Interleukin 15 variant

Abstract

There is provided a bacterium comprising an interleukin-15 (IL-15) variant, wherein in comparison to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Rα) interaction surface.

Description

[0001] INTERLEUKIN 15 VARIANT

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to interleukin-15 (IL-15) 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 15 (IL-15) and IL-15 agonist molecules are considered as promising immunotherapeutics because of their role in CD8 T cells and NK activation and proliferation (Guo, 2017; Robinson, 2017). Clinical trials are currently ongoing to test a combination therapy of intravesical BCG plus ANKTIVA® (former ALT-803, N803), an IL-15 super-agonist molecule (developed by ImmunityBio, former Aitor BioScience).

[0007] However, a challenge associated with the use of IL-15 as an immuno-stimulatory anti-cancer treatment, 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-15) 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 (Behar 2011 , Devi 2016, Nausch 2013).

[0009] The same limitations for cytokine expression efficiency observed in E. coli have also been observed in strains of Salmonella. In fact, it has been observed previously by the inventors of the present invention that the expression of human IL-15 in Salmonella Typhi ZH9 from the temperature inducible LPR promoter, in cultures grown at the suboptimal inducing temperature of 37°C, results in accumulation of IL-15 in insoluble aggregates.

[0010] 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.

[0011] 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. SUMMARY OF INVENTION

[0012] The present invention provides IL-15 variants with both optimised solubility and preserved bioactivity. The inventors of the present invention have surprisingly identified certain combinations of mutations in IL-15 that result in IL-15 variants which are more soluble than wild-type IL-15, and retain functionality. 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.

[0013] Accordingly, in a first aspect of the invention, there is provided a bacterium comprising an interleukin-15 (IL-15) variant, wherein in comparison to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface.

[0014] In a second aspect of the invention, there is provided an interleukin-15 (IL-15) variant, wherein wild-type IL-15 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-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, and wherein the one or more amino acid modifications comprise any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophobic substitution at amino acid position 49; iii) a hydrophobic-to-hydrophilic substitution at amino acid position 52; and / or iv) a serine-to-proline substitution at amino acid position 75; preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL- 15Ra) interaction surface.

[0015] In a third aspect of the invention, there is provided a nucleic acid molecule encoding the IL-15 variant as defined in the second aspect of the invention. In a fourth aspect of the invention, there is provided a bacterium comprising the IL-15 variant according to the second aspect 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 the bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule 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 bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third aspect of the invention, 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 bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third 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 structure of an interleukin-15 (IL-15, PDB ID: 4GS7) variant, indicating amino acid modifications in a surface region adjacent to the IL-15Ra interaction surface. Also shown are IL-15Ra, IL-2Rp (IL-15Rp), and GammaC (YC).

[0022] Figure 2 shows a schematic representation of IL-15 trans-presentation and biological function. Figure 3 shows the sequence-structure relationship of wild-type IL-15 and its interaction with cognate ligands. Short and long (mammalian) signal peptide sequences of pre-IL-15 (A), mature IL-15 (B), and IL-15 tertiary structure (C) are shown.

[0023] Figure 4 shows aggregation propensity and hydrophobicity score of IL-15 calculated using AggreScan and AggreScan 3D.

[0024] Figure 5 shows a summary of IL-15 and IL-15 agonists used in immunotherapy.

[0025] Figure 6 shows the structure of an IL-15 variant, indicating amino acid positions 45 and / or 49 and / or 52. Also shown are IL-15Ra, I L-2Rp, and GammaC (yc).

[0026] Figures 7A-7C show the structure of IL-15 in quaternary complex with IL-15Ra, IL-15R , and GammaC (yc) (PDB: 4GS7).

[0027] Figures 8A-8D demonstrate the solubility and functionality of different IL-15 variants. (A) shows the total number of soluble variants obtained through different mutagenesis strategies. (B) shows the solubility score distribution for each of the mutagenesis strategies, with in silico designs showing an overall higher success rate and epPCR variants showing the higher spread. (C) shows the total number of active variants obtained through different mutagenesis strategies. (D) shows the activity measurement as Relative Luminescence Units (RLU) from IL-15 Bioassay Cells (Promega) reporter cell line distributions for each mutagenesis strategy.

[0028] Figures 9A-9C demonstrate the biological activity of IL-15 / SAg variants using three independent assays (IL-15 Bioassay Cells, NK proliferation assay, and IFNy production assay).

[0029] Figures 10A-10B show protein purification data confirming the isolation of different IL-15 variants and IL-15 SAg variants. Figures 11A and 11B show pre-clinical candidate selection of IL-15 variants. The results of incubating a gradient of purified IL-15 variants for 6 hours with a reporter cell line are shown.

[0030] Figure 12 shows in vitro human cell assays. The results of incubating IL-15 variants with primary NK cells for 24 hours at 71 nM (experiment 1) and 58 nM (experiment 2) are shown.

[0031] Figure 13 shows the results of IL-15 variant mediated NK activation in mouse splenocytes after incubation with IL-15 variants for 72 hours.

[0032] DETAILED DESCRIPTION

[0033] The present invention provides optimised IL-15 variants (and / or super agonists thereof, i.e., SAg variants) which are both soluble and functional, and are therefore optimal candidates for expression in efficient secretion systems which allow the production and secretion of said IL-15 variants and SAg variants.

[0034] Accordingly, in a first aspect the invention provides a bacterium comprising an interleukin-15 (IL-15) variant, wherein in comparison to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface (Figure 1).

[0035] 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. Interleukin-15 (IL-15) is a four alpha-helix bundle cytokine belonging to the same family as IL-2, IL-4, IL-7, IL-9, and IL-21. IL-15 is constitutively expressed by a wide variety of cells including dendritic cells, monocytes, macrophages, bone marrow stromal cells and intestinal epithelial cells. Cells expressing IL-15 also express its high affinity receptor-a, IL-15Ra. Unlike most cytokines, which are secreted in soluble form, IL-15 has a unique form of expression whereby it is expressed in association with its high affinity receptor, IL-15Ra, and is shuttled as a heterodimeric complex to the surface of IL-15-producing cells. IL-15 can also be secreted as a soluble IL-15 independently from IL-15Ra. The IL-15 / IL-15Ra complex is a cell surface complex which efficiently stimulates neighbouring cells through the I L-2 / I L-15Rp and GammaC (yc) complex (I L-15R(3y) via a mechanism of trans-presentation (Figure 2). IL-15 preferentially stimulates NK (natural killer) and CD8+ T cells activation, proliferation and cytolytic activity (Guo, 2007; Santana-Carrero, 2019).

[0036] IL-15 trans-presentation mediates cells responses during homeostasis. There is evidence that the I L-15Ra / l L-15 complexes are cleaved from the surface of presenting cells in response to numerous types of immune stimulation such as total body irradiation, TLR stimulation, virus infections, CD40 stimulation, type I IFNs (IFN-I) and activation of the stimulator of interferon genes (STING) pathway, thus releasing a soluble heterodimeric I L-15 / I L-15Ra complex (Bergamaschi, 2012; Anthony, 2016).

[0037] Human IL-15 is a protein of 162 amino-acid (18,086 kDa) containing 4 helices, 2 disulphide bonds and 1 glycosylated site (Figure 3). It has two isoforms produced by alternatively spliced transcripts: one form with a short signal peptide (SSP), which contains a 21 amino acid leader peptide and remains in the cell cytosol, and one other with a long signal peptide (LSP), which contains a 48 amino acid signal peptide that allows for secretion of the protein into the extracellular environment (Saeed and Revell, 2001 ; Duitman, 2008). This secretion occurs through independent export into the Golgi Apparatus of the signal and the receptor alpha, followed by high affinity interaction between signal and receptor, and export into the environment of the complex via the receptor alpha. The signal peptide is cleaved always on the same position, resulting in the same mature form of IL-15, possibly through the highly conserved sequence N-GLPKTEA / NW-C, although SSP IL-15 is already shown to be bioactive, indicating certain flexibility to the presence of N-terminus peptides (Bergamaschi, 2009). The mature IL-15 is then folded in a four-helix bundle motif (the four a-helices are structured together lengthwise in antiparallel orientation), with 16-21 amino acids per a-chain, where alpha chain a1 interacts with a3 and a2 with a4 (interactions between helices represented as dashed lines in Figure 3, with key residues involved in proteinprotein interactions being highlighted). There is a disulphide bond that stabilises this latter interaction between Cys42-Cys85, and a second one between Cys35- Cys88. There are two highly disordered regions that loop between a1 a2 (loop 1- 2) and a3 a4 (loop 3-4).

[0038] The crystal structure of IL-15 co-coordinated to receptors I L-2Rp, yc, and IL-15Ra; has been resolved (Ring, 2012). This shows that the signalling protein interacts with IL-2Rp via polar interactions with helices a1 and a3, with the interactions IL15_D8:IL-2Rp {H133, Y134}, I L15_D61 {I L2R[3_{K71 } and IL15_N65:IL-2Rp {R42, Q70, Y134} being essential to stabilise this interaction, consistent with previously published mutagenesis studies (Eisenman, 2002; Pettit 1997). Other amino acids involved in stabilising the interaction would be I68, L69, K10, K11 and S7.

[0039] On the other hand, interactions with yc show a more general interaction, without a highly determined chemical signature, highlighting the promiscuity of yc to interact with multiple cytokines. Interaction with yc occurs via chains a1 and a4, with Q108, M109, and N112 being the key amino acids involved in this interaction. An additional site of contact is made with the extended alpha chain to compensate for the smaller size of I L-15.

[0040] Finally, interaction with IL-15Ra has been found to occur through a2. Chirifu et al (Chirifu, 2007) describe the interactions between IL-15 and IL-15Ra to be weak electrostatic (Van der Waals) interactions, and identified the following amino acids in the protein-protein interaction: D22, T24, Y26, E46, Q48, E53, E87, E90. A disulphide bond between C42-C88 is thought to stabilise contact with IL-15Ra and confer rigidity to the molecule.

[0041] The amino acid sequence of IL-15 has been analysed by the inventors of the present invention with AggreScan, ProtScale, and AggreScan3D to identify potential aggregation sites, which would be responsible for protein aggregation and formation of inclusion bodies upon recombinant overexpression in E. coli or Salmonella Typhi. The regions with higher propensity to aggregate are those forming the alpha helices (Figure 4), as these are the regions involved in proteinprotein interactions. Both rational design and directed evolution approaches discussed below aim to minimise these hydrophobic interactions without altering IL-15 binding properties to its receptors.

[0042] Given its role in the stimulation of proliferation and cytotoxic function of CD8 T cells and NK cells, IL-15 has been widely tested as a cancer immunotherapeutic agent. The efficacy of IL-15 is limited by the short half-life of the protein in vivo. A number of IL-15 variants have been generated to improve protein stability and efficacy.

[0043] Figure 5 depicts five (1-5) different IL-15-based agents that are used to stimulate cytotoxic T cell and NK cell responses. In order of increasing IL-15 signal strength / duration, (1) rlL-15 was the first form of IL-15 to be examined in vivo. When administered, rlL-15 is believed to predominantly bind to cell surface IL- 15Ra where it is trans-presented to IL-15 responsive cells. (2) Heterodimeric IL- 15 is the natural form of IL-15 that is cleaved from cells and can stimulate responses independent of cell interactions. Heterodimeric hlL-15 is being produced as a therapeutic by Novartis. (3) RLI (Cytune Pharmaceuticals) is a fusion protein consisting of IL-15 linked to the sushi (cytokine-binding) domain of the IL-15Ra that can act as a soluble IL-15 agonist. (4) IL-15 / IL-15Ra-Fc complexes are generated by mixing commercially available IL-15Ra-Fc chimeric fusion protein with rlL-15 and have been used extensively in preclinical studies. (5) ANKTIVA® (former ALT-803, N-803) (ImmunityBio, former Aitor Pharmaceutical) is an IL-15 superagonist consisting of mutated IL-15 (asparagine at position 72 within the mature IL-15 amino acid sequence replaced with an aspartic residue), which has an increased affinity for CD122-expressing immune cells, linked to the sushi domain of the IL-15Ra that is fused to an Fc fragment. Signal strength and duration of these IL-15-based agents increases with increased abundance of agonist, increased in vivo half-life (imparted by IL-15 binding to IL-15Ra and presence of an Fc fragment), dimerization of the agonists, and increased affinity for the IL-2Rp / yC complex (imparted by IL-15 binding to the sushi domain and the mutation present in ALT-803).

[0044] There is a clear need to develop IL-15 variants which are soluble and exhibit biological activity. 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 or unchanged form. In one embodiment, the wild-type IL-15 is mature (i.e., unmodified) IL-15. As used herein, the term “parental IL-15” refers to the baseline sequence from which all IL-15 variants according to the present invention are derived. The parental IL-15 is also referred to as IL-15M40 or M40 (SEQ ID NO: 16), is derived from wild-type IL-15, which comprises an amino acid sequence according to SEQ ID NO: 1. The difference between wildtype IL-15 and M40 IL-15 is the presence of an amino acid modification in which serine at amino acid position 75 of SEQ ID NO: 1 is substituted for a proline (S75P). As such, the S75P mutation gives rise to SEQ ID NO: 16 (parental) from SEQ ID NO: 1 (wild-type). Accordingly, in some embodiments, the IL-15 variant comprises an amino acid modification at position 75. In preferred embodiments, the IL-15 variant comprises a serine-to-proline substitution at position 75 (S75P). Any of the IL-15 variants described herein may feature the S75P mutation. The S75P mutation can provide increased solubility of the IL-15 variant to further stabilise the IL-15 variant and therefore optimise its expression in bacteria.

[0045] In comparison to “wild-type” IL-15, the term “interleukin variant” refers to an IL-15 molecule which, in comparison to wild-type IL-15, comprises one or more amino acid modifications. 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.

[0046] 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.

[0047] 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.

[0048] IL-15 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-15 variant may preferably comprise one, two, or three amino acid modifications.

[0049] In accordance with a first aspect of the invention, there is provided a bacterium comprising an IL-15 variant, wherein in comparison to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface.

[0050] The amino acid modifications will preferably be present in a surface region of the IL-15 variant. As used herein, the term “surface region” refers to the part of a protein which is exposed to solvent. In the overall three-dimensional tertiary structure of the protein, the protein surface is commonly referred to as the solvent- accessible region 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. The amino acid modifications on the IL-15 variant of the present invention will preferably involve those amino acids which are exposed to the solvent and are on the outer / exterior surface of the protein. Modifications to amino acids on the protein surface often implicate protein-protein interactions, as protein surfaces typically comprise protein-protein interfaces (i.e., the molecular surfaces through which proteins make contact and interact with one another). The protein surface may comprise large, flat interfaces, and / or smaller concavities or even single residues which may form an anchoring site for one or more interacting proteins. The protein surfaces of interacting proteins may be shape and / or chemically complementary. As would be understood by a person of skill in the art, modifications to or around the protein surface which implicates protein-protein interfaces can impact proteinprotein interactions. In the context of IL-15, protein-protein interactions may include the interaction between IL-15 and a receptor. For example, IL-15 may interact with any of its receptors, IL-15Ra, IL-15Rp and yc. Such interactions occur at the surface of IL-15 in a receptor interaction surface.

[0051] Accordingly, the surface region is a region adjacent to a receptor interaction surface of the IL-15 variant.

[0052] 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 of IL-15 which is involved in coordinating, interacting with and binding to a receptor (i.e., protein-protein interactions). There may be a precise group of amino acids which directly bind the receptor. In a preferred embodiment, the receptor interaction surface is an IL- 15 receptor interaction surface. In a more preferred embodiment, the receptor interaction surface is an IL-15 receptor alpha (IL-15Ra) interaction surface. The interaction surface is a surface 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. An example of a protein domain is the sushi domain of the IL-15Ra receptor. The interaction surface is a surface 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. An example of a protein domain is the sushi domain of the IL- 15Ra receptor.

[0053] In relation to SEQ ID NO: 1 , the “receptor interaction surface” is defined as those amino acids at amino acid positions D8, D61 and N65 (with other amino acids involving I68, L69, K10, K11 and S7) for IL-15Rp (i.e., IL-15 a1 and a3); Q108, M109 and N112 foryc (i.e., IL-15 a1 and a4); and D22, T24, Y26, E46, Q48, E53, E87, E90 for IL-15Ra (i.e., IL-15 a2). The “receptor interaction surface” is therefore well characterised in the art. The inventors of the present invention have surprisingly identified a region of IL- 15 in which amino acids may be modified in order to generate IL-15 variants with increased solubility and maintained biological activity. The amino acids to be modified are present in a surface region adjacent to the receptor interaction surface of IL-15. These amino acids were selected to be modified because they form part of the space available in the IL-15 structure that is not needed for interaction with IL-15Rp or yc.

[0054] In particular, the inventors of the present invention have surprisingly identified that the following amino acid positions may be modified within this region of IL-15 (i.e. , SEQ ID NO: 1): L45, Q48, V49, L52, H60 and N71. None of these amino acids interact with I L-15Rp or yc. I L-15Rp or yc are expressed on the surface of the target cells to be activated (NK cells and CD8+ T cells). Accordingly, the “region adjacent to the receptor interaction surface” can be described as any region of IL- 15 in which amino acids do not interact with IL-15Rp or yc. Such interactions include any covalent bond and electrostatic interaction, including Van der Waals interactions and hydrogen bonds. However, the amino acids modified herein may interact with IL-15Ra. For example, within IL-15 (i.e., SEQ ID NO: 1), L45, V49 and L52 have been described as in Van der Waals contact with IL-15Ra, and Q48 has been described as forming a hydrogen bond with IL-15Ra (Chirifu et al, 2007). Further, a mutation of N71 within IL-15 (N71 D, in the ALT-803 IL-15 superagonist) is known in the art to contribute to the activity of IL-15. Therefore, as used herein, the “region adjacent to the receptor interaction surface” may comprise amino acids within the IL-15 a2.

[0055] Without wishing to be bound by theory, the amino acids which are present the “region adjacent” to the receptor interaction surface could play a role in mediating protein-protein interactions. Therefore, the term “region adjacent” may refer to surface amino acids which are in proximity to the receptor (e.g., IL-15Ra) interaction surface and may interact with IL-15Ra but do not interact with IL-15Rp or yc. Indeed, certain modifications may therefore act to enhance the interaction between IL-15 and a receptor (e.g., I L-15Ra), for example by increasing the affinity of binding. Further, modifications which alter the composition properties of amino acids on the surface of IL-15 may impact the solubility of IL-15.

[0056] Accordingly, in a preferred embodiment, the amino acid modifications will be present adjacent a surface region of the IL-15 variant which is adjacent to a receptor (e.g., I L-15Ra) interaction surface of the IL-15 variant, whereby the amino acid modifications enhance an interaction between the IL-15 variant and the receptor. For example, an interaction between IL-15 and IL-15Ra may be enhanced in that there is a lower rate constant of ligand dissociation (Kotf) (i.e., high stability of IL-15 / IL-15Ra complexes), a higher rate constant of ligand association (Kon) (i.e., fast recognition of IL-15Ra by IL-15 and vice versa), and a lower equilibrium dissociation constant (Kd) (i.e., a high affinity interaction). Kdis the ratio between KOff and Kon(Kd = K0ff / K0n). Other means for characterising binding kinetics will be apparent to those in the art.

[0057] In accordance with the first aspect of the invention, there is provided a bacterium comprising an IL-15 variant, wherein in comparison to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface. As detailed herein, the wild-type IL-15 may comprise mature IL-15, or 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-15 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.

[0058] In accordance with the second aspect of the invention, there is provided an interleukin-15 (IL-15) variant, wherein wild-type IL-15 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-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, and wherein the one or more amino acid modifications comprise any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophobic substitution at amino acid position 49; iii) a hydrophobic-to-hydrophilic substitution at amino acid position 52; and / or iv) a serine-to-proline substitution at amino acid position 75; preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface. The IL-15 variant comprises an amino acid modification at one or more of amino acid positions 45 and / or 49 and / or 52, or any combination thereof (Figure 6).

[0059] The IL-15 variant 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).

[0060] In another embodiment, the IL-15 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.

[0061] In accordance with the invention, the IL-15 variant may be modified such that, in comparison to SEQ ID NO: 1 , the amino acid modifications occur at one or more of amino acid positions 45 and / or 49 and / or 52. These amino acid positions are located in a surface region adjacent to a receptor interaction surface of the IL-15 variant (preferably an interleukin 15 receptor alpha (IL-15Ra) interaction surface). The IL-15 variant may comprise a single amino acid modification at amino acid position 45; a single amino acid modification at amino acid position 49; a single amino acid modification at amino acid position 52; two amino acid modifications at amino acid positions 45 and 49; two amino acid modifications at amino acid positions 45 and 52; two amino acid modifications at amino acid positions 49 and 52; or three amino acid modifications at amino acid positions 45, 49, and 52. Any combination of these modifications is intended to be included. It will be understood that an IL-15 variant comprising any combination of amino acid mutations at amino acid positions 45 and / or 49 and / or 52 will retain at least 70% sequence identity to SEQ ID NO: 1.

[0062] In accordance with any aspect of the invention, the IL-15 variant may comprise an amino acid modification at amino acid position 75. The amino acid modification may be present with any other modifications or combinations thereof described herein. For example, the IL-15 variant may comprise amino acid modifications at any of amino acid positions 45, 49, 52 and / or 75. For example, amino acid positions 75 and 45; amino acid positions 75 and 49; amino acid positions 75 and 52; amino acid positions 75, 45 and 49; amino acid positions 75, 45 and 52; amino acid positions 75, 49 and 52; or amino acid positions 75, 45, 49 and 52. Preferably, the IL-15 variant may comprise amino acid modifications at amino acid positions 75 and 45; amino acid positions 75, 45 and 49; amino acid positions 75, 45 and 52; amino acid positions 75, 49 and 52.

[0063] In some embodiments, the IL-15 variant may comprise amino acid modifications whereby non-conservative amino acid substitutions are performed. In such embodiments, a hydrophobic amino acid naturally present in the wild-type sequence is replaced with a hydrophilic amino acid, or vice versa. In the case where a hydrophobic amino acid is in a solvent-exposed position on the three- dimensional protein structure, the protein may be destabilised. Therefore, mutation of such hydrophobic residues can serve to stabilise the protein.

[0064] In other embodiments, the IL-15 variant may comprise amino acid modifications whereby conservative amino acid substitutions are performed. In such embodiments, 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.

[0065] 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. “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 included by the term “hydrophobic amino acid”. “Hydrophilic amino acids” are amino acids which possess hydrophilic side chains, i.e., side chains that 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 included by the term “hydrophilic amino acid”.

[0066] In accordance with the invention, the IL-15 variant may comprise amino acid modifications at one or more of amino acid positions 45 and / or 49 and / or 52. In relation to SEQ ID NO: 1 , the wild-type amino acid at position 45 is leucine, the wild-type amino acid at position 49 is valine, and the wild-type amino acid at position 52 is leucine.

[0067] In a preferred embodiment of any aspect of the invention, the amino acid modification at amino acid position 45 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.

[0068] The wild-type amino acid at amino acid position 45 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 45 to be a leucine-to-serine substitution or a leucine-to-threonine substitution. Without wishing to be bound by theory, this non-conservative amino acid modification could act to alter the physiochemical properties of the region in which the amino acid at position 45 resides. Specifically, the region may become more hydrophilic and therefore an IL-15 variant with such an amino acid modification may become more soluble. This alteration in physiochemical properties of the amino acid which is in a surface region adjacent to receptor interaction surface (e.g., an interleukin 15 receptor alpha (IL-15Ra) interaction surface) of the IL-15 variant may also serve to enhance an interaction between the IL-15 variant and a receptor. Therefore, IL- 15 variants with such an amino acid modification may be more soluble in bacterial chassis than wild-type IL-15, while also exhibiting biological activity.

[0069] In another preferred embodiment, the amino acid modification at amino acid position 49 is a hydrophobic-to-hydrophobic 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 another hydrophobic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for another hydrophobic amino acid. For example, any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan, may be substituted for any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan. However, the amino acid modification at amino acid position 49 may alternatively be a hydrophobic-to-hydrophilic substitution, preferably a valine- to-serine substitution.

[0070] The wild-type amino acid at amino acid position 49 in relation to SEQ ID NO: 1 is valine. The preference of the present invention is for the hydrophobic-to- hydrophobic substitution at amino acid position 49 to be a valine-to-alanine substitution. The inventors of the present invention have surprisingly found that the hydrophobic-to-hydrophobic mutation of V49 enhances the properties of an IL-15 variant. Without wishing to be bound by theory, this relatively conservative amino acid modification could act to optimise the region in which the amino acid at position 49 resides. Specifically, alanine is a small amino acid with a molecular weight of 89.1 g / mol and a small methyl (-CH3) side chain, whereas valine is a larger amino acid with a molecular weight of 117.1 g / mol and a bulkier isopropyl side chain (-CH(CH3)2). Such a substitution to a smaller / less bulky amino acid could serve to optimise the protein-protein interface for IL-15 / receptor interactions. Further, alanine is less hydrophobic than valine, therefore this alteration in the physiochemical properties of an amino acid which is in a surface region adjacent to receptor interaction surface of the IL-15 variant may also serve to increase the solubility of the IL-15 receptor. By removing the hydrophobicity of valine in such a substitution, the inventors of the present invention were able to assess the impact on solubility / stability of the IL-15 variant. Therefore, IL-15 variants with such an amino acid modification may have optimised solubility and functionality in comparison to wild-type IL-15.

[0071] In another preferred embodiment, the amino acid modification at amino acid position 52 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 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 amino acid position 52 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 52 to be a leucine-to-lysine substitution or a leucine-to-arginine substitution. Without wishing to be bound by theory, this non-conservative amino acid modification could act to alter the physiochemical properties of the region in which the amino acid at position 52 resides. Specifically, the region may become more hydrophilic and therefore an IL-15 variant with such an amino acid modification may become more water- soluble. This alteration in physiochemical properties in of amino acid which is in a surface region adjacent to a receptor interaction surface (e.g., an interleukin 15 receptor alpha (IL-15Ra) interaction surface) of the IL-15 variant may also serve to enhance an interaction between the IL-15 variant and a receptor. Therefore, IL-15 variants with such an amino acid modification may be more soluble than wild-type IL-15, while also preserving biological activity.

[0073] The amino acids at positions 45, 49 and 52 may, in some embodiments, not be substituted for bulky amino acids. Amino acids at positions 45, 49 and 52 are adjacent to the receptor binding surface, therefore substituting these residues for bulkier residues may cause steric hindrance that could interfere with receptor binding. Aspartate, glutamate, asparagine and glutamine are examples of bulky amino acids. Therefore, in some embodiments, the amino acid modifications at amino acid positions 45, 49 and 52 may not be substituted for any of aspartate, glutamate, asparagine or glutamine. As detailed herein, the IL-15 variant according to the invention may comprise an amino acid modification at amino acid position 75. The amino acid modification may be present with any other modification described herein. The preference of the present invention is for the amino acid modification at amino acid position 75 to be a serine-to-proline substitution (S75P). The serine-to-proline modification at amino acid position 75 has been described in the art (Behar, 2011). The inventors of the present invention selected the S75P IL-15 mutant (parental / M40 IL-15) as a starting sequence for generating IL-15 variants as the S75P IL-15 mutant was observed to be more soluble than unmodified IL-15 (Behar, 2011). An IL-15 variant comprising the S75P amino acid modification may have a sequence according to SEQ ID NO: 16, or a sequence comprising at least 70% identity to SEQ ID NO: 16. In another embodiment, the IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 16; at least 80% sequence identity to SEQ ID NO: 16; at least 85% sequence identity to SEQ ID NO: 16; at least 90% sequence identity to SEQ ID NO: 16; at least 91 % sequence identity to SEQ ID NO: 16; at least 92% sequence identity to SEQ ID NO: 16; at least 93% sequence identity to SEQ ID NO: 16; at least 94% sequence identity to SEQ ID NO: 16; at least 95% sequence identity to SEQ ID NO: 16; at least 96% sequence identity to SEQ ID NO: 16; at least 97% sequence identity to SEQ ID NO: 16; at least 98% sequence identity to SEQ ID NO: 16; at least 99% sequence identity to SEQ ID NO: 16; or at least 100% sequence identity to SEQ ID NO: 16, wherein the % sequence identity to SEQ ID NO: 16 retains the amino acid modifications at amino acid position 75. 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.

[0074] Accordingly, in a preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45 and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise (i) a leucine-to-serine substitution at amino acid position 45; and (ii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 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 45 and 75.

[0075] In another embodiment, the IL-15 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 45 and 75.

[0076] In another preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophobic substitution at amino acid position 49, and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise (i) a leucine-to-serine substitution at amino acid position 45, (ii) a valine-to-alanine substitution at amino acid position 49, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 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 45, 49 and 75.

[0077] In another embodiment, the IL-15 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 45, 49 and 75.

[0078] In another preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophilic substitution at amino acid position 52, and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise (i) a leucine-to-serine substitution at amino acid position 45, (ii) a leucine-to-lysine substitution at amino acid position 52, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise 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 45, 52 and 75.

[0079] In another embodiment, the IL-15 variant may comprise 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 45, 52 and 75.

[0080] In another preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophilic substitution at amino acid position 52, and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise (i) a leucine-to-serine substitution at amino acid position 45, (ii) a leucine-to-arginine substitution at amino acid position 52, and (iii) a serine-to- proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise 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 45, 52 and 75.

[0081] In another embodiment, the IL-15 variant may comprise 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 45, 52 and 75.

[0082] In another preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophobic substitution at amino acid position 49, and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise (i) a leucine-to-threonine substitution at amino acid position 45, (ii) a valine-to-alanine substitution at amino acid position 49, and (iii) a serine-to- proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO:

[0083] 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

[0084] In another embodiment, the IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 6; at least 80% sequence identity to SEQ ID NO: 6; at least 85% sequence identity to SEQ ID NO: 6; at least 90% sequence identity to SEQ ID NO: 6; at least 91 % sequence identity to SEQ ID NO: 6; at least 92% sequence identity to SEQ ID NO: 6; at least 93% sequence identity to SEQ ID NO: 6; at least 94% sequence identity to SEQ ID NO: 6; at least 95% sequence identity to SEQ ID NO: 6; at least 96% sequence identity to SEQ ID NO: 6; at least 97% sequence identity to SEQ ID NO: 6; at least 98% sequence identity to SEQ ID NO: 6; at least 99% sequence identity to SEQ ID NO: 6; or at least 100% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

[0085] In another preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise a hydrophobic-to- hydrophobic substitution at amino acid position 49, a hydrophobic-to-hydrophilic substitution at amino acid position 52, and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, an IL-15 variant according to any aspect of the invention may comprise (i) a valine-to-alanine substitution at amino acid position 49, (ii) a leucine-to-arginine substitution at amino acid position 52, and (iii) a serine-to- proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO:

[0086] 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52 and 75. In another embodiment, the IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 7; at least 80% sequence identity to SEQ ID NO: 7; at least 85% sequence identity to SEQ ID NO: 7; at least 90% sequence identity to SEQ ID NO: 7; at least 91 % sequence identity to SEQ ID NO: 7; at least 92% sequence identity to SEQ ID NO: 7; at least 93% sequence identity to SEQ ID NO: 7; at least 94% sequence identity to SEQ ID NO: 7; at least 95% sequence identity to SEQ ID NO: 7; at least 96% sequence identity to SEQ ID NO: 7; at least 97% sequence identity to SEQ ID NO: 7; at least 98% sequence identity to SEQ ID NO: 7; at least 99% sequence identity to SEQ ID NO: 7; or at least 100% sequence identity to SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52 and 75.

[0087] In addition to any amino acid modifications at any of amino acid positions 45, 49, and / or 52, and 75, there is also provided IL-15 variants with one or more amino acid modifications at amino acid positions 48, 60, and / or 71. In one embodiment, there is provided an IL-15 variant comprising a hydrophilic-to-hydrophobic substitution at amino acid position 48, preferably a glutamine-to-isoleucine substitution or a glutamine-to-valine substitution at amino acid position 48. In another embodiment, there may be an IL-15 variant comprising a hydrophilic-to- hydrophobic substitution at amino acid position 60, preferably a histidine-to-valine substitution at amino acid position 60. In another embodiment, there may be an IL-15 variant comprising a hydrophilic-to-hydrophobic substitution at amino acid position 70, preferably an asparagine-to-isoleucine substitution at amino acid position 70. It will be understood that any of these modifications may be present in combination with any of the other amino acid modifications described herein, including those amino acid modifications at amino acid positions 45, 49 and / or 52. In particular, there may be an IL-15 variant comprising amino acid modifications at amino acid positions 75 and 45; amino acid positions 75, 45 and 48; amino acid positions 75, 45 and 60; amino acid positions 75, 45 and 71 ; amino acid positions 75, 48 and 49; amino acid positions 75, 48 and 52; or any combinations thereof. As detailed herein, there is provided an IL-15 variant comprising one or more amino acid modifications, wherein said amino acid modifications occur in a surface region adjacent to a receptor (e.g., IL-15Ra) interaction surface of the IL-15 variant (i.e., in comparison to SEQ ID NO: 1 , at one or more of amino acid positions 45 and / or 49 and / or 52). The receptor interaction surface of the IL-15 variant may interact with or bind to IL-15 receptors. There exist three IL-15 receptors: IL-15Rp, GammaC (yc), and IL-15Ra. IL-15Rp may also be known as IL-2Rp. These receptors form complexes with IL-15 via electrostatic interactions (Figure 7). In the sequence of the parental IL-15 (SEQ ID NO: 16), the amino acids with the potential to be modified to enhance stability of the complex are shown in bold and underlined.

[0088] SEQ ID NO: 16

[0089] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0090] The inventors of the present invention have surprisingly found that the residues as shown in the sequence above are present in an exposed region of IL-15. Analysis of the sequence revealed in particular that lysine-41 , leucine-45, glutamine-48, and leucine-52 are aggregation prone and therefore good candidates for amino acid modifications which enhance solubility and preserve function.

[0091] Therefore, in accordance with a second aspect of the invention, there is provided an interleukin-15 (IL-15) variant, wherein wild-type IL-15 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-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, and wherein the one or more amino acid modifications comprise any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophobic substitution at amino acid position 49; iii) a hydrophobic-to-hydrophilic substitution at amino acid position 52; and / or iv) a serine-to-proline substitution at amino acid position 75; preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface.

[0092] In a particularly preferred embodiment, the hydrophobic-to-hydrophilic substitution at amino acid position 45 is a leucine-to-serine or leucine-to-threonine substitution.

[0093] In another particularly preferred embodiment, the hydrophobic-to-hydrophilic substitution at amino acid position 52 is a leucine-to-lysine or leucine-to-arginine substitution.

[0094] In another particularly preferred embodiment, the hydrophobic-to-hydrophobic substitution at amino acid position 49 is a valine-to-alanine or valine-to-serine substitution.

[0095] The IL-15Ra receptor contains protein-binding motifs known in the art as “sushi domains”. In accordance with the invention, an IL-15 variant of any aspect of the invention detailed herein may be fused to an IL-15R molecule, in particular, an IL- 15Ra molecule and / or the IL-15Ra sushi domain. The IL-15Ra molecule is a transmembrane protein, which has hydrophobic transmembrane segment, and can often be flexible and unstable. This makes expression of the entire IL-15Ra molecule challenging. Therefore, the preference of the present invention is to fuse the IL-15 variant to the IL-15Ra sushi domain, as opposed to the entire IL-15Ra molecule. The I L-15Ra sushi domain is soluble and also the functional part of the receptor, meaning that only the sushi domain is required for functionality and is also easier to express alone. As used herein, the term “fused” refers to any joining of two or more proteins. Proteins may be fused via covalent attachment, for example via a linker, and can be produced via the expression of a nucleic acid molecule comprising the nucleic acid sequences of the two or more proteins. In one embodiment, a fusion protein may comprise the sushi domain of an IL-15Ra molecule followed by a linker followed by any of the IL-15 variants described herein (IL-15Ra sushi:linker: I L-15 variant), i.e. , in some embodiments, the fusion is made to the amino (N) terminus of the IL-15 variant. The IL-15 variant may (or may not) be fused to a sushi domain of an IL-15Ra molecule. Fusion of the sushi domain to the IL-15 variant generates a super agonist (SAg) molecule. Any other suitable proteins or polypeptides may also be joined to the IL-15 variant and / or I L-15 / IL-15Ra sushi domain complex.

[0096] Any of the IL-15 variants detailed herein according to any aspect of the invention may be fused to an IL-15Ra molecule or sushi domain thereof. Such a fusion is referred to herein as a “super agonist”, “SAg”, “SAg variant” or “IL-15 super agonist”. As used herein, the term “super agonist” refers to a type of cytokine which is capable of inducing a maximal immune response greater than that of the agonist (i.e., the IL-15 variant) itself. The super agonists may be less soluble than their IL-15 variant counterparts.

[0097] Any of the IL-15 variants detailed herein may be fused to an IL-15Ra molecule or the sushi domain of an IL-15Ra molecule to form their super agonist counterpart. For example, in one embodiment, there is provided an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7, fused to an IL-15Ra molecule or the sushi domain of an IL-15Ra molecule via a GS linker such as that according to SEQ ID NO: 17 to form a super agonist. In another embodiment, there is provided an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7, or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 80% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 91% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 94% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 95% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 96% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 97% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 98% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 99% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; and / or at least 100% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7, (wherein the % sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7 retains the amino acids at the relevant amino acid positions) fused to an IL-15Ra molecule or the sushi domain of an IL-15Ra molecule to form a super agonist. Preferably, the IL-15 variant is fused to the IL-15Ra sushi domain.

[0098] The term “relevant amino acid positions” is intended to encompass the amino acid modifications as described for each SEQ ID NO herein. For example, the amino acid modifications at the relevant amino acid positions for SEQ ID NO: 2 would be the amino acid modifications at amino acid positions 45 and 75.

[0099] In a preferred embodiment, the IL-15 super agonist may comprise an amino acid sequence according to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, or an amino acid sequence comprising at least 70% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13.

[0100] SEQ ID NO: 8 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 2 (i.e., the SAg01 super agonist which comprises GBL01). SEQ ID NO: 9 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 3 (i.e., the SAg15 super agonist which comprises GBL15). SEQ ID NO: 10 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 4 (i.e., the SAg17 super agonist which comprises GBL17). SEQ ID NO: 11 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 5 (i.e., the SAg18 super agonist which comprises GBL18). SEQ ID NO: 12 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 6 (i.e., the SAg25 superagonist which comprises GBL25). SEQ ID NO: 13 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 7 (i.e., the SAg50 superagonist which comprises GBL50). These super agonist sequences utilise the GS linker according to SEQ ID NO: 17, however, it will be appreciated by the skilled person that any suitable linker could be utilised in joining the IL-15 variant with an IL-15Ra molecule or the sushi domain of an IL-15Ra molecule.

[0101] Accordingly, in another embodiment, there is provided an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, or any combination thereof, or an amino acid sequence having or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs:

[0102] 8, 9, 10, 11 , 12, and / or 13; at least 80% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least 85% sequence identity to SEQ ID NOs: 8, 9, 10, 11 ,

[0103] 12, and / or 13; at least 90% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 91 % sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least 92% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least 93% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least

[0104] 94% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least 95% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least 96% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least 97% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least 98% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; at least 99% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; and / or at least 100% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, (wherein the % sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13 retains the amino acids at the relevant amino acid positions).

[0105] However, the preference is for the IL-15 super agonist may comprise an amino acid sequence according to any one of SEQ ID NOs: 9, 11, 12 and / or 13, or an amino acid sequence comprising at least 70% sequence identity with any one of SEQ ID NOs: 9, 11 , 12 and / or 13 (wherein the % sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13 retains the amino acid modifications at the relevant amino acid positions), as it was surprisingly found by the inventors of the present invention that these super agonists were more soluble than super agonists of wildtype IL-15. In another embodiment, the IL-15 super agonist may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 80% sequence identity to SEQ ID NOs: 9, 11, 12 and / or 13; at least 85% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 90% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 91 % sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 92% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 93% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 94% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 95% sequence identity to SEQ ID NOs: 9, 11, 12 and / or 13; at least 96% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 97% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 98% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; at least 99% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13; and / or at least 100% sequence identity to SEQ ID NOs: 9, 11 , 12 and / or 13, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions.

[0106] Such a fusion may be achieved by directly joining two or more proteins together (i.e. , a direct fusion protein), and / or indirectly joining two or more proteins via any suitable form of linkage. The linkage may be a covalent linkage. However, direct joining without a linker can lead to misfolding of the fusion protein, low solubility, or impaired bioactivity. Therefore, in a preferred embodiment, the IL-15 variant is fused to the sushi domain of an IL-15Ra molecule via a linker, such as a flexible polypeptide linker. Flexible linkers, for example, those which make use of serine and glycine residues, allow the joined proteins to move relative to one another. The length of the linker may be increased to prevent steric interference between the proteins. A short linker may be approximately 4 to 6 amino acids in length, a medium length linker may be approximately 6 to 14 amino acids in length, and a long linker may be approximately 14 to 21 or more amino acids in length. The linker may be a naturally occurring linker or a synthetic linker. The linker may adopt secondary protein conformations such as alpha helices and / or beta strands / coils / bends. In a more preferred embodiment, the linker is a glycineserine linker (GS linker). GS linkers primarily comprise stretches of glycine and serine residues, for example, (Ser-Gly-Gly-Gly-Gly)n, or (Ser-Gly-Gly-Gly)n. Any proportion of glycine and serine residues may be utilised, and other amino acids may be present, such as leucine or glutamine. The copy number ‘n’ may be adjusted to alter the length of the GS linker to achieve appropriate separation of the joined proteins, which can impact the functionality and interaction of said joined proteins. The copy number ‘n’ typically ranges between 1 and 6. However, the preferred GS linker of the present invention may comprise the sequence below:

[0107] SEQ ID NO: 17

[0108] SGGGSGGGGSGGGGSGGGGSGGGSLQA

[0109] In a preferred embodiment, the linker utilised to fuse the IL-15 variant with an IL- 15Ra molecule or the sushi domain of an IL-15Ra molecule comprises an amino acid sequence according to SEQ ID NO: 17, or amino acid sequence comprising at least 70%, at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 17.

[0110] While the preference of the present invention is to use flexible GS linkers, other flexible linkers may be used, for example those which contain additional amino acids such as threonine, alanine, lysine and / or glutamate. Further, flexible linkers consisting purely of glycine or serine may also be appropriate, for example a (Gly)s linker.

[0111] In addition to the joining of an IL-15Ra sushi domain, there may be any other suitable protein or polypeptide joined to the IL-15 variant and / or the IL-15 super agonist. 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, 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). 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-15 variant and / or the IL-15 super agonist 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-15 variant and / or the IL-15 super agonist to a particular intracellular or extracellular location. 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.

[0112] Human IL-15 is produced as two isoforms by alternative splicing of mRNA transcripts (which occurs only in mammalian cells, not bacterial cells). In humans, one isoform comprises a 21 amino acid short signal peptide (SSP) (SEQ ID NO: 15) and remains cytoplasmic. The other isoform comprises a 48 amino acid long signal peptide (LSP) (SEQ ID NO: 14) which allows for its secretion into the extracellular environment.

[0113] For expression in bacteria, a bacterial signal peptide will be required in order to secrete the IL-15 variant out of the bacterial cytosol and into, for example, the periplasm. In a preferred embodiment, the signal peptide directs the IL-15 variant into the periplasm of the bacteria in a rapid and robust manner. Therefore, the preference of the present invention is for the signal peptide to be a (bacterial) periplasmic signal peptide. 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. There is no universal signal peptide which can translocate any protein efficiently, and therefore the inventors of the present invention have also screened for optimal signal peptides for each IL-15 variant.

[0114] As will be appreciated 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 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 cytosol is a reducing environment which does not favour disulphide bond formation. Therefore, it can be seen that directing the cytokines (IL-15 variants and super agonists thereof) to the periplasm via signal peptides is beneficial to the production and correct folding of these proteins, and ultimate secretion in situ.

[0115] 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- 15 variants or super agonists thereof according to the present invention may be a SEC or TAT 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 Gramnegative 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-15 variant or IL-15 super agonist However, the IL-15 variant or IL-15 super agonist 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-15 variant or IL-15 super agonist.

[0116] From the periplasm, the IL-15 variant or super agonist can then be prompted into the supernatant or other extracellular environment. In fusing a periplasmic signal peptide to any of the IL-15 variants and / or the IL-15 super agonists described herein, if expressed within a bacterial cell, the IL-15 variants and / or the IL-15 super agonists can be transported efficiently to the periplasm of the bacterial cell in order to achieve proper folding and subsequent secretion of the IL-15 variants and / or the IL-15 super agonists 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-15 variants and / or the IL-15 super agonists. For example, upon infection and invasion of a host (e.g., eukaryotic) cell by Gramnegative bacteria expressing the IL-15 variants or super agonists of the present invention, the IL-15 variant or super agonist may be transferred into the cytoplasm of the host cell where it may achieve for example a therapeutic immunostimulatory effect.

[0117] In one embodiment, the signal peptide is fused to the IL-15 variant and / or super agonist 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-15 variant or super agonist thereof) which is to be secreted (i.e., signal peptide:spacer:sushi:linker:IL-15 variant, and / or signal peptide:spacer:IL-15 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.

[0118] In accordance with a third aspect of the invention, there is provided a nucleic acid molecule encoding the interleukin-15 (IL-15) variant according to the second aspect of the invention. The invention also provides a nucleic acid molecule encoding an IL-15 variant comprised by the bacteria according to the first aspect of the invention. As used herein, the term “nucleic acid molecule” refers to a nucleic acid molecule, including 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. Specifically, the nucleic acid molecule may be expressed in a bacterial cell, preferably, a Gramnegative 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, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, or any combination thereof (preferably any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13) or an amino acid sequence comprising at least 70% identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16 retains the amino acid modifications at the relevant amino acid positions. In some embodiments, expression of the IL-15 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.

[0119] 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. 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 single-stranded 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 noncompartmentalised 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.

[0120] 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.

[0121] 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, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, or any combination thereof. In a preferred embodiment, the nucleic acid molecule encodes any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 9, 11 , 12, 13, and / or 16, or any combination thereof. In a more preferred embodiment, the nucleic acid molecule encodes any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 9, 11 , 12, and / or 13, or any combination thereof. In another embodiment, the nucleic acid molecule encodes an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, or a sequence having at least 70% identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16 retains the amino acid modifications at the relevant amino acid positions.

[0122] In another embodiment, there may be provided a nucleic acid molecule comprising a sequence encoding an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 80% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 85% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 90% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 91 % sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 92% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 93% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 94% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 95% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 96% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 97% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 98% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16; at least 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9,

[0123] 10, 11 , 12, 13, and / or 16; and / or at least 100% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16 retains the amino acid modifications at the relevant amino acid positions.

[0124] 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, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13, or an amino acid sequence comprising at least 70% identity to any of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10,

[0125] 11 , 12, and / or 13; at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7,

[0126] 8, 9, 10, 11 , 12, and / or 13; at least 80% sequence identity to SEQ ID NOs: 2, 3,

[0127] 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 91 % sequence identity to SEQ I D NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 94% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,

[0128] 12, and / or 13; at least 95% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8,

[0129] 9, 10, 11 , 12, and / or 13; at least 96% sequence identity to SEQ ID NOs: 2, 3, 4,

[0130] 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 97% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 98% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; at least 99% sequence identity to SEQ I D NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13; and / or at least 100% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and / or 13, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions.

[0131] In a fourth aspect of the invention, there is provided a bacterium comprising the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third aspect of the invention. As would be understood by the skilled person, interleukins play a diverse role in eliciting an immune response. Given the role of IL-15 in the stimulation of proliferation and cytotoxic function of CD8 T cells and NK cells, the therapeutic potential of IL-15 is apparent. The inventors of the present invention have surprisingly found that certain mutations to the amino acid structure of IL-15 can generate IL-15 variants which are optimised in terms of solubility without impacting biological activity. Further, the super agonist counterparts of these IL-15-variants can have the potential to elicit maximal and durable immune responses against numerous diseases in a subject. Therefore, in accordance with a fifth aspect of the invention, there is provided a bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third aspect of the invention, for use as a therapy. Any of the IL-15 variants and / or IL-15 super agonists disclosed herein in any combination have uses in therapy.

[0132] 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 a bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule 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.

[0133] 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.

[0134] 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, or a neoplasia, tumour, cancer or metastasis that is progressing, worsening, stabilized or in remission.

[0135] 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, oesophagus, 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.

[0136] 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, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, endometrial cancer, skin cancer or sarcoma. More preferably, the neoplastic disease may be associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular cancer melanoma, oesophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer or breast cancer.

[0137] In some embodiments, the neoplastic disease is a malignant neoplastic disease.

[0138] In some embodiments the neoplastic disease is not a benign neoplastic disease.

[0139] In one preferred embodiment, 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.

[0140] In another preferred embodiment of the present invention, the IL-15 variant or IL- 15 super agonist is to be administered intratumourally, intravesically, intravenously, intraperitoneally, or orally. In a most preferred embodiment, the IL- 15 variant or IL-15 super agonist is administered intratumourally. However, it is also contemplated that other methods of administration may be used in some cases. Therefore, in certain instances the IL-15 variants or IL-15 super agonists of the present invention may be administered by injection, instillation, infusion, continuous infusion, intradermally, intraarterially, intralesionally, 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 via local instillation, intra-peritoneally, intrapleurally, intravesically, peritumoral injection, intratumoural injection or oral administration.

[0141] 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-15 variant or IL-15 super agonist directly into the neoplastic disease of the subject.

[0142] 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.

[0143] Aside from direct administration of any of the IL-15 variants and / or super agonists thereof disclosed herein, in order to achieve optimal and efficient expression, folding, and secretion of the IL-15 variants or super agonists thereof, there is provided herein a bacterium comprising any of the IL-15 variants and / or IL-15 variants super agonists disclosed herein, or any combination thereof, or the nucleic acid molecule encoding said variants. The IL-15 variants and / or super agonists thereof disclosed herein 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-15 variants and / or super agonists thereof may be administered together with the bacteria, whereby the bacteria comprising the IL-15 variant and / or super agonist thereof or nucleic acid molecule encoding the I L-15 variant and / or super agonist thereof are administered and express and secrete the IL-15 variants and / or super agonists thereof. I n some embodiments, the bacterium may be a Gram-negative bacterium. In other embodiments, the bacterium may be a Gram-positive bacterium.

[0144] Any suitable bacterium capable of comprising an IL-15 variant and / or super agonist thereof, or a nucleic acid molecule encoding an IL-15 variant and / or super agonist thereof 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.

[0145] For instance, there may be provided a bacterium, such as a 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, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, ora sequence having at least 70% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 75% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 80% sequence identity to SEQ I D NOs: 1,2,3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, and / or 16; at least 85% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 90% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 91% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 92% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 93% sequence identity to SEQ I D NOs: 1,2,3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, and / or 16; at least 94% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 95% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 96% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 97% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 98% sequence identity to SEQ I D NOs: 1,2,3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, and / or 16; at least 99% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, and / or 16; and / or at least 100% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ I D NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 retains the amino acid modifications at the relevant amino acid positions. Preferably, the 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, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13. Any suitable bacterium capable of comprising the IL-15 variant may be utilised according to the invention. However, in a particularly preferred embodiment, the bacterium is a Gram-negative bacterium.

[0146] 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 noncoding 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] Such a bacterium will be engineered to express the any of the IL-15 variants and / or super agonists thereof 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 of the 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.

[0152] For example, there is provided a bacterium, such as a Gram-negative bacterium, comprising an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, and / or 16, more preferably any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16, even more preferably any one of SEQ ID NOs:

[0153] 2, 3, 4, 5, 6, and / or 7, 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 bacterium, such as a Gramnegative bacterium, comprising an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16, or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 80% sequence identity to SEQ ID NOs: 2, 3, 4,

[0154] 5, 6, 7, and / or 16; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 91 % sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 94% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 95% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 96% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 97% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 98% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 99% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; and / or at least 100% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions.

[0155] In another preferred embodiment, there may be provided a bacterium, such as a Gram-negative bacterium, comprising an IL-15 super agonist comprising an amino acid sequence according to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, or an amino acid sequence comprising at least 70% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions. In an even more preferred embodiment, the bacteria may comprise an IL-15 super agonist comprising an amino acid sequence according to any one of SEQ ID NOs: 9, 11 , 12 and / or 13. In another embodiment, there may be provided a bacterium, such as a Gramnegative bacterium, comprising an IL-15 super agonist comprising an amino acid sequence according to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, or an amino acid sequence having or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 80% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 85% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 90% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 91% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 92% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 93% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 94% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 95% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 96% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 97% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 98% sequence identity to SEQ ID NOs: 8, 9 10, 11 , 12, and / or 13; at least 99% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13; and / or at least 100% sequence identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions. As will be understood by the skilled person, a bacterium, such as a Gram-negative bacterium, comprising a nucleic acid molecule encoding for any of the amino acid sequences described herein is also intended to be included.

[0156] The bacterium comprising an IL-15 variant and / or IL-15 super agonist (SAg variants) thereof may be referred to herein as a composition unless specified otherwise. The IL-15 variants and / or SAg variants thereof 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.

[0157] 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.

[0158] Accordingly, the combination of the IL-15 variant and / or IL-15 super agonist and bacterium, where the IL-15 variant and / or IL-15 super agonist is expressed and secreted by the bacterium, is synergistic and will provide an enhanced immune response in a subject. The IL-15 variant or IL-15 super agonist of the present invention, delivered by a bacterium, such as 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 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.

[0159] A bacterium, such as a Gram-negative engineered to express the any of the IL-15 variants and / or super agonists thereof according to any aspect of 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-15 variants and / or super agonists thereof are particularly suitable for the treatment of cancer in vivo.

[0160] The amount of the bacterium administered to the subject is sufficient to deliver a therapeutically effective amount of the IL-15 variant and / or IL-15 super agonist 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-15 variant and / or IL-15 super agonist thereof to be expressed and secreted, and the medical history of the subject to be treated.

[0161] 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.

[0162] 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.

[0163] The baseline measurement may be derived from untreated subjects.

[0164] 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.

[0165] The bacterium 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-15 variant to be delivered, as well as the specific indication to be treated.

[0166] The bacterium 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 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-15 variant or super agonist thereof and / or 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.

[0167] In a preferred embodiment of the present invention, the bacterium described herein is administered intratumourally, intravesically, intravenously, intraperitoneally, or orally administered. In a most preferred embodiment, the bacterium is administered intratumourally or orally. 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 injection, instillation, infusion, continuous infusion, intradermally, intraarterially, intralesionally, 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).

[0168] 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 intratumoural injection may not be the method of administration of choice. 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 with the invention, there is provided a bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule 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. Further, in a sixth aspect of the invention, there is provided a use of bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third aspect of the invention, in the manufacture of a medicament for a therapy.

[0169] Where the disease to be treated is a neoplastic disease, the bacterium, and the IL-15 variant and / or SAg variant thereof 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-15 variant and / or IL-15 super agonist with other known cancer therapies. Preferably, the bacterium expressing the IL-15 variant and / or IL-15 super agonist 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-15 variant and / or SAg variant thereof 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-15 variant and / or IL-15 super agonist may be administered separately from, simultaneously with or sequentially to (prior to and / or following) the other known cancer therapy.

[0170] 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.

[0171] 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; endometroid 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.

[0172] 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, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular (liver) 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, colorectal cancer, head and neck cancer or breast cancer.

[0173] In some embodiments, the neoplastic disease is a malignant neoplastic disease.

[0174] In some embodiments the neoplastic disease is not a benign neoplastic disease.

[0175] In one preferred embodiment, 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. 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 bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third aspect of the invention.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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 hyperproliferative 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.

[0180] 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.

[0181] 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.

[0182] The bacterium expressing an IL-15 variant and / or IL-15 super agonist 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 Gram-negative 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.

[0183] The composition is intended to enhance an immune response of a subject. Accordingly, the bacterium expressing an IL-15 variant and / or IL-15 super agonist may be provided as 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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-15, IL-16, IL-17, IL-18, IL-21) or chemokines (e.g. CXCL9, CXCL10, CXCL11 , CCL5, CCL2, CX3CL1).

[0188] The bacterium of the vaccine composition herein disclosed may include any one of, or any combination of, the features of the bacterium herein disclosed.

[0189] 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.

[0190] 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.

[0191] The invention is further described with reference to the following non-limiting examples:

[0192] EXAMPLES evaluation.

[0193] Variant design

[0194] Cytokine variants were designed following three approaches:

[0195] • Targeted in silico screenings: Computational analysis of cargo modelled against a reported crystal structure in complex with critical receptors (PDB ID: 4GS7). Analysis considered improvements in solubility and stability without losing activity. This resulted in five defined variants containing multiple mutations that were gene synthesized by IDT.

[0196] • Semi-rational candidates: Use of computational tools (e.g., Aggrescan3D) to identify aggregation-prone amino acids, followed by amino acid mutations of these positions only. These resulted in two libraries, depending on the method used to generate the mutants: a 72- member library where each variant contains 1 or 2 defined amino acid changes with respect to IL-15M40 and a pool of variants with 729 variants, each having from 0 -6 mutations (random). More than 85% of the variants in the pool had 3 - 6 mutations. The first library was gene synthesized by IDT and the second one by TWIST.

[0197] • Random candidates: Generation of random mutants via error-prone PCR using the Genemorph II kit (Agilent) and according to manufacturer specifications to create two pools, one with fragments expected to have 0 - 2 mutations and one with fragments expected to have 2 - 4 mutations.

[0198] Solubility screen

[0199] Each of the fragments generated during the design phase were cloned into a solubility reporter plasmid, pBRED, derived from the system reported by Foit et al. (2009 Molecular Cell 36 861-871) which is based on the survival of bacterial cells in penicillin antibiotics (e.g., carbenicillin, ampicillin, penicillin V) as a function of the stability of a protein of interest. pBRED contains a beta-lactamase 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. Survival of each experimental strain was tested either in liquid media or in solid. Both methods produced consistent and reproducible results, with the liquid assay providing a faster, easier to interpret, readout.

[0200] A first iteration screened the parental sequence, IL-15M40, which was found to yield carbenicillin sensitive cells at a concentration of 1 mg / mL. The protein mScarlet, which is known to be stable, was used as positive control or benchmark value.

[0201] Four of the five sequences in the targeted library (1) were found to be more stable than the parental control. In the semi-rational libraries, from the 72 variants designed to contain 1-2 amino acid mutations, most single (75%) and double amino acid replacements (72%) were found to improve stability. The pool generated with 85% of variants to contain 3-6 mutations yielded 94 / 176 positive sequences after screening (53%), which were sequenced. Sample mutation number was normally distributed with mean 4, and mutations at positions 45 and 49 were found to be the most prevalent. Finally, a total of 42 / 176 (24%) variants generated by error-prone PCR were positive in the assay. The final pool of positive candidates contained 169 sequences.

[0202] Activity Screen

[0203] Cytokine variants found to be more stable than the parental sequence (IL-15M40) were evaluated for activity using three assays: • Promega IL-15 kit: Rapid assay using an IL-15 Bioassay Cell (Promega) kit. Allows a sigmoidal assessment of activity, suitable for high throughput assays. First screening assay performed on the soluble candidates. In this assay, reporter cells were added to wells containing immobilised cytokine protein on their bottom and incubated as recommended by the manufacturer for 6 h. After incubation, substrate was added into the plate following manufacturer recommendations and luminescence measured on a Tecan plate reader.

[0204] • NK expansion assay: Bespoke assay based on a 4-day incubation at 37 °C and 5 % CO2 of NK cells isolated at Prokarium after induction with candidates (see below). Positive activity results in proliferation of the NK cells, measured as higher Optical Density (OD). Offers a more accurate output of the activity of the candidates, albeit the output range being narrow. Second screening assay performed on soluble candidates found positive with the Bioassay Cell (Promega) Kit.

[0205] • IFN-gamma (IFN-y) production assay: Bespoke assay based on a 1-day incubation of NK cells isolated at Prokarium after induction with candidates (see below). Positive activity results in production of Interferon gamma by the NK cells, which is then quantified by ELISA. As the NK Expansion Assay, offers a more accurate output of the activity of the candidates. Third and last screening assay performed on soluble candidates found positive with the Bioassay Cell (Promega) Kit and the NK Expansion Assay.

[0206] The 169 variants obtained by screening for solubility were transferred into a protein expression plasmid that constitutively expressed each cargo of interest with a C-terminal His tag. A volume of 4 mL of cells containing each plasmid were grown to OD6oo ~ 1.0 and spun down. Culture supernatant was discarded, and cell pellet re-suspended in 200 pL of NPI buffer (50mM NaH2PO4, 300mM NaCI) supplemented with 20 mM imidazole. The cells were then lysed on a PIXUL® Multi-Sample Sonicator. The insoluble fraction was separated from the soluble protein content by centrifugation for 20 min at 12000xg and loaded into Ni- NTA HisSorb (QIAGEN) or HisPur (ThermoFisher) plates. Proteins were allowed to bind for 1 h at room temperature. Plates were then washed with NPI supplemented with 50 mM imidazole three times and with PBS (37 mM NaCI, 2.7 mM KCI, 8 mM Na2HPC>4, and 2 mM KH2PO4) two times to remove excess imidazole. Plates were then used to perform activity assays as described above.

[0207] Only variants obtained from the library of sequences with 1-2 amino acid mutations were found to be active after screening them against the IL-15 Bioassay Cells.

[0208] A total of 438 IL-15 variants were screened. 169 of these variants were found to be more soluble than the reference sequence by between 2- and 40-fold (Figures 8Aand 8B). Of these soluble variants, 13 were also found to be biologically active using high-throughput screening Bioassay Cells (Promega) bioassays (Figures 8C and 8D; Table 1). Seven variants listed in Table 1 were selected for further experimentation (GBL01 , GBL15, GBL17, GBL18, GBL25, GBL50). variants have the S75P mutation. Parental IL-15M40 (IL-15 S75P) has a solubility score of 1.0, and mScarlet has a solubility score of 19.7 ± 0.8. Example 2: Generation of IL-15 super agonists (SAg) using IL-15Rg sushi domain.

[0209] Super agonist counterparts of the 13 IL-15 soluble and functional variants were generated by fusion to IL-15Ra (sushi domain) via a Gly-Ser flexible linker (ILR) (Table 2). Table 2. Solubility of different IL-15 variants fused with IL-15Ra sushi domain.

[0210] As previously described, the super agonists were challenged with the antibiotic challenge assay after being cloned into the reporter plasmid pBRED, using the super agonist of parental IL-15M40 as reference and the fusion of mScarlet to the IL-15Ra-ILR construct as positive control.

[0211] Super agonists were found to be less soluble than their IL-15 counterpart, requiring mid and low assay conditions to identify soluble partners. 5 out of 13 (38%) of super agonists were found to be more soluble than super agonists with parental IL-15. These soluble super agonists were SAg15, SAg17, SAg18, SAg25, and SAg50.

[0212] Further validation of the 13 candidate IL-15 variants and super agonists SAg15, SAg18, SAg25, and SAg50 was carried out using the IL-15 Bioassay Cells (Figure 9A), primary NK proliferation assay (Figure 9B), and the interferon gamma release assay (Figure 9C).

[0213] All IL-15 variants but GBL34 and GBL43 resulted in reproducible measurements in the Bioassay Cell (Promega) kit. This assay suggested that SAg15, SAg18, and SAg25 may be biologically active (Figure 9A).

[0214] For the proliferation assay, the same sample extracts were used as for the bioassay. To evidence differences, samples were normalised to the values of mScarlet negative control. Induction of NK cells with different IL-15 / SAg cargoes mirrored the results obtained in the Bioassay Cells (Promega) Assay (Figure 9B).

[0215] The IFN-y production assay results were consistent with the other experiments and allowed better quantification of activity than the proliferation (Figure 9C).

[0216] Exam ole 4: Protein purification of IL-15 variants and IL-15 / Saq variants.

[0217] The variants GBL01 , GBL18, GBL25, SAg18, and SAg25 were selected to undergo more thorough characterisation. The genes encoding for these were placed on the expression plasmid that allowed fusion with a His C-terminal tag and expressed in E coli. A total of 1 L of culture per variant was centrifuged and the resulting pellet was lysed by sonication. The soluble fraction was purified by Ni-NTA chromatography, yielding purified His-tagged IL-15, IL-15 variants, and super agonists (Figure 10B). Protein was then quantified by HPLC / MS, using commercial IL-15 as sample to calculate a standard curve. Purified protein concentrations (Table 3) were calculated using a standard curve of IL-15 concentration against MS peak area (Figure 10A).

[0218] Table 3. Concentrations of purified proteins obtained via Ni-NTA affinity chromatography.

[0219] Example 5 In vitro and ex vivo characterisation of recombinant IL-15 variants

[0220] The activity of recombinant, purified IL-15 variants was determined by testing a gradient of concentrations (from 10’2- 2.5 nM) using the Bioassay Cells (Promega) assay. IL-15 variants were incubated for 6 hours with a reporter cell line which expresses Iuc2 in response to IL-15 signalling. Results in Figure 11A are shown as mean fold induction (sample / blank) of n=3 independent replicates (+ / -SD). Figure 11 B shows INF-y release. Wild-type IL-15 protein showed the greatest activity, evident from the lowest EC50, GBL18 and GBL01 have the greatest activity of the candidates, and SAg18 and Sag25 do not induce activation of the Bioassay reporter cell line (Figure 11 A; Table 4). While this showed that IL- 15 wild-type was the protein with highest activity, that of GBL1 and GBL18 was comparable. Variant GBL25 was found to require approximately 2x higher concentrations to activate luminescence.

[0221] Table 4. EC50 of each IL-15 variant, calculated as the mean of three independent experiments. The results of Figure 11 were validated by incubating known concentrations of the purified variants with NK cells for 24 hours and measuring the resulting concentration of IFN-y in the supernatant (Figure 12). In Figure 12, primary human NK cells were isolated from PBMC using negative selection with magnetic microbeads. Purified IL-15 variants were incubated at 71 nM (Experiment 1) and 58 nM (Experiment 2) with primary NK cells for 24 hours. Results in Figure 12 displayed as mean + / -SEM from stimulation replicates of a single experiment representative of 3 experiments performed with 2 different cell donors. Supernatants were then assessed for the presence of IFN-y using a human duoset IFN-y ELISA. Peprotech is a recombinant IL-15 which is commercially available. All candidates induced IFN-y production from primary NK cells. The cells stimulated with WT IL-15 were the highest IFN-y-producing samples, with similar concentrations obtained from NK cells stimulated with SAg25, and followed by those stimulated with GBL01 and GBL18.

[0222] Finally, the variants were used to stimulate NK cells in mouse (naive balb / c) splenocytes for 72 hours. The splenocytes were then stained and assessed for NK cell and NK activation markers via flow cytometry (Table 5; Figure 13). GBL01 and GBL18 had comparable behaviour and were the most effective variants. In some measurements, these were also improved upon wild-type measurements.

[0223] Table 5. Markers utilised in ex vivo assays to analyse mouse cell survival, proliferation, activation and effector molecule expression. Example 6: Expression of IL-15 in ZH9 Salmonella strains in tumours for prolonged periods.

[0224] Due to difficulties in detecting IL-15 cargo proteins, IL-15 gene expression from Salmonella was used to assess pharmacokinetics.

[0225] Mice bearing subcutaneous MC38 tumours were injected intratumorally with a PBS control, a ZH9 Salmonella strain, or a candidate IL-15 variant-expressing ZH9 variant strain. Tissues were harvested at the indicated timepoints after treatment, and RNA was extracted, and copies of the Sa / mone / / a-derived IL-15 genes were enumerated by TaqMan quantitative PCR. Gene copies are normalised for tumour volume.

[0226] The results of this experiment show that the il-15 mRNA can be detected from Salmonella for prolonged periods (>7 days) in tumour tissue following intratumoural administration.

[0227] In addition, wild-type human IL-15 was expressed in ZH9 Salmonella. Western blot analysis indicated that wild-type human IL-15 was detected mainly in the bacterial insoluble fraction.

[0228] Example 7: In vivo bladder tumour model studies showed improved efficacy for IL-15 variants versus chassis strain

[0229] Female C57BL / 6 mice were inoculated intravesically (IVES) with MB49-luc tumour cells and treated with various Salmonella strains (PBS control, ZH9 Salmonella strain, or a candidate IL-15 variant-expressing ZH9 variant strain). The survival of these mice was monitored for 60 days. In all cases, mice injected with candidate IL-15 variant-expressing ZH9 variant strains showed greater median survival as compared to mice injected with ZH9 Salmonella strain (i.e., strains lacking the IL-15 variant).

[0230] The invention may be described as in any of the following embodiments. Embodiment 1. An interleukin-15 (IL-15) variant, wherein in comparison to wildtype IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface.

[0231] Embodiment 2. The IL-15 variant according to Embodiment 1 , wherein wild-type IL-15 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.

[0232] Embodiment s. The IL-15 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 45 and / or 49 and / or 52, or any combination thereof.

[0233] Embodiment 4. An interleukin-15 (IL-15) variant, wherein the IL-15 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 , and wherein in comparison to SEQ ID NO: 1 , the IL-15 variant comprises an amino acid modification at one or more of amino acid positions 45 and / or 49 and / or 52, or any combination thereof.

[0234] Embodiment 5. The IL-15 variant according to any one of Embodiments 2 to 4, wherein the IL-15 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.

[0235] Embodiment 6. The IL-15 variant according to any one of Embodiments 2 to 5, wherein the IL-15 variant comprises an amino acid modification at amino acid position 75.

[0236] Embodiment 7. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 45 is a hydrophobic-to-hydrophilic substitution. Embodiment s. The IL-15 variant according to Embodiment 7, wherein the hydrophobic-to-hydrophilic substitution is a leucine-to-serine substitution or a leucine-to-threonine substitution.

[0237] Embodiment 9. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 49 is a hydrophobic-to-hydrophobic substitution.

[0238] Embodiment 10. The IL-15 variant according to Embodiment 9, wherein the hydrophobic-to- hydrophobic substitution is a valine-to-alanine substitution.

[0239] Embodiment 11. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 52 is a hydrophobic-to-hydrophilic substitution.

[0240] Embodiment 12. The IL-15 variant according to Embodiment 11 , wherein the hydrophobic-to-hydrophilic substitution is a leucine-to-lysine substitution or a leucine-to-arginine substitution.

[0241] Embodiment 13. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 75 is a serine-to-proline substitution.

[0242] Embodiment 14. The IL-15 variant according to Embodiment 13, wherein the IL- 15 variant comprising the serine-to-proline substitution at amino acid position 75 comprises a sequence according to SEQ ID NO: 16, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 16, wherein the % sequence identity to SEQ ID NO: 16 retains the amino acid modifications at amino acid position 75.

[0243] Embodiment 15. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the serine-to-proline substitution at amino acid position 75. Embodiment 16. The IL-15 variant according to Embodiment 15, wherein the IL- 15 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:

[0244] 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 45 and 75.

[0245] Embodiment 17. The IL-15 variant according to Embodiment 16, wherein the IL- 15 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 45 and 75.

[0246] Embodiment 18. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the valine-to-alanine substitution at amino acid position 49; and iii) the serine-to-proline substitution at amino acid position 75.

[0247] Embodiment 19. The IL-15 variant according to Embodiment 18, wherein the IL- 15 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:

[0248] 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 45, 49 and 75.

[0249] Embodiment 20. The IL-15 variant according to Embodiment 19, wherein the IL- 15 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 45, 49 and 75.

[0250] Embodiment 21. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the leucine-to-lysine substitution at amino acid position 52; and iii) the serine-to-proline substitution at amino acid position 75.

[0251] Embodiment 22. The IL-15 variant according to Embodiment 21 , wherein the IL- 15 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:

[0252] 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 45, 52 and 75.

[0253] Embodiment 23. The IL-15 variant according to Embodiment 22, wherein the IL- 15 variant comprises 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 SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 45, 52 and 75.

[0254] Embodiment 24. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the leucine-to-arginine substitution at amino acid position 52; and iii) the serine-to-proline substitution at amino acid position 75.

[0255] Embodiment 25. The IL-15 variant according to Embodiment 24, wherein the IL- 15 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:

[0256] 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45, 52 and 75.

[0257] Embodiment 26. The IL-15 variant according to Embodiment 25, wherein the IL- 15 variant comprises 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 SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45, 52 and 75.

[0258] Embodiment 27. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the leucine-to-threonine substitution at amino acid position 45; and ii) the valine-to-alanine substitution at amino acid position 49; and iii) the serine-to-proline substitution at amino acid position 75.

[0259] Embodiment 28. The IL-15 variant according to Embodiment 27, wherein the IL- 15 variant comprises an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

[0260] Embodiment 29. The IL-15 variant according to Embodiment 28, wherein the IL- 15 variant comprises 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 SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

[0261] Embodiment 30. The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the valine-to-alanine substitution at amino acid position 49; and ii) the leucine-to-arginine substitution at amino acid position 52; and iii) the serine-to-proline substitution at amino acid position 75.

[0262] Embodiment 31. The IL-15 variant according to Embodiment 30, wherein the IL- 15 variant comprises an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52 and 75.

[0263] Embodiment 32. The IL-15 variant according to Embodiment 31 , wherein the IL- 15 variant comprises 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 SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52 and 75.

[0264] Embodiment 33. The IL-15 variant according to any preceding Embodiment, wherein the IL-15 variant is fused to an interleulkin-15 receptor alpha (IL-15Ra) molecule.

[0265] Embodiment 34. The IL-15 variant according to any preceding Embodiment, wherein the IL-15 variant is fused to a sushi domain of an interleulkin-15 receptor alpha (IL-15Ra) molecule.

[0266] Embodiment 35. The IL-15 variant according to Embodiment 33 or 34, wherein the IL-15 variant is fused via a linker, preferably wherein the linker is a glycine-serine linker.

[0267] Embodiment 36. The IL-15 variant according to Embodiment 35, wherein the linker comprises a sequence according to SEQ ID NO: 17, or a sequence comprising at least 70% identity to SEQ ID NO: 17.

[0268] Embodiment 37. The IL-15 variant according to any one of Embodiments 33 to 36, wherein the IL-15 variant comprises a sequence according to any of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, or a sequence comprising at least 70% identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, wherein the % sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, retains the amino acid modifications at the relevant amino acid positions.

[0269] Embodiment 38. The IL-15 variant according to Embodiment 37, wherein the IL- 15 variant comprises an amino acid sequence having 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: 8, 9, 10, 11 , 12, and / or 13, wherein the % sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, retains the amino acid modifications at the relevant amino acid positions.

[0270] Embodiment 39. The IL-15 variant according to any preceding Embodiment, wherein the IL-15 variant is fused to a signal peptide.

[0271] Embodiment 40. The IL-15 variant according to Embodiment 39, wherein the signal peptide is a periplasmic signal peptide.

[0272] Embodiment 41. The IL-15 variant according to Embodiment 39 or 40, wherein the signal peptide is fused to the IL-15 variant via a spacer sequence, preferably wherein the spacer sequence is between 1 to 5 amino acids in length.

[0273] Embodiment 42. The IL-15 variant according to any preceding Embodiment, for use as a therapy.

[0274] Embodiment 43. The IL-15 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.

[0275] Embodiment 44. The IL-15 variant for use according to Embodiment 43, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

[0276] Embodiment 45. The IL-15 variant for use according to Embodiment 44, 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, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, endometrial cancer, skin cancer or sarcoma.

[0277] Embodiment 46. The IL-15 variant for use according to any one of Embodiments 42 to 45, wherein the IL-15 variant is administered to the subject via local instillation, intra-peritoneally, intrapleurally, intravesically, intravaginally, peritumoral injection, intratumoural injection or orally. Embodiment 47. A nucleic acid molecule encoding the IL-15 variant as defined in any one of Embodiments 1 to 41 .

[0278] Embodiment 48. The nucleic acid molecule according to Embodiment 47, wherein the nucleic acid molecule encodes an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, or a sequence having at least 70% identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ I D NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16 retains the amino acid modifications at the relevant amino acid positions.

[0279] Embodiment 49. The nucleic acid molecule according to Embodiment 48, wherein the IL-15 variant comprises an amino acid sequence having 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, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16 retains the amino acid modifications at the relevant amino acid positions.

[0280] Embodiment 50. The nucleic acid molecule according to any one of Embodiments 47 to 49, wherein the nucleic acid molecule comprises a DNA molecule.

[0281] Embodiment 51. The nucleic acid molecule according to any one of Embodiments 47 to 50, wherein the nucleic acid molecule comprises an RNA molecule.

[0282] Embodiment 52. The nucleic acid molecule according to Embodiment 51 , wherein the RNA molecule comprises an mRNA molecule.

[0283] Embodiment 53. A Gram-negative bacterium comprising the IL-15 variant according to any one of Embodiments 1 to 41 , or the nucleic acid molecule according to any one of Embodiments 47 to 52.

[0284] Embodiment 54. The Gram-negative bacterium according to Embodiment 53, wherein the Gram-negative bacterium is a live attenuated Gram-negative bacterium. Embodiment 55. The Gram-negative bacterium according to Embodiment 54, wherein the live attenuated Gram-negative bacterium is a Salmonella spp.

[0285] Embodiment 56. The Gram-negative bacterium according to Embodiment 55, wherein the live attenuated Gram-negative bacterium is Salmonella enterica, preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi.

[0286] Embodiment 57. The Gram-negative bacterium according to Embodiment 56, wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi ZH9.

[0287] Embodiment 58. The Gram-negative bacterium according to any one of

[0288] Embodiments 53 to 57, for use as a therapy.

[0289] Embodiment 59. The Gram-negative bacterium according to any one of

[0290] Embodiments 53 to 58, for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0291] Embodiment 60. The Gram-negative bacterium for use according to Embodiment

[0292] 59, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

[0293] Embodiment 61 . The Gram-negative bacterium for use according to Embodiment

[0294] 60, 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.

[0295] Embodiment 62. The Gram-negative bacterium for use according to any one of Embodiments 53 to 61 , wherein the Gram-negative bacterium is administered to the subject via local instillation, intra-peritoneally, intrapleurally, intravenously, intravesically, peritumoral injection, intratumoural injection, or orally. Embodiment 63. Use of the IL-15 variant as defined in any one of Embodiments 1 to 41 , or the nucleic acid molecule as defined in any one of Embodiments 47 to 52, or the Gram-negative bacterium as defined in any one of Embodiments 53 to 62, in the manufacture of a medicament for a therapy. Embodiment 64. The use according to Embodiment 63, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0296] Embodiment 65. A method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject IL-15 variant as defined in any one of Embodiments

[0297] 1 to 41 , or the nucleic acid molecule as defined in any one of Embodiments 47 to 52, or the Gram-negative bacterium as defined in any one of Embodiments 53 to 62.

[0298] SEQUENCES FORMING PART OF THE DESCRIPTION

[0299] SEQ ID NO: 1 (wild-type / unmodified IL-15)

[0300] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI

[0301] HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0302] SEQ ID NO: 2 (GBL01)

[0303] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISLESGDASI

[0304] HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0305] SEQ ID NO: 3 (GBL15)

[0306] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQAISLESGDASI

[0307] HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0308] SEQ ID NO: 4 (GBL17)

[0309] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISKESGDASI

[0310] HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0311] SEQ ID NO: 5 (GBL18)

[0312] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISRESGDASI

[0313] HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0314] SEQ ID NO: 6 (GBL25)

[0315] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLTELQAISLESGDASI

[0316] HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0317] SEQ ID NO: 7 (GBL50)

[0318] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQAISRESGDASI

[0319] HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 8 (SAg01)

[0320] MITCPPPMSVE HADI WKS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATWAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLSELQVISLESGDASIHDTVENLI ILANNSLPSNGWTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0321] SEQ ID NO: 9 (SAg15)

[0322] MITCPPPMSVE HADI KS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLSELQAISLESGDASIHDTVENLI ILANNSLSSNGWTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0323] SEQ ID NO: 10 (SAg17)

[0324] MITCPPPMSVE HADI WKS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATWAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLSELQVISKESGDASIHDTVENLI ILANNSLSSNGWTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0325] SEQ ID NO: 11 (SAg18)

[0326] MITCPPPMSVE HADI WKS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATWAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLSELQVISRESGDASIHDTVENLI ILANNSLPSNGWTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0327] SEQ ID NO: 12 (SAg25)

[0328] MITCPPPMSVE HADI WKS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATWAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLTELQAISLESGDASIHDTVENLI ILANNSLPSNGWTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0329] SEQ ID NO: 13 (SAg50) MITCPPPMSVE HADI KS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT

[0330] PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL

[0331] YTESDVHPSCKVTAMKCFLLELQAISRESGDASIHDTVENLI ILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 14 (LSP)

[0332] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA

[0333] SEQ ID NO: 15 (SSP)

[0334] MVLGTIDLCSCFSAGLPKTEA

[0335] SEQ ID NO: 16 (parental / M40 IL-15) NWWISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI

[0336] HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0337] SEQ ID NO: 17 (exemplary GS linker) SGGGSGGGGSGGGGSGGGGSGGGSLQA

[0338] REFERENCES

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[0341] Behar, G., V. Sole, A. Defontaine, M. Maillasson, A. Quemener, Y. Jacques & C. Tellier (2011) Evolution of interleukin-15 for higher E. coli expression and solubility. Protein Eng Des Sei. 24(3): 283-290.

[0342] Bergamaschi, C., J. Bear, M. Rosati, R. K. Beach, C. Alicea, et al (2012) Circulating IL-15 exists as heterodimeric complex with soluble IL-15Ra in human and mouse serum. Blood. 120(1): e1-8.

[0343] Bergamaschi, C., R. Jalah, V. Kulkarni, M. Rosati, G. M. Zhang, C. et al (2009) Secretion and biological activity of short signal peptide IL-15 is chaperoned by IL- 15 receptor alpha in vivo. J Immunol. 183(5): 3064-3072.

[0344] Chirifu, M., C. Hayashi, T. Nakamura, S. Toma, T. Shuto, H. Kai, Y. Yamagata, S. J. Davis & S. Ikemizu (2007) Crystal structure of the I L-15-I L-15Ralpha complex, a cytokine-receptor unit presented in trans. Nat Immunol. 8(9): 1001-1007. de Marco, A. (2009) Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microbial Cell Factories. 8.

[0345] Devi, N., Adivitiya & Y. P. Khasa (2016) A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hlL-7 in E. coli fed-batch culture. Appl Microbiol Biotechnol. 100(23): 9979-9994.

[0346] Duitman, E. H., Z. Orinska, E. Bulanova, R. Paus & S. Bulfone-Paus (2008) How a cytokine is chaperoned through the secretory pathway by complexing with its own receptor: lessons from interleukin-15 (IL-15) / IL-15 receptor alpha. Mol Cell Biol. 28(15): 4851-4861.

[0347] Eisenman, J., M. Ahdieh, C. Beers, K. Brasel, M. K. Kennedy, T. Le, T. P. Bonnert, R. J. Paxton & L. S. Park (2002) Interleukin-15 interactions with interleukin-15 receptor complexes: characterization and species specificity. Cytokine. 20(3): 121-129.

[0348] Ferrer-Miralles N., Domingo-Espin J., Corchero J.L., Vazquez E., & Villaverde A. (2009) Microbial factories for recombinant pharmaceuticals. Microbial Cell Factories. 8.

[0349] Guo, Y, L. Luan, N. K. Patil & E. R. Sherwood (2017) Immunobiology of the IL- 15 / 1 L-15Ra complex as an antitumor and antiviral agent. Cytokine Growth Factor Rev 38: 10-21.

[0350] Kuhlman, T. E. & E. C. Cox (2010) Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids. Res 38(6): e92.

[0351] Nausch, H., J. Huckauf, R. Koslowski, U. Meyer, I. Broer & H. Mikschofsky (2013) Recombinant production of human interleukin 6 in Escherichia coli. PLoS One 8(1): e54933.

[0352] Park, Y, A. Espah Borujeni, T. E. Gorochowski, J. Shin & C. A. Voigt (2020) Precision design of stable genetic circuits carried in highly-insulated E. coli genomic landing pads. Mol Syst Biol 16(8): e9584.

[0353] Pettit, D. K., T. P. Bonnert, J. Eisenman, S. Srinivasan, R. Paxton, et al (1997) Structure-function studies of interleukin 15 using site-specific mutagenesis, polyethylene glycol conjugation, and homology modeling. J Biol Chem 272(4): 2312-2318.

[0354] Ring, A. M., J. X. Lin, D. Feng, S. Mitra, M. Rickert, et al (2012) Mechanistic and structural insight into the functional dichotomy between IL-2 and IL-15. Nat Immunol 13(12): 1187-1195. Robinson, T. O. & K. S. Schluns (2017) The potential and promise of IL-15 in immuno-oncogenic therapies. Immunol Lett 190: 159-168.

[0355] Saeed, S. & P. A. Revell (2001) Production and distribution of interleukin 15 and its receptors (IL-15Ralpha and IL-R2beta) in the implant interface tissues obtained during revision of failed total joint replacement. Int J Exp Pathol 82(3): 201 -209.

[0356] Santana Carrero, R. M., F. Beceren-Braun, S. C. Rivas, S. M. Hegde, A. Gangadharan, et al (2019) IL-15 is a component of the inflammatory milieu in the tumor microenvironment promoting antitumor responses. Proc Natl Acad Sci U S A 116(2): 599-608.

Claims

CLAIMS1. A bacterium comprising an interleukin-15 (IL-15) variant, wherein in comparison to wild-type I L-15, the I L-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin-15 receptor alpha (IL-15Ra) interaction surface.

2. The bacterium according to claim 1 , wherein the bacterium is a live attenuated bacterium.

3. The bacterium according to claim 2, wherein bacterium is a live attenuated Gram-negative bacterium.

4. The bacterium according to claim 3, wherein the live attenuated Gramnegative 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.

5. The bacterium according to any preceding claim, wherein wild-type IL-15 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: 16.

6. The bacterium according to claim 5, wherein the IL-15 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 .

7. The bacterium according to claim 5 or 6, wherein in comparison to SEQ ID NO: 1 , the amino acid modifications occur at one or more of amino acid positions 45 and / or 49 and / or 52, or any combination thereof.

8. The bacterium according to any preceding claim, wherein the IL-15 variant comprises an amino acid modification at amino acid position 75, preferably wherein the amino acid modification at amino acid position 75 is a serine-to-proline substitution.

9. The bacterium according to claim 8, wherein the IL-15 variant comprising the serine-to-proline substitution at amino acid position 75 comprises a sequence according to SEQ ID NO: 16, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 16, wherein the % sequence identity to SEQ ID NO: 16 retains the amino acid modifications at amino acid position 75.

10. The bacterium according to any of claims 7 to 9, wherein the amino acid modification at amino acid position 45 is a hydrophobic-to-hydrophilic substitution, preferably wherein the hydrophobic-to-hydrophilic substitution is a leucine-to- serine substitution or a leucine-to-threonine substitution.11 . The bacterium according to any of claims 7 to 10, wherein the amino acid modification at amino acid position 49 is a hydrophobic-to-hydrophobic substitution, preferably wherein the hydrophobic-to- hydrophobic substitution is a valine-to-alanine substitution.

12. The bacterium according to any of claims 7 to 11 , wherein the amino acid modification at amino acid position 52 is a hydrophobic-to-hydrophilic substitution, preferably wherein the hydrophobic-to-hydrophilic substitution is a leucine-to- lysine substitution or a leucine-to-arginine substitution.

13. The bacterium according to any of claims 7 to 12, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the serine-to-proline substitution at amino acid position 75.

14. The bacterium according to claim 13, wherein the IL-15 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 45 and 75.

15. The bacterium according to claim 14, wherein the IL-15 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 45 and 75.

16. The bacterium according to any of claims 7 to 15, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the valine-to-alanine substitution at amino acid position 49; and iii) the serine-to-proline substitution at amino acid position 75.

17. The bacterium according to claim 16, wherein the IL-15 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 45, 49 and 75.

18. The bacterium according to claim 17, wherein the IL-15 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 45, 49 and 75.

19. The bacterium according to any of claims 7 to 18, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the leucine-to-lysine substitution at amino acid position 52; andiii) the serine-to-proline substitution at amino acid position 75.

20. The bacterium according to claim 19, wherein the IL-15 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 45, 52 and 75.21 . The bacterium according to claim 20, wherein the IL-15 variant comprises 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 SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 45, 52 and 75.

22. The bacterium according to any of claims 7 to 21 , wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the leucine-to-arginine substitution at amino acid position 52; and iii) the serine-to-proline substitution at amino acid position 75.

23. The bacterium according to claim 22, wherein the IL-15 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 45, 52 and 75.

24. The bacterium according to claim 23, wherein the IL-15 variant comprises 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 SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45, 52 and 75.

25. The bacterium according to any of claims 7 to 24, wherein the IL-15 variant comprises: i) the leucine-to-threonine substitution at amino acid position 45; and ii) the valine-to-alanine substitution at amino acid position 49; and iii) the serine-to-proline substitution at amino acid position 75.

26. The bacterium according to claim 25, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

27. The bacterium according to claim 26, wherein the IL-15 variant comprises 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 SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

28. The bacterium according to any of claims 7 to 27, wherein the IL-15 variant comprises: i) the valine-to-alanine substitution at amino acid position 49; and ii) the leucine-to-arginine substitution at amino acid position 52; and iii) the serine-to-proline substitution at amino acid position 75.

29. The bacterium according to claim 28, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52 and 75.

30. The bacterium according to claim 29, wherein the IL-15 variant comprises 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 SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52 and 75.

31. The bacterium according to any preceding claim, wherein the IL-15 variant is fused to an interleulkin-15 receptor alpha (IL-15Ra) molecule.

32. The bacterium according to any preceding claim, wherein the IL-15 variant is fused to a sushi domain of an interleulkin-15 receptor alpha (IL-15Ra) molecule.

33. The bacterium according to claim 31 or 32, wherein the IL-15 variant is fused via a linker, preferably wherein the linker is a glycine-serine linker.

34. The bacterium according to claim 33, wherein the linker comprises a sequence according to SEQ ID NO: 17, or a sequence comprising at least 70% identity to SEQ ID NO: 17.

35. The bacterium according to any one of claims 31 to 34, wherein the IL-15 variant comprises a sequence according to any of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, or a sequence comprising at least 70% identity to SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, wherein the % sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, retains the amino acid modifications at the relevant amino acid positions.

36. The bacterium according to claim 35, wherein the IL-15 variant comprises an amino acid sequence having 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: 8, 9, 10, 11 , 12, and / or 13, wherein the % sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11 , 12, and / or 13, retains the amino acid modifications at the relevant amino acid positions.

37. The bacterium according to any preceding claim, wherein the IL-15 variant is fused to a signal peptide.

38. The bacterium according to claim 37, wherein the signal peptide is a periplasmic signal peptide.

39. The bacterium according to claim 37 or 38, wherein the signal peptide is fused to the IL-15 variant via a spacer sequence, preferably wherein the spacer sequence is between 1 to 5 amino acids in length.

40. The bacterium according to any preceding claim, wherein the IL-15 variant is encoded by a nucleic acid molecule.

41. The bacterium according to claim 40, wherein the nucleic acid molecule encodes an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, or a sequence having at least 70% identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 retains the amino acid modifications at the relevant amino acid positions.

42. The bacterium according to claim 41 , wherein the IL-15 variant comprises an amino acid sequence having 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, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, and / or 16 retains the amino acid modifications at the relevant amino acid positions.

43. The bacterium according to any of claims 40 to 42, wherein the nucleic acid molecule comprises a DNA molecule.

44. The bacterium according to any of claims 40 to 43, wherein the nucleic acid molecule comprises a RNA molecule, preferably wherein the RNA molecule comprises an mRNA molecule.

45. An interleukin-15 (IL-15) variant, wherein wild-type IL-15 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-15 variant comprises one or more amino acidmodifications in a surface region adjacent to a receptor interaction surface, and wherein the one or more amino acid modifications comprise any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophobic substitution at amino acid position 49; iii) a hydrophobic-to-hydrophilic substitution at amino acid position 52; and / or iv) a serine-to-proline substitution at amino acid position 75; preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface.

46. The IL-15 variant according to claim 45, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 45 is a leucine-to-serine or leucine- to-threonine substitution.

47. The IL-15 variant according to claim 45 or 46, wherein the hydrophobic- to-hydrophilic substitution at amino acid position 52 is a leucine-to-lysine or leucine-to-arginine substitution.

48. The IL-15 variant according to any of claims 45 to 47, wherein the hydrophobic-to-hydrophobic substitution at amino acid position 49 is a valine-to- alanine or valine-to-serine substitution.

49. A nucleic acid molecule encoding the IL-15 variant as defined in any of claims 45 to 48.

50. The nucleic acid molecule according to claim 49, wherein the nucleic acid molecule encodes an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 and / or 16, or a sequence having at least 70% identity to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 and / or 16 retains the amino acid modifications at the relevant amino acid positions.51 . The nucleic acid molecule according to claim 50, wherein the IL-15 variant comprises an amino acid sequence having 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: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 and / or 16, wherein the % sequence identity to any one of SEQ I D NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 and / or 16 retains the amino acid modifications at the relevant amino acid positions.

52. The nucleic acid molecule according to any of claims 49 to 51 , wherein the nucleic acid molecule comprises a DNA molecule.

53. The nucleic acid molecule according to any of claims 49 to 52, wherein the nucleic acid molecule comprises an RNA molecule, preferably wherein the RNA molecule comprises an mRNA molecule.

54. A bacterium comprising the IL-15 variant according to any of claims 45 to 48, or the nucleic acid molecule according to any of claims 49 to 53.

55. The bacterium according to claim 54, wherein the bacterium is a live attenuated bacterium.

56. The bacterium according to claim 55, wherein bacterium is a live attenuated Gram-negative bacterium.

57. The bacterium according to claim 56, wherein the live attenuated Gramnegative 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.

58. The bacterium according to any of claims 1 to 44 or 54 to 57, or the IL-15 variant according to any of claims 45 to 48, or the nucleic acid molecule according to any of claims 49 to 53, for use as a therapy.

59. The bacterium, IL-15 variant or nucleic acid molecule for use according to claim 58, wherein the therapy is a treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

60. The bacterium, IL-15 variant or nucleic acid molecule for use according to claim 59, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

61. The bacterium, IL-15 variant or nucleic acid molecule for use according to claim 60, 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.

62. The bacterium, IL-15 variant or nucleic acid molecule for use according to claim 61 , wherein the bacterium, IL-15 variant or nucleic acid molecule is administered to the subject via local instillation, intra-peritoneally, intrapleurally, intravenously, intravesically, peritumoral injection, intratumoural injection, or orally.

63. Use of the bacterium according to any of claims 1 to 44 or 54 to 57, or the IL-15 variant according to any of claims 45 to 48, or the nucleic acid molecule according to any of claims 49 to 53, in the manufacture of a medicament for a therapy.

64. The use according to claim 63, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

65. A method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject the bacterium according to any of claims 1 to 44 or 54 to 57, or the IL- 15 variant according to any of claims 45 to 48, or the nucleic acid molecule according to any of claims 49 to 53.

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