ii) 51 and / or 60.

[0013] In a second aspect of the invention, there is provided an IL-18 variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at amino acid positions 51 and 60.

[0014] In a third aspect of the invention, there is provided a nucleic acid molecule encoding the IL-18 variant according to the first and / or second aspects of the invention.

[0015] In a fourth aspect of the invention, there is provided a bacterium comprising the IL-18 variant according to the first and / or second aspects of the invention, or the nucleic acid molecule according to the third aspect of the invention.

[0016] In a fifth aspect of the invention, there is provided an IL-18 variant according to the first and / or second aspects of the invention, or the nucleic acid molecule according to the third aspect of the invention, or the bacterium according to the third aspect of the invention, for use as a therapy.

[0017] In a sixth aspect of the invention, there is provided a use of the IL-18 variant according to the first and / or second aspects of the invention, or the nucleic acid molecule according to the third aspect of the invention, or the bacterium according to the fourth aspect of the invention, for use in the manufacture of a medicament for a therapy.

[0018] In a seventh aspect of the invention, there is provided a method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject the IL-18 variant according to the first and / or second aspects of the invention, or the nucleic acid molecule according to the third aspect of the invention, or the bacterium according to the fourth aspect of the invention.

[0019] BRIEF DESCRIPTION OF DRAWINGS

[0020] The invention is described with reference to the accompanying drawings, wherein:

[0021] Figure 1 shows the crystal structure of interleukin-18 (IL-18, PDB ID: 3WO3), indicating amino acid modifications in a hydrophobic pocket adjacent to a receptor interaction surface. Also shown are IL-18Ra, I L-18Rp, and IL-18BP.

[0022] Figure 2 shows the crystal structure of IL-18 (PDB ID:3WO3), indicating amino acid positions 9, 46, 48, 51 , 60, 80, 110 and 111. Also shown are IL-18Ra, IL- 18Rp, and IL-18BP (PDB ID: 7AL7), which was superimposed on the 3WO3 structure using PyMol.

[0023] Figure 3 shows the crystal structure of IL-18 bound to IL-18Ra and IL-18Rp (3WO3), highlighting the interacting residues of IL-18 for each receptor as described in Novick, 1999 and Yuan, 2014. Figure 4 shows the AggreScan3D aggregation score per amino acid of an IL-18 sequence based on the indicated crystal structures.

[0024] Figure 5 shows the three-dimensional hydrophobicity protein surface of IL-18 in complex with IL-18 receptor (IL-18R) and IL-18 binding protein (IL-18BP) with residues L09, I46, I48, and I80 indicated. L09, I46, I48 cluster in an exposed hydrophobic region.

[0025] Figures 6A to 6C demonstrate the relative solubility (where 1 is the reference IL- 18 amino acid sequence) and activity (normalised to be between 0 (sfGFP) and 1 (reference IL-18 amino acid sequence)) of IL-18 variants and wild-type IL-18.

[0026] Figure 7 shows the effect each mutation (at amino acid positions 9, 46, 48 and 80, labelled A to D following their position in the sequence) has on the solubility of the IL-18 variants, with sign arbitrarily positive for amino acid changes towards a positive charge and negative for amino acid changes towards a negative charge. The substitution of AA48 for a negatively charged amino acid has the largest effect (factor C).

[0027] Figure 8 shows the effect of each mutation (labelled A to D following their position in the sequence), with sign arbitrarily positive for amino acid changes towards a positive charge and negative for amino acid changes towards a negative charge, on the protein signalling activity in in vitro cell based assays. Replacement of AA9 for a positive amino acid could result in higher activity (factor A).

[0028] Figures 9A to 9C show the design and solubility of decoy resistant IL-18 variants which show increasing binding independence from IL-18BP. (A) Crystal structure of IL-18 (PDB ID: 3WO3) highlighting amino acids selected for screening decoyresistance (M51 , M60, S105, D110, N111) and those selected for solubility improvement (L09, I46, I48, I80). (B) Experimental strategy to identify decoy- resistant IL-18 variants soluble in bacteria. Briefly, variants were generated via combinatorial assembly of three fragments that contained different mutations (see 9A), creating a total of 128 variants fused in-frame with a tri-partite beta-lactamase reporter protein (pBRED). These were transformed in bacteria (E. coli DHWbeta) and assayed for solubility on 1 mg mL-1of carbenici Hi n , which results in death of reporter bacteria if variant is not soluble and growth if it is soluble. (C) Solubility score of different potential decoy-resistant IL-18 variants clustered by the fragments used. Missing gaps are not assayed variants. Note that the most soluble group is that with F2D-H, which corresponds to those containing mutation M2 (I48D, bottom models - I48 did not hinder protein activity).

[0029] Figures 10A and 10B show the experimental set up and screening to determine the decoy-resistance of IL-18. (A) Briefly, candidate variants were transferred to a CFPE plasmid, where the gene of interest is expressed from a T7 promoter and HiBit tagged (C-terminus). These plasmids were used as reagent in cell-free reactions to produce the HiBit-tagged protein product, which was incubated with IL-18BP variant that had previously been attached to a Ni-NTA resin at the bottom of a 96-well plate. Samples were then washed to remove unbound protein and subjected to HiBit Assay. Samples showing no luminescence were deemed decoy-resistance and were subsequently produced as HiBit-tag proteins. These were bound to the bottom of 96-well plates (HisSorb® NiNTA) and used to stimulate HEK-Blue™ cells. (B) (Left) Positive (IL18-WT) and negative (IL18-DR) controls confirmed that HiBit signal is only detected in proteins bound to IL-18BP. (Right, Top) HiBit signal measurements of each variant after binding to IL-18BP. (Right, Bottom) HiBit signal measurements of each variant after protein production and before binding to IL-18BP.

[0030] Figures 11A and 11B show the results of two independent activity assays of decoy resistant IL-18 variants.

[0031] Figures 12A to 12E show the results of protein purification of IL-18 variants. (A) SDS-PAGE protein gel stained with Coomassie after running purified protein extracts of a pilot experiment performed to identify optimal expression conditions, where temperature (indicated below) and IPTG concentrations (indicated above in mM) were varied. Protein IL-18V59 was expressed from a bespoke expression plasmid under T7 promoter control. (B) SDS-PAGE showing the final purified protein variants expressed using optimal conditions of 30 °C and 0.02 mM IPTG. (C) Absolute concentration of each variant determined by HPLC-MS after protein purification. (D) Solubility scores obtained after antibiotic challenge assay (E) Correlation between solubility score and absolute concentration from purified protein.

[0032] Figures 13A and 13B show titration curves for comparison of activity of different IL-18 variants versus wild-type IL-18, and IL-18BP titration.

[0033] DETAILED DESCRIPTION

[0034] The present invention provides optimised IL-18 variants which are both soluble and functional and are therefore optimal candidates for efficient secretion systems which allow the production, correct folding and secretion of said IL-18 variants. The IL-18 variants described herein are also found to be decoy-resistant in that they will preferentially bind IL-18 receptor (IL-18R) over they decoy receptor, IL- 18 binding protein (IL-18BP), i.e., the variants described herein are resistant to IL- 18BP and have independent binding to IL-18R from IL-18BP. In comparison to the prior art, the inventors of the present invention have surprisingly found that amino acid modifications to the amino acids at positions 51 and 60 of IL-18 are all that is required to achieve decoy resistance in the IL-18 variant. Further, the inventors of the present invention have surprisingly found the amino acid modification at amino acid position 48 allows for IL-18 variants comprising this modification to be solubilised (i.e., stabilised) while retaining similar biological activity levels to that of wild-type IL-18.

[0035] Accordingly, in a first aspect the invention provides an interleukin-18 (IL-18) variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at the following amino acid positions: i) 48; and ii) 51 and / or 60 (Figure 1).

[0036] As used herein, the term “interleukin” (IL) refers to any member of the glycoprotein family which is expressed by leukocytes (white blood cells) and is involved in regulating immune responses. Interleukins may also be referred to as cytokines. Interleukins have diverse roles in the generation of immune cells, including immune cell activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. Interleukins can have pro- and antiinflammatory properties. Interleukins make up a large group of proteins which have the capacity to elicit several reactions in cells and tissues by binding to receptors known as interleukin receptors in order to trigger downstream signalling from the receptor.

[0037] Interleukin-18 (IL-18) is a pro-inflammatory cytokine from the IL-1 family that holds a pivotal role in innate and adaptive immunity by influencing diverse immune cells including T-cells, macrophages, and dendritic cells (Dinarello, 2001). The influence of IL-18 emerges through interactions with IL-18 receptors (IL-18Ra and IL-18Rp), which initiate immune cascades (Dinarello, 2001 ; Goyal, 2022). The binding specificity of IL-18 to IL-18Ra depends on critical amino acids, including E42 and K89, with two distinct binding sites (Site I and Site II), while I L-18Rp has a lesser interaction with a third Site III (Novick, 1999; Yuan, 2014). This activation is balanced by IL-18 binding protein (IL-18BP), which restrains IL-18's pro- inflammatory actions through higher affinity (Kd400 pM) than IL-18 receptors (Kd69 nM) and abundant serum presence (Kim, 2000; Kim, 2001 ; Novick, 2001). Additionally, IL-18BP's up to four isoforms refine its regulatory role (Cheung, 2005). This intricate interplay underscores immune modulation complexity with therapeutic potential for inflammatory disorders, autoimmune disorders, and cancer (Dinarello, 2001).

[0038] The potential of IL-18 in immunological cancer treatment has been reported. Feng et al. (Feng et al, 2020) observed that upregulated expression of IL-18 in colon cancer patients was correlated with favourable prognosis, whereas decreased expression was correlated with increased tumour size. Exogenous IL-18 supplementation synergized with PD-1 -dependent immunosuppression (Terme, 2011) and other cytokines (Terme, 2011 ; Schaller, 2002) to enhance anti-tumour responses, or when delivered through microbial carriers (Fensterle, 2008). Conversely, some tumours overexpress IL-18BP (Zhang, 2020; Detry 2022), inactivating IL-18 treatment. This has prompted the evolution of a new class of engineered "Decoy-Resistant IL-18" (DR-18), which preferentially bind IL-18Ra over IL-18BP (Zhou et al, 2020; WO 2019 / 051015A1). Notably, elevated IL-18 levels correlate with poor prognosis in certain cases, such as advanced gastric cancer (Yao et al, 2020; Ye et al, 2007).

[0039] In the context of microbe-induced cancer treatments, expressing IL-18 in bacterial chassis has yielded partial activity (Fensterle, 2008), but advances like DR-18 are more promising. Challenges remain, including the optimisation of protein expression in bacteria of both IL-18 and DR-18.

[0040] There is a clear need to easily develop IL-18 variants which are soluble, biologically active and IL-18BP resistant. As such, the inventors of the present invention have generated IL-18 variants which can be delivered to a disease site, allowing sustained activity and reducing issues derived from systemic toxicities and short half-life. As used herein, the term “wild-type” (“WT”) is to be given its usual meaning in the art, referring to a protein or gene encoding a protein found in its natural, non-mutated, unmodified, or unchanged form. In one embodiment, the wild-type IL-18 is mature (i.e., unmodified) IL-18. The wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1. In comparison to “wild-type” IL-18, the term “interleukin variant” refers to an IL-18 molecule which, in comparison to wild-type IL-18, comprises one or more amino acid modifications. The wild-type IL-18 may also be referred to as parental IL-18, as the IL-18 variants of the present invention are derived from this baseline sequence.

[0041] As used herein, the term “amino acid modifications” refers to any change or mutation to a wild-type amino acid sequence. An amino acid modification may or may not alter the structure, function, and / or physiochemical properties of the protein. Amino acid modifications may include, but are not limited to deletions, substitutions, and insertions. Deletion mutations involve the loss of an amino acid, resulting in a frameshift or decrease in the length of the amino acid sequence. Insertion mutations involve the addition of an amino acid, resulting in a frameshift or increase in the length of the amino acid sequence. Substitution mutations involve the replacement of one amino acid for another. Substitution mutations can be conservative or non-conservative. As would be understood by the skilled person, amino acids can be grouped broadly according to their properties (i.e. , size, bulkiness, charge, hydrophobicity, polarity, etc). Similarity or dissimilarity between amino acids can also be calculated using substitution matrices and physicochemical distances. A conservative amino acid substitution involves the replacement or exchange of one amino acid for another amino acid with similar properties. For example, a substitution of a hydrophobic amino acid for another hydrophobic amino acid (e.g., a valine-to- alanine substitution) would constitute a conservative amino acid substitution. A non-conservative or radical amino acid substitution involves the replacement or exchange of one amino acid for another amino acid with different or dissimilar properties. For example, a substitution of a hydrophobic amino acid for a hydrophilic amino acid (e.g., a leucine-to-serine substitution) would constitute a non-conservative amino acid substitution. Non-conservative amino acid substitutions may be more likely to be associated with changes in protein structure and function compared to conservative amino acid substitutions.

[0042] Several methods are well known in the art for inducing amino acid modifications. As will be readily understood by the skilled person, a nucleic acid sequence encoding the modified or mutated amino acid sequence of interest may be generated by several means (i.e., de novo synthesis) which are known in the art. Such a gene may be introduced into an expression vector or expression system which supports expression of the gene and translation of the messenger RNA (mRNA). The gene introduced into the expression system may be a heterologous gene (i.e., an exogenous gene), meaning that the gene is derived from a cell type originating from a different organism to the recipient expression system. Methods for heterologous expression of recombinant proteins will be well known to those skilled in the art. The expression system of the present invention is a bacterial expression system, preferably a bacterial cell, such as a Salmonella strain.

[0043] The inventors of the present invention have surprisingly identified a number of amino acid modifications which can increase the solubility of IL-18 while retaining its biological activity. It has also been surprisingly found by the inventors of the present invention that increasing the number of certain amino acid modifications in the IL-18 sequence results in higher independence from IL-18BP in that the variants described herein are resistant to the decoy receptor IL-18BP. However, only a certain number of amino acid modifications are required to achieve decoy resistance. In particular, in comparison to the prior art, the inventors of the present invention have surprisingly found that amino acid modifications to the amino acids at positions 51 and 60 of IL-18 are all that is required to achieve decoy resistance, and adding an amino acid modification to amino acid position 111 is preferable (i.e., modifications at positions 51 , 60 and 111).

[0044] As used herein, the terms “decoy-resistance”, “IL-18BP resistance”, “IL-18BP independence” and “binding independence” may be used interchangeably and refer to an IL-18 variant which has reduced or no binding to IL-18BP (i.e., a decoy- resistant IL-18 variant). IL-18BP binds to IL-18 and prevents the binding of IL-18 to the receptor, IL-18R, thereby inhibiting immune signalling pathways downstream of IL-18R. Elevation of IL-18BP levels has been identified in several different diseases, including cancer. The inventors of the present invention have surprisingly identified certain amino acid modifications and combinations thereof which prevent, block, or reduce the binding of IL-18BP to the IL-18 variant. As such, the IL-18 variants of the present invention are ‘independent’ from or ‘resistant’ to IL-18BP, and thus preferentially bind IL-18R over IL-18BP. The preference for IL-18R and resistance to IL-18BP can allow for the activation of immune signalling pathways and immune responses downstream of IL-18R. For example, by inhibiting the binding of IL-18BP, the decoy-resistant IL-18 variants are free to bind to IL-18R and thus activate, for example IL-18-induced INF- gamma production, resulting in increased T-helper type 1 immune responses.

[0045] IL-18 variants according to the invention may comprise one or more, two or more, three or more, four or more, five or more, or more than five amino acid modifications. However, the IL-18 variant will preferably comprise two, three, four, or five amino acid modifications.

[0046] In accordance with a first aspect of the invention, there is provided an interleukin- 18 (IL-18) variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at the following amino acid positions: i) 48; and ii) 51 and / or 60.

[0047] In the context of the present invention, the region adjacent to a receptor interaction surface of the IL-18 variant may be an exposed hydrophobic pocket and / or a surface region.

[0048] Accordingly, in one embodiment, the region adjacent to a receptor interaction surface may be an exposed hydrophobic pocket of the IL-18 variant. As used herein, the terms “hydrophobic pocket”, “hydrophobic cavity”, and “hydrophobic binding site” are used interchangeably and refer to an exposed three-dimensional region or cavity within the IL-18 variant structure which primarily comprises hydrophobic amino acid residues. The hydrophobic pocket is adjacent to a receptor interaction surface of the IL-18 variant, and is not in direct contact with the IL-18 receptor or the IL-18 binding protein. As such, the amino acids present in this pocket which are to be modified are not predicted to interact with the IL- 18R (see Figures 1-3). Figure 1 shows the crystal structure of IL-18 in complex with IL-18Ra and I L-18Rp, and the positioning of amino acids to be modified within the pocket. The preferred amino acids to be modified within the hydrophobic pocket are at amino acid positions 9, 46 and 48, most preferably 48. The hydrophobic amino acids have a low affinity for water and cluster in said pocket to form a hydrophobic environment which excludes water or the surrounding aqueous environment. Hydrophobic pockets are often a localised region in the protein structure which can accommodate other hydrophobic molecules including other hydrophobic amino acids (i.e. , from the same or another protein), lipids or lipid moieties, or other organic molecules. In some instances, the hydrophobic pocket may act as a docking site or interaction site for ligands.

[0049] In other embodiments, the region adjacent to a receptor interaction surface may be a surface region. As used herein, the term “surface region” refers to the part of a protein which is exposed to solvent. In the overall three-dimensional structure of the protein, the protein surface is commonly referred to as the solvent- accessible surface of the protein. For a soluble protein, the protein surface will be predominantly composed of hydrophilic amino acid residues, whereas the core (i.e. , the interior) of the protein will be composed of hydrophobic residues which are packed together away from the solvent. Modifications to amino acids which are on or near the surface of the protein may alter the solubility of a protein. The preferred amino acid to be modified at the surface region of the IL-18 variant are the amino acids at amino acid positions 51 , 60, 80, 105, 110, and / or 111 of the IL- 18 variant.

[0050] Figure 2 shows the crystal structure of IL-18 in complex with IL-18Ra and I L-18Rp (Figure 2A and B) and IL-18BP (Figure 2C) (PyMol). In Figure 2A, the selected amino acids to be modified within the hydrophobic pocket (amino acid positions 9, 46, and 48) are shown, labelled with the amino acid code and their position relative to the start of the mature polypeptide chain. The hydrophobic surface of IL-18 is overlayed with the protein cartoon representation. In Figure 2B, the crystal structure of I L-18 in complex with I L-18Ra and I L-18Rp has that of I L-18 and I L- 18BP superimposed. The selected amino acids to be modified for solubility are labelled with the amino acid code and their position relative to the start of the mature polypeptide chain (amino acid positions 9, 46, and 48). The amino acids previously identified to contribute to binding to IL-18BP are also highlighted and their code and positions also specified (amino acid positions 51 , 60, 80, 110, 111 and 112). Figure 2C shows the preferred amino acids to be modified (amino acid positions 48, 51 , and 60), and the additional desirable amino acids (amino acid positions 9 and 11).

[0051] Figure 3 shows the crystal structure of IL-18 in complex with IL-18Ra and IL-18Rp, and the residues which interact with IL-18Ra (amino acid positions 5, 13, 17, 31 , 32, 33, 59, 93, and 132) and IL-18Rp (amino acid positions 51 , 60, 105, 108, 109, 110, 111 , 112, 145, 147 and 150).

[0052] Modifications to amino acids on the protein surface may implicate protein-protein interactions. Protein surfaces may comprise protein-protein interfaces (i.e., the molecular surfaces through which subunits of multi-protein complexes make contact and interact with one another). These interfaces may comprise large, flat interfaces, and / or smaller concavities or pockets or even single residues which may form an anchoring site for one or more interacting proteins. The proteinprotein interface will be shape and / or chemically complementary.

[0053] In the context of IL-18, protein-protein interactions include the interaction between IL-18 and its receptor (IL-18Ra and I L-18Rp) and IL-18 and its decoy receptor, IL- 18BP. The IL-18 / 1 L-18R and IL-18 / IL-18BP interactions occur at the surface in a receptor interaction surface of IL-18. As used herein, the terms “receptor interaction surface”, “receptor binding surface”, “interaction surface”, “binding surface”, and “ligand binding surface” are used interchangeably and refer to the surface which is involved in coordinating, interacting with and binding to an IL-18 receptor. Preferably, the receptor interaction surface is a surface for interacting with IL-18 receptors. More preferably, the IL-18 receptor is an IL-18R (IL-18Ra and IL-18Rp) and / or IL-18BP (IL-18 decoy receptor). There may be a defined group of amino acids which directly bind the IL-18 receptors.

[0054] Two IL-18 sites, site I and site II, contact IL-18Ra. Site I is located on a side of the core barrel of the p-trefoil structure, and site II is at the top of the p-barrel. Site III of IL-18 recognises I L-18Rp. Site III also recognises IL-18BP. The key amino acids in site I include R13, D17, E31 , D32, M33, D37, and D132. The key amino acids in site II include L5, K53, M60, and K93. The key amino acids in site III include G108, H109, D110, K112, M150, R147, and G14 (Tsutsumi, 2014). Therefore, it can be seen that the “receptor interaction surface” is well characterised in the art. For example, Tsutsumi et al. (2014) show the residues involved in interacting with IL-18Ra and I L-18Rp, and Detry et al. (2022) show the interaction surface between IL-18 and IL-18BP. None of the amino acids modified herein to alter the solubility of IL-18 are involved in any interactions with IL-18R or IL-18BP. In contrast, some of the amino acids modified herein to achieve decoy resistance of IL-18 may be involved in interactions with IL-18R and IL-18BP. Further, IL-18 is non-glycosylated, and the majority of the glycosylation patterns are in the receptors (for instance, IL-18BPs are heavily glycosylated).

[0055] The interaction surface is a region, as opposed to a domain. Protein domains are distinct functional and / or structural units within a protein that are self-stabilising and independent from the rest of the protein. Amino acids which are in a region adjacent to the receptor interaction surface may play a role in mediating protein-protein interactions (i.e. , they are adjacent to the residues which interact with the receptors). As described herein, the “region” may be an exposed hydrophobic pocket and / or a surface region.

[0056] As used herein, the term “region adjacent” refers to those amino acids in region which may be adjacent to, yet far away from, the amino acids which form the IL- 18 receptor and / or IL-18BP interaction surface (see Figure 3). Given the mutation of select amino acids disclosed herein, the skilled person will readily appreciate that the term “region adjacent to the receptor interaction surface” intends to cover amino acids in a region of IL-18 which may, or may not be, involved in coordinating, effecting or maintaining the interactions between IL-18 and IL-18R or IL-18BP. For instance, as described herein, the region adjacent to a receptor interaction surface of the IL-18 variant may be an exposed hydrophobic pocket and / or a surface region. The amino acids in the exposed hydrophobic pocket may not participate in interactions with IL-18R or IL-18BP, whereas the amino acids in the surface region may participate in interactions with IL-18R and / or IL-18BP. Therefore, the skilled person will readily understand that the term “region adjacent to a receptor interaction surface” intends to cover any amino acids that are known to participate in interactions (Detry, 2022; Tsutsumi, 2014), but also those that may be a distance from the interaction surface and may not participate in interactions.

[0057] Certain modifications may act to disrupt the interaction between IL-18 and IL- 18BP, for example by decreasing the affinity of binding or blocking binding all together. The amino acid modifications can block, prevent, or decrease the likelihood of an interaction between the IL-18 variant and IL-18BP. The modifications will preferably allow the IL-18 variant to be resistant to IL-18BP binding, and / or to preferably bind IL-18R over IL-18BP. The interaction between IL-18 and IL-18BP may be prevented in that there is a higher rate constant of ligand dissociation (Kotf) (i.e., low stability of IL-18 / IL-18BP complexes), a lower rate constant of ligand association (Kon) (i.e., slow or no recognition of IL-18BP by IL-18 and vice versa), and a higher equilibrium dissociation constant (Kd) (i.e., a low affinity interaction or no interaction). Kdis the ratio between Kotf and Kon(Kd = Koff / Kon). Other means for characterising binding kinetics will be apparent to those in the art.

[0058] Further, modifications which alter the composition properties of amino acids within IL-18 may impact the solubility of IL-18, without impacting the biological activity of IL-18.

[0059] In accordance with the first aspect of the invention, there is provided an interleukin-18 (IL-18) variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at the following amino acid positions: i) 48; and ii) 51 and / or 60. As detailed herein, the wild-type IL-18 may comprise mature IL-18, i.e., an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% identity to SEQ ID NO: 1. The IL-18 variant may also be present with other protein sequences such as signal sequences, localisation sequences, peptide tags, peptide linkers, other interleukins or receptors thereof, other immunostimulatory proteins, or any combinations thereof.

[0060] The IL-18 variant according to the invention may comprise an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1. As used herein, the term “sequence identity” and “sequence homology” are interchangeable and refers to the number of identical residues over a defined length into a given alignment. To calculate % sequence identity of any of the sequences herein disclosed, sequence comparison software may be used, for example, using the default settings on the BLAST software package (V2.10.1).

[0061] In another embodiment, the IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 1 ; at least 80% sequence identity to SEQ ID NO: 1 ; at least 85% sequence identity to SEQ ID NO: 1 ; at least 90% sequence identity to SEQ ID NO: 1 ; at least 91 % sequence identity to SEQ ID NO: 1 ; at least 92% sequence identity to SEQ ID NO: 1 ; at least 93% sequence identity to SEQ ID NO: 1 ; at least 94% sequence identity to SEQ ID NO: 1 ; at least 95% sequence identity to SEQ ID NO: 1 ; at least 96% sequence identity to SEQ ID NO: 1 ; at least 97% sequence identity to SEQ ID NO: 1 ; at least 98% sequence identity to SEQ ID NO: 1 ; or at least 99% sequence identity to SEQ ID NO: 1 , wherein the % sequence identity to SEQ ID NO: 1 retains the amino acid modifications at the relevant amino acid positions.

[0062] In another embodiment, the IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 1 ; at least 80% sequence identity to SEQ ID NO: 1 ; at least 85% sequence identity to SEQ ID NO: 1 ; at least 90% sequence identity to SEQ ID NO: 1 ; at least 91 % sequence identity to SEQ ID NO: 1 ; at least 92% sequence identity to SEQ ID NO: 1 ; at least 93% sequence identity to SEQ ID NO: 1 ; at least 94% sequence identity to SEQ ID NO: 1 ; at least 95% sequence identity to SEQ ID NO: 1 ; at least 96% sequence identity to SEQ ID NO: 1 ; at least 97% sequence identity to SEQ ID NO: 1 ; at least 98% sequence identity to SEQ ID NO: 1 ; at least 99% sequence identity to SEQ ID NO: 1 ; or at least 100% sequence identity to SEQ ID NO: 1 , wherein the % sequence identity to SEQ ID NO: 1 retains the amino acid modifications at relevant amino acid positions.

[0063] In accordance with a first aspect of the invention, the IL-18 variant may be modified such that, in comparison to SEQ ID NO: 1 , the amino acid modifications occur at amino acid positions 48; and 51 and / or 60. For instance, there may be an IL-18 variant comprising amino acid modifications at amino acid positions 48 and 60; 48 and 51 ; or 48, 51 and 60.

[0064] The inventors of the present invention have surprisingly found that amino acid modifications to the amino acids at positions 51 and 60 of IL-18 are all that is required to achieve decoy resistance in the IL-18 variant, and that an amino acid modification at amino acid position 48 allows for IL-18 variants comprising this modification to be solubilised (i.e., stabilised) while retaining similar biological activity levels to that of wild-type IL-18.

[0065] Therefore, in a preferred embodiment, the IL-18 variant may comprise amino acid modifications at amino acid positions 48 and 60. In another preferred embodiment, the IL-18 variant may comprise amino acid modifications at amino acid positions 48, 51 and 60.

[0066] In addition to the modifications at amino acid positions 48 and 51 and / or 60, the IL-18 variant may further comprise a single amino acid modification at any one of amino acid positions 9, 46, 80, 105, 110, or 111; two amino acid modifications at any two of amino acid positions 9, 46, 80, 105, 110, and / or 111; three amino acid modifications at any three of amino acid positions 9, 46, 80, 105, 110, and / or 111 ; four amino acid modifications at any four of amino acid positions 9, 46, 80, 105, 110, and / or 111 ; five amino acid modifications at any five of amino acid positions 9, 46, 48, 105, 110, and / or 111 ; or six amino acid modifications at all of amino acid positions 9, 46, 80, 105, 110, and 111. The amino acid modifications may further occur at any of amino acid positions 9, 46, 80, 105, 110, and / or 111 , or any combination thereof. These amino acid positions are located in a region adjacent to a receptor interaction surface of the IL-18 variant. Amino acid positions 9, 46, and 48 are in a region which is a hydrophobic pocket of the IL-18 variant. The remaining amino acid positions are in a region which is a surface region of the IL- 18 variant, that includes Site II or nearby the I L-18 / IL-18R alpha interface.

[0067] In a preferred embodiment, the IL-18 variant may comprise amino acid modifications at amino acid positions 48 and 60; 48, 51 and 60; 48, 51 , 60 and 111 ; or 9, 48, 51 , 60 and 111. The IL-18 variants comprising mutations at these combinations of amino acid positions were surprisingly found by the inventors of the present invention to be soluble, active and decoy resistant. It will be understood that an IL-18 variant comprising any of the modifications disclosed herein will retain at least 70% sequence identity to SEQ ID NO: 1 .

[0068] In some embodiments, the IL-18 variant may comprise amino acid modifications whereby non-conservative amino acid substitutions are performed. In such embodiments, a wild-type amino acid is replaced by another amino acid with different properties to that of the wild-type amino acid. For example, a hydrophobic amino acid naturally present in the wild-type sequence is replaced with a hydrophilic amino acid, or vice versa. Another example of a nonconservative amino acid substitution may involve a smaller amino acid naturally present in the wild-type sequence being replaced with a larger / bulkier amino acid, or vice versa. Another example of a non-conservative amino acid substitution may involve a polar amino acid naturally present in the wild-type sequence being replaced with a non-polar amino acid, or vice versa. In other embodiments, the IL- 18 variant may comprise amino acid modifications whereby conservative amino acid substitutions are performed. In such embodiments, a wild-type amino acid is replaced by another amino acid with similar or the same properties to that of the wild-type amino acid. For example, a hydrophobic amino acid naturally present in the wild-type sequence is replaced with another hydrophobic amino acid, or a hydrophilic amino acid naturally present in the wild-type sequence is replaced with another hydrophilic amino acid. Another example of a conservative amino acid substitution may involve an amino acid naturally present in the wild-type sequence being replaced with another amino acid of a similar size (mass / bulkiness etc). Another example of a conservative amino acid substitution may involve a polar amino acid naturally present in the wild-type sequence being replaced with another polar amino acid, or a non-polar amino acid naturally present in the wildtype sequence being replaced with another non-polar amino acid.

[0069] As used herein, the terms “hydrophobic” and “hydrophilic” are to be given their usual meanings in the art, that is having a weak affinity and strong affinity for water, respectively. As used herein, the terms “polar” and “non-polar” are to be given their usual meanings in the art, and polar amino acids may have hydrophilic side chains, whereas non-polar amino acids may have hydrophobic side chains. “Hydrophobic amino acids” are amino acids which possess hydrophobic side chains, i.e. , side chains that do not interact with (repel) water. Hydrophobic side chains are typically or predominantly composed of carbon and hydrogen atoms. Hydrophobic amino acids are generally non-polar and therefore do not dissolve in polar solvents such as water. Hydrophobic amino acids that are naturally occurring may include glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (lie), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). However, any non-naturally occurring, synthetic, or modified amino acid with hydrophobic properties is also intended to be covered by the term “hydrophobic amino acid”. “Hydrophilic amino acids” are amino acids which possess hydrophilic side chains, i.e., side chains that can interact with (attract) water. Hydrophilic side chains are typically composed of oxygen, nitrogen and sulphur atoms. Hydrophilic amino acids are generally polar and therefore can dissolve in polar solvents such as water via the formation of hydrogen bonds. Hydrophilic amino acids that are naturally occurring may include serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), glutamine (Gin), and tyrosine (Tyr). Other naturally occurring hydrophilic amino acids may include arginine (Arg), histidine (His), lysine (Lys), aspartate (Asp), and glutamate (Glu), as these amino acids have electrically charged side chains. Arginine, histidine and lysine have an overall positive charge, whereas aspartate and glutamate have an overall negative charge. However, any non-naturally occurring, synthetic, or modified amino acid with hydrophilic properties is also intended to be covered by the term “hydrophilic amino acid”.

[0070] In accordance with the first aspect of invention, the IL-18 variant may comprise amino acid modifications at amino acid positions i) 48; and ii) 51 and / or 60. The IL-18 variant may further comprise amino acid modifications at one or more of amino acid positions 9, 46, 80, 105, 110, and / or 111 , or any combination thereof. In relation to SEQ ID NO: 1 , the wild-type amino acid at position 9 is leucine, the wild-type amino acid at position 46 is isoleucine, the wild-type amino acid at position 48 is isoleucine, the wild-type amino acid at position 51 is methionine, the wild-type amino acid at position 60 is methionine, the wild-type amino acid at position 80 is isoleucine, the wild-type amino acid at position 110 is aspartate, and the wild-type amino acid at position 111 is asparagine. Table 1 shows the wild-type amino acids and their preferred mutations. able 1. Amino acids within IL-18 and their modifications.

[0071] In a preferred embodiment of the invention, the amino acid modification at amino acid position 9, 46, 48, and / or 80 is a hydrophobic-to-hydrophilic substitution. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of a hydrophobic amino acid for a hydrophilic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for any naturally occurring or non-naturally occurring hydrophilic amino acid. For example, any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan, may be substituted for any one of serine, threonine, cysteine, asparagine, glutamine, tyrosine, arginine, histidine, lysine, aspartate, or glutamate.

[0072] The wild-type amino acid at position 48 in relation to SEQ ID NO: 1 is isoleucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 48 to be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. In a more preferred embodiment, the substitution at amino acid position 48 is an isoleucine-to-aspartate substitution. The inventors of the present invention have surprisingly found that the amino acid modification at amino acid position 48 allows for IL-18 variants comprising this modification to be solubilised (i.e., stabilised) while retaining similar biological activity levels to that of wild-type IL-18 (Figure 6C). IL-18 variants comprising an amino acid modification at amino acid position 48 were found to be approximately 15 times more stable than wild-type IL-18 and at least two-thirds as active as wildtype IL-18 (Figure 13 shows that the variants were found to be as active as wildtype).

[0073] The wild-type amino acid at amino acid position 9 in relation to SEQ ID NO: 1 is leucine. The preference of the present invention is for the hydrophobic-to- hydrophilic substitution at amino acid position 9 to be a leucine-to-serine substitution or a leucine-to-aspartate substitution. In a more preferred embodiment, the substitution at amino acid position 9 is leucine-to-serine substitution (see Figure 8, factor A). The inventors of the present invention have surprisingly found that the amino acid modification at amino acid position 9 results in IL-18 variants which are more stable than wild-type IL-18, and consistently 25% more active than wild-type IL-18 (see Figure 6C).

[0074] The wild-type amino acid at position 46 in relation to SEQ ID NO: 1 is isoleucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 46 to be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. In a more preferred embodiment, the substitution at amino acid position 46 is an isoleucine-to-aspartate substitution.

[0075] The wild-type amino acid at position 80 in relation to SEQ ID NO: 1 is isoleucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 80 to be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. In a more preferred embodiment, the substitution at amino acid position 80 is an isoleucine-to-aspartate substitution.

[0076] Without wishing to be bound by theory, these non-conservative amino acid modifications could act to alter the physiochemical properties of the region in which the amino acids at positions 9, 46, 48, and / or 80 reside. Specifically, the region may become more hydrophilic which allows an IL-18 variant with such amino acid modifications may become more soluble / stable. These alterations may also serve to block or prevent the binding of IL-18BP. Therefore, IL-18 variants with such amino acid modifications may be more soluble than wild-type IL-18, while exhibiting biological activity and IL-18BP decoy resistance.

[0077] In another preferred embodiment, the amino acid modification at amino acid position 51 involves substitution of a wild-type amino acid for a polar amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for a polar amino acid, including any one of lysine, threonine, asparagine, glutamine, tyrosine, serine, arginine, histidine, aspartate, and glutamate. The wild-type amino acid at amino acid position 51 in relation to SEQ ID NO: 1 is methionine. The preference of the present invention is for the amino acid substitution at amino acid position 51 to be a methionine-to-lysine substitution or a methionine-to-threonine substitution. In a more preferred embodiment, the substitution at amino acid position 51 is a methionine-to-threonine substitution. Without wishing to be bound by theory, this amino acid modification could act to optimise the region in which the amino acid at position 51 resides such that an interaction with IL-18BP is prevented or blocked. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wildtype IL-18.

[0078] In another preferred embodiment, the amino acid modification at amino acid position 60 substitution of a large hydrophobic wild-type amino acid for another large amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for any large amino acid, such as leucine or lysine.

[0079] The wild-type amino acid at amino acid position 60 in relation to SEQ ID NO: 1 is methionine. The preference of the present invention is for the amino acid substitution at amino acid position 60 to be a methionine-to-lysine substitution or a methionine-to-leucine substitution. In a more preferred embodiment, the substitution at amino acid position 60 is a methionine-to-lysine substitution. Without wishing to be bound by theory, this amino acid modification could act to prevent or block an interaction with IL-18BP. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wild-type IL-18.

[0080] In another preferred embodiment, the amino acid modification at amino acid position 105 involves substitution of a wild-type amino acid for a polar amino acid, of a wild-type amino acid for a polar amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for a polar amino acid, including any one of lysine, threonine, cysteine, asparagine, glutamine, tyrosine, serine, arginine, histidine, aspartate, and glutamate. The wild-type amino acid at amino acid position 105 in relation to SEQ ID NO: 1 is serine. The preference of the present invention is for the amino acid substitution at amino acid position 105 to be a serine-to-aspartate substitution or a serine-to- asparagine substitution. In a more preferred embodiment, the substitution at amino acid position 105 is a serine-to-aspartate substitution. Without wishing to be bound by theory, this amino acid modification could act to optimise the region in which the amino acid at position 105 resides such that an interaction with IL- 18BP is prevented or blocked. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wild-type IL-18.

[0081] In another preferred embodiment, the amino acid modification at amino acid position 110 involves substitution of a wild-type amino acid for (a) a polar amino acid, or (b) a neutral amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for a polar amino acid, including any one of lysine, threonine, cysteine, asparagine, glutamine, tyrosine, serine, arginine, histidine, aspartate, and glutamate, or a neutral amino acid, such as glycine.

[0082] The wild-type amino acid at amino acid position 110 in relation to SEQ ID NO: 1 is aspartate. The preference of the present invention is for the amino acid substitution at amino acid position 110 to be an aspartate-to-serine substitution or an aspartate-to-lysine substitution, or an aspartate-to-glycine substitution. In a more preferred embodiment, the substitution at amino acid position 110 is an aspartate-to-lysine substitution. Without wishing to be bound by theory, this amino acid modification could act to optimise the region in which the amino acid at position 110 resides such that an interaction with IL-18BP is prevented or blocked. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wild-type IL-18.

[0083] In another preferred embodiment, the amino acid modification at amino acid position 111 involves substitution of a wild-type amino acid for (a) a positively charged amino acid, or (b) a neutral amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for a positively charged amino acid, including any one of arginine, histidine or lysine, or a neutral amino acid, such as glycine.

[0084] The wild-type amino acid at amino acid position 111 in relation to SEQ ID NO: 1 is asparagine. The preference of the present invention is for the amino acid substitution at amino acid position 111 to be an asparagine-to-arginine substitution or an asparagine-to-histidine substitution, or an asparagine-to-glycine substitution. In a more preferred embodiment, the substitution at amino acid position 111 is an asparagine-to-histidine substitution. Without wishing to be bound by theory, this amino acid modification could act to optimise the region in which the amino acid at position 111 resides such that an interaction with IL-18BP is prevented or blocked. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wild-type IL-18.

[0085] It will be understood that any of the amino acid modifications herein disclosed may be present in any combination with any of the other amino acid modifications herein disclosed. For example, an IL-18 variant according to the present invention may comprise one or more amino acid modifications at any of amino acid positions 9, 46, 48, 51 , 60, 80, 105, 110, and / or 111 , or any combination thereof. As detailed herein, in a preferred embodiment, the IL-18 variant may comprise amino acid modifications at amino acid positions 48 and 60 (V17); 48, 51 , and 60 (V25); 48, 51 , 60, and 111 (V27); or 9, 48, 51 , 60, and 111 (V59). Surprisingly, activity assays have reproducibly demonstrated that the IL-18 variants denoted V17, V25, V27 and V59 are biologically active (see Figures 1-3).

[0086] Accordingly, in a preferred embodiment, an IL-18 variant according to the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 48 and a substitution of a wild-type amino acid for a polar amino acid at amino acid position 60. In an even more preferred embodiment, an IL-18 variant according to the invention may comprise (i) the isoleucine-to-aspartate substitution at amino acid position 48; and (ii) the methionine-to-lysine substitution or a methionine-to-leucine substitution at amino acid position 60. In this embodiment, the IL-18 variant may comprise an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 48 and 60.

[0087] In another embodiment, the IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 2; at least 80% sequence identity to SEQ ID NO: 2; at least 85% sequence identity to SEQ ID NO: 2; at least 90% sequence identity to SEQ ID NO: 2; at least 91 % sequence identity to SEQ ID NO: 2; at least 92% sequence identity to SEQ ID NO: 2; at least 93% sequence identity to SEQ ID NO: 2; at least 94% sequence identity to SEQ ID NO: 2; at least 95% sequence identity to SEQ ID NO: 2; at least 96% sequence identity to SEQ ID NO: 2; at least 97% sequence identity to SEQ ID NO: 2; at least 98% sequence identity to SEQ ID NO: 2; at least 99% sequence identity to SEQ ID NO: 2; or at least 100% sequence identity to SEQ ID NO: 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 48 and 60.

[0088] In a particularly preferred embodiment, an IL-18 variant according to the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 48, a substitution of a wild-type amino acid for a polar amino acid at amino acid position 51 , and a substitution of a wild-type amino acid for a large amino acid at amino acid position 60. In an even more preferred embodiment, an IL-18 variant according to the invention may comprise (i) the isoleucine-to-aspartate substitution at amino acid position 48; and (ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and (iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60. In this embodiment, the IL-18 variant may comprise an amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 48, 51 , and 60.

[0089] In another embodiment, the IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 3; at least 80% sequence identity to SEQ ID NO: 3; at least 85% sequence identity to SEQ ID NO: 3; at least 90% sequence identity to SEQ ID NO: 3; at least 91 % sequence identity to SEQ ID NO: 3; at least 92% sequence identity to SEQ ID NO: 3; at least 93% sequence identity to SEQ ID NO: 3; at least 94% sequence identity to SEQ ID NO: 3; at least 95% sequence identity to SEQ ID NO: 3; at least 96% sequence identity to SEQ ID NO: 3; at least 97% sequence identity to SEQ ID NO: 3; at least 98% sequence identity to SEQ ID NO: 3; at least 99% sequence identity to SEQ ID NO: 3; or at least 100% sequence identity to SEQ ID NO: 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 48, 51 , and 60.

[0090] In another preferred embodiment, an IL-18 variant according to the invention comprises a hydrophobic-to-hydrophilic substitution at amino acid position 48, a substitution of a wild-type amino acid for a polar amino acid at amino acid position 51 , a substitution of a wild-type amino acid for a large amino acid at amino acid position 60, and a substitution of a wild-type amino acid for a positively charged amino acid or a neutral amino acid at amino acid position 111 . In an even more preferred embodiment, an IL-18 variant according to the invention comprises (i) the isoleucine-to-aspartate substitution at amino acid position 48; and (ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and (iii) the methionine-to-lysine substitution or methionine-to- leucine substitution at amino acid position 60; and (iv) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111. In this embodiment, the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 48, 51 , 60, and 111.

[0091] In another embodiment, the IL-18 variant comprises an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 4; at least 80% sequence identity to SEQ ID NO: 4; at least 85% sequence identity to SEQ ID NO: 4; at least 90% sequence identity to SEQ ID NO: 4; at least 91 % sequence identity to SEQ ID NO: 4; at least 92% sequence identity to SEQ ID NO: 4; at least 93% sequence identity to SEQ ID NO: 4; at least 94% sequence identity to SEQ ID NO: 4; at least 95% sequence identity to SEQ ID NO: 4; at least 96% sequence identity to SEQ ID NO: 4; at least 97% sequence identity to SEQ ID NO: 4; at least 98% sequence identity to SEQ ID NO: 4; at least 99% sequence identity to SEQ ID NO: 4; or at least 100% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 48, 51 , 60, and 111.

[0092] In another preferred embodiment, an IL-18 variant according to the invention comprises a hydrophobic-to-hydrophilic substitution at amino acid position 9, a hydrophobic-to-hydrophilic substitution at amino acid position 48, a substitution of a wild-type amino acid for a polar amino acid at amino acid position 51 , a substitution of a wild-type amino acid for a large amino acid at amino acid position 60, and a substitution of a wild-type amino acid for a positively charged amino acid or a neutral amino acid at amino acid position 111. In an even more preferred embodiment, an IL-18 variant according to the invention comprises (i) the leucine- to-serine substitution at amino acid position 9; and (ii) the isoleucine-to-aspartate substitution at amino acid position 48; and (iii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and (iv) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and (v) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111 . In this embodiment, the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 9, 48, 51 , 60, and 111.

[0093] In another embodiment, the IL-18 variant comprises an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 5; at least 80% sequence identity to SEQ ID NO: 5; at least 85% sequence identity to SEQ ID NO: 5; at least 90% sequence identity to SEQ ID NO: 5; at least 91 % sequence identity to SEQ ID NO: 5; at least 92% sequence identity to SEQ ID NO: 5; at least 93% sequence identity to SEQ ID NO: 5; at least 94% sequence identity to SEQ ID NO: 5; at least 95% sequence identity to SEQ ID NO: 5; at least 96% sequence identity to SEQ ID NO: 5; at least 97% sequence identity to SEQ ID NO: 5; at least 98% sequence identity to SEQ ID NO: 5; at least 99% sequence identity to SEQ ID NO: 5; or at least 100% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 9, 48, 51 , 60, and 111.

[0094] As described herein, in contrast to what is known in the art, the inventors of the present invention have surprisingly found that amino acid modifications to the amino acids at positions 51 and 60 of IL-18 are all that is required to achieve decoy resistance in the IL-18 variant. Therefore, in accordance with a second aspect of the invention, there is provided an IL-18 variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at amino acid positions 51 and 60. The amino acid modifications at amino acid positions 51 and 60 may as be described as for the first aspect of the invention. For the avoidance of doubt, in a preferred embodiment, the amino acid modification at amino acid position 51 may involve a substitution of a wild-type amino acid for a polar amino acid. In a more preferred embodiment, the amino acid modification at amino acid position 51 may be a methionine-to-lysine substitution or a methionine-to-threonine substitution, preferably a methionine-to- threonine substitution. In another preferred embodiment, the amino acid modification at amino acid position 60 may involve substitution of a wild-type amino acid for a large amino acid. In a more preferred embodiment, the amino acid modification at amino acid position 60 may be a methionine-to-lysine substitution or a methionine-to-leucine substitution, preferably a methionine-to- lysine substitution.

[0095] As such, a decoy resistant IL-18 variant may comprise the following decoy resistance mutations: M51 K or M51T and M60L or M60K, preferably M51T and M60K. It is envisaged that there may be any suitable protein or polypeptide joined to the IL-18 variant. Such examples may include, but are not limited to, signal peptides, antibody fragments, antibodies, bi- or multi-specific antibodies, tissue-targeting sequences (such as the NGR (Asn-Gly-Arg) tripeptide), chemokines, or interleukins particularly those that stimulate cell mediated immunity (e.g., IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17, IL-18, IL-21) or chemokines (e.g. CXCL9, CXCL10, CXCL11 , CCL5, CCL2, CX3CL1).

[0096] In particular, in order to mount a strong immune response, for example, in the tumour microenvironment, the therapeutic bacteria must be able to produce and secrete immuno-stimulatory cytokines in situ. Therefore, in a preferred embodiment, the IL-18 variant is fused to a signal peptide. As used herein, the term “signal peptide” refers to any signal sequence, localisation sequence or other polypeptide sequence which directs the IL-18 variant to a particular intracellular or extracellular location. For expression in bacteria, a bacterial signal peptide will be required in order to secrete the IL-18 variant out of the bacterial cytosol and into, for example, the periplasm. The addition of small (15 to 25 amino acid) sequences, known as signal peptides, to a protein of interest can be used to direct to a desired location.

[0097] As will be known by the skilled person, the periplasm is a concentrated matrix in the space defined between the inner plasma membrane and the bacterial outer membrane of Gram-negative bacteria. The (bacterial) periplasm is an oxidative compartment, and is ideal to form disulphide bonds, which are ubiquitous in human cytokines. There are also chaperones in the periplasm which assist and promote the formation of disulphide bonds. In contrast, the (bacterial) cytosol is a reducing environment which does not favour disulphide bond formation. Therefore, it can be seen that directing the cytokines (IL-18 variants) to the periplasm via signal peptides is beneficial to the production and correct folding of these proteins, and ultimate secretion in situ. The present use of a periplasmic signal peptide for exporting recombinant proteins from the bacterial cytosol has the advantage of avoiding the formation of inclusion bodies and therefore avoids the need for additional and laborious requirements of solubilising and refolding the proteins from inclusion bodies, thus allowing for high yield / secretion of the present cytokines.

[0098] The preference of the present invention is for the signal peptide to be a (bacterial) periplasmic signal peptide. The signal peptide directs the IL-18 variant into the periplasm of the bacteria in a rapid and robust manner. A periplasmic signal peptide is a polypeptide sequence which allows for a protein to which the signal peptide is attached to be transported, directed, localised, or targeted to the periplasm of a bacterial cell.

[0099] In bacteria, there are to major pathways for exporting proteins across the bacterial plasma membrane: the secretion (SEC) pathway or the twin-arginine translocation (TAT) pathway. The SEC pathway allows unfolded protein to cross the inner membrane (IM), whereas the TAT pathway allows crossing of proteins folded in the cytoplasm. Therefore, the signal peptide as used for exporting any of the IL- 18 variants according to the present invention may be a SEC or TAT periplasmic signal peptide. However, the SEC pathway allows the protein to fold in the appropriate environment, therefore in a preferred embodiment, the signal peptide is a SEC signal peptide. The SEC pathway is shared by both Gram-negative and Gram-positive bacteria. In Gram-negative bacteria, as a result of the SEC pathway, proteins are secreted into the periplasm. In Gram-positive bacteria, as a result of the SEC pathway, proteins are secreted directly into the external environment. In some embodiments, the signal peptide used to direct proteins to the periplasm is N-terminally fused to the IL-18 variant. However, the IL-18 variant could be exported extracellularly via other signal peptides, such as Type I signal peptides. Therefore, in other embodiments, the signal peptide is C-terminally fused to the IL-18 variant. There is no universal signal peptide which can translocate any protein efficiently, and therefore the inventors of the present invention have also created optimal signal peptides for each IL-18 variant.

[0100] From the periplasm, the IL-18 variant can then be prompted into the supernatant or other extracellular environment. In fusing a periplasmic signal peptide to any of the IL-18 variants described herein, it is envisaged that, if expressed within a bacterial cell, the IL-18 variants would be transported efficiently to the periplasm of the bacterial cell in order to achieve efficient secretion of the IL-18 variants from the bacterial cell. Such secretion would enable the delivery of these molecules to a site of interest where they are needed, including to a site of disease in a subject who may benefit from such an enhanced immune response induced by the IL-18 variants. For example, upon infection and invasion of a host (e.g., eukaryotic) cell by bacteria, such as Gram-negative bacteria, expressing the IL-18 variants of the present invention, the IL-18 variant may be transferred into the cytoplasm of the host cell where it may achieve for example a therapeutic immunostimulatory effect.

[0101] In another embodiment, the signal peptide isfused to the IL-18 variant via a spacer sequence. As used herein, the terms “spacer” and “spacer sequence” may be used interchangeably and refer to any amino acid sequence which is to be located between the signal peptide and the cargo (IL-18 variant) which is to be secreted (i.e., signal peptide:spacer:IL-18 variant). Preferably, the spacer is a very short amino acid sequence, typically 1 to 5 amino acids in length (e.g., one amino acid, two amino acids, three amino acids, four amino acids, four amino acids or five amino acids in length). Any suitable sequence of amino acids may be utilised as a spacer. Even more preferably, the spacer is present immediately after an AXA domain of the signal peptide. The AXA domain of the signal peptide is recognised by a signal peptidase which cleaves the signal peptide from the mature protein. The purpose of the spacer sequence is to improve the export rate of the unfolded protein into the periplasm, and to improve cleavage of the signal peptide such that the mature protein is released.

[0102] As described herein, in comparison to the prior art, the inventors of the present invention have surprisingly found that amino acid modifications to the amino acids at positions 51 and 60 of IL-18 are all that is required to achieve decoy resistance in the IL-18 variant. Further, the inventors of the present invention have surprisingly found the amino acid modification at amino acid position 48 allows for IL-18 variants comprising this modification to be solubilised (i.e., stabilised) while retaining similar biological activity levels to that of wild-type IL-18. Of the different residues tested for altering the solubility of IL-18 variants, mutation of amino acid 48 was surprisingly found to yield the greatest enhancement of solubility. Therefore, in a most preferred embodiment, and in accordance with the second aspect of the invention, the IL-18 variant may comprise amino acid modifications at amino acid positions 48, and 51 and / or 60. For instance, the IL-18 variant may comprise an amino acid modification at amino acid position 48. The IL-18 variant may comprise amino acid modifications at amino acid positions 48 and 51. The IL-18 variant may comprise amino acid modifications at amino acid positions 48 and 60. The IL-18 variant may comprise amino acid modifications at amino acid positions 48, 51 and 60.

[0103] In this embodiment, the amino acid modification at amino acid position 48 may be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution Preferably, the amino acid modification at amino acid position 48 may be an isoleucine-to-aspartate substitution. The amino acid modification at amino acid position 51 may involve substitution of a wild-type amino acid for a polar amino acid. Preferably, the amino acid modification at amino acid position 51 may be a methionine-to-lysine substitution or a methionine-to-threonine substitution, more preferably a methionine-to-threonine substitution. The amino acid modification at amino acid position 60 may involve substitution of a wild-type amino acid for a large amino acid. Preferably, the amino acid modification at amino acid position 60 may be a methionine-to-lysine substitution or a methionine-to-leucine substitution, more preferably a methionine-to-lysine substitution.

[0104] In a more preferred embodiment, the IL-18 variant according to the second aspect of the invention may comprise an I48D mutation and an M60K mutation. In another preferred embodiment, the IL-18 variant according to the second aspect of the invention may comprise an I48D mutation and an M51T mutation. In another more preferred embodiment, the IL-18 variant according to the second aspect of the invention may comprise an I48D mutation and an M51T mutation and an M60K mutation.

[0105] In accordance with a third aspect of the invention, there is provided a nucleic acid molecule encoding the interleukin-18 (IL-18) variant according to the first and / or second aspects of the invention. As used herein, the term “nucleic acid molecule” refers to a nucleic acid construct (i.e. , DNA and / or RNA), generated recombinantly or synthetically, comprising a series of specified nucleic acid elements that permit the expression of a particular nucleic acid sequence in a host cell. The nucleic acid molecule may be an isolated nucleic acid molecule. Specifically, it is envisaged that the nucleic acid molecule will be expressed in a bacterial cell, preferably, a Gram-negative bacterial cell. The nucleic acid molecule may comprise a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, or any combination thereof (preferably any one of SEQ ID NOs: 2, 3, 4, and / or 5) or an amino acid sequence comprising at least 70% identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5 retains the amino acid modifications at the relevant amino acid positions. In some embodiments, expression of the IL-18 variants can be controlled via the modulation of promoter spatio-temporal activation and strength i.e., using promoters that activate at particular locations (e.g., inside the cell or extracellularly), particular times, or in response to particular chemicals.

[0106] In one embodiment, the nucleic acid molecule is a DNA molecule. As used herein, the terms “DNA” and “deoxyribonucleic acid” are given their usual meaning in the art and are used interchangeably to refer to nucleic acids composed of thymine, adenine, guanine, and cytosine deoxyribonucleic acid bases. These terms and concepts will be well known to those in the art.

[0107] In another embodiment, the nucleic acid molecule is an RNA molecule. As used herein, the terms “RNA” and “ribonucleic acid” are given their usual meaning in the art and are used interchangeably to refer to nucleic acids composed of uracil, adenine, guanine, and cytosine ribonucleic acid bases. These terms and concepts will be well known to those in the art. Types of RNA molecules include, for example, mRNA, siRNA, shRNA, miRNA, tRNA, and rRNA. In a preferred embodiment, the RNA molecule is an mRNA molecule. As used herein, the terms “mRNA” and “messenger RNA” are used interchangeably and refer to a singlestranded RNA molecule involved in protein synthesis, which is transcribed from DNA and translated into an amino acid sequence. Bacterial mRNA molecules are transcribed from DNA that is non-compartmentalised and translated in the cytosol coupled to transcription. These terms and concepts will be well known to those in the art. Eukaryotic mRNA molecules are transcribed from DNA in the nucleus of a eukaryotic cell, and subsequently exported from the nucleus into the cytoplasm of the eukaryotic cell, where translation of the mRNA molecule into proteins takes place.

[0108] The nucleic acid may be expressed in a host cell, such as a bacterial cell which has been engineered or modified to express said nucleic acid. Alternatively, the nucleic acid may be expressed in a eukaryotic cell. In this embodiment, the nucleic acid (e.g., RNA) may be comprised within a bacterial cell, which can then be delivered to a eukaryotic cell. An RNA molecule would have a nucleotide- encoding structure which, in the case of mRNA, allows for transcription within the bacterium itself, and then upon invasion and replication of the bacteria in a target eukaryotic cell, protein translation and expression within the eukaryotic cell.

[0109] The nucleic acid molecule may comprise a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4 and / or 5, or an amino acid sequence comprising at least 70% identity to any of SEQ ID NOs: 1 , 2, 3, 4 and / or 5, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions.

[0110] In another embodiment, there may be provided a nucleic acid molecule may comprise a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 80% sequence identity to SEQ I D NOs: 1 , 2, 3, 4, and / or 5; at least 85% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 90% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 91 % sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 92% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 93% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 94% sequence identity to SEQ I D NOs: 1 , 2, 3, 4, and / or 5; at least 95% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 96% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 97% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 98% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; and / or at least 100% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions.

[0111] In a most preferred embodiment, the nucleic acid molecule may comprise a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% identity to any of SEQ ID NOs: 2, 3, 4 and / or 5; at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 80% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 91 % sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 94% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 95% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 96% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 97% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 98% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 99% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; and / or at least 100% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions.

[0112] As described herein and as would be understood by the skilled person, interleukins play a diverse role in eliciting an immune response. Given the role of IL-18 in the stimulation of proliferation and cytotoxic function of CD8 T cells and NK cells, the therapeutic potential of IL-18 is apparent. The inventors of the present invention have surprisingly found that certain mutations to the amino acid sequence of IL-18 generates IL-18 variants which are optimised in terms of solubility and without impacting biological activity and are resistant to the decoy receptor IL-18BP. Therefore, in accordance with a fifth aspect of the invention, there is provided an IL-18 variant according to the first and / or second aspects of the invention, or the nucleic acid molecule according to the third aspect of the invention, or the bacterium according to the fourth aspect of the invention, for use in therapy. Any of the IL-18 variants according to the present invention, in any combination, have uses in therapy.

[0113] Due to the wide-spread involvement of interleukins in several different signalling pathways (including both pro-inflammatory and anti-inflammatory signalling), it will be understood that the present invention has implications in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of several diseases. In a preferred embodiment the disease is a human disease. Diseases may include, but are not limited to, an infectious disease, a cardiovascular disease, a neurodegenerative disease, a gastrointestinal disease, a respiratory disease, a renal disease, a liver disease, an autoimmune disease, an inflammatory disease, or a genetic disorder. In a preferred embodiment, the disease may be a neoplastic disease. Therefore, the invention provides an IL-18 variant according to the first and / or second aspects of the invention, or the nucleic acid molecule according to the third aspect of the invention, or the bacterium according to the third aspect of the invention, for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0114] The terms "tumour," "cancer", “malignancy” and "neoplasia" are used interchangeably and refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation, or survival of a normal counterpart cell, e.g., a cell proliferative or differentiative disorder. Typically, the growth is uncontrolled. The term "malignancy" refers to invasion of nearby tissue. The term "metastasis" refers to spread or dissemination of a tumour, cancer or neoplasia to other sites, locations, or regions within the subject, in which the sites, locations or regions are distinct from the primary tumour or cancer.

[0115] In one embodiment, the neoplastic disease may be a solid cancer and / or a haematological malignancy. Neoplasia, tumours, and cancers include benign, malignant, metastatic, and non-metastatic types, and include any stage (I, II, III, IV orV) or grade (G1 , G2, G3, etc.) of neoplasia, tumour, or cancer, ora neoplasia, tumour, cancer, or metastasis that is progressing, worsening, stabilized or in remission.

[0116] Cancers that may be treated according to the invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestines, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to the following: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumour, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumour, malignant; thecoma, malignant; granulosa cell tumour, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumour, malignant; lipid cell tumour, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumour; Mullerian mixed tumour; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumour, malignant; phyllodes tumour, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumour of bone; Ewing's sarcoma; odontogenic tumour, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumour; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumour, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. Preferably, the solid cancer and / or the haematological malignancy may be a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, mesothelioma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma. Even more preferably, the neoplastic disease may be associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular (liver) cancer, melanoma, oesophageal cancer, gastric cancer, ovarian cancer, endometrial cancer, colorectal cancer, head and neck cancer or breast cancer.

[0117] More preferably, the malignant tumour may be associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial carcinoma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

[0118] In some embodiments, the neoplastic disease is a malignant neoplastic disease.

[0119] In some embodiments the neoplastic disease is not a benign neoplastic disease.

[0120] In a preferred embodiment of the present invention, the IL-18 variant is to be administered intratumourally, intravesically, intravenously, intraperitoneally, or orally administered. In a most preferred embodiment, the IL-18 variant is administered orally or intratumourally. However, it is also contemplated that other methods of administration may be used in some cases. Therefore, in certain instances the IL-18 variants of the present invention may be administered orally or by injection, instillation, infusion, continuous infusion, intradermally, intraarterially, intralesionally, peritumourally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival mucosally, intrapericardially, intraumbilically, intraocularally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, inhalation (e.g. aerosol inhalation), via a catheter, via a lavage, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990). However, the preference of the present invention is for administration to be oral or via local instillation, intra-peritoneally, intrapleurally, intravesically, intravaginally, peritumoral injection, or intratumoural injection.

[0121] As used herein, the term “instillation” refers to the composition being introduced into the relevant anatomical site and remaining there for a specific amount of time before being drained, voided, or withdrawn. As used herein, the term “intraperitoneally” refers to an injection of the composition into the peritoneum of the subject. As used herein, the term “intrapleurally” refers to an injection of the composition into the pleura, or a pleural cavity, of the subject. As used herein, the term “intravesically” refers to an injection or instillation of the composition via a catheter into the urinary bladder of the subject. As used herein, the term “peritumoral injection” refers to, where the disease in question is a neoplastic disease, an injection of the composition around the site of the neoplastic disease. As used herein, the term “intratumoural injection” refers to, where the disease in question is a neoplastic disease, an injection of the IL-18 variant directly into the neoplastic disease of the subject.

[0122] It is understood that the specific method of administration of the composition may depend on the disease to be treated, for example, both the location and type of disease to be treated. For example, if it is desirable that a large surface area of a body cavity is treated, for example, a pleural cavity of a subject, then administration via instillation may be most appropriate. Alternatively, if the disease is located within the peritoneal cavity, administration via intra-peritoneal injection may be most appropriate. Yet further, if the disease to be prevented and / or treated is for example, a haematological malignancy, it is noted that intra-tumoural injection may not be the method of administration of choice.

[0123] Aside from direct administration of any of the IL-18 variants according to the present invention, in order to achieve optimal and efficient expression, folding and secretion of the IL-18 variants, there is provided in accordance with a fourth aspect of the invention a bacterium comprising any of the IL-18 variants of the present invention, or any combination thereof, or a nucleic acid molecule encoding said variants. The IL-18 variants may be expressed in and secreted by a suitable expression system, and isolated for direct administration to a subject in need thereof. However, in a preferred embodiment, the IL-18 variants may be administered together with the bacteria, whereby the bacteria are administered and express and secrete the IL-18. In some embodiments, the bacterium may be a Gram-negative bacterium. In other embodiments, the bacterium may be a Gram-positive bacterium.

[0124] Any suitable bacterium capable of comprising an IL-18 variant, or a nucleic acid molecule encoding an IL-18 variant according to the present invention may be utilised. However, in a particularly preferred embodiment, there is provided a Gram-negative bacterium, comprising a nucleic acid construct according to the present invention.

[0125] For instance, there may be provided a bacterium, such as Gram-negative bacterium, comprising a nucleic acid molecule comprising a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4 and / or 5, or an amino acid sequence comprising at least 70% identity to any of SEQ ID NOs: 1 , 2, 3, 4 and / or 5, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions. In another embodiment, there may be provided a bacterium, such as Gram-negative bacterium comprising a nucleic acid molecule comprising a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 80% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 85% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 90% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 91% sequence identity to SEQ I D NOs: 1 , 2, 3, 4, and / or 5; at least 92% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 93% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 94% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 95% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 96% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 97% sequence identity to SEQ I D NOs: 1 , 2, 3, 4, and / or 5; at least 98% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; and / or at least 100% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions. Preferably, the Gram-negative bacterium may comprise a nucleic acid molecule comprising a sequence encoding an amino acid sequence according to any one of any of SEQ ID NOs: 2, 3, 4, and / or 5.

[0126] Any suitable bacterium capable of comprising the IL-18 variant may be utilised according to the invention. However, in a particularly preferred embodiment, the bacterium is a Gram-negative bacterium.

[0127] Any Gram-negative bacterium capable of expressing and secreting heterologous interleukins as disclosed herein. Examples of Gram-negative bacteria for use in the present invention include, but are not limited to, Salmonella, Escherichia coli, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia and Yersinia. Preferably, the Gram-negative bacterium will be a live attenuated Gram-negative bacterium. As used herein, the term “attenuated” in the context of the present invention, refers to the alteration of a microorganism to reduce its pathogenicity, rendering it harmless to the host, whilst maintaining its viability. This method is commonly used in the development of vaccines due to its ability to elicit a highly specific immune response whilst maintaining an acceptable safety profile. Development of such vaccines may involve a number of methods, examples include, but are not limited to, passing the pathogens under in vitro conditions until virulence is lost, chemical mutagenesis and genetic engineering techniques. Such an attenuated microorganism is preferably a live attenuated microorganism, although non-live attenuated microorganisms are also disclosed. As used herein, the term “inactivating mutations” refers to modifications of the natural genetic code of a particular gene or gene promoter associated with that gene, such as modification by changing the nucleotide code or deleting sections of nucleotide or adding non- coding nucleotides or non-natural nucleotides, such that the particular gene is either not transcribed or translated appropriately or is expressed into a non-active protein such that the gene’s natural function is abolished or reduced to such an extent that it is not measurable. Thus, the mutation of the gene inactivates that gene’s function or the function of the protein which that gene encodes.

[0128] In other embodiments, the bacteria may be Gram-positive bacteria. Any Grampositive bacterium capable of expressing and secreting heterologous cytokines as disclosed herein may be utilised. Examples of Gram-positive bacteria for use in the present invention include, but are not limited to, Bacillus, Clostridium, Corynebacterium, Listeria, and Gardnerella. Again, the preference is for the bacteria to be live attenuated.

[0129] As used herein, the term “non-natural bacterium or bacteria” refers to bacterial (prokaryotic) cells that have been genetically modified or “engineered” such that it is altered with respect to the naturally occurring cell. Such genetic modification may for example be the incorporation of additional genetic information into the cell, modification of existing genetic information or indeed deletion of existing genetic information. This may be achieved, for example, by way of transfection of a recombinant plasmid into the cell or modifications made directly to the bacterial genome. Additionally, a bacterial cell may be genetically modified by way of chemical mutagenesis, for example, to achieve attenuation, the methods of which will be well known to those skilled in the art. As such, the term “non-natural bacterium or bacteria” may refer to both recombinantly modified and non- recombinantly modified strains of bacteria. As used herein, the term “recombinant”, “recombinant strain” or “recombinant bacteria” are used interchangeably and, in the context of the present invention, refers to a strain of bacteria that has undergone genetic engineering such that the bacterial DNA has been altered by the introduction of new DNA. Recombinant DNA methods commonly involve the introduction of new DNA via a vector, for example, a plasmid. Such methods are well known to those skilled in the art. Use of recombinant strains of bacteria may confer advantageous properties to the bacterial strain, such as prolonged activity, eliciting a stronger immune response in a subject, or introduction of a desired molecule.

[0130] Preferably, the Gram-negative bacteria of the present invention may be enteric bacteria. Examples of enteric bacteria include, but are not limited to, Enterobacteriaceae (such as Escherichia coli (E. coli), Klebsiella, Proteus, and Enterobacter), Salmonella, Shigella, Yersinia, and Campylobacter jejuni.

[0131] Preferably, the Gram-negative bacterium is a Salmonella species. Examples of Salmonella species for use in the present invention are Salmonella enterica and Salmonella bongori. Salmonella enterica can be further sub-divided into different serotypes or serovars. Examples of said serotypes or serovars for use in the present invention are Salmonella enterica Typhi, Salmonella enterica Paratyphi A, Salmonella enterica Paratyphi B, Salmonella enterica Paratyphi C, Salmonella enterica Typhimurium and Salmonella enterica Enteritidis. In a preferred embodiment, the live attenuated Gram-negative bacterium is any attenuated, non- pathogenic, Salmonella enterica serovar Typhi or Typhimurium strain. In a most preferred embodiment, the live attenuated Gram-negative bacterium is Salmonella enterica Typhi. In an even more preferred embodiment, the live attenuated bacterium is Salmonella enterica Typhi ZH9 (also referred to as M01ZH09). Derivatives or variants of the Salmonella enterica Typhi ZH9 strain are also intended to be included, including genetically modified variants.

[0132] Such a bacterium will be engineered to express the any of the IL-18 variants disclosed herein. Numerous methods and techniques for genetically engineering bacterial strains will be well known to the person skilled in the art. These techniques include those required for introducing heterologous genes into the bacteria either via chromosomal integration or via the introduction of a stable autosomal self-replicating genetic element. Exemplary methods for genetically modifying (also referred to as "transforming" or “engineering”) bacterial cells include bacteriophage infection, transduction, conjugation, lipofection or electroporation. Other techniques, including CRISPR and CRISPR associated proteins (such as Cas9), CasCLOVER, TALENs, retrons, homing endonucleases or mega-nucleases, zinc-finger nucleases, and transposon-based methods are also intended to be included. A general discussion on these and other methods for genetic engineering in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); which are hereby incorporated by reference.

[0133] For example, there is provided a bacteria, such as Gram-negative bacteria comprising an IL-18 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, more preferably SEQ ID NOs: 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% sequence identity to any of these sequences, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions. In another embodiment, there may be provided a bacteria, such as Gram-negative bacteria comprising an I L-18 variant comprising an amino acid sequence according to any one of SEQ I D NOs: 1 , 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 80% sequence identity to SEQ I D NOs: 1 , 2, 3, 4, and / or 5; at least 85% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 90% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 91 % sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 92% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 93% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 94% sequence identity to SEQ I D NOs: 1 , 2, 3, 4, and / or 5; at least 95% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 96% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 97% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 98% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; and / or at least 100% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions. In a preferred embodiment, the bacteria, such as Gram-negative bacteria comprise an IL-18 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 80% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 91% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 94% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 95% sequence identity to SEQ ID NOs:

[0134] 2, 3, 4, and / or 5; at least 96% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 97% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 98% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 99% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; and / or at least 100% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions.

[0135] In a most preferred embodiment, there is provided a bacterium comprising an IL- 18 variant comprising an amino acid sequence according to SEQ ID NOs: 2 and / or

[0136] 3.

[0137] As will be understood by the skilled person, a bacterium, such as a Gram-negative bacterium, comprising a nucleic acid molecule comprising a sequence encoding any of the amino acid sequences described herein (i.e., SEQ ID NOs: 1-5 and / or any combination thereof) is also included.

[0138] The bacterium comprising an IL-18 variant or nucleic acid molecule encoding an IL-18 variant may be referred to herein as a composition unless specified otherwise. The IL-18 variants will prompt an enhanced immune response and the bacterium itself will additionally elicit strong humoral and cellular immune responses. As used herein, the term "immune response" refers to the action of cellular components of the immune system, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells. As used herein, the term “cellular components of the immune system” refers to immunocytes such as lymphocytes, such as T and B lymphocytes, gamma-delta T cells, and NK cells, which may recognize specific antigens, such as prion, viral, bacterial, yeast, fungal, parasite, tumour- associated or tumour-specific antigens, or other antigens associated with a particular disease, disorder, or condition. Other immunocytes we refer to include white blood cells, which may be granulocytes or agranulocytes. Examples of immunocytes include neutrophils, eosinophils, basophils, lymphocytes, monocytes, and macrophages. Dendritic cells, microglia, and other antigen- presenting cells are also included within this definition.

[0139] The immune response may be, amongst others, a systemic immune response, a local immune response, an innate immune response, an adaptive immune response, a memory immune response, a primary and / or secondary immune response, a specific and / or non-specific immune response, immune cell activation, proliferation, and / or differentiation or the like, or any combinations thereof. As used herein, the terms “systemic immune response” and “systemic immunity” are used interchangeably and refers to a widespread immune response throughout the body of a subject directed against the eliciting agent, as well as widespread non-specific immune activation, as opposed to a local, spatially restricted response. Such a response involves a complex interaction between the different cells of the immune response, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by the above cells or the liver, and in the context of the present invention is thought to prime the immune system of the subject, such that the subject is more responsive to the second composition comprising the live attenuated bacteria, when the second composition is administered locally to the site of the neoplastic disease. A “systemic immune response” may therefore be measured and quantified via the analysis of various different immune cell types, including, but not limited to, neutrophils, monocytes, dendritic cells, T cells (e.g., CD4+ and / or CD8+ T cells) and natural killer cells. The methods by which these effects can be measured are well known to those skilled in the art, for example, flow cytometry. A “systemic immune response” may also be measured and quantified by the presence of antibodies, including but not limited to IgG and IgA isotype antibodies. The methods by which these antibodies can be measured are well known to those skilled in the art, for example, ELISA. Accordingly, the first composition may act to prime (used interchangeably with “condition”, “boost”, “amplify”, “enhance”, “improve”, “augment” or “promote”) the immune response of a subject following administration of the second composition.

[0140] Accordingly, the combination of the IL-18 variant and bacterium, where the IL-18 variant is expressed and secreted by the bacterium, is synergistic and will provide an enhanced immune response in a subject. The IL-18 of the present invention, delivered by a Gram-negative bacterium as defined herein (i.e., a composition), would enable the subject’s immune system to mount an effective immune response to a disease, which is potent and durable and can result in enhanced therapeutic benefit. The decoy-resistance of the IL-18 variants will further act to allow an enhanced and durable immune response by preventing a dampening of the immune response by IL-18BP. The immune response initiated by the administration of the composition may be of a therapeutic level in itself or be of a sub-therapeutic level requiring the subsequent administration of further dose of the composition to exert a therapeutic effect. Therefore, in accordance with a fifth aspect of the invention, there is provided a Gram-negative bacterium for use in therapy.

[0141] A bacteria, such as Gram-negative bacteria, engineered to express the any of the IL-18 variants according to the invention would act as therapeutic strains. Therefore, the bacteria disclosed herein may be used in therapy. These therapeutic strains may be administered to a subject in need of such therapy. The term "treatment" or "therapy" refers to administering to a subject an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (e.g., a disease), the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a disease, or otherwise arrest or inhibit further development of the disease, condition, or disorder in a statistically significant manner. As used herein, the term "subject" is intended to include human and non-human animals (i.e., any member of the animal kingdom). Preferred subjects include humans (i.e., human patients in need of enhancement of an immune response). The methods are particularly suitable for treating patients (preferably human patients) having a disorder that can be treated by augmenting the immune response. In a particular embodiment, IL-18 variants are particularly suitable for the treatment of cancer in vivo.

[0142] The amount of the Gram-negative bacterium administered to the subject is sufficient to deliver a therapeutically effective amount of the IL-18 variant to the subject. The skilled person will readily understand that the precise amount to be administered will be dependent on a number of factors, for example, the disease to be treated, the IL-18 variant to be expressed and secreted, and the medical history of the subject to be treated.

[0143] The terms "therapeutically effective amount" and "effective amount" refer to a sufficient amount of an agent to provide the desired biological or therapeutic result. That result can be reduction, amelioration, palliation, lessening, delaying, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to cancer, an effective amount may comprise an amount sufficient to cause a tumour to shrink and / or to decrease the growth rate of the tumour (such as to suppress tumour growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development or prolong survival or induce stabilisation of the cancer or tumour. In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay recurrence. A therapeutically effective amount can be administered in one or more administrations. The therapeutically effective amount of the drug or combination may result in one or more of the following: (i) reduce the number of cancer cells; (ii) reduce tumour size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; (v) inhibit tumour growth; (vi) prevent or delay occurrence and / or recurrence of tumour; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0144] For example, for the treatment of tumours, a "therapeutically effective dosage" may induce tumour shrinkage by at least about 5% relative to baseline measurement, such as at least about 10%, or about 20%, or about 60% or more. The baseline measurement may be derived from untreated subjects.

[0145] A therapeutically effective amount of a therapeutic compound can decrease tumour size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0146] The Gram-negative bacterium described herein may be administered at a dose of between 105and 1012CFU, where CFU is a colony-forming unit. For example, suitable doses may be between 105and 106CFU, 105and 107CFU, 105and 108CFU, 105and 109CFU, 105and 1010CFU, 105and 1011CFU, 106and 107CFU, 106and 108CFU, 106and 109CFU, 106, and 1010CFU, 106and 1011CFU, 106and 1012CFU, 107and 108CFU, 107and 109CFU, 107and 1010CFU, 107and 1011CFU, 107and 1012CFU, 108and 109CFU, 108and 1010CFU, 108and 1011CFU, 108and 1012CFU, 109and 1010CFU, 109and 1011CFU, 109and 1012CFU, 1010and 1011CFU, 1010and 1012CFU, or 1011and 1012CFU. The composition may be administered in a single dose or in multiple doses. The specific number of doses to be administered are understood to be dependent on the IL-18 variant to be delivered, as well as the specific indication to be treated.

[0147] The Gram-negative bacterium described herein may be administered in any form in which is considered to be within close proximity of the disease. As used herein, the term “close proximity” is intended to refer to the area or region surrounding the disease out to a distance. For example, in one embodiment the term “close proximity” may refer to the area / region extending up to 10 mm, up to 5 mm, or up to 2.5 mm for example, from the boundary of the disease. The distance at which the composition is administered from the site of the disease will allow for the biological effects (for example, recruitment and activation of various immune cell types) to have an effect on the local environment of the disease in question, whilst having minimal / no effect on tissue situated in unrelated areas of the body. Additionally, the composition may be administered “in” or “on” the tissue or surrounding tissue. As such, the term “local administration” refers to any context in which the bacteria, such as Gram-negative bacteria may come into contact with the disease or into contact with the immediately surrounding tissue to have the desired effect. As used herein, the terms “locally” and “administered locally” are used interchangeably and in the context of the present invention refer to the way in which the IL-18 variant and / or Gram-negative bacterium are administered to a subject. As such, the resulting immune response produced in the subject is also said to be local to the site of the disease, that is, it is not a widespread systemic immune response.

[0148] In a preferred embodiment of the present invention, the bacterium, such as a Gram-negative bacterium, described herein is administered intratumourally, intravesically, intravenously, intraperitoneally, or orally administered. In a most preferred embodiment, the live attenuated bacterium is administered intratumourally. However, it is also contemplated that other methods of administration may be used in some cases. Therefore, in certain instances the bacterium of the present invention may be administered by orally or by injection, instillation, infusion, continuous infusion, intradermally, intraarterially, intralesionally, peritumourally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival mucosally, intrapericardially, intraumbilically, intraocularally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, inhalation (e.g. aerosol inhalation), via a catheter, via a lavage, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990). However, the preference of the present invention is for administration to be oral or via local instillation, intra-peritoneally, intrapleurally, intravesically, intravaginally, peritumoral injection, or intratumoural injection. It is understood that the specific method of administration of the bacterium described herein may depend on the disease to be treated, for example, both the location and type of disease to be treated. For example, if it is desirable that a large surface area of a body cavity is treated, for example, a pleural cavity of a subject, then administration via instillation may be most appropriate. Alternatively, if the disease is located within the peritoneal cavity, administration via intraperitoneal injection may be most appropriate. Yet further, if the disease to be prevented and / or treated is for example, a haematological malignancy, it is noted that intra-tumoural injection may not be the method of administration of choice.

[0149] Due to the wide-spread involvement of interleukins in several different signalling pathways (including both pro-inflammatory and anti-inflammatory signalling), it will be understood that the present invention has implications in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of several diseases. In a preferred embodiment the disease is a human disease. Diseases may include, but are not limited to, an infectious disease, a cardiovascular disease, a neurodegenerative disease, a gastrointestinal disease, a respiratory disease, a renal disease, a liver disease, an autoimmune disease, an inflammatory disease, or a genetic disorder. In a preferred embodiment, the disease may be a neoplastic disease. Therefore, in accordance the invention, there is provided a bacterium, such as a Gram-negative bacterium for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0150] Further, in a sixth aspect of the invention, there is provided a use of the IL-18 variant according to the first and / or second aspects of the invention, or the nucleic acid molecule according to the third aspect of the invention, or the bacterium according to the third aspect of the invention, in the manufacture of a medicament for a therapy.

[0151] Where the disease to be treated is a neoplastic disease, the bacterium, such as a Gram-negative bacterium and the IL-18 variant may be administered in combination with another therapy. It is widely known that the causes of neoplastic diseases are multifaceted and diverse, often leading to prevention and treatment strategies comprising multiple therapies to achieve optimum results. As such, the present invention may involve combining the bacterium expressing the IL-18 variant with other known cancer therapies. Preferably, the bacterium expressing the IL-18 variant may be administered in combination with an immunotherapy, radiotherapy, chemotherapy, or an anti-cancer agent. The term “anti-cancer agent” as used herein refers to any agent that is effective in killing cancer cells, halting the division of cancer cells, or helps to prevent the recurrence of cancer cells, but is not considered to be an immunotherapy, radiotherapy, or chemotherapy. In a preferred embodiment, the bacterium expressing the IL-18 variant may be administered in combination with an immunotherapy. Preferably, the immunotherapy may comprise a checkpoint inhibitor, an antigen specific T cell, an adoptive T cell therapy, a therapeutic antibody, a cancer vaccine, or any other engineered cellular immunotherapy. The bacterium expressing the IL-18 variant may be administered separately from, simultaneously with or sequentially to (prior to and / or following) the other known cancer therapy.

[0152] In one embodiment, the neoplastic disease may be a solid cancer and / or a haematological malignancy. Neoplasia, tumours, and cancers include benign, malignant, metastatic and non-metastatic types, and include any stage (I, II, III, IV or V) or grade (G1 , G2, G3, etc.) of neoplasia, tumour, or cancer, or a neoplasia, tumour, cancer or metastasis that is progressing, worsening, stabilized or in remission.

[0153] Cancers that may be treated according to the invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestines, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to the following: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumour, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumour, malignant; thecoma, malignant; granulosa cell tumour, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumour, malignant; lipid cell tumour, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumour; Mullerian mixed tumour; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumour, malignant; phyllodes tumour, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumour of bone; Ewing's sarcoma; odontogenic tumour, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumour; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumour, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0154] Preferably, the solid cancer and / or the haematological malignancy may be a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, mesothelioma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma. Even more preferably, the neoplastic disease may be associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular (liver) cancer, melanoma, oesophageal cancer, gastric cancer, ovarian cancer, endometrial cancer, colorectal cancer, head and neck cancer or breast cancer. More preferably, the malignant tumour may be associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial carcinoma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

[0155] In some embodiments, the neoplastic disease is a malignant neoplastic disease.

[0156] In some embodiments the neoplastic disease is not a benign neoplastic disease.

[0157] In another preferred embodiment, the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, or sarcoma.

[0158] In a seventh aspect of the invention, there is provided a method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject the IL-18 variant according to the first and / or second aspects of the invention, or the nucleic acid molecule according to the third aspect of the invention, or the bacterium according to the third aspect of the invention The present invention therefore also provides for a method treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject. As such, the method of the present invention may be used to reduce or inhibit metastasis of a primary tumour or cancer to other sites, or the formation or establishment of metastatic tumours or cancers at other sites distal from the primary tumour or cancer thereby inhibiting or reducing tumour or cancer relapse or tumour or cancer progression. Accordingly, the present invention provides a detectable or measurable improvement in a condition of a given subject, such as alleviating or ameliorating one or more adverse (physical) symptoms or consequences associated with the presence of a cell proliferative or cellular hyperprol iterative disorder, neoplasia, tumour or cancer, or metastasis, i.e. , a therapeutic benefit or a beneficial effect. A therapeutic benefit or beneficial effect is any objective or subjective, transient, temporary, or long-term improvement in the condition or pathology, or a reduction in onset, severity, duration or frequency of an adverse symptom associated with or caused by cell proliferation or a cellular hyperprol iterative disorder such as a neoplasia, tumour or cancer, or metastasis. It may lead to improved survival. A satisfactory clinical endpoint of a treatment method in accordance with the invention is achieved, for example, when there is an incremental or a partial reduction in severity, duration or frequency of one or more associated pathologies, adverse symptoms or complications, or inhibition or reversal of one or more of the physiological, biochemical or cellular manifestations or characteristics of cell proliferation or a cellular hyperprol iterative disorder such as a neoplasia, tumour or cancer, or metastasis. A therapeutic benefit or improvement therefore may include, but is not limited to, destruction of target proliferating cells (e.g., neoplasia, tumour or cancer, or metastasis) or ablation of one or more, most or all pathologies, adverse symptoms or complications associated with or caused by cell proliferation or the cellular hyperprol iterative disorder such as a neoplasia, tumour or cancer, or metastasis. However, a therapeutic benefit or improvement need not be a cure or complete destruction of all target proliferating cells (e.g., neoplasia, tumour or cancer, or metastasis) or ablation of all pathologies, adverse symptoms or complications associated with or caused by cell proliferation or the cellular hyperprol iterative disorder such as a neoplasia, tumour or cancer, or metastasis. For example, partial destruction of a tumour or cancer cell mass, or a stabilization of the tumour or cancer mass, size or cell numbers by inhibiting progression or worsening of the tumour or cancer, can reduce mortality and prolong lifespan even if only for a few days, weeks or months, even though a portion or the bulk of the tumour or cancer mass, size or cells remain.

[0159] Specific non-limiting examples of therapeutic benefit include a reduction in neoplasia, tumour or cancer, or metastasis volume (size or cell mass) or numbers of cells, inhibiting or preventing an increase in neoplasia, tumour or cancer volume (e.g., stabilizing), slowing or inhibiting neoplasia, tumour or cancer progression, worsening or metastasis, or inhibiting neoplasia, tumour or cancer proliferation, growth or metastasis. An invention method may not take effect immediately. For example, treatment may be followed by an increase in the neoplasia, tumour or cancer cell numbers or mass, but over time eventual stabilization or reduction in tumour cell mass, size or numbers of cells in a given subject may subsequently occur.

[0160] Additional adverse symptoms and complications associated with neoplasia, tumour, cancer, and metastasis that can be inhibited, reduced, decreased, delayed or prevented include, for example, nausea, lack of appetite, lethargy, pain and discomfort. Thus, a partial or complete decrease or reduction in the severity, duration or frequency of an adverse symptom or complication associated with or caused by a cellular hyperprol iterative disorder, an improvement in the subject’s quality of life and / or well-being, such as increased energy, appetite, psychological well-being, are all particular non-limiting examples of therapeutic benefit.

[0161] A therapeutic benefit or improvement therefore can also include a subjective improvement in the quality of life of a treated subject. In an additional embodiment, a method prolongs or extends lifespan (survival) of the subject. In a further embodiment, a method improves the quality of life of the subject.

[0162] A therapeutic benefit may also include the prevention of recurrence of neoplasia, tumour, cancer, and metastasis, for example, wherein said neoplasia, tumour, cancer, and metastasis has been surgically or chemically ablated.

[0163] The bacterium, such as a Gram-negative bacterium, expressing an IL-18 variant of the present invention will typically be administered to the subject in a composition that comprises an effective amount of the bacterium, such as a Gramnegative bacterium, for example, the Salmonella serovar Typhi ZH9 strain, and further comprises a pharmaceutically acceptable carrier / adjuvant / diluent or excipient. The phrases "pharmaceutically” and “pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Such preparations will be known to those skilled in the art. Moreover, for animal (e.g., human or any other member of the animal kingdom) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards, as applicable.

[0164] The composition may be intended to enhance an immune response of a subject. Accordingly, the composition (i.e., bacterium expressing an IL-18 variant of the present invention) may be a vaccine or vaccine composition. Such terms are used interchangeably and refer to a biological preparation in which the subject produces an immune response to said biological preparation, therefore providing active acquired immunity to a particular infectious disease, for example, a disease caused by a Salmonella spp. In the context of the present invention, the vaccine may contain an agent, or “foreign” agent, that resembles the infection-causing bacteria, which is a weakened or killed form of said bacteria, or any portion of, or fragment of, a bacteria protein, capsule, DNA or RNA. Such a foreign agent would be recognised by a vaccine-receiver’s immune system, which in turn would destroy said agent and develop “memory” against the bacteria, inducing a level of lasting protection against future bacterial infections from the same or similar viruses. Through the route of vaccination, including those vaccine compositions of the present invention, it is envisaged that once the vaccinated subject again encounters the same bacteria or bacterial isolate of which said subject was vaccinated against, the individual’s immune system may thereby recognise said bacteria or bacterial isolate and elicit a more effective defence against infection. The active acquired immunity that is induced in the subject as a result of the vaccine may be humoral and / or cellular in nature. The vaccine composition may further comprise an adjuvant, a pharmaceutically acceptable carrier or excipient.

[0165] As used herein, "pharmaceutically acceptable carrier / adjuvant / diluent / excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavouring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Examples include, but are not limited to disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulphate, calcium chloride, sucrose, borate buffer, sterile saline solution (0.9 % NaCI) and sterile water.

[0166] Suitable aqueous and non-aqueous carriers that may be employed in the vaccine compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0167] The vaccine compositions herein disclosed may further contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of presence of unwanted microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin. The vaccine composition may also optionally include additional therapeutic agents, known to be efficacious in, for example, infectious disease or neoplastic disease. Accordingly, the vaccine composition herein disclosed may also comprise antiretroviral drugs, antibiotics, antifungals, antiparasitics and anticancer agents.

[0168] The vaccine composition may also comprise additional components intended for enhancing an immune response. Examples of such additional components include but are not limited to; aluminium salts such as aluminium hydroxide, aluminium oxide and aluminium phosphate, oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (e.g., mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), muramyldipeptides, Immune Stimulating Complexes (the "Iscoms" as disclosed in EP 109 942, EP 180564 and EP 231 039), saponins, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, polyols, the Ribi adjuvant system (see, for instance, GB-A-2 189 141 ), vitamin E, Carbopol, interferons (e.g., IFN- alpha, IFN-gamma, or IFN-beta), or other (unmodified) interleukins particularly those that stimulate cell mediated immunity (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-18, IL-16, IL-17, IL-18, IL-21) or chemokines (e.g. CXCL9, CXCL10, CXCL11 , CCL5, CCL2, CX3CL1).

[0169] The bacterium of the vaccine composition herein disclosed may include any one of, or any combination of, the features of the bacterium herein disclosed.

[0170] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

[0171] As used herein, "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" can mean a range of up to 20%. When particular values are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

[0172] The invention is further described with reference to the following non-limiting examples: EXAMPLES

[0173] Example 1: Identification of aggregation-prone amino acids with Aggrescan 3D and validation of selection through computation of hydrophobic surface with PyMol

[0174] Cytokine variants were obtained following a semi-rational experimental design, where computational tools (e.g., Aggrescan3D) were used to identify aggregation- prone amino acids (four amino acids identified: L9, I46, I48, I80). Computation of the protein surface with PyMol was used to distinguish the hydrophobic and hydrophilic areas (Figure 5). This computation revealed that three quarters of the aggregation-prone residues identified by Aggrescan 3D (Figure 4) can be found in a hydrophobic pocket that does not interact with IL-18 receptor (top ribbon) or IL- 18BP (lower ribbon).

[0175] Mutations for these amino acids were selected, with two possible variants positively charged, polar residue (K) or negatively charged, polar residue (D). Variants were designed to contain up to two different mutations per sequence, resulting in a 32-member library where each variant contains 1 or 2 defined amino acid changes with respect to wild-type IL-18 (see below, SEQ ID NO: 1 , mature, human IL-18, Uniprot ID: Q13478). The library was gene synthesized by IDT as eBlock library.

[0176] The following amino acid sequence was used as reference:

[0177] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTI FI ISMYKDSQPRGMAVT IS VKCEKISTLSCENKI I SFKEMNPPDNIKDTKSDI IFFQRSVPGHDNKMQFESSSYEGYFLACEKE RDLFKLILKKEDELGDRSIMFTVQNED

[0178] Each of the fragments generated during the design phase were cloned into a solubility reporter plasmid, named pBRED, derived from the system reported by Foit et al. (2009 Molecular Cell 36 861-871) and that is based on survival of bacterial cells to penicillin antibiotics (e.g., carbenicillin, ampicillin, penicillin V) as a function of the stability of a protein of interest. This contains a beta-lactamase (tpbla) gene split in two domains (alpha and omega) that can independently fold. Two flexible linkers between these domains allows fusion of any protein cargo so that the final protein is configured as: Blaaipha-linker-cargo-linker-BlaOmega. If the cargo is stable / soluble in nature, the alpha and omega sub-domains can work together to degrade the penicillin-based antibiotic. On the other hand, if the cargo is unstable / insoluble and degraded, the two elements are split, and reporter bacteria cannot survive to the antibiotic.

[0179] The baseline Carbenicillin antibiotic concentration required to abolish growth when IL-18 was fused to thetpbla gene was found to be 1 mg / mL. Antibiotic challenge assays at 1 mg / mL of Carbenicillin identified few variants with higher solubility than wild-type IL-18 (Figure 6A). The protein mScarlet, known to be stable, was used as positive control or benchmark value.

[0180] Cytokine variants were evaluated for activity using a cell-based assay with reporter HEK-Blue™ IL-18 Cells (Invivogen). Protein variants were synthesized using a cell-free extract prepared from E. coli Shuttle strain and plasmid where the gene of the variant was downstream a T7 promoter and fused to a C-terminus 6xHis-tag, which was used to tether the synthesized protein to the surface of a Ni- NTA HisSorb plate (Qiagen). Unspecific products were removed from the plate by washing with NPI-50 buffer (50 mM NaH2PO4, 300 mM NaCI, 50 mM imidazole, pH 8.0) and posteriorly with PBS (137 mM NaCI, 2.7 mM KCI, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4). HEK-Blue cells were added on top of this tethered protein and all subsequent steps were performed as specified by the manufacturer. Variant activity was consistent across different experiments (Figure 6B) and normalised between IL-18 wild-type (activity = 1.0) and sfGFP (activity = 0.0).

[0181] A comparison of the activity of IL-18 variants versus the solubility of IL-18 variants revealed that the solubilising amino acid modification at amino acid position 48 (Figure 6C) did not affect protein activity below the arbitrary threshold of 50% of IL-18 wild-type activity. Table 2 details the IL-18 variants and their amino acid modifications which lead to increased solubility without affecting protein activity below the set threshold. Variants with the amino acid modification at amino acid position 48 were found to be more soluble than wild-type IL-18, when the replacement was for asparagine (D), with activity levels at least 50% than those of wild-type IL-18 when this mutation was alone or in combination with the mutation in L9. Variants with an amino acid modification at amino acid position 9 were found to be 25% more active than wild-type IL-18 but similar solubility than wild-type IL-18.

[0182] Table 2. IL-18 variants and amino acid modifications when testing variant solubility.

[0183] Figure 7 shows the selection of solubility-enhancing mutations. The effect caused by each amino acid modification was evaluated based on overall change in charge (positive or negative). The amino acid modification at amino acid position 48 caused the greatest change (negative charge change). The other amino acid changes or interaction between changes were found not to have any impact on stability that was significantly different from a normal distribution.

[0184] Figure 8 shows the selection of mutations that do not affect activity of the IL-18 variant. The effect caused by each amino acid modification was evaluated based on overall change in charge (positive or negative). The amino acid modification at amino acid position 9 caused the greatest chance (negative charge change). The other amino acid changes nor interaction between changes were found to have any impact on activity that was significantly different from a normal distribution. Example 3: The design and solubility of decoy-resistant IL-18 variants

[0185] Figure 9A shows the 3D structure of IL-18, indicating the relevant amino acid positions previously identified, including 48 and 9. IL-18R is shown in the periphery of IL-18 (middle). Previously reported decoy-resistant amino acid modifications are also indicated at amino acid positions 51 , 60, 105, 110, and 111. Table 3 shows the decoy-resistance amino acid positions and their possible modification.

[0186] IL-18 was split into three fragments (F1 , F2 and F3), designed to contain Bbsl fragments upstream and downstream compatible with cloning on pBRED, and a large library of 128 IL-18 variant amino acid sequences was generated from a combinatorial assembly of different eBlocks (Figure 9B) via Bbsl-dependent Golden Gate. Note that the library was assembled directly to the antibiotic challenge plasmid, which was then transformed into reporter cell lines and assayed. This assay confirmed the stabilising effect of the amino acid modification at amino acid position 48 (Figure 9C), which was the main determinant of stability.

[0187] Table 3. Decoy resistant amino acid modifications in IL-18.

[0188] Example 4 Determining the resistance to IL-18BP (decoy resistance) of IL-18 variants and the activity of decoy resistant IL-18 variants (DR IL-18)

[0189] Figure 10A shows a schematic representation of the experimental set up for determining IL-18 variant decoy resistance and activity. Briefly, the previously assembled variants were extracted from pBRED by PCR amplification, which added compatible Bbsl restriction sites at the 5’ and 3’ ends. These fragments were loaded onto a cell-free expression plasmid and used to synthesize HiBit- tagged versions of each relevant protein variant. The variants were loaded into NiNTA HisSorb plates (Qiagen) that had been previously loaded with His-tagged IL-18BP protein, and the unspecific proteins washed. Then, the IL-18: IL-18BP interaction was measured by complementing the HiBit tag with NanoLuc and measuring the resulting luminescence after substrate addition. A positive result was determined when no luminescence was detected. Screening of the potential IL-18DR variants showed that several of those do not interact with IL-18BP (Figure 10B).

[0190] A sub-pool of 32 candidates, determined not to interact with IL-18BP, were transferred to a plasmid containing His6-tag instead of HiBit, and synthesized on cell-free extracts as previously described for binding into NiNTA HisSorb plates and used to determine their activity in the previously described cell-based activity assays.

[0191] Assays reproducibly showed activity in IL-18 variants labelled V25, V27, and V59 (Figure 11A and 11 B). Interestingly, these variants all share the same core mutations at positions 48, and 51 and / or 60 (Table 4), which are found close to each other in the 3D structure of IL-18.

[0192] Table 4. IL-18 variants and their amino acid modifications which demonstrated decoy-resistance and biological activity.

[0193] Example 5: Protein purification of decoy resistant IL-18 variants

[0194] The final protein variants selected (V17, V25, V27 and V59) were expressed in E. coli Shuffle from the cell-free expression plasmid in 1 L cultures of LB broth at 30°C and 200 rpm after induction with 0.02 mM IPTG for 5 h. These conditions were selected after a pilot study (Figure 12A) used to identify the optimal conditions for purification of the V59 IL-18 variant.

[0195] Bacterial pellets were lysed by sonication and the soluble fraction was purified by Ni-NTA chromatography, yielding purified protein aliquots, which were pooled after validation on SDS-PAGE. Figure 12B shows the final SDS-PAGE gel of the purified of wild-type IL-18, V17, V25, V27 and V59 IL-18 variants. Proteins were quantified by HPLC-MS against standard curves measured with samples of known concentration. Figure 12C and 12D show protein quantification (protein concentration) of wild-type IL-18, V17, V25, V27 and V59 IL-18 variants. Figure 12E shows the results of antibiotic challenge (ABC) assays using wild-type IL-18, V17, V25, V27 and V59 IL-18 variants and Figure 12F shows a comparison of protein concentration versus ABC assay results. As protein concentration increased, greater ABC scores were identified.

[0196] Example 6: Titration curves of decov resistant IL-18 variants

[0197] Figure 13A is a titration curve which shows a comparison of the activity of IL-18 variants V17, V25, V27 and V59 in comparison to wild-type IL-18.

[0198] Figure 13B is a series of titrations which show that the IL-18 variants are decoy- resistant. (i) The IL-18 variants were used to stimulate HEK-Blue™ IL-18 reporter cell lines. IL-18 variant activity was measured as a function of alkaline phosphatase activity (OD62o nm) at 3h after addition of reactant solution. Activity of the variants is comparable to that of IL-18wt. (ii) Example result of IL-18BP inhibition assay. Briefly, a known concentration of IL-18 variant (0.017, 0.17, or 0.71 nM) was incubated with various concentrations of IL-18BP protein before the activity assay. In this example, only IL-18wt responds to IL-18BP binding, (iii) Comparison of IL-18BP inhibition at all IL-18variant concentrations. The trendline indicated that only IL-18wt interacts with IL-18BP. (iv) Comparison of fold signal inhibition after incubation of each variant at various concentrations with 2.3 nM of IL-18BP.

[0199] The invention may be described as in any of the following embodiments. Embodiment 1. An interleukin-18 (IL-18) variant, wherein in comparison to wildtype IL-18, the IL-18 variant comprises one or more amino acid modifications in a region adjacent to a receptor interaction surface of the IL-18 variant.

[0200] Embodiment 2. The IL-18 variant according to Embodiment 1 , wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1.

[0201] Embodiment s. The IL-18 variant according to Embodiment 2, wherein in comparison to SEQ ID NO: 1 , the amino acid modifications occur at one or more of amino acid positions 9, 46, 48, 51 , 60, 80, 105, 110, and / or 111 , or any combination thereof.

[0202] Embodiment 4. An interleukin-18 (IL-18) variant, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at one or more of amino acid positions 9, 46, 48, 51 , 60, 80, 105, 110, and / or 111 , or any combination thereof.

[0203] Embodiment 5. The IL-18 variant according to any one of Embodiments 2 to 4, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1.

[0204] Embodiment 6. The IL-18 variant according to any preceding Embodiment, wherein the IL-18 variant preferentially binds an IL-18 receptor (IL-18Ra and IL- 18Rp) over an IL-18 binding protein (IL-18BP).

[0205] Embodiment 7. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 9, 46, 48, and / or 80 is a hydrophobic-to-hydrophilic substitution. Embodiment s. The IL-18 variant according to Embodiment 7, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 9 is a leucine-to- serine substitution or a leucine-to-aspartate substitution.

[0206] Embodiment 9. The IL-18 variant according to Embodiment 8, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 9 is a leucine-to- serine substitution.

[0207] Embodiment 10. The IL-18 variant according to any one of Embodiments 7 to 9, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 46 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

[0208] Embodiment 11. The IL-18 variant according to Embodiment 10, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 46 is an isoleucine- to-aspartate substitution.

[0209] Embodiment 12. The IL-18 variant according to any one of Embodiments 7 to 11 , wherein the hydrophobic-to-hydrophilic substitution at amino acid position 48 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

[0210] Embodiment 13. The IL-18 variant according to Embodiment 12, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 48 is an isoleucine- to-aspartate substitution.

[0211] Embodiment 14. The IL-18 variant according to any one of Embodiments 7 to 13, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 80 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

[0212] Embodiment 15. The IL-18 variant according to Embodiment 14, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 80 is an isoleucine- to-aspartate substitution.

[0213] Embodiment 16. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 51 involves substitution of a wild-type amino acid for a polar amino acid. Embodiment 17. The IL-18 variant according to Embodiment 16, wherein the amino acid modification at amino acid position 51 is a methionine-to-lysine substitution or a methionine-to-threonine substitution.

[0214] Embodiment 18. The IL-18 variant according to Embodiment 17, wherein the amino acid modification at amino acid position 51 is a methionine-to-threonine substitution.

[0215] Embodiment 19. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 60 involves substitution of a wild-type amino acid for a large amino acid.

[0216] Embodiment 20. The IL-18 variant according to Embodiment 19, wherein the amino acid modification at amino acid position 60 is a methionine-to-lysine substitution or a methionine-to-leucine substitution.

[0217] Embodiment 21. The IL-18 variant according to Embodiment 20, wherein the amino acid modification at amino acid position 60 is a methionine-to-lysine substitution.

[0218] Embodiment 22. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 105 involves substitution of a wild-type amino acid for a polar amino acid.

[0219] Embodiment 23. The IL-18 variant according to Embodiment 22, wherein the amino acid modification at amino acid position 105 is a serine-to-aspartate substitution or a serine-to-asparagine substitution.

[0220] Embodiment 24. The IL-18 variant according to Embodiment 23, wherein the amino acid modification at amino acid position 105 is a serine-to-aspartate substitution.

[0221] Embodiment 25. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 110 involves substitution of a wild-type amino acid for (a) a polar amino acid, or (b) a neutral amino acid. Embodiment 26. The IL-18 variant according to Embodiment 25, wherein the amino acid modification at amino acid position 110 is (a) an aspartate-to-serine substitution or an aspartate-to-lysine substitution, or (b) an aspartate-to-glycine substitution.

[0222] Embodiment 27. The IL-18 variant according to Embodiment 26, wherein the amino acid modification at amino acid position 110 is an aspartate-to-lysine substitution.

[0223] Embodiment 28. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 111 involves substitution of a wild-type amino acid for (a) a positively charged amino acid, or (b) a neutral amino acid.

[0224] Embodiment 29. The IL-18 variant according to Embodiment 28, wherein the amino acid modification at amino acid position 111 is (a) an asparagine-to-arginine substitution or an asparagine-to-histidine substitution, or (b) an asparagine-to- glycine substitution.

[0225] Embodiment 30. The IL-18 variant according to Embodiment 29, wherein the amino acid modification at amino acid position 111 is an asparagine-to-histidine substitution.

[0226] Embodiment 31. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or a methionine-to-leucine substitution at amino acid position 60, preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48 and the methionine-to-lysine substitution at amino acid position 60. Embodiment 32. The IL-18 variant according to Embodiment 31 , wherein the IL- 18 variant comprises an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO:

[0227] 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 48 and 60.

[0228] Embodiment 33. The IL-18 variant according to Embodiment 32, wherein the IL- 18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 48 and 60.

[0229] Embodiment 34. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60, preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , and the methionine-to-lysine substitution at amino acid position 60.

[0230] Embodiment 35. The IL-18 variant according to Embodiment 34, wherein the IL- 18 variant comprises an amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO:

[0231] 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 48, 51 , and 60. Embodiment 36. The IL-18 variant according to Embodiment 35, wherein the IL- 18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 48, 51 , and 60.

[0232] Embodiment 37. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and iv) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111 , preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , the methionine-to-lysine substitution at amino acid position 60, and the asparagine-to-histidine substitution at amino acid position 111.

[0233] Embodiment 38. The IL-18 variant according to Embodiment 37, wherein the IL- 18 variant comprises an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 48, 51 , 60, and 111.

[0234] Embodiment 39. The IL-18 variant according to Embodiment 38, wherein the IL- 18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 48, 51 , 60, and 111.

[0235] Embodiment 40. The IL-18 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-18 variant comprises: i) the leucine-to-serine substitution or leucine-to-aspartate substitution at amino acid position 9; and ii) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and iii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iv) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and v) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111 , preferably wherein the IL-18 variant comprises the leucine-to-serine substitution at amino acid position 9, the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , the methionine-to-lysine substitution at amino acid position 60, and the asparagine- to-histidine substitution at amino acid position 111.

[0236] Embodiment 41. The IL-18 variant according to Embodiment 40, wherein the IL- 18 variant comprises an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 9, 48, 51 , 60, and 111.

[0237] Embodiment 42. The IL-18 variant according to Embodiment 41 , wherein the IL- 18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 9, 48, 51 , 60, and 111.

[0238] Embodiment 43. The IL-18 variant according to any preceding Embodiment, wherein the IL-18 variant is fused to an interleulkin-18 receptor (IL-18R) molecule.

[0239] Embodiment 44. The IL-18 variant according to any preceding Embodiment, wherein the IL-18 variant is fused to a signal peptide.

[0240] Embodiment 45. The IL-18 variant according to Embodiment 44, wherein the signal peptide is a periplasmic signal peptide.

[0241] Embodiment 46. The IL-18 variant according to Embodiments 44 or 45, wherein the signal peptide is fused to the IL-18 variant via a spacer sequence, preferably wherein the spacer sequence is between 1 to 5 amino acids in length.

[0242] Embodiment 47. The IL-18 variant according to any preceding Embodiment, for use as a therapy.

[0243] Embodiment 48. The IL-18 variant according to any preceding Embodiment, for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0244] Embodiment 49. The IL-18 variant for use according to Embodiment 48, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

[0245] Embodiment 50. The IL-18 variant for use according to Embodiment 49, wherein the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma. Embodiment 51. The IL-18 variant for use according to any one of Embodiments 47 to 50, wherein the IL-18 variant is administered to the subject via local instillation, intra-peritoneally, intrapleurally, intravesically, peritumoral injection, intratumoural injection or orally.

[0246] Embodiment 52. A nucleic acid molecule encoding the IL-18 variant as defined in any one of Embodiments 1 to 46.

[0247] Embodiment 53. The nucleic acid molecule according to Embodiment 52, wherein the nucleic acid molecule comprises a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5 retains the amino acid modifications at the relevant amino acid positions.

[0248] Embodiment 54. The nucleic acid molecule according to Embodiment 53, wherein the nucleic acid molecule comprises a sequence encoding an amino acid sequence comprising at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5 retains the amino acid modifications at the relevant amino acid positions.

[0249] Embodiment 55. The nucleic acid molecule according to any one of Embodiments 52 to 54, wherein the nucleic acid molecule comprises a DNA molecule.

[0250] Embodiment 56. The nucleic acid molecule according to any one of Embodiments 52 to 55, wherein the nucleic acid molecule comprises an RNA molecule.

[0251] Embodiment 57. The nucleic acid molecule according to Embodiment 56, wherein the RNA molecule comprises an mRNA molecule.

[0252] Embodiment 58. A Gram-negative bacterium comprising the IL-18 variant according to any one of Embodiments 1 to 46, or the nucleic acid molecule according to any one of Embodiments 52 to 57. Embodiment 59. The Gram-negative bacterium according to Embodiment 58, wherein the Gram-negative bacterium is a live attenuated Gram-negative bacterium.

[0253] Embodiment 60. The Gram-negative bacterium according to Embodiment 59, wherein the live attenuated Gram-negative bacterium is a Salmonella spp.

[0254] Embodiment 61. The Gram-negative bacterium according to Embodiment 60, wherein the live attenuated Gram-negative bacterium is Salmonella enterica, preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi.

[0255] Embodiment 62. The Gram-negative bacterium according to Embodiment 61 , wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi ZH9.

[0256] Embodiment 63. The Gram-negative bacterium according to any one of

[0257] Embodiments 58 to 62, for use as a therapy.

[0258] Embodiment 64. The Gram-negative bacterium according to any one of

[0259] Embodiments 58 to 62, for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0260] Embodiment 65. The Gram-negative bacterium for use according to Embodiment

[0261] 64, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

[0262] Embodiment 66. The Gram-negative bacterium for use according to Embodiment

[0263] 65, wherein the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma. Embodiment 67. The Gram-negative bacterium for use according to any one of Embodiments 63 to 66, wherein the Gram-negative bacterium is administered to the subject via local instillation, intra-peritoneally, intrapleurally, intravesically, peritumoral injection, or intratumoural injection, or orally.

[0264] Embodiment 68. Use of the IL-18 variant as defined in any one of Embodiments 1 to 46, or the nucleic acid molecule as defined in any one of Embodiments 52 to 57, or the Gram-negative bacterium of as defined in any one of Embodiments 58 to 62, in the manufacture of a medicament for a therapy.

[0265] Embodiment 69. The use according to Embodiment 68, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0266] Embodiment 70. A method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject IL-18 variant as defined in any one of Embodiments 1 to 46, or the nucleic acid molecule as defined in any one of Embodiments 52 to 57, or the Gram-negative bacterium of as defined in any one of Embodiments 58 to 62.

[0267] Embodiment 71. A bacterium comprising an interleukin-18 (IL-18) variant, wherein in comparison to wild-type IL-18, the IL-18 variant comprises one or more amino acid modifications in a region adjacent to a receptor interaction surface of the IL- 18 variant.

[0268] Embodiment 72. The bacterium according to Embodiment 71 , wherein the bacterium is a live attenuated bacterium.

[0269] Embodiment 73. The bacterium according to Embodiment 72, wherein bacterium is a live attenuated Gram-negative bacterium.

[0270] Embodiment 74. The bacterium according to Embodiment 73, wherein the live attenuated Gram-negative bacterium is a Salmonella spp, preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica, more preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi, even more preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi ZH9.

[0271] Embodiment 75. The bacterium according to any preceding Embodiment, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1.

[0272] Embodiment 76. The bacterium according to Embodiment 75, wherein in comparison to SEQ ID NO: 1 , the amino acid modifications occur at one or more of amino acid positions 9, 46, 48, 51 , 60, 80, 105, 110, and / or 111 , or any combination thereof.

[0273] Embodiment 77. The bacterium according to preceding Embodiment, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1.

[0274] Embodiment 78. The bacterium according to any preceding Embodiment, wherein the IL-18 variant preferentially binds an I L-18 receptor (I L-18Ra and I L-18Rp) over an IL-18 binding protein (IL-18BP).

[0275] Embodiment 79. The bacterium according to Embodiment 76 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 9, 46, 48, and / or 80 is a hydrophobic-to-hydrophilic substitution.

[0276] Embodiment 80. The bacterium according to Embodiment 79, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 9 is a leucine-to- serine substitution or a leucine-to-aspartate substitution.

[0277] Embodiment 81. The bacterium according to Embodiment 80, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 9 is a leucine-to- serine substitution. Embodiment 82. The bacterium according to any one of Embodiments 79 to 81 , wherein the hydrophobic-to-hydrophilic substitution at amino acid position 46 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

[0278] Embodiment 83. The bacterium according to Embodiment 82, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 46 is an isoleucine- to-aspartate substitution.

[0279] Embodiment 84. The bacterium according to any one of Embodiments 79 to 83, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 48 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

[0280] Embodiment 85. The bacterium according to Embodiment 84, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 48 is an isoleucine- to-aspartate substitution.

[0281] Embodiment 86. The bacterium according to any one of Embodiments 79 to 85, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 80 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

[0282] Embodiment 87. The bacterium according to Embodiment 86, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 80 is an isoleucine- to-aspartate substitution.

[0283] Embodiment 88. The bacterium according to Embodiment 76 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 51 involves substitution of a wild-type amino acid for a polar amino acid.

[0284] Embodiment 89. The bacterium according to Embodiment 88, wherein the amino acid modification at amino acid position 51 is a methionine-to-lysine substitution or a methionine-to-threonine substitution.

[0285] Embodiment 90. The bacterium according to Embodiment 89, wherein the amino acid modification at amino acid position 51 is a methionine-to-threonine substitution. Embodiment 91. The bacterium according to Embodiment 76 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 60 involves substitution of a wild-type amino acid for a large amino acid.

[0286] Embodiment 92. The bacterium according to Embodiment 91 , wherein the amino acid modification at amino acid position 60 is a methionine-to-lysine substitution or a methionine-to-leucine substitution.

[0287] Embodiment 93. The bacterium according to Embodiment 92, wherein the amino acid modification at amino acid position 60 is a methionine-to-lysine substitution.

[0288] Embodiment 94. The bacterium according to Embodiment 76 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 105 involves substitution of a wild-type amino acid for a polar amino acid.

[0289] Embodiment 95. The bacterium according to Embodiment 94, wherein the amino acid modification at amino acid position 105 is a serine-to-aspartate substitution or a serine-to-asparagine substitution.

[0290] Embodiment 96. The bacterium according to Embodiment 95, wherein the amino acid modification at amino acid position 105 is a serine-to-aspartate substitution.

[0291] Embodiment 97. The bacterium according to Embodiment 76 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 110 involves substitution of a wild-type amino acid for (a) a polar amino acid, or (b) a neutral amino acid.

[0292] Embodiment 98. The bacterium according to Embodiment 97, wherein the amino acid modification at amino acid position 110 is (a) an aspartate-to-serine substitution or an aspartate-to-lysine substitution, or (b) an aspartate-to-glycine substitution.

[0293] Embodiment 99. The bacterium according to Embodiment 98, wherein the amino acid modification at amino acid position 110 is an aspartate-to-lysine substitution. Embodiment 100. The bacterium according to Embodiment 86 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 111 involves substitution of a wild-type amino acid for (a) a positively charged amino acid, or (b) a neutral amino acid.

[0294] Embodiment 101. The bacterium according to Embodiment 100, wherein the amino acid modification at amino acid position 111 is (a) an asparagine-to-arginine substitution or an asparagine-to-histidine substitution, or (b) an asparagine-to- glycine substitution.

[0295] Embodiment 102. The bacterium according to Embodiment 101 , wherein the amino acid modification at amino acid position 111 is an asparagine-to-histidine substitution.

[0296] Embodiment 103. The bacterium according to Embodiment 76 or any one of those pendent thereon, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or a methionine-to-leucine substitution at amino acid position 60, preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48 and the methionine-to-lysine substitution at amino acid position 60.

[0297] Embodiment 104. The bacterium according to Embodiment 103, wherein the IL- 18 variant comprises an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 48 and 60.

[0298] Embodiment 105. The bacterium according to Embodiment 104, wherein the IL- 18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 48 and 60.

[0299] Embodiment 106. The bacterium according to Embodiment 76 or any one of those pendent thereon, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60, preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , and the methionine-to-lysine substitution at amino acid position 60.

[0300] Embodiment 107. The bacterium according to Embodiment 106, wherein the IL- 18 variant comprises an amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 48, 51 , and 60.

[0301] Embodiment 108. The bacterium according to Embodiment 107, wherein the IL- 18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 48, 51 , and 60.

[0302] Embodiment 109. The IL-18 variant according to Embodiment 76 or any one of those pendent thereon, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and iv) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111 , preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , the methionine-to-lysine substitution at amino acid position 60, and the asparagine-to-histidine substitution at amino acid position 111.

[0303] Embodiment 110. The bacterium according to Embodiment 109, wherein the IL- 18 variant comprises an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 48, 51 , 60, and 111.

[0304] Embodiment 111. The bacterium according to Embodiment 110, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 48, 51 , 60, and 111.

[0305] Embodiment 112. The bacterium according to Embodiment 76 or any one of those pendent thereon, wherein the IL-18 variant comprises: i) the leucine-to-serine substitution or leucine-to-aspartate substitution at amino acid position 9; and ii) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and iii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iv) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and v) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111 , preferably wherein the IL-18 variant comprises the leucine-to-serine substitution at amino acid position 9, the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , the methionine-to-lysine substitution at amino acid position 60, and the asparagine- to-histidine substitution at amino acid position 111.

[0306] Embodiment 113. The bacterium according to Embodiment 112, wherein the IL- 18 variant comprises an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 9, 48, 51 , 60, and 111.

[0307] Embodiment 114. The bacterium according to Embodiment 113, wherein the IL- 18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 9, 48, 51 , 60, and 111.

[0308] Embodiment 115. The bacterium according to any preceding Embodiment, wherein the IL-18 variant is fused to an interleulkin-18 receptor (IL-18R) molecule.

[0309] Embodiment 116. The bacterium according to any preceding Embodiment, wherein the IL-18 variant is fused to a signal peptide. Embodiment 117. The bacterium according to Embodiment 116, wherein the signal peptide is a periplasmic signal peptide.

[0310] Embodiment 118. The bacterium according to Embodiments 116 or 117, wherein the signal peptide is fused to the IL-18 variant via a spacer sequence, preferably wherein the spacer sequence is between 1 to 5 amino acids in length.

[0311] SEQUENCES FORMING PART OF THE DESCRIPTION

[0312] SEQ ID NO: 1 (unmodified IL-18)

[0313] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRG

[0314] MAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS

[0315] SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0316] SEQ ID NO: 2 (V17)

[0317] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFDISMYKDSQPRG

[0318] KAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS

[0319] SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0320] SEQ ID NO: 3 (V25)

[0321] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFDISTYKDSQPRG

[0322] KAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS

[0323] SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0324] SEQ ID NO: 4 (V27)

[0325] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFDISTYKDSQPRG

[0326] KAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDHKMQFESS

[0327] SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0328] SEQ ID NO: 5 (V59)

[0329] YFGKLESKSSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFDISTYKDSQPRG

[0330] KAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDHKMQFESS

[0331] SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED REFERENCES

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Claims

CLAIMS1. An interleukin-18 (IL-18) variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at the following amino acid positions: i) 48; and ii) 51 and / or 60.

2. The IL-18 variant according to claim 1 , wherein in comparison to SEQ ID NO:1 , the IL-18 variant further comprises an amino acid modification at one or more of amino acid positions 9, 46, 80, 105, 110, and / or 111.

3. The IL-18 variant according to claim 1 or 2, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1.

4. The IL-18 variant according to any preceding claim, wherein the IL-18 variant preferentially binds to an IL-18 receptor over IL-18 binding protein.

5. The IL-18 variant according to any preceding claim, wherein the amino acid modification at amino acid position 48 is a hydrophobic-to-hydrophilic substitution.

6. The IL-18 variant according to claim 5, wherein the amino acid modification at amino acid position 48 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

7. The-18 variant according to claim 6, wherein the amino acid modification at amino acid position 48 is an isoleucine-to-aspartate substitution.

8. The IL-18 variant according to any preceding claim, wherein the amino acid modification at amino acid position 51 involves substitution of a wild-type amino acid for a polar amino acid.

9. The IL-18 variant according to claim 8, wherein the amino acid modification at amino acid position 51 is a methionine-to-lysine substitution or a methionine-to-threonine substitution.

10. The IL-18 variant according to claim 9, wherein the amino acid modification at amino acid position 51 is a methionine-to-threonine substitution.

11. The IL-18 variant according to any preceding claim, wherein the amino acid modification at amino acid position 60 involves substitution of a wild-type amino acid for a large amino acid.

12. The IL-18 variant according to claim 11 , wherein the amino acid modification at amino acid position 60 is a methionine-to-lysine substitution or a methionine-to-leucine substitution.

13. The IL-18 variant according to claim 12, wherein the amino acid modification at amino acid position 60 is a methionine-to-lysine substitution.

14. The IL-18 variant according to any of claims 2 to 13, wherein the amino acid modification at amino acid position 9, 46 and / or 80 is a hydrophobic-to- hydrophilic substitution.

15. The IL-18 variant according to claim 14, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 9 is a leucine-to-serine substitution or a leucine-to-aspartate substitution.

16. The IL-18 variant according to claim 15, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 9 is a leucine-to-serine substitution.

17. The IL-18 variant according to any one of claims 2 to 16, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 46 is an isoleucine- to-aspartate substitution or an isoleucine-to-lysine substitution.

18. The IL-18 variant according to claim 17, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 46 is an isoleucine-to-aspartate substitution.

19. The IL-18 variant according to any one of claims 2 to 18, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 80 is an isoleucine- to-aspartate substitution or an isoleucine-to-lysine substitution.

20. The IL-18 variant according to claim 19, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 80 is an isoleucine-to-aspartate substitution.

21. The IL-18 variant according to any of claims 2 to 20, wherein the amino acid modification at amino acid position 105 involves substitution of a wild-type amino acid for a polar amino acid.

22. The IL-18 variant according to claim 21 , wherein the amino acid modification at amino acid position 105 is a serine-to-aspartate substitution or a serine-to-asparagine substitution.

23. The IL-18 variant according to claim 22, wherein the amino acid modification at amino acid position 105 is a serine-to-aspartate substitution.

24. The IL-18 variant according to any of claims 2 to 23, wherein the amino acid modification at amino acid position 110 involves substitution of a wild-type amino acid for (a) a polar amino acid, or (b) a neutral amino acid.

25. The IL-18 variant according to claim 24, wherein the amino acid modification at amino acid position 110 is (a) an aspartate-to-serine substitution or an aspartate-to-lysine substitution, or (b) an aspartate-to-glycine substitution.

26. The IL-18 variant according to claim 25, wherein the amino acid modification at amino acid position 110 is an aspartate-to-lysine substitution.

27. The IL-18 variant according to any of claims 2 to 26, wherein the amino acid modification at amino acid position 111 involves substitution of a wild-type amino acid for (a) a positively charged amino acid, or (b) a neutral amino acid.

28. The IL-18 variant according to claim 27, wherein the amino acid modification at amino acid position 111 is (a) an asparagine-to-arginine substitution or an asparagine-to-histidine substitution, or (b) an asparagine-to- glycine substitution.

29. The IL-18 variant according to claim 28, wherein the amino acid modification at amino acid position 111 is an asparagine-to-histidine substitution.

30. The IL-18 variant according to any preceding claim, wherein the IL-18 variant comprises an amino acid mutation at amino acid positions 48 and 60.

31. The IL-18 variant according to claim 30, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or a methionine-to-leucine substitution at amino acid position 60, preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48 and the methionine-to-lysine substitution at amino acid position 60.

32. The IL-18 variant according to claim 31 , wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 48 and 60.

33. The IL-18 variant according to claim 32, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 48 and 60.

34. The IL-18 variant according to any preceding claim, wherein the IL-18 variant comprises an amino acid mutation at amino acid positions 48, 51 and 60.

35. The IL-18 variant according to claim 34, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60, preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , and the methionine-to-lysine substitution at amino acid position 60.

36. The IL-18 variant according to claim 35, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 48, 51 , and 60.

37. The IL-18 variant according to claim 36, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 48, 51 , and 60.

38. The IL-18 variant according to any of claims 2 to 37, wherein the IL-18 variant comprises an amino acid mutation at amino acid positions 48, 51 , 60 and 111.

39. The IL-18 variant according to claim 38, wherein the IL-18 variant comprises: i) the isoleucine-to-aspartate or isoleucine-to-lysine substitution at amino acid position 48; and ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and iv) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111 , preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , the methionine-to-lysine substitution at amino acid position 60, and the asparagine-to-histidine substitution at amino acid position 111.

40. The IL-18 variant according to claim 39, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 48, 51 , 60, and 111.

41. The IL-18 variant according to claim 40, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acidmodifications at amino acid positions 48, 51 , 60, and 111.

42. The IL-18 variant according to claim 38, wherein the IL-18 variant comprises: i) the leucine-to-serine substitution or leucine-to-aspartate substitution at amino acid position 9; and ii) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; and iii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51 ; and iv) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and v) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111 , preferably wherein the IL-18 variant comprises the leucine-to-serine substitution at amino acid position 9, the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , the methionine-to-lysine substitution at amino acid position 60, and the asparagine- to-histidine substitution at amino acid position 111.

42. The IL-18 variant according to claim 41 , wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 9, 48, 51 , 60, and 111.

43. The IL-18 variant according to claim 42, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 9, 48, 51 , 60, and 111.

44. An IL-18 variant, wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-18 variant comprises an amino acid modification at amino acid positions 51 and 60.

45. The IL-18 variant according to claim 44, wherein the amino acid modification at amino acid position 51 involves substitution of a wild-type amino acid for a polar amino acid, preferably wherein the amino acid modification at amino acid position 51 is a methionine-to-lysine substitution or a methionine-to- threonine substitution, more preferably a methionine-to-threonine substitution, and wherein the amino acid modification at amino acid position 60 involves substitution of a wild-type amino acid for a large amino acid, preferably wherein the amino acid modification at amino acid position 60 is a methionine-to- lysine substitution or a methionine-to-leucine substitution, more preferably a methionine-to-lysine substitution.

46. The IL-18 variant according to any preceding claim, wherein the IL-18 variant is fused to an interleulkin-18 receptor (IL-18R) molecule.

47. The IL-18 variant according to any preceding claim, wherein the IL-18 variant is fused to a signal peptide.

48. The IL-18 variant according to claim 47, wherein the signal peptide is a periplasmic signal peptide.

49. The IL-18 variant according to claims 47 or 48, wherein the signal peptide is fused to the IL-18 variant via a spacer sequence, preferably wherein the spacer sequence is between 1 to 5 amino acids in length.

50. A nucleic acid molecule encoding the IL-18 variant according to any of claims 1 to 49.51 . The nucleic acid molecule according to claim 50, wherein the nucleic acid molecule comprises a sequence encoding an amino acid sequence according toany one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5 retains the amino acid modifications at the relevant amino acid positions.

52. The nucleic acid molecule according to claim 51 , wherein the nucleic acid molecule comprises a sequence encoding an amino acid sequence comprising at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5 retains the amino acid modifications at the relevant amino acid positions.

53. The nucleic acid molecule according to any one of claims 50 to 52, wherein the nucleic acid molecule comprises a DNA molecule.

54. The nucleic acid molecule according to any one of claims 50 to 53, wherein the nucleic acid molecule comprises an RNA molecule.

55. The nucleic acid molecule according to claim 54, wherein the RNA molecule comprises an mRNA molecule.

56. A bacterium comprising the IL-18 variant according to any one of claims 1 to 49, or the nucleic acid molecule according to any one of claims 50 to 55.

57. The bacterium according to claim 56, wherein the Gram-negative bacterium is a live attenuated Gram-negative bacterium.

58. The bacterium according to claim 57, wherein the live attenuated Gramnegative bacterium is a Salmonella spp.

59. The bacterium according to claim 58, wherein the live attenuated Gramnegative bacterium is Salmonella enterica, preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi.

60. The bacterium according to claim 59, wherein the live attenuated Gramnegative bacterium is Salmonella enterica serovar Typhi ZH9.61 . The IL-18 variant according to any of claims 1 to 49, or the nucleic acid according to any of claims 50 to 55, or the bacterium according to any of claims 56 to 60, for use as a therapy.

62. The IL-18 variant, nucleic acid molecule or bacterium according to claim 61 , for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

63. The IL-18 variant, nucleic acid molecule or bacterium for use according to claim 62, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

64. The IL-18 variant, nucleic acid molecule or bacterium for use according to claim 63, wherein the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

65. The IL-18 variant, nucleic acid molecule or bacterium for use according to any one of claims 61 to 64, wherein the Gram-negative bacterium is administered to the subject via local instillation, intra-peritoneally, intrapleurally, intravesically, peritumoral injection, or intratumoural injection, or orally.

66. Use of the IL-18 variant according to any of claims 1 to 49, or the nucleic acid molecule according to any of claims 50 to 55, or the bacterium according to any of claims 56 to 60, in the manufacture of a medicament for a therapy.

67. The use according to claim 66, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

68. A method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering tothe subject IL-18 variant according to any of claims 1 to 49, or the nucleic acid molecule according to any of claims 50 to 55, or the bacterium according to any of claims 56 to 60.

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