Interleukin variant

Abstract

There is provided an interleukin-21 (IL-21) variant, wherein in comparison to wild-type IL-21 that 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, the IL-21 variant comprises an amino acid modification at one or more of the following amino acid positions: 29, 33, 36 and / or 71.

Description

[0001] INTERLEUKIN VARIANT

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to interleukin-21 (IL-21) 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 (40-80% 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 up to 27 intravesical, 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-21 (IL-21) molecules are considered as promising immunotherapeutics because of their role in CD8 T cells and NK activation and proliferation (Parrish- Novak et al, 2000).

[0007] However, a challenge associated with the use of IL-21 as an immuno-stimulatory anti-cancer treatment 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 due to the lack of post- translational modifications which play an integral role in protein folding, activity, and stability. 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 et al, 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-21) where there are disulphide bonds in the protein structure.

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

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

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

[0011] SUMMARY OF INVENTION

[0012] The present invention provides IL-21 variants with optimised solubility and preserved bioactivity. The inventors of the present invention have surprisingly identified certain combinations of mutations in IL-21 that result in IL-21 variants which are soluble and functional. 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 an interleukin-21 (IL-21) variant, wherein in comparison to wild-type IL-21 that 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, the IL-21 variant comprises an amino acid modification at one or more of the following amino acid positions: 29, 33, 36 and / or 71.

[0014] In a second aspect of the invention, there is provided a nucleic acid molecule encoding an IL-21 variant according to the first aspect of the invention.

[0015] In a third aspect of the invention, there is provided a bacterium comprising the IL- 21 variant according to the first aspect of the invention, or the nucleic acid molecule according to the second aspect of the invention.

[0016] In a fourth aspect of the invention, there is provided an IL-21 variant according to the first aspect of the invention, or the nucleic acid molecule according to the second aspect of the invention, or the bacterium according to the third aspect of the invention, for use in therapy.

[0017] In a fifth aspect of the invention, there is provided a use of the IL-21 variant according to the first aspect of the invention, or the nucleic acid molecule according to the second aspect of the invention, or the bacterium according to the third aspect of the invention, for use in the manufacture of a medicament for a therapy.

[0018] In a sixth 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 IL-21 variant according to the first aspect of the invention, or the nucleic acid molecule according to the second aspect of the invention, or the bacterium according to the third aspect of the invention. BRIEF DESCRIPTION OF DRAWINGS

[0019] The invention is described with reference to the accompanying drawings, wherein:

[0020] Figure 1 shows the crystal structure of IL-21 bound to IL-21 R (3TGX), overlaid with the computed hydrophobic surface. Amino acids I8, V24, V28, F31 , I66 and V69 are indicated (corresponding to 113, V29, V33, F36, 171 and V74 as the crystal structure residue labelling is shifted -5 amino acid positions relative to SEQ ID NO: 6, or -6 amino acid positions relative to SEQ ID NO: 1).

[0021] Figures 2A and 2B show the solubility and activity of IL-21 variants versus wildtype IL-21.

[0022] Figure 3 shows the experimental set up to evaluate variant activity.

[0023] Figure 4 shows the crystal structure of IL-21 in complex with IL-21 R, and the selected amino acids to be modified at amino acid positions V24, V28, F31 and I66 (corresponding to V29, V33, F36 and 171).

[0024] Figures 5A and 5B show the screening of purification conditions (A) and purification of IL-21 variants using His-based purification (B).

[0025] Figure 6 shows the His-based purification of IL-21 variants using the AKTA purification system.

[0026] Figure 7 shows an SDS-PAGE showing the final protein purification products of the four IL-21 variants.

[0027] Figures 8A and 8B show the results of cell-based assays (relative cell activation) using increasing concentrations of the four purified IL-21 variants versus the wildtype IL-21.

[0028] DETAILED DESCRIPTION The present invention provides optimised interleukin-21 (IL-21) variants which are both soluble and functional, and are therefore optimal candidates for efficient secretion systems which allow the production, correct folding and secretion of said IL-21 variants. The inventors of the present invention have surprisingly identified that certain combinations of mutations to certain aggregation-prone amino acids in IL-21 (Figure 1) results in IL-21 variants which are soluble, stable and functional.

[0029] Accordingly, in a first aspect the invention provides an interleukin-21 (IL-21) variant, wherein in comparison to wild-type IL-21 that 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, the IL-21 variant comprises an amino acid modification at one or more of the following amino acid positions: 29, 33, 36 and / or 71. These amino acid modifications may be present in a region adjacent to a receptor interaction surface of the IL-21 variant (Figure 1).

[0030] 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.

[0031] Interleukin-21 (IL-21) stands as a pivotal member of the cytokine family, exhibiting a myriad of pleiotropic actions that profoundly influence the differentiation and function of both lymphoid and myeloid cells (Parrish-Novak et al, 2000; Spolski et al, 2008). This multifaceted cytokine is primarily synthesized by natural killer (NK) T cells, T follicular helper (TFH) cells, and TH17 cells (Parrish-Novak et al, 2000; Ozaki et al, 2000). Structurally, IL-21 consists of four a-helical bundles, adopting the typical type I cytokine structure characterized by a four-helix bundle with an up-up-down-down topology (Ozaki et al, 2000; Hamming et al, 2012). IL-21 exercises its biological effects through a heterodimeric receptor complex comprising the IL-21 receptor (IL-21 R) and the common y chain (gammaC or ‘yC’), shared by several other cytokines including IL-2, IL-4, IL-7, IL-9, and IL-15 (Kovanen et al, 2004; Leonard et al, 2001 ; Asao et al, 2001). Upon IL-21 binding to its receptor, signalling cascades are initiated through the Jak-STAT pathway, mediating diverse cellular responses.

[0032] The interaction between IL-21 and IL-21 R is driven by the IL-21 R chain's high- affinity binding and stabilization of helix C in IL-21 upon receptor engagement. This interaction is mediated by specific residues in helices A and C, as well as a portion of the CD loop following helix C of IL-21. Notably, ten residues of IL-21 engage in polar interactions with eleven IL-21 R residues, while fourteen IL-21 residues establish van der Waals contacts with sixteen IL-21 R residues. Key contributors to the binding surface include Arg5, Arg9, and Gln12 of helix A, along with Arg76 and Lys73 of helix C in IL-21. Additionally, Met70 facilitates the interaction of Arg9 and Arg76 of IL-21 with Asp72 and Asp73 of IL-21 R, respectively.

[0033] One of the remarkable features of IL-21 is its ability to cooperate with other cytokines, amplifying the cytotoxicity of CD8+ T cells and facilitating the proliferation of these cells in the presence of antigens (Kovanen et al, 2004; Leonard et al, 2001 ; Asao et al, 2001 ; Zeng et al, 2005; Hinrichs et al, 2008). Functional IL-21 receptors are broadly expressed on various lympho- hematopoietic populations, including myeloid cells, allowing IL-21 to exert its effects on a wide range of cell types. In the context of B cell immunoglobulin responses, IL-21 plays a significant role, enhancing IgE induction when combined with IL-4 (Jin et al, 2004; Bryant et al, 2007). Paradoxically, IL-21 can stimulate B cell proliferation and differentiation, but also possesses the capability to induce B cell apoptosis, particularly in the absence of a T cell signal or in the presence of Toll-like receptor (TLR) signals (Jin et al, 2004). IL-21 is not only vital for CD8+ T cell proliferation and functional responses but also enhances the cytotoxic activity of both CD8+ T cells and NK cells (Zeng et al, 2005). This positions IL-21 as a potential candidate for anti-tumour therapy. Studies in murine models have demonstrated that IL-21 , either alone or in combination with other cytokines like IL-15, inhibits tumour growth, leading to regression of melanoma and fibrosarcoma (Zeng et al, 2005; Wang et al, 2003), with a substantial contribution from NK cells and CD8+ T cells. Unlike its counterpart IL-2, IL-21 does not induce the expansion of regulatory T cells (TReg cells), thereby preserving the activity of expanded antitumor CD8+ T cells (Sivakumar et al, 2013).

[0034] The unique ability of IL-21 to enhance the ability of NK cells to lyse antibody- coated tumour cells adds another layer of complexity to its anti-tumour potential (Roda et al, 2006). IL-21's diverse and intricate roles in regulating immune responses, coupled with its potential in anti-tumour therapy, make it a promising target for further research and therapeutic development.

[0035] There is a clear need to develop variants of IL-21 which are optimally soluble and functional / biologically active, and can therefore be successfully expressed in, and secreted from, an expression system.

[0036] 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, nonmutated, unmodified, or unchanged form. In one embodiment, the wild-type IL-21 is mature (i.e., unmodified) IL-21. The wild-type IL-21 typically comprises an amino acid sequence according to SEQ ID NO: 1 orSEQ ID NO: 6. In comparison to “wild-type” IL-21 , the terms “interleukin variant”, “variant” and “IL-21 variant” are interchangeable and refer to an IL-21 molecule which, in comparison to wild-type IL-21 , comprises one or more amino acid modifications. The wild-type IL-21 may also be referred to as parental IL-21 , as the IL-21 variants of the present invention are derived from this baseline sequence.

[0037] As used herein, the term “amino acid modifications” refers to any change or mutation to a wild-type amino acid sequence. An amino acid modification may or may not alter the structure, function, and / or physiochemical properties of the protein. Amino acid modifications may include, but are not limited to deletions, substitutions, and insertions. Deletion mutations involve the loss of an amino acid, resulting in a frameshift or decrease in the length of the amino acid sequence. Insertion mutations involve the addition of an amino acid, resulting in a frameshift or increase in the length of the amino acid sequence. Substitution mutations involve the replacement of one amino acid for another. Substitution mutations can be conservative or non-conservative. As would be understood by the skilled person, amino acids can be grouped broadly according to their properties (i.e., size, bulkiness, charge, hydrophobicity, polarity, etc). Similarity or dissimilarity between amino acids can also be calculated using substitution matrices and physicochemical distances. A conservative amino acid substitution involves the replacement or exchange of one amino acid for another amino acid with similar properties. For example, a substitution of a hydrophobic amino acid for another hydrophobic amino acid (e.g., a valine-to-alanine substitution) would constitute a conservative amino acid substitution. A non-conservative or radical amino acid substitution involves the replacement or exchange of one amino acid for another amino acid with different or dissimilar properties. For example, a substitution of a hydrophobic amino acid for a hydrophilic amino acid (e.g., a leucine-to-serine substitution) would constitute a non-conservative amino acid substitution. Non- conservative amino acid substitutions are more likely to be associated with changes in protein structure and function compared to conservative amino acid substitutions.

[0038] 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.

[0039] The inventors of the present invention have surprisingly identified a number of amino acid modifications which can increase the solubility of IL-21 while retaining its biological activity. In particular, the inventors of the present invention have surprisingly found that amino acid modifications to amino acids at positions 29, 33, 37 and / or 71 of IL-21 can generate an IL-21 variant which is soluble, stable and has similar activity levels to that of wild-type IL-21.

[0040] IL-21 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-21 variant preferably comprises two amino acid modifications.

[0041] In accordance with a first aspect of the invention, there is provided an interleukin- 21 (IL-21) variant, wherein in comparison to wild-type IL-21 that 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, the IL-21 variant comprises an amino acid modification at one or more of the following amino acid positions: 29, 33, 36 and / or 71.

[0042] In one embodiment, the region adjacent to a receptor interaction surface may be a surface region. As used herein, the term “surface region” refers to the part of a protein which is exposed to solvent. In the overall three-dimensional structure of the protein, the protein surface is commonly referred to as the solvent-accessible surface of the protein. For a soluble protein, the protein surface will be predominantly composed of hydrophilic amino acid residues, whereas the core (i.e. , the interior) of the protein will be composed of hydrophobic residues which are packed together away from the solvent. Modifications to amino acids which are on or near the surface of the protein may alter the solubility of a protein, especially modifications which substitute hydrophobic for hydrophilic amino acids. The amino acids to be modified at the surface region of the IL-21 variant are the amino acids at amino acid positions 13, 29, 33, and / or 36. In a preferred embodiment of the invention, the amino acids to be modified are the amino acids at amino acid positions 29, 33, and / or 36. In other embodiments, the region adjacent to a receptor interaction surface is not a surface region. The amino acids at amino acid positions 71 and / or 74 are not present on the surface of IL-21 .

[0043] Figure 1 shows the crystal structure of IL-21 in complex with IL-21 R. The aggregation-prone amino acids are labelled with the amino acid code and their position relative to the start of the mature polypeptide chain. The hydrophobic surface of IL-21 is overlaid with the protein cartoon representation. The amino acids that may be modified (amino acid positions 13, 29, 33, 36, 71 and / or 74) are shown.

[0044] Modifications to amino acids on the protein surface may implicate protein-protein interactions. Protein surfaces may comprise protein-protein interfaces (i.e., the molecular surfaces through which subunits of multi-protein complexes make contact and interact with one another). These interfaces may comprise large, flat interfaces, and / or smaller concavities or pockets or even single residues which may form an anchoring site for one or more interacting proteins. The proteinprotein interface may be shape and / or chemically complementary. Other nonsurface regions may also implicate protein-protein interactions, for example pockets or cavities within the three-dimensional protein structure which form binding sites or interaction pockets.

[0045] In the context of IL-21 , protein-protein interactions may include the interaction between IL-21 and its receptor (IL-21 R) together with gamma C. The IL-21 / IL- 21 R interaction occurs at the surface of IL-21 in a receptor interaction surface. 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 I L-21 which is involved in coordinating, interacting with and binding to a receptor. Preferably, the receptor interaction surface is a surface for interacting with an IL-21 receptor, more preferably IL- 21 R / GammaC. There may be a defined group of amino acids which directly bind the receptor. Amino acids which are in a region adjacent to the receptor interaction surface may therefore play a role in mediating protein-protein interactions with the receptor (i.e. , they are adjacent to the residues which interact with the receptors). As used herein, the term “adjacent” refers to surface amino acids which are proximal to the receptor interaction surface (see Figure 1).

[0046] Certain modifications may act to enhance the interaction between IL-21 and IL- 21 R, for example by increasing the affinity of binding. The interaction IL-21 and IL-21 R may be enhanced in that there is a lower rate constant of ligand dissociation (Kotf) (i.e., high stability of IL-21 / IL-21 R complexes), a higher rate constant of ligand association (Kon) (i.e., fast recognition of IL-21 by IL-21 R and vice versa), and a lower equilibrium dissociation constant (Kd) (i.e., a high affinity interaction or no 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.

[0047] Further, as described herein, modifications which alter the composition properties of amino acids within IL-21 , especially those which alter the hydrophobicity of the protein, may impact the solubility of IL-21. The inventors of the present invention have surprisingly identified mutations within IL-21 that increase the solubility / stability of IL-21 without impacting the biological activity of IL-21.

[0048] In accordance with the invention, there is provided an interleukin-21 (IL-21) variant, wherein in comparison to wild-type IL-21 , the IL-21 variant comprises one or more amino acid modifications in a region adjacent to a receptor interaction surface of the IL-21 variant. As detailed herein, the wild-type IL-21 may comprise mature IL-21 , i.e., an amino acid sequence according to SEQ ID NOs: 1 or 6, or an amino acid sequence comprising at least 70% identity to SEQ ID NOs: 1 or 6. A sequence comprising at least 70% identity to SEQ ID NO: 6 encompasses SEQ ID NO: 1. In the context of the present invention, “wild-type IL-21” may also refer to the parental sequence of IL-21 (SEQ ID NO: 1). By “parental”, we intend the baseline sequence from which the variant sequences are derivable. SEQ ID NO: 1 is the wild-type IL-21 with an additional methionine residue at the start of the sequence to facilitate its expression in bacteria. SEQ ID NO: 6 is the wild-type IL- 21 without the additional methionine residue. Therefore, the IL-21 variants can be derived from SEQ ID NO: 1 or SEQ ID NO: 6, or any amino acid sequence comprising at least 70% identity thereto.

[0049] SEQ ID NO: 1 and SEQ ID NO: 6 are shown below. The amino acid modifications at amino acid positions 13, 29, 33, 36, 71 and 74 which may be present as described herein are highlighted in bold and underlined. The numbering of the amino acid positions as defined in the claims are given in relation to SEQ ID NO: 6. Therefore, the numbering of the amino acid positions in relation to SEQ ID NO: 1 are shifted +1 relative to SEQ ID NO: 6 due to the presence of an additional methionine residue at the beginning of SEQ ID NO: 1.

[0050] SEQ ID NO: 1

[0051] MHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA QLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFK SLLQKMIHQHLSSRTHGSEDS

[0052] SEQ ID NO: 6

[0053] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS LLQKMIHQHLSSRTHGSEDS

[0054] The IL-21 variant may comprise an amino acid sequence comprising at least 70% sequence identity to SEQ ID NOs: 1 or 6. 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).

[0055] In one embodiment, the IL-21 variant may comprise an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 6; 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; or at least 99% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the one or more amino acid modifications at the relevant amino acid positions as defined in the claims.

[0056] In another embodiment, the IL-21 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 1 ; at least 80% sequence identity to SEQ ID NO: 1 ; at least 85% sequence identity to SEQ ID NO: 1 ; at least 90% sequence identity to SEQ ID NO: 1 ; at least 91% sequence identity to SEQ ID NO: 1 ; at least 92% sequence identity to SEQ ID NO: 1 ; at least 93% sequence identity to SEQ ID NO: 1 ; at least 94% sequence identity to SEQ ID NO: 1 ; at least 95% sequence identity to SEQ ID NO: 1 ; at least 96% sequence identity to SEQ ID NO: 1 ; at least 97% sequence identity to SEQ ID NO: 1 ; at least 98% sequence identity to SEQ ID NO: 1 ; or at least 99% sequence identity to SEQ ID NO: 1 , wherein the % sequence identity to SEQ ID NO: 1 retains the one or more amino acid modifications at the relevant amino acid positions as defined in the claims.

[0057] In accordance with the invention, the IL-21 variant may be modified such that, in comparison to SEQ ID NO: 6, the amino acid modifications occur at one or more of amino acid positions 13, 29, 33, 36, 71 and / or 74, or any combination thereof. These amino acid positions are located in a region adjacent to a receptor interaction surface of the IL-21 variant. In comparison to SEQ ID NO: 1 , the amino acid modifications occur at one or more of amino acid positions 14, 30, 34, 37, 72 and / or 75, or any combination thereof.

[0058] In a preferred embodiment, the IL-21 variant may comprise one or more amino acid modifications at any of amino acid positions 29, 33, 36, and / or 71 . However, in a most preferred embodiment, the IL-21 variant may comprise two amino acid modifications at amino acid positions 29 and 36; 33 and 71 ; and / or 36 and 71. It will be understood that an IL-21 variant comprising any of the modifications disclosed herein will retain at least 70% sequence identity to SEQ ID NO: 6.

[0059] In some embodiments, the IL-21 variant may comprise amino acid modifications whereby non-conservative amino acid substitutions are performed. In such embodiments, a wild-type amino acid is replaced by another amino acid with different properties to that of the wild-type amino acid. For example, a hydrophobic amino acid naturally present in the wild-type sequence is replaced with a hydrophilic amino acid, or vice versa.

[0060] In other embodiments, the IL-21 variant may comprise amino acid modifications whereby conservative amino acid substitutions are performed. In such embodiments, a wild-type amino acid is replaced by another amino acid with similar or the same properties to that of the wild-type amino acid. For example, a hydrophobic amino acid naturally present in the wild-type sequence is replaced with another hydrophobic amino acid, or where a hydrophilic amino acid naturally present in the wild-type sequence is replaced with another hydrophilic amino acid.

[0061] As used herein, the terms “hydrophobic” and “hydrophilic” are to be given their usual meanings in the art, that is having a weak affinity and strong affinity for water, respectively. As used herein, the terms “polar” and “non-polar” are to be given their usual meanings in the art, and polar amino acids may have hydrophilic side chains, whereas non-polar amino acids may have hydrophobic side chains.

[0062] “Hydrophobic amino acids” are amino acids which possess hydrophobic side chains, i.e. , side chains that do not interact with (repel) water. Hydrophobic side chains are typically or predominantly composed of carbon and hydrogen atoms. Hydrophobic amino acids are generally non-polar and therefore do not dissolve in polar solvents such as water. Hydrophobic amino acids that are naturally occurring may include glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (lie), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). However, any non-naturally occurring, synthetic, or modified amino acid with hydrophobic properties is also intended to be covered by the term “hydrophobic amino acid”.

[0063] “Hydrophilic amino acids” are amino acids which possess hydrophilic side chains, i.e., side chains that can interact with (attract) water. Hydrophilic side chains are typically composed of oxygen, nitrogen, and sulphur atoms. Hydrophilic amino acids are generally polar and therefore can dissolve in polar solvents such as water via the formation of hydrogen bonds. Hydrophilic amino acids that are naturally occurring may include serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), glutamine (Gin), and tyrosine (Tyr). Other naturally occurring hydrophilic amino acids may include arginine (Arg), histidine (His), lysine (Lys), aspartate (Asp), and glutamate (Glu), as these amino acids have electrically charged side chains. Arginine, histidine, and lysine have an overall positive charge, whereas aspartate and glutamate have an overall negative charge. However, any non-naturally occurring, synthetic, or modified amino acid with hydrophilic properties is also intended to be covered by the term “hydrophilic amino acid”.

[0064] In accordance with the invention, the IL-21 variant may comprise amino acid modifications at one or more of amino acid positions 13, 29, 33, 36, 71 and / or 74, or any combination thereof. In relation to SEQ ID NO: 6, the wild-type amino acid at position 13 is isoleucine (I), the wild-type amino acid at position 29 is valine (V), the wild-type amino acid at position 33 is valine (V), the wild-type amino acid at position 36 is phenylalanine (F), the wild-type amino acid at position 71 is isoleucine (I), and the wild-type amino acid at position 74 is valine (V).

[0065] In a preferred embodiment of the invention, the amino acid modifications at amino acid positions 13, 29, 33, 36, 71 and 74 is a hydrophobic-to-hydrophilic substitution. 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.

[0066] Table 1 shows the wild-type amino acids of IL-21 and their possible modifications / mutations. It should be noted that the amino acid numbering given (113, V29, V33, F36, 171 and V74) is derived from the mature protein ( SEQ ID NO: 6). The numbering in SEQ ID NO: 1 is shifted +1 amino acid position relative to SEQ ID NO: 6 due to an additional methionine at the beginning of the sequence according to SEQ ID NO: 1 which facilitates its translation in bacteria. able 1. Amino acids within IL-21 and their modifications.

[0067] The wild-type amino acid at amino acid position 13 in relation to SEQ ID NO: 6 is isoleucine. The preference of the present invention is for the hydrophobic-to- hydrophilic substitution at amino acid position 13 to be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. Either of these modifications may be selected, however, in a more preferred embodiment, the substitution at amino acid position 13 is an isoleucine-to-aspartate substitution.

[0068] The wild-type amino acid at position 29 in relation to SEQ ID NO: 6 is valine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 29 to be a valine-to-threonine substitution or a valine-to-aspartate substitution. In a more preferred embodiment, the substitution at amino acid position 29 is a valine-to-threonine substitution.

[0069] The wild-type amino acid at position 33 in relation to SEQ ID NO: 6 is valine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 33 to be a valine-to-threonine substitution or a valine-to-aspartate substitution. Either of these modifications may be selected, however, in a more preferred embodiment, the substitution at amino acid position 33 is a valine-to-threonine substitution.

[0070] The wild-type amino acid at position 36 in relation to SEQ ID NO: 6 is phenylalanine. The preference of the present invention is for the hydrophobic-to- hydrophilic substitution at amino acid position 36 to be a phenylalanine-to- glutamine substitution or a phenylalanine-to-lysine substitution. In a more preferred embodiment, the substitution at amino acid position 36 is a phenylalanine-to-lysine substitution.

[0071] The wild-type amino acid at position 71 in relation to SEQ ID NO: 6 is isoleucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 71 to be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. In a more preferred embodiment, the substitution at amino acid position 71 is an isoleucine-to-lysine substitution.

[0072] The wild-type amino acid at position 74 in relation to SEQ ID NO: 6 is valine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 74 to be a valine-to-threonine substitution or a valine-to-aspartate substitution. Either of these modifications may be selected, however, in a more preferred embodiment, the substitution at amino acid position 74 is a valine-to-threonine substitution.

[0073] Without wishing to be bound by theory, these non-conservative amino acid modifications could act to alter the physiochemical properties of the region in which these amino acids reside. Specifically, the region may become more hydrophilic which allows an IL-21 variant with such amino acid modifications to become more soluble / stable. These alterations should not substantially impact the biological activity of IL-21 , therefore resulting in soluble IL-21 variants with similar levels of biological activity to wild-type IL-21 .

[0074] It will be understood that any of the amino acid modifications herein disclosed may be present in any combination with any of the other amino acid modifications herein disclosed. For example, an IL-21 variant according to the present invention may comprise one or more amino acid modifications at any of amino acid positions 13, 29, 33, 36, 71 and / or 74, or any combination thereof.

[0075] Table 2 summarises the possible combinations of modifications / mutations present in each of the four IL-21 variants described above.

[0076] Table 2. Four different IL-21 variants and their preferred mutations.

[0077] As detailed herein, in a preferred embodiment, the IL-21 variant may comprise amino acid modifications at amino acid positions 29 and 36 (IL-21 V36); 33 and 71 (IL-21 V52, IL-21 V58); and / or 36 and 71 (IL-21 V62). Four variants (IL-21 V36, IL-21V52, IL-21V58 and IL-21V62; Table 2) were found to be more soluble than wild-type IL-21 , and have similar activity to wild-type IL-21 (Figures 2 and 3).

[0078] Accordingly, in a preferred embodiment, an IL-21 variant according to the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid positions 29 and 36. In an even more preferred embodiment, an IL-21 variant according to the invention may comprise (i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 29 and (ii) the phenylalanine-to-lysine substitution or phenylalanine-to-glutamine substitution at amino acid position 36. In a most preferred embodiment, the IL-21 variant may comprise the valine-to-threonine substitution at amino acid position 29 and the phenylalanine-to-lysine substitution at amino acid position 36. In this embodiment, the IL-21 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 29 and 36. In another embodiment, the IL-21 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 29 and 36.

[0079] In another preferred embodiment, an IL-21 variant according to the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid positions 33 and 71. In an more preferred embodiment, an IL-21 variant according to the invention may comprise (i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33 and (ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 .

[0080] In one even more preferred embodiment, the IL-21 variant may comprise the valine-to-threonine substitution at amino acid position 33 and the isoleucine-to- lysine substitution at amino acid position 71. In this embodiment, the IL-21 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 33 and 71 .

[0081] In another embodiment, the IL-21 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 33 and 71.

[0082] In another even more preferred embodiment, the IL-21 variant may comprise the valine-to-aspartate substitution at amino acid position 33 and the isoleucine-to- lysine substitution at amino acid position 71 . In this embodiment, the IL-21 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 33 and 71 .

[0083] In another embodiment, the IL-21 variant comprises an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 4; at least 80% sequence identity to SEQ ID NO: 4; at least 85% sequence identity to SEQ ID NO: 4; at least 90% sequence identity to SEQ ID NO: 4; at least 91% sequence identity to SEQ ID NO: 4; at least 92% sequence identity to SEQ ID NO: 4; at least 93% sequence identity to SEQ ID NO: 4; at least 94% sequence identity to SEQ ID NO: 4; at least 95% sequence identity to SEQ ID NO: 4; at least 96% sequence identity to SEQ ID NO: 4; at least 97% sequence identity to SEQ ID NO: 4; at least 98% sequence identity to SEQ ID NO: 4; at least 99% sequence identity to SEQ ID NO: 4; or at least 100% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 33 and 71.

[0084] In another preferred embodiment, an IL-21 variant according to the invention may comprise a hydrophobic-to-hydrophilic substitution at amino acid positions 36 and 71 . In an more preferred embodiment, an IL-21 variant according to the invention may comprise (i) the phenylalanine-to-lysine substitution or phenylalanine-to- glutamine substitution at amino acid position 36 and (ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71. In an even more preferred embodiment, the IL-21 variant may comprise the phenylalanine-to-lysine substitution at amino acid position 36 and the isoleucine- to-lysine substitution at amino acid position 71. In this embodiment, the IL-21 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 36 and 71 .

[0085] In another embodiment, the IL-21 variant comprises an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 5; at least 80% sequence identity to SEQ ID NO: 5; at least 85% sequence identity to SEQ ID NO: 5; at least 90% sequence identity to SEQ ID NO: 5; at least 91% sequence identity to SEQ ID NO: 5; at least 92% sequence identity to SEQ ID NO: 5; at least 93% sequence identity to SEQ ID NO: 5; at least 94% sequence identity to SEQ ID NO: 5; at least 95% sequence identity to SEQ ID NO: 5; at least 96% sequence identity to SEQ ID NO: 5; at least 97% sequence identity to SEQ ID NO: 5; at least 98% sequence identity to SEQ ID NO: 5; at least 99% sequence identity to SEQ ID NO: 5; or at least 100% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 36 and 71.

[0086] The IL-21 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.

[0087] It is envisaged that there may be any suitable protein or polypeptide joined to the IL-21 variant. Such examples may include, but are not limited to, signal peptides, antibody fragments, antibodies, bi- or multi-specific antibodies, tissue-targeting sequences (such as the NGR (Asn-Gly-Arg) tripeptide), chemokines, or interleukins particularly those that stimulate cell mediated immunity (e.g., IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17, IL-18, IL-21) or chemokines (e.g. CXCL9, CXCL10, CXCL11 , CCL5, CCL2, CX3CL1).

[0088] 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-21 variant is fused to a signal peptide. As used herein, the term “signal peptide” refers to any signal sequence, localisation sequence or other polypeptide sequence which directs the IL-21 variant to a particular intracellular or extracellular location. For expression in bacteria, a bacterial signal peptide will be required in order to secrete the IL-21 variant out of the bacterial cytosol and into, for example, the periplasm. The addition of small (15 to 25 amino acid) sequences, known as signal peptides, to a protein of interest can be used to direct to a desired location.

[0089] As will be known by the skilled person, the periplasm is a concentrated matrix in the space defined between the inner plasma membrane and the bacterial outer membrane of Gram-negative bacteria. The (bacterial) periplasm is an oxidative compartment, and is ideal to form disulphide bonds, which are ubiquitous in human cytokines. There are also chaperones in the periplasm which assist and promote the formation of disulphide bonds. In contrast, the (bacterial) cytosol is a reducing environment which does not favour disulphide bond formation. Therefore, it can be seen that directing the cytokines (IL-21 variants) to the periplasm via signal peptides is beneficial to the production and correct folding of these proteins, and ultimate secretion in situ. The present use of a periplasmic signal peptide for exporting recombinant proteins from the bacterial cytosol has the advantage of avoiding the formation of inclusion bodies and therefore avoids the need for additional and laborious requirements of solubilising and refolding the proteins from inclusion bodies, thus allowing for high yield / secretion of the present cytokines.

[0090] The preference of the present invention is for the signal peptide to be a (bacterial) periplasmic signal peptide. The signal peptide directs the IL-21 variant into the periplasm of the bacteria in a rapid and robust manner. A periplasmic signal peptide is a polypeptide sequence which allows for a protein to which the signal peptide is attached to be transported, directed, localised, or targeted to the periplasm of a bacterial cell.

[0091] 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- 21 variants according to the present invention may be a SEC or TAT periplasmic signal peptide. However, the SEC pathway allows the protein to fold in the appropriate environment, therefore in a preferred embodiment, the signal peptide is a SEC signal peptide. The SEC pathway is shared by both Gram-negative and Gram-positive bacteria. In Gram-negative bacteria, as a result of the SEC pathway, proteins are secreted into the periplasm. In Gram-positive bacteria, as a result of the SEC pathway, proteins are secreted directly into the external environment. In some embodiments, the signal peptide used to direct proteins to the periplasm is N-terminally fused to the IL-21 variant. However, the IL-21 variant could be exported extracellularly via other signal peptides, such as Type I signal peptides. Therefore, in other embodiments, the signal peptide is C-terminally fused to the IL-21 variant. There is no universal signal peptide which can translocate any protein efficiently, and therefore the inventors of the present invention have also created optimal signal peptides for each IL-21 variant.

[0092] From the periplasm, the IL-21 variant can then be prompted into the supernatant or other extracellular environment. In fusing a periplasmic signal peptide to any of the IL-21 variants described herein, if expressed within a bacterial cell, the IL- 21 variants can be transported efficiently to the periplasm of the bacterial cell in order to achieve efficient secretion of the IL-21 variants from the bacterial cell. Such secretion would enable the delivery of these molecules to a site of interest where they are needed, including to a site of disease in a subject who may benefit from such an enhanced immune response induced by the IL-21 variants. For example, upon infection and invasion of a host (e.g., eukaryotic) cell by bacteria, such as Gram-negative bacteria, expressing the IL-21 variants of the present invention, the IL-21 variant may be transferred into the cytoplasm of the host cell where it may achieve for example a therapeutic immunostimulatory effect.

[0093] In another embodiment, the signal peptide may be fused to the IL-21 variant via a spacer sequence. As used herein, the terms “spacer” and “spacer sequence” may be used interchangeably and refer to any amino acid sequence that is to be located between the signal peptide and the cargo (IL-21 variant) that is to be secreted (i.e. , signal peptide:spacer:IL-21 variant, i.e. , the spacer is located at the N terminus of the 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.

[0094] In accordance with a second aspect of the invention, there is provided a nucleic acid molecule encoding the interleukin-21 (IL-21) variant according to any aspect of the invention. The nucleic acid molecule may be an isolated nucleic acid molecule. As used herein, the term “nucleic acid molecule” refers to a nucleic acid construct (i.e., DNA and / or RNA), generated recombinantly or synthetically, comprising a series of specified nucleic acid elements that permit the expression of a particular nucleic acid sequence in a host cell. Specifically, the nucleic acid molecule may be expressed in a bacterial cell, preferably, a Gram-negative bacterial cell. The nucleic acid molecule may preferably comprise a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5 thereof (preferably any one of SEQ ID NOs: 2, 3, 4, and / or 5), or any combination thereof, or an amino acid sequence comprising at least 70% identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5 retains the one or more amino acid modifications at the relevant amino acid positions (as defined in the claims). In some embodiments, expression of the IL-21 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.

[0095] 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.

[0096] In another embodiment, the nucleic acid molecule is an RNA molecule. As used herein, the terms “RNA” and “ribonucleic acid” are given their usual meaning in the art and are used interchangeably to refer to nucleic acids composed of uracil, adenine, guanine, and cytosine ribonucleic acid bases. These terms and concepts will be well known to those in the art. Types of RNA molecules include, for example, mRNA, siRNA, shRNA, miRNA, tRNA, and rRNA. In a preferred embodiment, the RNA molecule is an mRNA molecule. As used herein, the terms “mRNA” and “messenger RNA” are used interchangeably and refer to a singlestranded RNA molecule involved in protein synthesis, which is transcribed from DNA and translated into an amino acid sequence. Bacterial mRNA molecules are transcribed from DNA that is non-compartmentalised and translated in the cytosol coupled to transcription. These terms and concepts will be well known to those in the art. Eukaryotic mRNA molecules are transcribed from DNA in the nucleus of a eukaryotic cell, and subsequently exported from the nucleus into the cytoplasm of the eukaryotic cell, where translation of the mRNA molecule into proteins takes place.

[0097] 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.

[0098] The nucleic acid molecule may comprise a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4 and / or 5, or an amino acid sequence comprising at least 70% identity to any of SEQ ID NOs: 1 , 2, 3, 4 and / or 5, wherein the % sequence identity retains the one or more amino acid modifications at the relevant amino acid positions (as defined in the claims).

[0099] In a most preferred embodiment, the nucleic acid molecule may comprise a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% identity to any of SEQ ID NOs: 2, 3, 4 and / or 5; at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 80% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 91 % sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 94% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 95% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 96% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 97% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 98% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 99% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; and / or at least 100% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity retains the one or more amino acid modifications at the relevant amino acid positions (as defined in the claims).

[0100] As described herein and as would be understood by the skilled person, interleukins play a diverse role in eliciting an immune response. Given the role of IL-21 in the stimulation of proliferation and cytotoxic function of CD8 T cells and NK cells, the therapeutic potential of IL-21 is apparent. The inventors of the present invention have surprisingly found that certain mutations to the amino acid sequence of IL-21 generates IL-21 variants which are optimised in terms of solubility and without impacting biological activity. Therefore, in accordance with a fourth aspect of the invention, there is provided an IL-21 variant according to the first aspect of the invention, or the nucleic acid molecule according to the second aspect of the invention, or a bacterium according to the third aspect of the invention for use in therapy. Any of the IL-21 variants according to the present invention, alone or in any combination, have use in therapy.

[0101] 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 an IL-21 variant according to the first aspect of the invention, or the nucleic acid molecule according to the second aspect of the invention, or the bacterium according to the third aspect of the invention, for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0102] 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.

[0103] 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.

[0104] 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, cervix, vulva, 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.

[0105] Preferably, the solid cancer and / or the haematological malignancy may be a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular cancer, liver cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, vulvar cancer, ovarian cancer, endometrial 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 cancer, liver cancer, melanoma, oesophageal cancer, gastric cancer, ovarian cancer, cervical cancer, vulvar cancer, endometrial cancer, colorectal cancer, head and neck cancer or breast cancer. In another preferred embodiment, the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, endometrial cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

[0106] In a preferred embodiment of the invention, the IL-21 variant is to be administered intratumourally, intravesically, intravaginally, intravenously, intraperitoneally, or orally administered. In a most preferred embodiment, the IL-21 variant 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-21 variants of the present invention may be administered by injection, instillation, infusion, continuous infusion, intradermally, intraarterially, intralesionally, peritumourally, intrarectally, intramuscularly, subcutaneously, subconjunctival mucosally, intrapericardially, intraumbilically, intraocularally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, i ntranasally , 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). In a more preferred embodiment, administration is to be oral or via local instillation, intra-peritoneally, intrapleurally, intravesically, i ntravaginally , peritumoral injection, or intratumoural injection.

[0107] As used herein, the terms “oral administration” or “orally administered” refer to the route of administration in which the composition is introduced to a subject through the mouth. The composition may be swallowed and processed via the subject’s digestive system. 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 “intra-peritoneally” 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 “intravaginally” refers to the application of the composition inside the vagina. The application may be via a vaginal tablet, cream, solution, gel, suppository, ring, instillation, or injection. 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-21 variant directly into the neoplastic disease of the subject.

[0108] It is understood that the specific method of administration 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.

[0109] Aside from direct administration of any of the IL-21 variants according to the present invention, in order to achieve optimal and efficient expression, folding, and secretion of the IL-21 variants, there is provided in accordance with a third aspect of the invention, a bacterium comprising any of the IL-21 variants of the present invention, or any combination thereof, or the nucleic acid molecule according to a second aspect of the invention. The IL-21 variants may be expressed in and secreted by a suitable expression system and isolated for direct administration to a subject in need thereof. However, in a preferred embodiment, the IL-21 variant may be administered together with the bacteria, whereby the bacteria comprising the IL-21 variant or nucleic acid molecule encoding the IL-21 variant are administered and express and secrete the IL-21. In some embodiments, the bacterium may be a Gram-negative bacterium. In other embodiments, the bacterium may be a Gram-positive bacterium.

[0110] Any suitable bacterium capable of comprising an IL-21 variant, or a nucleic acid molecule encoding an IL-21 variant according to the present invention may be utilised. However, in a particularly preferred embodiment, there is provided a Gram-negative bacterium, comprising a nucleic acid construct according to the present invention.

[0111] For instance, there may be provided a bacterium, such as Gram-negative bacterium, comprising a nucleic acid molecule comprising a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4 and / or 5, or an amino acid sequence comprising at least 70% identity to any of SEQ ID NOs: 1 , 2, 3, 4 and / or 5, wherein the % sequence identity retains the one or more amino acid modifications at the relevant amino acid positions as defined in the claims. In another embodiment, there may be provided a bacterium, such as Gram-negative bacterium, comprising a nucleic acid molecule comprising a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% identity to any of SEQ ID NOs: 2, 3, 4 and / or 5; at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 80% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 91 % sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 94% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 95% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 96% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 97% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 98% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; at least 99% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5; and / or at least 100% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity retains the one or more amino acid modifications at the relevant amino acid positions as defined in the claims.

[0112] Any suitable bacterium capable of comprising the IL-21 variant may be utilised according to the invention. However, in a particularly preferred embodiment, the bacterium is a Gram-negative bacterium.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] Such a bacterium will be engineered to express the any of the IL-21 variants disclosed herein. Numerous methods and techniques for genetically engineering bacterial strains will be well known to the person skilled in the art. These techniques include those required for introducing heterologous genes into the bacteria either via chromosomal integration or via the introduction of a stable autosomal self-replicating genetic element. Exemplary methods for genetically modifying (also referred to as "transforming" or “engineering”) bacterial cells include bacteriophage infection, transduction, conjugation, lipofection or electroporation. Other techniques, including CRISPR and CRISPR associated proteins (such as Cas9), CasCLOVER, TALENs, retrons, homing endonucleases or mega-nucleases, zinc-finger nucleases, and transposon-based methods are also intended to be included. A general discussion on these and other methods for genetic engineering in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); which are hereby incorporated by reference.

[0119] For example, there is provided a bacterium, such as a Gram-negative bacterium, comprising an IL-21 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, preferably any one of SEQ ID NOs: 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% sequence identity to any of these sequences, wherein the % sequence identity retains the one or more amino acid modifications at the relevant amino acid positions as defined in the claims.

[0120] In another embodiment, there may be provided a bacterium, such as a Gramnegative bacterium, comprising an IL-21 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 1 , 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 80% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 85% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 90% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 91% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 92% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 93% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 94% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 95% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 96% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 97% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 98% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; at least 99% sequence identity to SEQ ID NOs: 1 , 2, 3, 4, and / or 5; and / or 100% sequence identity to SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity retains the one or more amino acid modifications at the relevant amino acid positions as defined in the claims.

[0121] In a most preferred embodiment, there is provided a bacterium comprising an IL- 21 variant comprising an amino acid sequence according to SEQ ID NOs: 2, 3, 4, and / or 5.

[0122] As will be understood by the skilled person, a bacterium, such as a Gram-negative bacterium, comprising a nucleic acid molecule comprising a sequence encoding any of the amino acid sequences described herein (preferably SEQ ID NOs: 2-5 and / or any combination thereof) is also included.

[0123] The bacterium comprising an IL-21 variant may be referred to herein as a composition unless specified otherwise. The IL-21 variants will prompt an enhanced immune response and the bacterium itself will additionally elicit strong humoral and cellular immune responses. As used herein, the term "immune response" refers to the action of cellular components of the immune system, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells. As used herein, the term “cellular components of the immune system” refers to immunocytes such as lymphocytes, such as T and B lymphocytes, gamma-delta T cells, and NK cells, which may recognize specific antigens, such as prion, viral, bacterial, yeast, fungal, parasite, tumour-associated or tumour-specific antigens, or other antigens associated with a particular disease, disorder, or condition. Other immunocytes we refer to include white blood cells, which may be granulocytes or agranulocytes. Examples of immunocytes include neutrophils, eosinophils, basophils, lymphocytes, monocytes, and macrophages. Dendritic cells, microglia, and other antigen-presenting cells are also included within this definition.

[0124] 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 may prime the immune system of the subject. 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, immune response of a subject may be primed (used interchangeably with “condition”, “boost”, “amplify”, “enhance”, “improve”, “augment” or “promote”).

[0125] Accordingly, the combination of the IL-21 variant and bacterium, where the IL-21 variant is expressed and secreted by the bacterium, is synergistic and will provide an enhanced immune response in a subject. The IL-21 of the present invention, delivered by a Gram-negative bacterium as defined herein (i.e., a composition), would enable the subject’s immune system to mount an effective immune response to a disease, which is potent and durable and can result in enhanced therapeutic benefit. The immune response initiated by the administration of the composition may be of a therapeutic level in itself or be of a sub-therapeutic level requiring the subsequent administration of further dose of the composition to exert a therapeutic effect. Therefore, in accordance with a fourth aspect of the invention, there is provided a bacterium for use in therapy.

[0126] Bacteria, such as Gram-negative bacteria, engineered to express the any of the IL-21 variants according to the invention 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 to prevent the recurrence 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 nonhuman 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-21 variants are particularly suitable for the treatment of cancer in vivo.

[0127] The amount of the Gram-negative bacterium administered to the subject is sufficient to deliver a therapeutically effective amount of the IL-21 variant to the subject. The skilled person will readily understand that the precise amount to be administered will be dependent on a number of factors, for example, the disease to be treated, the IL-21 variant to be expressed and secreted, and the medical history of the subject to be treated. 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.

[0128] For example, for the treatment of tumours, a "therapeutically effective dosage" may induce tumour shrinkage by at least about 5% relative to baseline measurement, such as at least about 10%, or about 20%, or about 60% or more. The baseline measurement may be derived from untreated subjects.

[0129] 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.

[0130] The Gram-negative bacterium described herein may be administered at a dose of between 105and 1012CFU, where CFU is a colony-forming unit. For example, suitable doses may be between 105and 106CFU, 105and 107CFU, 105and 108CFU, 105and 109CFU, 105and 1010CFU, 105and 1011CFU, 106and 107CFU, 106and 108CFU, 106and 109CFU, 106, and 1010CFU, 106and 1011CFU, 106and 1012CFU, 107and 108CFU, 107and 109CFU, 107and 1010CFU, 107and 1011CFU, 107and 1012CFU, 108and 109CFU, 108and 1010CFU, 108and 1011CFU, 108and 1012CFU, 109and 1010CFU, 109and 1011CFU, 109and 1012CFU, 1010and 1011CFU, 1010and 1012CFU, or 1011and 1012CFU. The composition may be administered in a single dose or in multiple doses. The specific number of doses to be administered are understood to be dependent on the IL-21 variant to be delivered, as well as the specific indication to be treated.

[0131] The Gram-negative bacterium described herein may be administered in any form in which is considered to be within close proximity of the disease. As used herein, the term “close proximity” is intended to refer to the area or region surrounding the disease out to a distance. For example, in one embodiment the term “close proximity” may refer to the area / region extending up to 10 mm, up to 5 mm, or up to 2.5 mm for example, from the boundary of the disease. The distance at which the composition is administered from the site of the disease will allow for the biological effects (for example, recruitment and activation of various immune cell types) to have an effect on the local environment of the disease in question, whilst having minimal / no effect on tissue situated in unrelated areas of the body. Additionally, the composition may be administered “in” or “on” the tissue or surrounding tissue. As such, the term “local administration” refers to any context in which the bacteria, such as Gram-negative bacteria, may come into contact with the disease or into contact with the immediately surrounding tissue to have the desired effect. As used herein, the terms “locally” and “administered locally” are used interchangeably and in the context of the present invention refer to the way in which the IL-21 variant and / or Gram-negative bacterium are administered to a subject. As such, the resulting immune response produced in the subject is also said to be local to the site of the disease, that is, it is not a widespread systemic immune response. In a preferred embodiment of the present invention, the bacterium, such as a Gram-negative bacterium, described herein is administered intratumourally, intravesically, intravaginally, intravenously, intraperitoneally, or orally administered. In a most preferred embodiment, the live attenuated bacterium is administered intratumourally. However, it is also contemplated that other methods of administration may be used in some cases. Therefore, in certain instances the bacterium of the present invention may be administered by injection, instillation, infusion, continuous infusion, intradermally, peritumourally, 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). In a more preferred embodiment, administration is to be oral or via local instillation, intra-peritoneally, intrapleurally, intravesically, intravaginally, peritumoral injection, or intratumoural injection.

[0132] It is understood that the specific method of administration of the bacterium described herein may depend on the disease to be treated, for example, both the location and type of disease to be treated. For example, if it is desirable that a large surface area of a body cavity is treated, for example, a pleural cavity of a subject, then administration via instillation may be most appropriate. Alternatively, if the disease is located within the peritoneal cavity, administration via intraperitoneal injection may be most appropriate. Yet further, if the disease to be prevented and / or treated is for example, a haematological malignancy, it is noted that intra-tumoural injection may not be the method of administration of choice.

[0133] 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, such as a Gram-negative bacterium, for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0134] Further, in a fifth aspect of the invention, there is provided a use of the IL-21 variant according to the first aspect of the invention, or the nucleic acid molecule according to the second aspect of the invention, or the bacterium according to the third aspect of the invention, in the manufacture of a medicament for a therapy.

[0135] Where the disease to be treated is a neoplastic disease, the bacterium, such as a Gram-negative bacterium, and the IL-21 variant may be administered in combination with another therapy. It is widely known that the causes of neoplastic diseases are multifaceted and diverse, often leading to prevention and treatment strategies comprising multiple therapies to achieve optimum results. As such, the present invention may involve combining the bacterium expressing the IL-21 variant with other known cancer therapies. Preferably, the bacterium expressing the IL-21 variant may be administered in combination with an immunotherapy, radiotherapy, chemotherapy, or an anti-cancer agent. The term “anti-cancer agent” as used herein refers to any agent that is effective in killing cancer cells, halting the division of cancer cells, or helps to prevent the recurrence of cancer cells, but is not considered to be an immunotherapy, radiotherapy, or chemotherapy. In a preferred embodiment, the bacterium expressing the IL-21 variant may be administered in combination with an immunotherapy. Preferably, the immunotherapy may comprise a checkpoint inhibitor, an antigen specific T cell, an adoptive T cell therapy, a therapeutic antibody, a cancer vaccine, or any other engineered cellular immunotherapy. The bacterium expressing the IL-21 variant may be administered separately from, simultaneously with or sequentially to (prior to and / or following) the other known cancer therapy. In one embodiment, the neoplastic disease may be a solid cancer and / or a haematological malignancy. Neoplasia, tumours, and cancers include benign, malignant, metastatic and non-metastatic types, and include any stage (I, II, III, IV or V) or grade (G1 , G2, G3, etc.) of neoplasia, tumour, or cancer, or a neoplasia, tumour, cancer or metastasis that is progressing, worsening, stabilized or in remission.

[0136] 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, cervix, vulva, 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. More preferably, the malignant tumour may be associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial carcinoma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

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

[0140] In a sixth 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 IL-21 variant according to the first aspect of the invention, or the nucleic acid molecule according to the second aspect of the invention, or the bacterium according to the third aspect of the invention. The present invention therefore also provides for a method treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject. As such, the method of the present invention may be used to reduce or inhibit metastasis of a primary tumour or cancer to other sites, or the formation or establishment of metastatic tumours or cancers at other sites distal from the primary tumour or cancer thereby inhibiting or reducing tumour or cancer relapse or tumour or cancer progression. Accordingly, the present invention provides a detectable or measurable improvement in a condition of a given subject, such as alleviating or ameliorating one or more adverse (physical) symptoms or consequences associated with the presence of a cell proliferative or cellular hyperproliferative 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 hyperproliferative 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 hyperproliferative 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 hyperproliferative 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.

[0141] 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.

[0142] 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.

[0143] Additional adverse symptoms and complications associated with neoplasia, tumour, cancer, and metastasis that can be inhibited, reduced, decreased, delayed or prevented include, for example, nausea, lack of appetite, lethargy, pain and discomfort. Thus, a partial or complete decrease or reduction in the severity, duration or frequency of an adverse symptom or complication associated with or caused by a cellular hyperprol iterative disorder, an improvement in the subject’s quality of life and / or well-being, such as increased energy, appetite, psychological well-being, are all particular non-limiting examples of therapeutic benefit.

[0144] 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.

[0145] 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.

[0146] The bacterium, such as Gram-negative bacterium, expressing an IL-21 variant of the present invention will typically be administered to the subject in a composition that comprises an effective amount of the bacterium, such as Gram-negative bacterium, for example, the Salmonella serovar Typhi ZH9 strain, and further comprises a pharmaceutically acceptable camer / 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.

[0147] The composition may be intended to enhance an immune response of a subject. Accordingly, the composition (i.e., a bacterium expressing an IL-21 variant of the present invention) may be a vaccine or vaccine composition. Such terms are used interchangeably and refer to a biological preparation in which the subject produces an immune response to said biological preparation, therefore providing active acquired immunity to a particular infectious disease, for example, a disease caused by a Salmonella spp. In the context of the present invention, the vaccine may contain an agent, or “foreign” agent, that resembles the infection-causing bacteria, which is a weakened or killed form of said bacteria, or any portion of, or fragment of, a bacteria protein, capsule, DNA or RNA. Such a foreign agent would be recognised by a vaccine-receiver’s immune system, which in turn would destroy said agent and develop “memory” against the bacteria, inducing a level of lasting protection against future bacterial infections from the same or similar viruses. Through the route of vaccination, including those vaccine compositions of the present invention, it is envisaged that once the vaccinated subject again encounters the same bacteria or bacterial isolate of which said subject was vaccinated against, the individual’s immune system may thereby recognise said bacteria or bacterial isolate and elicit a more effective defence against infection. The active acquired immunity that is induced in the subject as a result of the vaccine may be humoral and / or cellular in nature. The vaccine composition may further comprise an adjuvant, a pharmaceutically acceptable carrier or excipient.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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).

[0152] The bacterium of the vaccine composition herein disclosed may include any one of, or any combination of, the features of the bacteria herein disclosed. 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.

[0153] 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.

[0154] The invention is further described with reference to the following non-limiting examples:

[0155] EXAMPLES

[0156] Example 1: Identification of aggregation-prone amino acids with Aggrescan 3D and validation of selection through computation of surface with Pymol

[0157] Cytokine variants were obtained following a semi-rational experimental design, where computational tools (e.g., Aggrescan3D) were used to identify aggregation- prone amino acids (six amino acids identified: 113, V29, V33, F36, 171 , V74; Table 3). Computation of the protein surface with PyMol was used to distinguish the hydrophobic and hydrophilic areas (Figure 1).

[0158] Table 3. Computation of aggregation score per amino acid.

[0159] Mutations for these amino acids were selected based on two possibilities: either positive or polar charge (e.g., Threonine or Lysin), or negatively charged (e.g., Aspartic Acid or Glutamic Acid). Variants were designed to contain up to two different mutations per sequence, resulting in a 72-member library where each variant contains 1 or 2 defined amino acid changes with respect to wild-type IL-21 (SEQ ID NO: 1 , mature, human IL-21 , PMID: Q9HBE4). The library was gene synthesized by IDT as eBlock library.

[0160] The following amino acid sequence was used as reference:

[0161] SEQ ID NO 6:

[0162] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERI INVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS LLQKMIHQHLSSRTHGSEDS

[0163] Example 2: Determining the solubility and activity of IL-21 variants

[0164] Each of the fragments generated during the design phase were cloned into a solubility reporter plasmid, named pBRED, derived from the system reported by Foit et al. (2009 Molecular Cell 36 861-871) and that is based survival of bacterial cells to penicillin antibiotics (e.g., carbenici Hi n , ampicillin, penicillin V) as a function of the stability of a protein of interest. This contains a beta-lactamase (tpbla) gene split in two domains (alpha and omega) that can independently fold. Two flexible linkers between these domains allows fusion of any protein cargo so that the final protein is configured as: Blaaipha-linker-cargo-linker-BlaOmega. If the cargo is stable / soluble in nature, the alpha and omega sub-domains 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 the reporter bacteria cannot survive to the antibiotic.

[0165] The baseline carbenici Hi n antibiotic concentration required to abolish growth when IL-21 was fused to thetpbla gene was found to be 1 mg / mL. Antibiotic challenge assays at 1 mg / mL of carbenici I li n identified few variants with higher solubility than wild-type IL-21 (Figure 2A). The protein mScarlet, known to be stable, was used as positive control or benchmark value.

[0166] Cytokine variants were evaluated for activity using a cell-based assay with reporter in B9 cells, a mouse B cell hybridoma line. Protein variants were synthesized using a cell-free extract prepared from E. coli Shuttle strain and plasmid where the gene of the variant was downstream a T7 promoter and fused to a C-terminus 6xHis-tag, which was used to tether the synthesized protein to the surface of a Ni-NTA HisSorb plate (Qiagen). Unspecific products were removed from the plate by washing with NPI-50 buffer (50 mM NaH2PO4, 300 mM NaCI, 50 mM imidazole, pH 8.0) and posteriorly with PBS (137 mM NaCI, 2.7 mM KCI, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4). B9 cells were stimulated of the cells (50,000 / well) IL-21 candidates over a 48-hour period. IL-6, a cytokine required for the survival and proliferation of the B9 cells was included as a positive control, in addition to a commercially produced, recombinant human IL-21 protein. Additional controls included the buffers used in the elution of the test candidates (NPI, NPI- 20 and NPI-50; described above). Post-stimulation, proliferation of both stimulated and unstimulated cells were measured using the Cell Counting Kit-8 (CCK-8) assay. Briefly, stimulated B9 cells were incubated with the CCK-8 reagent, which is transformed into an orange product by the viable cells. Product yields were evaluated at 1-, 3-, 6- and 24-hours post-incubation with the CCK-8 reagent by measurement of absorbance. Variant activity was highly dependent on B cell donor across different experiments (Figure 2B) but variants IL-21 V36, IL-21V52, IL-21V58, and IL-21V62 were consistently showing the same activity levels than wild-type IL-21. Figure 3 summarises the experimental set up utilised to evaluate variant activity, and Figure 4 summarises the selection of solubility-enhancing mutations. Example 3: Protein purification of IL-21 variants

[0167] A pilot study aimed at identifying optimal protein production conditions by modifying temperature (30 or 37 °C) and isopropyl beta-D-1 - thiogalactopyranoside (IPTG) concentrations (0, 0.02, 0.2 mM) failed to identify an optimal combination for IL-21 production in E. coli Shuffle (Figure 5A). A subsequent screening (Figure 5B) revealed that proteins accumulated overnight (16 hours) at low temperatures (18 °C), which were then used for the purification of the IL-21 variants (V36, V52, V58 and V62) in E. coli Shuffle from the cell-free expression plasmid in small-scale (1 L) cultures of LB broth. Cultures were induced when OD6oo reached approximately 0.4 in 200 rpm and 37 °C conditions, which were then shifted to the cooler temperatures identified as optimal.

[0168] The soluble fraction was lysed by sonication in NPI-20 (100 mL), bound to a Ni- NTA column and eluted by using a constant gradient of imidazole. Figure 6 shows the SDS-PAGE gels of the purified wild-type, V36 and V52 variants, which revealed two peaks potentially containing IL-21 (peak A and peak B) in all the variants. Validation through Mass Spectrometry revealed peak B as the correct peak and allowed quantification. The results of quantification of peak B are shown in Table 4.

[0169] Table 4. Concentration of components within peak B.

[0170] Proteins were then pooled and dialysed against NPI buffer to remove the excess of imidazole. Figure 7 shows the final SDS-PAGE of final protein purification products (wild-type, V36, V52, V58 and V62 IL-21). Example 4: Cell-based assays of wild-type IL-21 and IL-21 variant activity

[0171] Purified protein stocks were used to induce B9 cells, using the same conditions as previously described, but using a gradient of 0.78-200 ng / pL of each variant. Titration curves depicted in Figures 8A (IL21V36 and IL-21 V58) and 8B (IL-21 V52 and IL-21V62) show that the purified variants exhibited consistent activity across all proteins tested. All variants were not significantly different to wild-type IL-21. The IL-21V52 pattern indicated that this variant may be slightly better than wildtype.

[0172] The invention may be described as in any of the following embodiments:

[0173] Embodiment 1. An interleukin-21 (IL-21) variant, wherein in comparison to wildtype IL-21 , the IL-21 variant comprises one or more amino acid modifications in a region adjacent to a receptor interaction surface of the IL-21 variant.

[0174] Embodiment 2. The IL-21 variant according to Embodiment 1 , wherein wild-type IL-21 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.

[0175] Embodiment s. The IL-21 variant according to Embodiment 2, wherein in comparison to SEQ ID NO: 6, the amino acid modifications occur at one or more of amino acid positions 13, 29, 33, 36, 71 , and / or 74, or any combination thereof.

[0176] Embodiment 4. An interleukin-21 (IL-21) variant, said variant comprising an amino acid sequence corresponding to the sequence according to SEQ ID NO: 6, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 6, wherein in comparison to SEQ ID NO: 6, the IL-21 variant comprises an amino acid modification at one or more of amino acid positions 13, 29, 33, 36, 71 , and / or 74, or any combination thereof.

[0177] Embodiment 5. The IL-21 variant according to any one of Embodiments 2 to 4, wherein the IL-21 variant comprises an amino acid sequence according to SEQ ID NO: 6, 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: 6.

[0178] Embodiment 6. The IL-21 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modifications are hydrophobic-to- hydrophilic substitutions.

[0179] Embodiment ?. The IL-21 variant according to Embodiment 6, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 13 is an isoleucine- to-aspartate substitution or an isoleucine-to-lysine substitution.

[0180] Embodiment s. The IL-21 variant according to Embodiment 7, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 13 is an isoleucine- to-aspartate substitution.

[0181] Embodiment 9. The IL-21 variant according to any one of Embodiments 6 to 8, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 29 is a valine-to-threonine substitution or a valine-to-aspartate substitution.

[0182] Embodiment 10. The IL-21 variant according to Embodiment 9, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 29 is a valine-to- threonine substitution.

[0183] Embodiment 11. The IL-21 variant according to any one of Embodiments 6 to 10, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 33 is a valine-to-threonine substitution or a valine-to-aspartate substitution.

[0184] Embodiment 12. The IL-21 variant according to any one of Embodiments 6 to 10, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 36 is a phenylalanine-to-glutamine substitution or a phenylalanine-to-lysine substitution.

[0185] Embodiment 13. The IL-21 variant according to Embodiment 12, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 36 is a phenylalanine-to-lysine substitution. Embodiment 14. The IL-21 variant according to any one of Embodiments 6 to 13, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 71 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

[0186] Embodiment 15. The IL-21 variant according to Embodiment 14, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 71 is an isoleucine- to-lysine substitution.

[0187] Embodiment 16. The IL-21 variant according to any one of Embodiments 6 to 15, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 74 is a valine-to-threonine substitution or a valine-to-aspartate substitution.

[0188] Embodiment 17. The IL-21 variant according to Embodiment 15, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 74 is a valine-to- threonine substitution.

[0189] Embodiment 18. The IL-21 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-21 variant comprises: i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 29; and ii) the phenylalanine-to-lysine substitution or phenylalanine-to- glutamine substitution at amino acid position 36, preferably wherein the IL-21 variant comprises the valine-to-threonine substitution at amino acid position 29 and the phenylalanine-to-lysine substitution at amino acid position 36.

[0190] Embodiment 19. The IL-21 variant according to Embodiment 18, wherein the IL- 21 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 29 and 36. Embodiment 20. The IL-21 variant according to Embodiment 19, wherein the IL-21 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 29 and 36.

[0191] Embodiment 21 . The IL-21 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-21 variant comprises: i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the valine-to-threonine substitution at amino acid position 33 and the isoleucine-to-lysine substitution at amino acid position 71.

[0192] Embodiment 22. The IL-21 variant according to Embodiment 21 , wherein the IL- 21 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 33 and 71 .

[0193] Embodiment 23. The IL-21 variant according to Embodiment 22, wherein the IL- 21 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 33 and 71 .

[0194] Embodiment 24. The IL-21 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-21 variant comprises: i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the valine-to-aspartate substitution at amino acid position 33 and the isoleucine-to-lysine substitution at amino acid position 71.

[0195] Embodiment 25. The IL-21 variant according to Embodiment 24, wherein the IL- 21 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 33 and 71 .

[0196] Embodiment 26. The IL-21 variant according to Embodiment 25, wherein the IL- 21 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 33 and 71 .

[0197] Embodiment 27. The IL-21 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-21 variant comprises: i) the phenylalanine-to-lysine substitution or phenylalanine-to- glutamine substitution at amino acid position 36; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the phenylalanine-to-lysine substitution at amino acid position 36 and the isoleucine-to-lysine substitution at amino acid position 71.

[0198] Embodiment 28. The IL-21 variant according to Embodiment 27, wherein the IL- 21 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 36 and 71 .

[0199] Embodiment 29. The IL-21 variant according to Embodiment 28, wherein the IL-21 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 36 and 71 .

[0200] Embodiment 30. The IL-21 variant according to any preceding Embodiment, wherein the IL-21 variant is fused to a signal peptide.

[0201] Embodiment 31. The IL-21 variant according to Embodiment 30, wherein the signal peptide is a periplasmic signal peptide.

[0202] Embodiment 32. The IL-21 variant according to Embodiments 30 or 31 , wherein the signal peptide is fused to the IL-21 variant via a spacer sequence, preferably wherein the spacer sequence is between 1 to 5 amino acids in length.

[0203] Embodiment 33. The IL-21 variant according to any preceding Embodiment, for use as a therapy.

[0204] Embodiment 34. The IL-21 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.

[0205] Embodiment 35. The IL-21 variant for use according to Embodiment 34, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

[0206] Embodiment 36. The IL-21 variant for use according to Embodiment 35, wherein the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma. Embodiment 37. The IL-21 variant for use according to any one of Embodiments 34 to 36, wherein the IL-21 variant is administered to the subject orally or via local instillation, intra-peritoneally, intravaginally, intrapleurally, intravesically, peritumoral injection or intratumoural injection.

[0207] Embodiment 38. A nucleic acid molecule encoding the IL-21 variant as defined in any one of Embodiments 1 to 32.

[0208] Embodiment 39. The nucleic acid molecule according to Embodiment 38, wherein the nucleic acid molecule comprises a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% identity to any one of SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 2, 3, 4, and / or 5 retains the one or more amino acid modifications at the relevant amino acid positions.

[0209] Embodiment 40. The nucleic acid molecule according to Embodiment 39, wherein the nucleic acid molecule comprises a sequence encoding an amino acid sequence comprising at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 2, 3, 4, and / or 5 retains the one or more amino acid modifications at the relevant amino acid positions.

[0210] Embodiment 41 . The nucleic acid molecule according to any one of Embodiments 38 to 40, wherein the nucleic acid molecule comprises a DNA molecule.

[0211] Embodiment 42. The nucleic acid molecule according to any one of Embodiments 38 to 41 , wherein the nucleic acid molecule comprises an RNA molecule.

[0212] Embodiment 43. The nucleic acid molecule according to Embodiment 42, wherein the RNA molecule comprises an mRNA molecule.

[0213] Embodiment 44. A Gram-negative bacterium comprising the IL-21 variant according to any one of Embodiments 1 to 32, or the nucleic acid molecule according to any one of Embodiments 38 to 43. Embodiment 45. The Gram-negative bacterium according to Embodiment 44, wherein the Gram-negative bacterium is a live attenuated Gram-negative bacterium.

[0214] Embodiment 46. The Gram-negative bacterium according to Embodiment 45, wherein the live attenuated Gram-negative bacterium is a Salmonella spp.

[0215] Embodiment 47. The Gram-negative bacterium according to Embodiment 46, wherein the live attenuated Gram-negative bacterium is Salmonella enterica, preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi.

[0216] Embodiment 48. The Gram-negative bacterium according to Embodiment 47, wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi ZH9.

[0217] Embodiment 49. The Gram-negative bacterium according to any one of

[0218] Embodiments 44 to 48, for use as a therapy.

[0219] Embodiment 50. The Gram-negative bacterium according to any one of

[0220] Embodiments 44 to 49, for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0221] Embodiment 51. The Gram-negative bacterium for use according to Embodiment

[0222] 50, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

[0223] Embodiment 52. The Gram-negative bacterium for use according to Embodiment

[0224] 51 , wherein the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, vulvar cancer, endometrial cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma. Embodiment 53. The Gram-negative bacterium for use according to any one of Embodiments 50 to 52, wherein the Gram-negative bacterium is administered to the subject orally or via local instillation, intra-peritoneally, intravaginally, intrapleurally, intravesically, peritumoral injection or intratumoural injection.

[0225] Embodiment 54. Use of the IL-21 variant as defined in any one of Embodiments 1 to 32, or the nucleic acid molecule as defined in any one of Embodiments 38 to 43, or the Gram-negative bacterium of as defined in any one of Embodiments 44 to 48, in the manufacture of a medicament for a therapy.

[0226] Embodiment 55. The use according to Embodiment 54, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0227] Embodiment 56. A method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject IL-21 variant as defined in any one of Embodiments 1 to 32, or the nucleic acid molecule as defined in any one of Embodiments 38 to 43, or the Gram-negative bacterium of as defined in any one of Embodiments 44 to 48.

[0228] Embodiment 57. A bacterium comprising an interleukin-21 (IL-21) variant, wherein in comparison to wild-type IL-21 , the IL-21 variant comprises one or more amino acid modifications in a region adjacent to a receptor interaction surface of the IL- 21 variant.

[0229] Embodiment 58. The bacterium according to Embodiment 57, wherein the bacterium is a live attenuated bacterium.

[0230] Embodiment 59. The bacterium according to Embodiment 58, wherein bacterium is a live attenuated Gram-negative bacterium.

[0231] Embodiment 60. The bacterium according to Embodiment 59, wherein the live attenuated Gram-negative bacterium is a Salmonella spp, preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica, more preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi, even more preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi ZH9.

[0232] Embodiment 61. The bacterium according to any of Embodiments 57 to 60, wherein wild-type IL-21 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.

[0233] Embodiment 62. A bacterium comprising an interleukin-21 (IL-21) variant, wherein in comparison to wild-type IL-21 , the IL-21 variant comprises one or more amino acid modifications in a region adjacent to a receptor interaction surface of the IL- 21 variant.

[0234] Embodiment 63. The bacterium according to Embodiment 62, wherein the bacterium is a live attenuated bacterium.

[0235] Embodiment 64. The bacterium according to Embodiment 63, wherein bacterium is a live attenuated Gram-negative bacterium.

[0236] Embodiment 65. The bacterium according to Embodiment 64, wherein the live attenuated Gram-negative bacterium is a Salmonella spp, preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica, more preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi, even more preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi ZH9.

[0237] Embodiment 66. The bacterium according to any of Embodiments 62 to 65, wherein wild-type IL-21 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.

[0238] Embodiment 67. The bacterium according to Embodiment 66, wherein in comparison to SEQ ID NO: 6, the amino acid modifications occur at one or more of amino acid positions 13, 29, 33, 36, 71 , and / or 74, preferably 29, 33, 36 and / or 71. Embodiment 68. The bacterium according to Embodiment 67, wherein the IL-21 variant comprises an amino acid sequence according to SEQ ID NO: 6, 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: 6.

[0239] Embodiment 69. The bacterium according to any of Embodiments 62 to 68, wherein the amino acid modifications are hydrophobic-to-hydrophilic substitutions.

[0240] Embodiment 70. The bacterium according to Embodiment 69, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 13 is an isoleucine- to-aspartate substitution or an isoleucine-to-lysine substitution.

[0241] Embodiment 71. The bacterium according to Embodiment 70, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 13 is an isoleucine- to-aspartate substitution.

[0242] Embodiment 72. The bacterium according to any of Embodiments 62 to 71 , wherein the hydrophobic-to-hydrophilic substitution at amino acid position 29 is a valine-to-threonine substitution or a valine-to-aspartate substitution.

[0243] Embodiment 73. The bacterium according to Embodiment 72, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 29 is a valine-to- threonine substitution.

[0244] Embodiment 74. The bacterium according to any of Embodiments 62 to 73, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 33 is a valine-to-threonine substitution or a valine-to-aspartate substitution.

[0245] Embodiment 75. The bacterium according to any of Embodiments 62 to 74, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 36 is a phenylalanine-to-glutamine substitution or a phenylalanine-to-lysine substitution.

[0246] Embodiment 76. The bacterium according to Embodiment 75, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 36 is a phenylalanine-to-lysine substitution. Embodiment 77. The bacterium according to any of Embodiments 62 to 76, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 71 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

[0247] Embodiment 78. The bacterium according to Embodiment 77, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 71 is an isoleucine- to-lysine substitution.

[0248] Embodiment 79. The bacterium according to any of Embodiments 62 to 78, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 74 is a valine-to-threonine substitution or a valine-to-aspartate substitution.

[0249] Embodiment 80. The bacterium according to Embodiment 79, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 74 is a valine-to- threonine substitution.

[0250] Embodiment 81. The bacterium according to any of Embodiments 62 to 80, wherein the IL-21 variant comprises: i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 29; and ii) the phenylalanine-to-lysine substitution or phenylalanine-to- glutamine substitution at amino acid position 36, preferably wherein the IL-21 variant comprises the valine-to-threonine substitution at amino acid position 29 and the phenylalanine-to-lysine substitution at amino acid position 36.

[0251] Embodiment 82. The bacterium according to Embodiment 81 , wherein the IL-21 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 29 and 36. Embodiment 83. The bacterium according to Embodiment 82, wherein the IL-21 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 29 and 36.

[0252] Embodiment 84. The bacterium according to any of Embodiments 62 to 83, wherein the IL-21 variant comprises: i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the valine-to-threonine substitution at amino acid position 33 and the isoleucine-to-lysine substitution at amino acid position 71.

[0253] Embodiment 85. The bacterium according to Embodiment 84, wherein the IL-21 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 33 and 71 .

[0254] Embodiment 86. The bacterium according to Embodiment 85, wherein the IL-21 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 33 and 71 .

[0255] Embodiment 87. The bacterium according to any of Embodiments 62 to 88, wherein the IL-21 variant comprises: i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the valine-to-aspartate substitution at amino acid position 33 and the isoleucine-to-lysine substitution at amino acid position 71.

[0256] Embodiment 88. The bacterium according to Embodiment 87, wherein the IL-21 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 33 and 71 .

[0257] Embodiment 89. The bacterium according to Embodiment 88, wherein the IL-21 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 33 and 71 .

[0258] Embodiment 90. The bacterium according to any of Embodiments 62 to 89, wherein the IL-21 variant comprises: i) the phenylalanine-to-lysine substitution or phenylalanine-to- glutamine substitution at amino acid position 36; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the phenylalanine-to-lysine substitution at amino acid position 36 and the isoleucine-to-lysine substitution at amino acid position 71.

[0259] Embodiment 91. The bacterium according to Embodiment 90, wherein the IL-21 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 36 and 71 .

[0260] Embodiment 92. The bacterium according to Embodiment 91 , wherein the IL-21 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 36 and 71 .

[0261] Embodiment 93. The bacterium according to any of Embodiments 62 to 92, wherein the IL-21 variant is fused to a signal peptide. Embodiment 94. The bacterium according to Embodiment 93, wherein the signal peptide is a periplasmic signal peptide.

[0262] Embodiment 95. The bacterium according to Embodiments 93 or 94, wherein the signal peptide is fused to the IL-21 variant via a spacer sequence, preferably wherein the spacer sequence is between 1 to 5 amino acids in length.

[0263] SEQUENCES FORMING PART OF THE DESCRIPTION

[0264] SEQ ID NO: 1 (parental IL-21)

[0265] MHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA

[0266] QLKSANTGNNERI INVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFK

[0267] SLLQKMIHQHLSSRTHGSEDS

[0268] SEQ ID NO: 2 (V36)

[0269] MHKSSSQGQDRHMIRMRQLIDIVDQLKNYTNDLVPEKLPAPEDVETNCEWSAFSCFQKA

[0270] QLKSANTGNNERI INVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFK

[0271] SLLQKMIHQHLSSRTHGSEDS

[0272] SEQ ID NO: 3 (V52)

[0273] MHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLTPEFLPAPEDVETNCEWSAFSCFQKA

[0274] QLKSANTGNNERKINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFK

[0275] SLLQKMIHQHLSSRTHGSEDS

[0276] SEQ ID NO: 4 (V58)

[0277] MHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLDPEFLPAPEDVETNCEWSAFSCFQKA

[0278] QLKSANTGNNERKINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFK

[0279] SLLQKMIHQHLSSRTHGSEDS

[0280] SEQ ID NO: 5 (V62)

[0281] MHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEELPAPEDVETNCEWSAFSCFQKA

[0282] QLKSANTGNNERKINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFK

[0283] SLLQKMIHQHLSSRTHGSEDS SEQ ID NO: 6 (mature IL-21)

[0284] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ

[0285] LKSANTGNNERI INVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS

[0286] LLQKMIHQHLSSRTHGSEDS

[0287] REFERENCES

[0288] Alhunaidi, O. and A. R. Zlotta (2019) "The use of intravesical BCG in urothelial carcinoma of the bladder." Ecancermedicalscience. 13: 905.

[0289] Asao H, Okuyama C, Kumaki S, Ishii N, Tsuchiya S, Foster D, et al. “Cutting edge: the common gamma-chain is an indispensable subunit of the IL-21 receptor complex.” J Immunol. 2001 ; 167(1): 1-5.

[0290] Bryant VL, Ma CS, Avery DT, Li Y, Good KL, Corcoran LM, et al. “Cytokine- mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells.” J Immunol. 2007; 179(12):8180-90. de Marco, A. (2009) “Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia col i.” Microbial Cell Factories. 8.

[0291] Devi, N., Adivitiya and 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.

[0292] Ferrer-Miralles N., Domingo-Espin J., Corchero J.L., Vazquez E., & Villaverde A. (2009) “Microbial factories for recombinant pharmaceuticals.” Microbial Cell Factories. 8.

[0293] Hamming OJ, Kang L, Svensson A, Karlsen JL, Rahbek-Nielsen H, Paludan SR, et al. “Crystal structure of interleukin-21 receptor (IL-21 R) bound to IL-21 reveals that sugar chain interacting with WSXWS motif is integral part of IL-21 R.” J Biol Chem. 2012;287(12):9454-60.

[0294] Hinrichs CS, Spolski R, Paulos CM, Gattinoni L, Kerstann KW, Palmer DC, et al. „IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy.” Blood. 2008;111 (11):5326-33. Jin H, Carrio R, Yu A, Malek TR. “Distinct activation signals determine whether IL- 21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis.” J Immunol. 2004; 173(1 ):657-65.

[0295] Kovanen PE, Leonard WJ. “Cytokines and immunodeficiency diseases: critical roles of the gamma(c)-dependent cytokines interleukins 2, 4, 7, 9, 15, and 21 , and their signaling pathways.” Immunol Rev. 2004;202:67-83.

[0296] Leonard WJ. “Cytokines and immunodeficiency diseases.” Nat Rev Immunol. 2001 ;1 (3):200-8.

[0297] Ozaki K, Kikly K, Michalovich D, Young PR, Leonard WJ. “Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain.” Proc Natl Acad Sci U SA. 2000;97(21): 11439-44.

[0298] Parrish-Novak, J., Dillon, S.R., Nelson, A., Hammond, A., Sprecher, C., Gross, J.A., et al (2000) “Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.” Nature. 408 (6808): 57-63.

[0299] Roda JM, Parihar R, Lehman A, Mani A, Tridandapani S, Carson WE, 3rd. “Interleukin-21 enhances NK cell activation in response to antibody-coated targets.” J Immunol. 2006;177(1):120-9.

[0300] Sivakumar PV, Garcia R, Waggie KS, Anderson-Haley M, Nelson A, Hughes SD. “Comparison of vascular leak syndrome in mice treated with IL21 or IL2.” Comp Med. 2013;63(1): 13-21.

[0301] Spolski R, Leonard WJ. “Interleukin-21 : basic biology and implications for cancer and autoimmunity.” Annu Rev Immunol. 2008;26:57-79.

[0302] Wang G, Tschoi M, Spolski R, Lou Y, Ozaki K, Feng C, et al. “In vivo antitumor activity of interleukin 21 mediated by natural killer cells.” Cancer Res. 2003;63(24):9016-22. Zeng R, Spolski R, Finkelstein SE, Oh S, Kovanen PE, Hinrichs CS, et al. ..Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function.” J Exp Med. 2005;201 (1): 139-48.

Claims

CLAIMS1. An interleukin-21 (IL-21) variant, wherein in comparison to wild-type IL- 21 that 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, the IL-21 variant comprises an amino acid modification at one or more of the following amino acid positions: 29, 33, 36 and / or 71.

2. The IL-21 variant according to claim 1 , wherein the IL-21 variant comprises amino acid modifications at amino acid positions 29 and 36, 33 and 71 , or 36 and 71.

3. The IL-21 variant according to claim 1 or2, wherein in comparison to SEQ ID NO: 6, the IL-21 variant further comprises an amino acid modification at one or more of amino acid positions 13 and / or 74.

4. The IL-21 variant according to claim 1 to 3, wherein the IL-21 variant comprises an amino acid sequence according to SEQ ID NO: 6, 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: 6.

5. The IL-21 variant according to any preceding claim, wherein the amino acid modifications are hydrophobic-to-hydrophilic substitutions.

6. The IL-21 variant according to any preceding claim, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 29 is a valine-to- threonine substitution or a valine-to-aspartate substitution.

7. The IL-21 variant according to claim 6, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 29 is a valine-to-threonine substitution.

8. The IL-21 variant according to any preceding claim, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 33 is a valine-to- threonine substitution or a valine-to-aspartate substitution.

9. The IL-21 variant according to any preceding claim, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 36 is a phenylalanine-to-glutamine substitution or a phenylalanine-to-lysine substitution.

10. The IL-21 variant according to claim 9, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 36 is a phenylalanine-to-lysine substitution.

11. The IL-21 variant according to any preceding claim, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 71 is an isoleucine- to-aspartate substitution or an isoleucine-to-lysine substitution.

12. The IL-21 variant according to claim 11 , wherein the hydrophobic-to- hydrophilic substitution at amino acid position 71 is an isoleucine-to-lysine substitution.

13. The IL-21 variant according to any preceding claim, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 13 is an isoleucine- to-aspartate substitution or an isoleucine-to-lysine substitution.

14. The IL-21 variant according to claim 13, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 13 is an isoleucine-to-aspartate substitution.

15. The IL-21 variant according to any preceding claim, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 74 is a valine-to- threonine substitution or a valine-to-aspartate substitution.

16. The IL-21 variant according to claim 15, wherein the hydrophobic-to- hydrophilic substitution at amino acid position 74 is a valine-to-threonine substitution.

17. The IL-21 variant according to any preceding claim, wherein the IL-21 variant comprises:i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 29; and ii) the phenylalanine-to-lysine substitution or phenylalanine-to- glutamine substitution at amino acid position 36, preferably wherein the IL-21 variant comprises the valine-to-threonine substitution at amino acid position 29 and the phenylalanine-to-lysine substitution at amino acid position 36.

18. The IL-21 variant according to claim 17, wherein the IL-21 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 29 and 36.

19. The IL-21 variant according to claim 18, wherein the IL-21 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 29 and 36.

20. The IL-21 variant according to any preceding, wherein the IL-21 variant comprises: i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the valine-to-threonine substitution at amino acid position 33 and the isoleucine-to-lysine substitution at amino acid position 71.

21. The IL-21 variant according to claim 20, wherein the IL-21 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 33 and 71 .

22. The IL-21 variant according to claim 21 , wherein the IL-21 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 33 and 71 .

23. The IL-21 variant according to any preceding claim, wherein the IL-21 variant comprises: i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the valine-to-aspartate substitution at amino acid position 33 and the isoleucine-to-lysine substitution at amino acid position 71.

24. The IL-21 variant according to claim 23, wherein the IL-21 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 33 and 71 .

25. The IL-21 variant according to claim 24, wherein the IL-21 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 33 and 71 .

26. The IL-21 variant according to any preceding claim, wherein the IL-21 variant comprises: i) the phenylalanine-to-lysine substitution or phenylalanine-to- glutamine substitution at amino acid position 36; and ii) the isoleucine-to-lysine substitution or isoleucine-to-aspartate substitution at amino acid position 71 , preferably wherein the IL-21 variant comprises the phenylalanine-to-lysine substitution at amino acid position 36 and the isoleucine-to-lysine substitution at amino acid position 71.

27. The IL-21 variant according to claim 26, wherein the IL-21 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 36 and 71 .

28. The IL-21 variant according to claim 27, wherein the IL-21 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 36 and 71 .

29. The IL-21 variant according to any preceding claim, wherein the IL-21 variant is fused to a signal peptide.

30. The IL-21 variant according to claim 29, wherein the signal peptide is a periplasmic signal peptide.

31. The IL-21 variant according to claims 29 or 30, wherein the signal peptide is fused to the IL-21 variant via a spacer sequence, preferably wherein the spacer sequence is between 1 to 5 amino acids in length.

32. A nucleic acid molecule encoding the IL-21 variant as defined in any one of claims 1 to 31.

33. The nucleic acid molecule according to claim 32, wherein the nucleic acid molecule comprises a sequence encoding an amino acid sequence according to any one of SEQ ID NOs: 2, 3, 4, and / or 5, or an amino acid sequence comprising at least 70% identity to any one of SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 2, 3, 4, and / or 5 retains the one or more amino acid modifications at the relevant amino acid positions.

34. The nucleic acid molecule according to claim 33, wherein the nucleic acid molecule comprises a sequence encoding an amino acid sequence comprising at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 3, 4, and / or 5, wherein the % sequence identity to any one of SEQ ID NOs: 2, 3, 4, and / or 5 retains the one or more amino acid modifications at the relevant amino acid positions.

35. The nucleic acid molecule according to any one of claims 32 to 34, wherein the nucleic acid molecule comprises a DNA molecule.

36. The nucleic acid molecule according to any one of claims 32 to 35, wherein the nucleic acid molecule comprises an RNA molecule.

37. The nucleic acid molecule according to claim 36, wherein the RNA molecule comprises an mRNA molecule.

38. A bacterium comprising the IL-21 variant according to any one of claims 1 to 31 , or the nucleic acid molecule according to any one of claims 32 to 37.

39. The bacterium according to claim 38, wherein the bacterium is a live attenuated Gram-negative bacterium.

40. The bacterium according to claim 39, wherein the live attenuated Gramnegative bacterium is a Salmonella spp.41 . The bacterium according to claim 40, wherein the live attenuated Gramnegative bacterium is Salmonella enterica, preferably wherein the live attenuated Gram-negative bacterium is Salmonella enterica serovar Typhi.

42. The bacterium according to claim 41 , wherein the live attenuated Gramnegative bacterium is Salmonella enterica serovar Typhi ZH9.

43. The IL-21 variant according to any of claims 1 to 31 , or the nucleic acid molecule according to any of claims 32 to 37, or the bacterium according to any of claims 38 to 42, for use as a therapy.

44. The IL-21 variant, nucleic acid molecule or bacterium for use according to claim 43, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

45. The IL-21 variant, nucleic acid molecule or bacterium for use according to claim 44, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

46. The IL-21 variant, nucleic acid molecule or bacterium for use according to claim 45, wherein the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

47. The IL-21 variant, nucleic acid molecule or bacterium for use according to any one of claims 43 to 46, wherein the IL-21 variant, nucleic acid molecule or bacterium is administered to the subject orally or via local instillation, intraperitoneally, intravaginally, intrapleurally, intravesically, peritumoral injection or intratumoural injection.

48. Use of the IL-21 variant according to any of claims 1 to 31 , or the nucleic acid molecule according to any of claims 32 to 37, or the bacterium according to any of claims 38 to 42, in the manufacture of a medicament for a therapy.

49. The use according to claim 48, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

50. A method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject IL-21 variant IL-21 variant according to any of claims 1 to 31 , or the nucleic acid molecule according to any of claims 32 to 37, or the bacterium according to any of claims 38 to 42.

Images