Nucleic acid construct

Abstract

The present invention provides a nucleic acid construct encoding two or more of an interleukin-15 (IL-15) variant, an interleukin-18 (IL-18) variant, an interleukin-21 (IL-21) variant, and CXCL9, wherein in comparison to wild-type interleukins, the interleukin variants comprise one or more amino acid modifications in a region adjacent to a receptor interaction surface of the interleukin variants.

Description

[0001] NUCLEIC ACID CONSTRUCT

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to nucleic acid constructs encoding multiple cytokines.

[0004] BACKGROUND

[0005] Evidence supporting the use of immuno-stimulatory molecules, such as cytokines, in the treatment of cancer has grown significantly in recent years. In particular, interleukin 15 (IL-15) and IL-15 agonist molecules, interleukin-18 (IL-18) molecules and interleukin-21 (IL-21) molecules are considered as promising immunotherapeutics because of their role in CD8+ T-cell and Natural Killer (NK) cell activation and proliferation (Guo, 2017; Robinson, 2017; Atallah-Yunes, 2022; Parrish-Novak et al, 2000).

[0006] Cytokines can be produced naturally by mammalian cells, or engineered using recombinant DNA technology for expression in bacterial systems. Expression in bacteria as microbial immunotherapy (i.e. , microbial living cures) has been shown to be effective as post-surgery treatment to reduce the incidence of tumour recurrence, with BCG treatment being the current standard of care for certain cancer types, in particular for NMIBC patients (Alhunaidi, 2019). BCG therapy has limitations including the efficacy range (40-60% of patients will experience tumour recurrence within 5 years of surgery), adverse effects and safety concerns, complex manufacturing requirements, and a long treatment regimen (patients undergo 27 intra-vesical, transurethral instillations over 36 months). To improve efficacy, microbial immunotherapy together with co-immunostimulatory molecules has been proposed.

[0007] A challenge associated with the use of cytokines, such as IL-15, IL-18 and IL-21, as an immuno-stimulatory anti-cancer treatment, especially in the context of microbial living cures, is their 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 theformation 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 expression and delivery of cytokines by 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 where there are disulphide bonds in the protein structure.

[0008] A further challenge specific to IL-18 as an immuno-stimulatory anti-cancer treatment is the upregulation of a high-affinity decoy receptor, IL-18 binding protein (IL-18BP) in cancers which blocks downstream immuno-stimulatory signalling pathways.

[0009] Attempts to improve cytokine expression levels and solubility in bacteria such as E. coli have included lowering the cultivation temperature, co-expression of chaperones, in vitro re-solubilisation and refolding protocols, use of weak promoters, fusion to solubility enhancer tags, adding signal peptides for periplasmic localisation or N-terminal fusion to carrier for extracellular export (Devi 2016; Nausch 2013; Behar2011).

[0010] 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. In fact, it has been observed previously by the inventors of the present invention that the expression of human IL-15 in Salmonella Typhi ZH9from the temperature inducible LPR promoter, in cultures grown at the suboptimal inducing temperature of 37°C, results in accumulation of IL-15 in insoluble aggregates.

[0011] Due to the intricate and complex nature of the signalling networks that regulate the immune system, for cancer immunotherapies to be successful, the synergistic effect of multiple cytokines may be required to achieve effective therapeutic results. Synergy leverages the advantages of multiple agents to strengthen the overall therapeutic effect, for example by overcoming problems with tumour resistance, broadening the subject’s immune response and more effectively targeting the tumour microenvironment, leading to more efficient and durable responses by engaging the immune system in multiple ways. For example, the production and release of chemokines, such as CXCL-9, from the bacterial chassis may attract the appropriate immune cells, such as CD8+ T-cells and NK cells, to the tumour site as targets for delivered interleukins, such as IL-15, IL-18, or IL-21, that overcome the immunosuppressive environment and activate proliferation and cytotoxic activity of such immune cells to achieve efficacious antitumour properties.

[0012] However, expressing multiple recombinant proteins as therapeutic agents in a host cell (such as a therapeutic strain) typically results in excessive metabolic burden, which can impact both function and survivability of the therapeutic strain, and require extensive rounds of optimization. A host may suffer metabolic burden due to the excessive energy requirements for expressing various non-essential proteins that compete with essential resources, which can be worsened by the formation of inclusion bodies. When a host is subject to high metabolic demand, there are limits placed on the cell’s capacity to maintain normal transcriptional activity, which in turn may enhance metabolic burden. In particular, depletion of transcriptional resources has been identified as a major source of burden (Ceroni et al, 2018; Li and Rinas, 2020; Segall-Shapiro et al, 2014). Depletion of transcriptional resources would also be worsened with multiple promoters driving the expression of multiple heterologous genes. One solution to this problem is to combine all heterologous genes in a single polycistronic operon. However, thisapproach is limited in numerous ways. For example, merely combining multiple cytokines into a single nucleic acid could lead to unpredictable variations in their respective expression patterns due to distal interactions between the different genes within the operon, that can result in attenuation or occlusion of important genetic signatures (such as the ribosome binding site). Furthermore, it is not certain that each cytokine will be expressed in a predictable manner or at the same ratio.

[0013] As such, it can be seen that there is a need to develop circuits encoding optimal combinations of cytokines which can be optimally expressed in, and efficiently secreted by host cells, which are also able to bind their receptor to allow immunostimulatory signalling. Suitably designed circuits encoding these cytokines to allow for increasingly efficacious immunotherapies are therefore needed in the art.

[0014] SUMMARY OF INVENTION

[0015] The inventors of the present invention have discovered synergistic (Strengell et al, 2003) combinations of cytokine molecules, including both wild-type and variant forms of IL-15, IL-18, and IL-21, and CXCL9, which can be assembled in particular arrangements into a single transcriptional unit for expression from a single nucleic acid to be optimally expressed such as to stimulate immune cells and trigger higher production of interferon-gamma, thereby having the capacity to promote an enhanced immune response. The inventors of the present invention have identified certain modifications which optimise these cytokines, particularly for enhanced expression by and secretion from bacterial strains.

[0016] Accordingly, in a first aspect of the invention, there is provided a nucleic acid construct comprising polynucleotide sequences encoding two or more of: (i) an interleukin-15 (IL-15) molecule; (ii) an interleukin-18 (IL-18) molecule; (iii) an interleukin-21 (IL-21) molecule; and / or (iv) a CXCL9 molecule. In preferred embodiments, the nucleic acid construct comprises polynucleotide sequences encoding an IL-15 molecule and an IL-18 molecule.In a second aspect of the invention, there is provided a cell comprising the nucleic acid construct according to the first aspect of the invention.

[0017] In a third aspect of the invention, there is provided a nucleic acid construct according to the first aspect of the invention or a cell according to the second aspect of the invention for use as a therapy.

[0018] In a fourth aspect of the invention, there is provided a nucleic acid construct according to the first aspect of the invention or a cell according to the second aspect of the invention for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0019] In a fifth aspect of the invention, there is provided a use of a nucleic acid construct according to the first aspect of the invention or a cell according to the second aspect of the invention in the manufacture of a medicament for a therapy.

[0020] 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 a nucleic acid construct according to the first aspect of the invention or a cell according to the second aspect of the invention.

[0021] BRIEF DESCRIPTION OF DRAWINGS

[0022] The invention is described with reference to the accompanying drawings, wherein:

[0023] Figure 1 shows the crystal structures and the amino acid positions which may be modified within (A) IL-15 (PDB ID: 4GS7), (B) IL-18 (PDB ID: 3WO4, PDB ID: 7AL7 aligned over3WO4), and (C) IL-21 (PDB ID: 3TGX).

[0024] Figure 2 shows a schematic representation of the potential synergistic combinations of IL-15, IL-18, IL-21 and CXCL9 molecules on lymphocyte recruitment and activation through the secretion of the cytokines via bacterial strains containing multicytokine circuits.Figure 3 shows a schematic representation of the development of bacterial strains expressing the multicytokine circuits. Each desired cytokine or chemokine can be encoded into either a linker or an end module by Bbsl-dependent Golden Gate Assembly (GGA) onto appropriate recipient plasmids. Each module contains Bsal restriction sites compatible with multi-module assembly, and a ribozyme upstream each protein encoding gene. Multicytokine circuits (MCCs) can be created by combining the resulting linked modules with an end module. As one example, a MCC with IL-15, IL-18 and CXCL-9 can be assembled using linker modules for IL-15 and IL-18 and an end module with CXCL-9 on a Bsal-dependent GGA. The modules can be transferred into compatible vectors bearing different promoters through a Bsmbl-dependent GGA. MCCs can be tested either on plasmids or genomically integrated by evaluating the protein produced in the whole bacterial cell, periplasm, and supernatant by HPLC / MS quantification.

[0025] Figure 4 shows a heatmap of NK cell stimulation using different cytokine combinations exogenously added, and demonstrates that interferon-gamma (IFNy) production was higher when more than one cytokine is added.

[0026] Figure 5 shows the validation of cytokine delivery into the periplasm through the Sec-dependent pathway by combination of fusions of the mature beta-lactamase (b / a) gene with all native cytokine sequences for all cargoes (IL-15, IL-18, IL-21, CXCL9, and the Sushi domain of the IL-15 Receptor) and various signal peptides (SP). Bacterial strains bearing these constructs were challenged with increasing concentrations (mg / mL) of carbenicillin antibiotic, shown throughout (A)-(D), which is degraded by the gene product of bla (Bia). Higher amounts of Bia allow bacterial survival (measured as OD6oo) at higher concentrations of Carbenicillin.

[0027] Figure 6 shows the construction of benchmark circuits in which all cytokine variants are fused with the optimal signal peptide previously determined (e.g., pelB or malE for il-15) and assayed under different constitutive promoters with various strengths ( / .e., pro1 < proA < proB < proC) to establish the optimal conditions for expression either as single or multicytokine circuits.Figure 7 shows a schematic representation of the analysis of the impact the strength of the promoter has on single cytokine production. ZH9 Salmonella were transformed with constructs comprising various combinations of a signal peptide, a promoter and a cargo molecule (cytokine), then grown in lysogeny broth (LB). After cells reached OD6oo~1.0, bacteria were pelleted, separated from the supernatant, and then lysed. Both fractions (supernatant and lysed pellet) were subject to a blind, randomized HPLC / MS analysis.

[0028] Figure 8 shows the analysis of the impact of the PelB, MalE, OmpA, DsbA and PhoA signal peptides, the pro1, proA, proB, and proC promoters, and IL-15, CXCL9, IL-18 and IL-21 cargoes on bacterial growth.

[0029] Figure 9 shows that the production of CXCL9 increased linearly with promoter activity to a maximum threshold of 40 pg / 108bacteria in the cell pellet and approximately 2 pg / 108bacteria in the supernatant. RSU are based on the values assigned by Davis etal. (Nucleic Acids Res. 2011; 39(3): 1131-1141).

[0030] Figure 10 shows cargoes and signal peptides which may be selected to construct and characterise multicytokine circuits.

[0031] Figure 11 shows various circuits which may be constructed and their corresponding MC identifier codes.

[0032] Figure 12 shows a bacterial growth curve of ZH9 Salmonella expressing various multicytokine circuits described herein under constitutive promoters. The curve confirms that the addition of the multicytokine circuits in plasmids to ZH9 Salmonella does not impact bacterial growth in lysogeny broth (LB).

[0033] Figure 13 shows CXCL9 protein production in whole bacterial cell and bacterial periplasm, quantified by HPLC / MS from the bacterial cell pellet and bacterial periplasm from ZH9 cells expressing single and multicytokine circuits.

[0034] Figure 14 shows a schematic representation of the pSPEBIa (Signal Peptide Evolution + Bia) plasmid. Promoter proC drives expression of a mature betalactamase fusion within a low copy number plasmid. Any payload can be fused tobla via Bbsl Golden Gate reaction, replacing a FLAG tag stuffer sequence. Protein fusion from this construct will be retained in the cytosol. The protein fusion can be directed into the periplasm by replacement of stuffer sequence containing rbs34 and ATG-StrepTagll sequence for a signal peptide sequence containing an appropriate ribosome binding site.

[0035] Figure 15 demonstrates that the position of each cytokine in a multi-cytokine circuit can influence total expression and secretion levels. Relative luminescence units (RLU) per OD6oo of derived from a luciferase assay (Promega) where the luminescence is proportional to the presence of a HiBit tag placed at the C-terminus end of the indicated protein. Samples labelled as ‘single’ represent the control single cytokine circuit. Samples labelled with a three-digit sequence indicates the position of each cytokine in the construct, wherein T is IL15, ‘2’ is IL18, and ‘4’ is CXCL9.

[0036] Figure 16 shows the relative production one, two or three cytokines from IL-15 (black), IL-18 (grey) and / or CXCL-9 (white) in multi-cytokine circuits expressed in bacteria. Relative production is measured as relative luminescence units (RLU) through NanoGio Assay (Promega) detecting a C-terminal HiBit tag in supernatant of bacterial cultures grown in Lysogeny Broth for 5 h at 37 °C (OD600 ~ 1.0) for strains bearing circuits indicated on the x-axis under constitutive control. Each individual cytokine in the circuit was labelled with a HiBit tag (resulting in each circuit being built three times, each having a unique cytokine labelled), allowing to study the effect of increasing payload number and its position in the circuit sequence, which can be affected ~10-fold.

[0037] Figure 17 shows the relative production of IL-15 (black), IL-18 (grey) or CXCL-9 (white) as measured through NanoGio Assay (Promega) detecting a C-terminal HiBit tag in supernatant of bacterial cultures grown in Lysogeny Broth for 5 h at 37 °C (OD600 ~ 1.0) for strains bearing circuits indicated on the x-axis under either constitutive control (top) ora quorum sensing-based expression system (bottom).

[0038] Figure 18 demonstrates the correlation between production, measured as relative luminescence from a NanoGio Assay, of the same multi-cytokine circuits from aconstitutive promoter (x-axis) or a quorum sensing-based expression system (y-axis). The labelling of each point corresponds to encoded circuit name, where 1 = IL-15, 2 = IL-18, 3 = IL-21, 4 = CXCL9; H = preceding cytokine is HiBit-tagged. For example, 1H2 is an IL-15+IL-18 circuit where IL-15 is C-terminal HiBit tagged.

[0039] DETAILED DESCRIPTION

[0040] The inventors of the present invention have discovered synergistic combinations of cytokine molecules (IL-15, IL-18, and IL-21 and CXCL9), including both wildtype and variant forms thereof, which can be combined in order to achieve a synergistic effect and thus enhanced therapeutic benefits. The inventors have determined that these cytokines can be arranged in certain numbers and orders into a single transcriptional unit as a nucleic acid construct from which expression can be controlled in a refined manner and avoiding distal effects which disturb the expression of each cytokine.

[0041] The inventors have previously identified specific amino acid modifications within IL-15, IL-18, and IL-21 which enhance their stability for bacterial expression (while preserving bioactivity) and, in the case of IL-18, allow for the preferential binding of IL-18 receptors over IL-18 decoy receptors (IL-18 binding protein). These cytokines are ideal candidates for expression in, and secretion from, efficient expression systems such as bacteria, for example Gram-negative bacteria and Gram-positive bacteria. Therefore, the inventors have presently designed nucleic acid constructs featuring optimal combinations of cytokine variants, in addition to wild-type cytokines.

[0042] By combining polynucleotide sequences encoding these cytokine molecules into a single nucleic acid construct, the present invention also offers the advantage of enabling the integration of this unit into plasmids or chromosomes, while leaving other spaces free for the addition of other therapeutically advantageous agents which may be expressed together with the present cytokines.

[0043] Even further, the inventors have surprisingly discovered the optimal signal peptides which may be fused to each cytokine molecule to enhance their secretion / export from a host cell comprising the nucleic acid construct.Accordingly, in a first aspect of the invention, there is provided a nucleic acid construct comprising polynucleotide sequences encoding two or more of: an interleukin-15 (IL-15) molecule; an interleukin-18 (IL-18) molecule; an interleukin-21 (IL-21) molecule; and / or a CXCL9 molecule. As described herein, the term “cytokine” includes interleukins and chemokines. Cytokine molecules may be wild-type cytokines or cytokine variants. In comparison to wild-type interleukins, the interleukin variants comprise one or more amino acid modifications in a region adjacent to a receptor interaction surface of the interleukin variants.

[0044] As used herein, the term “nucleic acid construct” refers to a nucleic acid sequence, molecule or segment that may be artificially designed. The nucleic acid construct may be an isolated nucleic acid construct. The nucleic acid construct can be incorporated into a vector, such as a plasmid, that is used to deliver the nucleic acid construct into a target cell, such as a bacterial cell or a mammalian cell. The construct may be referred to as an expression construct. The nucleic acid construct may be integrated into the genome of the cell, such as a bacterial chromosome.

[0045] The nucleic acid construct may also be referred to as a “multicytokine circuit” or “MCC”, since the construct may comprise polynucleotide sequences encoding various combinations of cytokines as detailed herein. The nucleic acid construct of the present invention may encode two or more of: an IL-15 molecule, an IL-18 molecule, an IL-21 molecule, and / or a CXCL9 molecule. The term “encode” is given its usual meaning in the art, in that the nucleic acid construct comprises a polynucleotide sequence which corresponds to the amino acid sequence of the cytokine. When the nucleic acid sequences are expressed (i.e., if the construct is DNA, transcribed and translated, or if the construct is RNA, translated), the proteins corresponding to the encoded cytokines are produced. The polynucleotide sequences within the nucleic acid construct may be cloned from an existing sequence (e.g., using restriction enzymes to remove the sequence of interest from an organism’s DNA and amplifying the sequence using PCR) or developed synthetically. The nucleic acid construct can encode multiple cytokines as a single transcriptional unit; however, the cytokines can be expressed from thisunit as distinct proteins that are not physically joined. In addition to cytokines, there may also be present other components within the nucleic acid construct. For example, there may be present any components which are required to ensure the successful expression of the cytokines, such as ribosome binding sites, promoters, spacer sequences, linker sequences, restriction enzyme sites and the like.

[0046] Accordingly, the cytokine molecules are to be encoded by the nucleic acid construct. However, merely combining the cytokine molecules into one large circuit would lead to unpredictable variations in the expression of each cytokine due to distal interactions between the different genes within the overall construct. Such distal interactions can lead to attenuation or occlusion of important genetic signatures, as well as insufficient expression of certain cytokine molecules due to significant variation in the ratio of each cytokine expressed. The inventors have found that the ways in which cytokines are combined into a multicytokine circuit impacts the expression and secretion levels of each cytokine (Figure 15, 16, 17). Accordingly, the inventors of the present invention have carefully designed the present nucleic acid constructs by determining the optimal combinations and arrangements of each cytokine molecule such that numerous cytokine molecules can be encoded by a single transcriptional unit in distinct protein-encoding modules, but once transcribed, the modules encoding each cytokine effectively split apart from the overall construct such that each cytokine may be translated in a predictable manner at the same ratio.

[0047] By “single transcriptional unit”, we intend that the modules encoding the cytokine molecules are transcribed from a single promoter. Exemplary promoters include constitutive promoters such as, but not limited to, proA, proB, proC, prod or J23119), and promoters from quorum sensing systems such as, but not limited to, Lux, Tra, Rpa, and mammalian promoters such as, but not limited to CMV, EF1a, SV40, CAG, TRE, UAS, and ADH1. As used herein, the term “quorum sensing” will be understood by those skilled in the art and refers to the mechanism by which bacteria regulate gene expression in accordance with cell population density via the release and detection of signalling molecules (or ‘autoinducers’). Autoinducers are diffusible chemical signals produced by bacteria in response tochanges in the population density and employed to communicate within and between different species. Other suitable promoters will be known to those in the art. The use of a single promoter advantageously allows for refined and efficient control of expression, abrogating the need for multiple promoters to express multiple cytokines. The expression of the cytokine molecules from the same promoter also has the benefit of ensuring that the expression of the cytokines is not dependent on the availability of certain resources which are required to effect expression from various promoters, such as polymerases and ribosomes.

[0048] Within each module, there may be present the cytokine of choice, and other elements such as a ribosome binding site, a ribozyme sequence and / or a signal peptide sequence (Figure 2). The skilled person will be familiar with the cellular machinery required to effect expression from a gene. As used herein, the term “ribozyme” is given its usual meaning in the art, referring to an RNA molecule with catalytic activity and capable of RNA (i.e. , self) cleavage. Therefore, where each module in the nucleic acid construct comprises a ribozyme sequence, once the nucleic acid construct is transcribed from the single promoter into RNA, the ribozyme sequences present can catalyse self-cleavage of the RNA, thereby splitting the RNA transcript encoding all of the cytokines into separate segments of RNA, separately encoding each cytokine. Exemplary ribozyme sequences include LtsvJ, VtmoJ, riboJ and sscJ, given herein as SEQ ID NOs: 73, 74, 75 and 76, respectively. The nucleic acid construct may comprise a ribozyme sequences upstream of each independent module (i.e., the polynucleotide sequence encoding a cytokine molecule). A ribozyme sequence may be present upstream of each polynucleotide sequence encoding a cytokine molecule, at the junction between each polynucleotide sequence encoding a cytokine molecule.

[0049] Therefore, in some embodiments, the nucleic acid construct comprises polynucleotide sequences encoding two or more of an IL-15 molecule, an IL-18 molecule, an IL-21 molecule, and / or a CXCL9 molecule, and a ribozyme sequence. The ribozyme sequence may be positioned upstream of the polynucleotide sequence encoding the cytokine molecule. The nucleic acid construct may comprise a single ribozyme sequence, two ribozyme sequences, three ribozyme sequences, or four ribozyme sequences. The inventors havefound that inclusion of ribozyme sequences confers the advantage of enabling the nucleic acid constructs of the present invention to be transferred between different expression systems with the expectation that the output of expression will be directly proportional to the strength of the promoter. Promoter strength impacts the expression output (i.e., total protein produced), and may not impact the expression profile, thereby enabling expression of the encoded cytokines in a more predictable manner.

[0050] Accordingly, in preferred embodiments, the nucleic acid construct may be DNA. In other embodiments, the nucleic acid construct may be RNA. In a preferred embodiment, where the nucleic acid construct is RNA, the RNA is mRNA. In another preferred embodiment, the nucleic acid construct is DNA.

[0051] As used herein, the terms “RNA” and “ribonucleic acid” are used interchangeably, and 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, messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA(miRNA), transfer RNA (tRNA), self-amplifying RNA (saRNA) and ribosomal RNA (rRNA). Preferably, the RNA cargo molecule is an mRNA molecule. As used herein, the terms “mRNA” and “messenger RNA” are used interchangeably and refer to a single-stranded RNA molecule involved in protein synthesis.

[0052] As used herein, the terms “DNA” and “deoxyribonucleic acid” are used interchangeably, and refer to nucleic acids composed of thymine, adenine, guanine, and cytosine ribonucleic acid bases. 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. 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. RNA molecules are transcribed and translated within the bacterium itself.As used herein, the term “cytokine” is given its usual meaning in the art and refers to a broad classification of proteins involved in cell signalling that are secreted by various cell types. Cytokines play an important role in regulating the immune system and the immune response by acting in an autocrine fashion (i.e. , acting on the same cells that secreted them) or in a paracrine fashion (i.e., acting on nearby cells). Cytokines exert their function by binding to their cell surface receptors to activate downstream signalling cascades within the cell. The term “cytokine” is a general term for the multiple proteins that are encompassed within the classification. For the avoidance of doubt, the general term “cytokines” encompasses, amongst other molecules, “interleukins” and “chemokines”. Interleukins referred to herein in particular include IL-15, IL-18 and IL-21, and a chemokine referred to herein includes C-X-C motif chemokine ligand 9 (CXCL9). The person skilled in the art will be familiar with other interleukins and chemokines which may also be utilised.

[0053] A “molecule” refers to a cytokine, including wild-type cytokines and variant cytokines, thereby also encompassing wild-type interleukins and chemokines and variant interleukins and chemokines. A “variant”, as described herein, refers to refer to a molecule which, in comparison to the wild-type molecule, comprises one or more amino acid modifications. An interleukin molecule therefore encompasses wild-type interleukins, such as wild-type IL-15, wild-type IL-18, and wild-type IL-21, as well as interleukin variants, such as IL-15 variants, IL-18 variants, and IL-21 variants.

[0054] “Chemokines” are a small type of cytokine which can promote the chemotaxis of immune cells toward a target. CXCL9 is an example of a chemokine. Other examples include CXCL10, CXCL11, CCL5, CCL2, CX3CL1. CXCL9 (also known as monokine induced by gamma interferon or MIG) is a small cytokine belonging to the CXC chemokine family that plays role in inducing chemotaxis, promoting differentiation and multiplication of leukocytes, and can cause tissue extravasation.

[0055] “Interleukins” (IL) are another type of cytokine. IL-15, IL-18, and IL-21 are examples of interleukins. Other examples include 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-16, and IL-17. As used herein, the term “interleukin” (IL) refers to any member of the glycoprotein family 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, as well as proliferation, maturation, migration, and adhesion. Interleukins can have pro- and anti-inflammatory 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.

[0056] Interleukin-15 (IL-15) is a four alpha-helix bundle cytokine belonging to the same family as IL-2, IL-4, IL-7, IL-9 and IL-21. IL-15 is constitutively expressed by a wide variety of cells including dendritic cells, monocytes, macrophages, bone marrow stromal cells and intestinal epithelial cells. Cells expressing IL-15 also express its high affinity receptor-a, IL-15Ra. Unlike most cytokines, which are secreted in soluble form, IL-15 has a unique form of expression whereby it is expressed in association with its high affinity IL-15Ra and is shuttled as a heterodimeric complex to the surface of IL-15-producing cells. IL-15 can also be secreted as a soluble IL-15 independently from IL-15Ra. The IL-15 / IL-15Ra complex is a cell surface complex which efficiently stimulates neighbouring cells via the IL-2 / 15Rp and GammaC (yc) complex (IL-15Rpy) via a mechanism of trans-presentation. IL-15 preferentially stimulates NK (natural killer) and CD8+ T-cells activation, proliferation and cytolytic activity (Guo, 2007; Santana-Carrero, 2019).

[0057] IL-15 trans-presentation mediates cells responses during homeostasis. There is evidence that the I L-15Ra / l L-15 complexes are cleaved from the surface of presenting cells in response to numerous types of immune stimulation such as total body irradiation, TLR stimulation, virus infections, CD40 stimulation, type I IFNs (IFN-I) and activation of the stimulator of interferon genes (STING) pathway, thus releasing a soluble heterodimeric I L-15 / I L-15Ra complex (Bergamaschi, 2012; Anthony, 2016).Human IL-15 is a protein of 162 amino-acid (18,086 kDa) containing 4 helices, 2 disulphide bonds and 1 glycosylated site. It has two isoforms produced by alternatively spliced transcripts: one form with a short signal peptide (SSP), which contains a 21 amino acid leader peptide and remains in the cell cytosol, and one other with a long signal peptide (LSP), which contains a 48 amino acid signal peptide that allows for secretion of the protein into the extracellular environment (Saeed and Revell, 2001; Duitman, 2008). This secretion occurs through independent export into the Golgi Apparatus of the signal and the receptor alpha, followed by high affinity interaction between signal and receptor, and export into the environment of the complex via the receptor alpha. The signal peptide is cleaved always on the same position, resulting in the same mature form of IL-15, possibly through the highly conserved sequence N-GLPKTEA / NW-C, although SSP IL-15 is already shown to be bioactive, indicating certain flexibility to the presence of N-terminus peptides (Bergamaschi, 2009). The mature IL-15 is then folded in a four-helix bundle motif (the four a-helices are structured together lengthwise in antiparallel orientation), with 16-21 amino acids per a-chain, where alpha chain a1 interacts with a3 and a2 with a4. There is a disulphide bond that stabilises this latter interaction between Cys42-Cys85, and a second one between Cys35-Cys88. There are two highly disordered regions that loop between a1 a2 (loop 1-2) and a3 a4 (loop 3-4).

[0058] The crystal structure of IL-15 co-coordinated to receptors I L-2Rp, yc, and IL-15Ra has been resolved (Ring, 2012). This shows that the signalling protein interacts with I L-2Rp via polar interactions with helices a1 and a3, being the interactions IL15_D8:IL-2Rp {H133, Y134}, I L15_D61 {I L2Rp_{K71 } and IL15_N65:IL-2Rp {R42, Q70, Y134} being essential to stabilise this interaction, consistent with previously published mutagenesis studies (Eisenman, 2002; Pettit 1997). Other amino acids involved in stabilising the interaction would be I68, L69, K10, K11 and S7.

[0059] On the other hand, interactions with yc show a more general interaction, without a highly determined chemical signature, highlighting the promiscuity of yc to interact with multiple cytokines. Interaction occurs via chains a1 and a4, being Q108, M109, and N112 the key amino acids involved in this interaction. Anadditional site of contact is made with the extended alpha chain to compensate for the smaller size of I L-15.

[0060] Finally, interaction with IL-15Ra has been found to occur through a2. Chirifu and co-workers (Chirifu, 2007) describe the interactions to be weak electrostatic (Van der Waals) and identified the following amino acids in the protein-protein interaction: D22, T24, Y26, E46, Q48, E53, E87, E90. A disulphide bond between C42-C88 is thought to stabilise contact with IL-15Ra and confer rigidity to the molecule.

[0061] The amino acid sequence of IL-15 has been analysed by the inventors of the present invention with AggreScan, ProtScale, and AggreScan3D to identify potential aggregation sites, which would be responsible for protein aggregation and formation of inclusion bodies upon recombinant overexpression in E. coli or Salmonella Typhi. The regions with higher propensity to aggregate are those forming the alpha helices, as these are the regions involved in protein-protein interactions. Both rational design and directed evolution approaches discussed below would aim to minimise these hydrophobic interactions without altering IL-15 binding properties to its receptors.

[0062] Given its role in the stimulation of proliferation and cytotoxic function of CD8+ T-cells and NK cells, IL-15 has been widely tested as a cancer immunotherapeutic agent. The efficacy of IL-15 is limited by the short half-life of the protein in vivo. A number of IL-15 variants have been generated to improve protein stability and efficacy.

[0063] Interleukin-18 (IL-18), a pro-inflammatory cytokine from the IL-1 family, holds a pivotal role in innate and adaptive immunity by influencing diverse immune cells including T-cells, macrophages, and dendritic cells (Dinarello, 2001). The influence of IL-18 emerges through interactions with IL-18 receptors (IL-18Ra and IL-18Rp), which initiate immune cascades (Dinarello, 2001; Goyal, 2022). The binding specificity of IL-18 to IL-18Ra depends on critical amino acids, including E42 and K89, with two distinct binding sites (Site I and Site II), while I L-18Rp has a lesser interaction with a third Site III (Novick, 1999; Yuan, 2014). This activationis balanced by IL-18 binding protein (IL-18BP), which restrains IL-18's pro-inflammatory actions through higher affinity (Kd 400 pM) than IL-18 receptors (Kd 69 nM) and abundant serum presence (Kim, 2000; Kim, 2001; Novick, 2001). Additionally, IL-18BP's up to four isoforms refine its regulatory role (Cheung, 2005). This intricate interplay underscores immune modulation complexity with therapeutic potential for inflammatory, autoimmune disorders, and cancer (Dinarello, 2001).

[0064] The potential of IL-18 in immunological cancer treatment has been reported. Feng et al. observed that upregulated expression of IL-18 in colon cancer patients was correlated with favourable prognosis, whereas decreased expression was correlated with increased tumour size. Exogenous IL-18 supplementation synergized with PD-1 -dependent immunosuppression (Terme, 2011) and other cytokines (Terme, 2011; Schaller, 2002) to enhance anti-tumour responses, or when delivered through microbial carriers (Fensterle, 2008). Conversely, some tumours overexpress IL-18BP (Zhang, 2020; Detry 2022), inactivating IL-18 treatment. This has prompted the evolution of a new class of engineered "Decoy-Resistant IL-18" (DR-18), binding IL-18Ra but not IL-18BP (Zhou et al, 2020; WO 2019 / 051015A1). Notably, elevated IL-18 levels correlate with poor prognosis in certain cases, such as advanced gastric cancer (Yao et al, 2020; Ye et al, 2007). In the context of microbe-induced cancer treatments, expressing IL-18 in bacterial chassis has yielded partial activity (Fensterle, 2008), but advances like DR-18 are more promising. Challenges remain, including the optimisation of protein sequences and expression in bacteria of both IL-18 and DR-18. The inventors of the present invention have provided optimised IL-18 variants which are both soluble and functional, and are therefore optimal candidates for efficient secretion systems which allow the production, correct folding and secretion of said IL-18 variants. The IL-18 variants described herein are decoy-resistant in that they will preferentially bind IL-18 receptor (IL-18R) over they decoy receptor, IL-18 binding protein (IL-18BP), i.e. , the variants described herein are resistant to IL-18BP and have independent binding to IL-18R from IL-18BP. In comparison to the prior art, the inventors of the present invention have found that amino acid modifications tothe amino acids at positions 51 and 60 of IL-18 are all that is required to achieve decoy resistance in the IL-18 variant. Further, the inventors of the present invention have found the amino acid modification at amino acid position 48 allows for IL-18 variants comprising this modification to be solubilised (i.e., stabilised) while retaining similar biological activity levels to that of wild-type IL-18.

[0065] 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).

[0066] 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 (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.

[0067] 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.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).

[0068] 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 inhibits tumour growth, leading to regression of melanomas and fibrosarcomas (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).

[0069] 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.

[0070] As such, the inventors have previously generated a number of IL-15, IL-18 and IL-21 variants with improved protein stability and efficacy in comparison to the wild type interleukins, and in the case of the IL-18, variants which also IL-18BP resistant (W02025 / 068401 , WO2025 / 068413 and WO2025 / 109155 ). Yet, there is a need to further enhance the efficacy of immunotherapies using cytokines astherapeutic agents, in particular in a manner which allows for the efficient expression of such cytokines while also permitting the delivery of other therapeutic agents.

[0071] In accordance with a first aspect of the invention, there is provided a nucleic acid construct comprising polynucleotide sequences encoding two or more of an IL-15 molecule, an IL-18 molecule, an IL-21 molecule and / or a CXCL9 molecule. As described herein, an “interleukin molecule” may be a wild-type interleukin or an interleukin variant. In comparison to wild-type interleukins, the interleukin variants comprise one or more amino acid modifications in a region adjacent to a receptor interaction surface of the interleukin variants. For example, in one preferred embodiment, the nucleic acid construct may encode an IL-15 molecule and at least one of an IL-18 molecule, an IL-21 molecule and / or a CXCL9 molecule. Preferably, an IL-15 molecule and an IL-18 molecule.

[0072] As such, the nucleic acid construct comprises two or more cytokine molecules, each encoded by a polynucleotide sequence as a “module” or “sub-circuit” within the overall multicytokine circuit as detailed herein.

[0073] In another preferred embodiment, the nucleic acid construct encodes three or more of said cytokine molecules. For example, in a more preferred embodiment, the nucleic acid construct may encode an IL-15 molecule and at least two or more of an IL-18 molecule, an IL-21 molecule and / or a CXCL9 molecule.

[0074] The polynucleotide sequences encoding the IL-15 molecule, IL-18 molecule, IL-21 molecule and CXCL9 molecule within the nucleic acid construct may be present with polynucleotide sequences encoding other proteins and / or peptides such as signal sequences, localisation sequences, peptide tags, peptide linkers, other interleukins or receptors thereof, other immunostimulatory proteins, or any combinations thereof.

[0075] 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 baseline, natural, non-mutated or unchanged form.In some embodiments, the nucleic acid construct may encode wild-type cytokines, variant cytokines, or combinations thereof. For the avoidance of doubt, the nucleic acid construct may encode only wild-type cytokines, only cytokine variants, or a combination of wild-type cytokines and cytokine variants. The nucleic acid construct may encode more than one copy of each cytokine, including a wild-type and variant form thereof. Different cytokines may be present in wild-type and / or variant form. The various cytokine variants are described in detail herein.

[0076] As described herein, the wild-type IL-15 may be mature IL-15 (i.e., unmodified IL-15), and comprise an amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1. For example, at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. As used herein, the term “parental IL-15” refers to the baseline sequence from which all IL-15 variants are derived. The parental IL-15 is also referred to as “IL-15M40” or “M40” (SEQ ID NO: 4) and is derived from wild-type IL-15 (SEQ ID NO: 1). The difference between wild-type IL-15 and parental / M40 IL-15 is the presence of an amino acid modification in which serine at amino acid position 75 of SEQ ID NO: 1 is substituted for a proline. As such, the S75P mutation gives rise to SEQ ID NO: 4 from SEQ ID NO: 1. Where the nucleic acid construct encodes variants of IL-15, in some embodiments, the IL-15 variant comprises an amino acid modification at position 75. In preferred embodiments, the IL-15 variant comprises a serine-to-proline substitution at position 75 (S75P). Any of the IL-15 variants described herein may feature the S75P mutation. The S75P mutation can provide increased solubility of the IL-15 variant to further stabilise the IL-15 variant and therefore optimise its expression in bacteria. SEQ ID NO: 1 is shown below. The amino acids which may be modified (amino acid positions 45, 49, 52 and / or 75) in IL-15 as described herein are highlighted in bold and underlined.

[0077] SEQ ID NO: 1

[0078] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSThe wild-type IL-18 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. For example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2. SEQ ID NO: 2 is shown below. The amino acids which may be modified (amino acid positions 9, 46, 48, 51, 60, 80, 105, 110, and / or 111 (preferably 48, 51 and / or 60)) in IL-18 as described herein are highlighted in bold and underlined.

[0079] SEQ ID NO: 2

[0080] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRG MAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0081] The wild-type IL-21 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. For example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. SEQ ID NO: 3 is shown below together with SEQ ID NO: 72. SEQ ID NO: 72 represents wild-type IL-21 with an additional methionine residue at the N terminus which serves to enhance expression in bacteria. The amino acids which may be modified (amino acid positions 13, 29, 33, 36, 71 and / or 74) in IL-21 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: 3. Therefore, the numbering of the amino acid positions in relation to SEQ ID NO: 72 are shifted +1 relative to SEQ ID NO: 3 due to the presence of an additional methionine residue at the beginning of SEQ ID NO: 72.

[0082] SEQ ID NO: 3

[0083] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS LLQKMIHQHLSSRTHGSEDS

[0084] SEQ ID NO: 72MHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKA QLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFK SLLQKMIHQHLSSRTHGSEDS

[0085] CXCL9 comprises an amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 26. For example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26. SEQ ID NO: 26 is shown below.

[0086] SEQ ID NO: 26

[0087] MTPWRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSA DVKE L I KKWEKQVS QKKKQKNGKKHQKKKVLKVRKS QRS RQKKT T

[0088] 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).

[0089] In comparison to “wild-type” interleukins, the terms “interleukin variant” or “variant” refer to an interleukin molecule which, in comparison to the wild-type interleukin, comprises one or more amino acid modifications. As used herein, the term “amino acid modifications” refers to any change or mutation to a wild-type amino acid sequence. An amino acid modification may or may not alter the structure, function, and / or physiochemical properties of the protein. Amino acid modifications may include, but are not limited to deletions, substitutions, and insertions. Deletion mutations involve the loss of an amino acid, resulting in a frameshift or decrease in the length of the amino acid sequence. Insertion mutations involve the addition of an amino acid, resulting in a frameshift or increase in the length of the amino acid sequence. Substitution mutations involve the replacement of one amino acid for another. Substitution mutations can be conservative or non-conservative.As would be understood by the skilled person, amino acids can be grouped broadly according to their properties (i.e. , size, bulkiness, charge, hydrophobicity, polarity, etc). Similarity or dissimilarity between amino acids can also be calculated using substitution matrices and physicochemical distances. A conservative amino acid substitution involves the replacement or exchange of one amino acid for another amino acid with similar properties. For example, a substitution of a hydrophobic amino acid for another hydrophobic amino acid (e.g., a valine-to-alanine substitution) would constitute a conservative amino acid substitution. A non-conservative or radical amino acid substitution involves the replacement or exchange of one amino acid for another amino acid with different or dissimilar properties. For example, a substitution of a hydrophobic amino acid for a hydrophilic amino acid (e.g., a leucine-to-serine substitution) would constitute a non-conservative amino acid substitution. Non-conservative amino acid substitutions may be more likely to be associated with changes in protein structure and function compared to conservative amino acid substitutions.

[0090] Where the nucleic acid construct comprises an IL-15 variant, the IL-15 variant, in comparison to SEQ ID NO: 1, may comprise amino acid modifications at one or more of amino acid positions 45 and / or 49 and / or 52, or any combination thereof. Preferably, the IL-15 variant further comprises an amino acid modification at amino acid position 75. More preferably, the IL-15 variant comprises amino acid modifications at amino acid positions 45 and 75.

[0091] Where the nucleic acid construct comprises an IL-18 variant, the IL-18 variant, in comparison to SEQ ID NO: 2, may comprise amino acid modifications at one or more of amino acid positions (i) 48; and (ii) 51 and / or 60. The IL-18 variant may further comprise amino acid modifications at one or more of amino acid positions 9, 46, 80, 105, 110, and / or 111. The IL-18 variant, in comparison to SEQ ID NO: 2, may comprise amino acid modifications at amino acid positions 51 and 60. The amino acid modifications in an IL-18 variant, in comparison to SEQ ID NO: 2, may consist of amino acid modifications at amino acid positions 51 and 60.

[0092] Where the nucleic acid construct comprises an IL-21 variant, the IL-21 variant, in comparison to SEQ ID NO: 3, may comprise amino acid modifications at one ormore of amino acid positions 29, 33, 36 and / or 71. The IL-21 variant may further comprise amino acid modifications at amino acid positions 13 and / or 74.

[0093] 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. Preferably, the expression system of the present invention is a bacterial expression system, preferably a bacterial cell, such as a Salmonella strain.

[0094] Interleukin variants encoded by the nucleic acid construct may comprise one or more, two or more, three or more, four or more, five or more, or more than five amino acid modifications (i.e., modifications to the nucleic acid sequence which may give rise to modifications to the amino acid sequence). In comparison to wildtype interleukins, the interleukin variants comprise one or more amino acid modifications in a region adjacent to a receptor interaction surface of the interleukin variants. As used herein, the “region” adjacent to a receptor interaction surface of the interleukin may be an exposed hydrophobic pocket or a surface region.

[0095] The region may be surface region of the interleukin molecule. As used herein, the term “surface region” refers to the part of a protein which is exposed to solvent. In the overall three-dimensional tertiary structure of the protein, the protein surface is commonly referred to as the solvent-accessible region of the protein. For a soluble protein, the protein surface will be predominantly composed of hydrophilic amino acid residues, whereas the core (i.e., the interior) of the protein will becomposed of hydrophobic residues which are packed together away from the solvent. The amino acid modifications may involve those amino acids which are exposed to the solvent and are on the outer / exterior surface of the protein. Modifications to amino acids on the protein surface often implicate protein-protein interactions, as protein surfaces typically comprise protein-protein interfaces (i.e. , the molecular surfaces through which proteins make contact and interact with one another). The protein surface may comprise large, flat interfaces, and / or smaller concavities or even single residues which may form an anchoring site for one or more interacting proteins. The protein surfaces of interacting proteins will be shape and / or chemically complementary. As would be understood by a person of skill in the art, modifications to or around the protein surface which implicates protein-protein interfaces can impact protein-protein interactions.

[0096] The “region” in the context of IL-15 and IL-21 molecules is preferably a surface region. Additionally, in the context of IL-18 molecules, the term “region” may refer to an exposed hydrophobic pocket. In one embodiment, the region adjacent to a receptor interaction surface may be an exposed hydrophobic pocket of the IL-18 molecule. As used herein, the terms “hydrophobic pocket”, “hydrophobic cavity”, and “hydrophobic binding site” are used interchangeably and refer to an exposed three-dimensional region or cavity within the IL-18 molecule structure which primarily comprises hydrophobic amino acid residues. The hydrophobic pocket is adjacent to a receptor interaction surface of the IL-18 molecule, and is not in direct contact with the IL-18 receptor or the IL-18 binding protein. As such, the amino acids present in this pocket which are to be modified are not predicted to interact with the IL-18 receptor. With reference to SEQ ID NO: 2, the preferred amino acids to be modified within the hydrophobic pocket are the amino acids at amino acid positions 9, 46 and 48. The hydrophobic amino acids have a low affinity for water and cluster in said pocket to form a hydrophobic environment which excludes water or the surrounding aqueous environment. Hydrophobic pockets are often a localised region in the protein structure which can accommodate other hydrophobic molecules including other hydrophobic amino acids (i.e., from the same or another protein), lipids or lipid moieties, or other organic molecules. Insome instances, the hydrophobic pocket may act as a docking site or interaction site for ligands.

[0097] Irrespective of whether the “region” is a surface or a hydrophobic pocket, the “region” is to be adjacent to 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 region of interleukin variant which is involved in coordinating, interacting with and binding to a receptor (i.e. , protein-protein interactions). There may be a precise group of amino acids which directly bind the receptor. The receptor interaction surface may constitute an IL-15 / IL-18 / IL-21 receptor interaction surface, including IL-15Ra, IL-18Ra, IL-18Rp, IL-18BP (also known as IL-18DR) and IL-21 R. The receptor interaction surface is a region, as opposed to a domain. Protein domains are distinct functional and / or structural units within a protein that are self-stabilising and independent from the rest of the protein. An example of a protein domain is the sushi domain of the IL-15Ra receptor.

[0098] Within IL-15, with respect to SEQ ID NO: 1, the amino acids to be modified may be the amino acids at amino acid positions 45, 49 and / or 52. Accordingly, within IL-15, the surface region is a region adjacent to a receptor interaction surface of the IL-15 variant. The IL-15 variant may also feature a modification at amino acid position 75.

[0099] Within IL-18, with respect to SEQ ID NO: 2, the amino acids to be modified may be the amino acids at amino acid positions 9, 46, 48, 51 , 60, 80, 110 and / or 111 of the IL-18 variant. Preferably, the amino acids to be modified may be the amino acids at amino acid positions 48, 51 , and 60; or 51 and 60.

[0100] Within IL-21, with respect to SEQ ID NO: 3, the amino acids to be modified may be the amino acids at amino acid positions 13, 29, 33, 36, 71 and / or 74 (with the amino acids at amino acid positions 13, 29, 33, and 36 being those present at the surface region of IL-21). Preferably, the amino acids to be modified may be the amino acids at amino acid positions 29, 33, 36 and / or 71.In relation to SEQ ID NO: 1 (IL-15), a “receptor interaction surface” of IL-15 may be defined as those amino acids at amino acid positions D8, D61 and N65 (with other amino acids involving I68, L69, K10, K11 and S7) for IL-15Rp (i.e., IL-15 a1 and a3); Q108, M109 and N112 for yc (i.e., IL-15 a1 and a4); and D22, T24, Y26, E46, Q48, E53, E87, E90 for IL-15Ra (i.e., IL-15 a2). The “receptor interaction surface” is therefore well characterised in the art. The inventors of the present invention have identified a region of IL-15 in which amino acids may be modified in order to generate IL-15 variants with increased solubility and maintained biological activity. The amino acids to be modified are present in a surface region adjacent to the receptor interaction surface of IL-15. These amino acids were previously selected to be modified and form part of the space available in the IL-15 structure that is not needed for interaction with I L-15Rp or yc. In the context of IL-15, protein-protein interactions may include the interaction between IL-15 and a receptor. For example, IL-15 may interact with any of its receptors, IL-15Ra, IL-15Rp and yc. Such interactions occur at the surface of IL-15 in a receptor interaction surface. The inventors of the present invention have identified that the following amino acid positions may be modified within this region of IL-15 (i.e., SEQ ID NO: 1): L45, Q48, V49, L52, H60 and N71. None of these amino acids interact with I L-15Rp or yc. I L-15Rp or yc are expressed on the surface of the target cells to be activated (NK cells and CD8+ T-cells). Accordingly, the “region adjacent to the receptor interaction surface” in IL-15 can be described as any region of IL-15 in which amino acids do not interact with IL-15Rp or yc. Such interactions include any covalent bond and electrostatic interaction, including Van der Waals interactions and hydrogen bonds. However, the amino acids modified herein may interact with IL-15Ra. For example, within IL-15 (i.e., SEQ ID NO: 1), L45, V49 and L52 have been described as in Van der Waals contact with IL-15Ra, and Q48 has been described as forming a hydrogen bond with IL-15Ra (Chirifu et al, 2007). Further, a mutation of N71 within IL-15 (N71D, in the ALT-803 IL-15 superagonist) is known in the art to contribute to the activity of IL-15. Therefore, as used herein, the “region adjacent to the receptor interaction surface” in relation to IL-15 may comprise amino acids within the IL-15 a2.In relation to SEQ ID NO: 2 (IL-18), protein-protein interactions include the interaction between IL-18 and its receptor (IL-18Ra and IL-18Rp) and IL-18 and its decoy receptor, IL-18BP. The IL-18 / IL-18R and IL-18 / IL-18BP interactions occur at the surface of IL-18 in a receptor interaction surface. Preferably, the “receptor interaction surface” of IL-18 is a surface for interacting with IL-18 receptors, more preferably the IL-18 receptor is an IL-18R (IL-18Ra and I L-18Rp) and / or IL-18BP (IL-18 decoy receptor). There may be a defined group of amino acids which directly bind the IL-18 receptors. Two IL-18 sites, site I and site II, contact IL-18Ra. Site I is located on a side of the core barrel of the p-trefoil structure, and site II is at the top of the p-barrel. In relation to wild-type IL-18, the key amino acids in site I include R13, D17, E31, D32, M33, D37, and D132. The key amino acids in site II include L5, K53, M60, and K93. In site III of IL-18 which recognises IL-18Rp, key amino acids include G108, H109, D110, K112, M150, R147, and G14. The crystal structure of IL-18 in complex with IL-18Ra and IL-18Rp is known in the art. The residues which interact with IL-18Ra are those at amino acid positions 5, 13, 17, 31 , 32, 33, 59, 93, and 132, and the residues which interact with IL-18Rp are those at amino acid positions 51, 60, 105, 108, 109, 110, 111, 112, 145, 147 and 150. The residues involved in IL-18Rp interactions may be modified so as to reduce IL-18BP interactions and thereby achieve decoy resistance. The “receptor interaction surface” of IL-18 is therefore well characterised in the art. For example, Tsutsumi et al. (2014) show the residues involved in interacting with IL-18Ra and I L-18Rp, and Detry et al. (2022) show the interaction surface between IL-18 and IL-18BP. None of the amino acids modified herein to alter the solubility of IL-18 are involved in any interactions with IL-18R or IL-18BP. In contrast, some of the amino acids modified herein to achieve decoy resistance of IL-18 may be involved in interactions with IL-18R and IL-18BP.

[0101] In relation to SEQ ID NO: 3 (IL-21), protein -protein interactions may include the interaction between IL-21 and its receptor (IL-21 R) together with yc. The IL-21 / IL-21 R interaction occurs at the surface of IL-21 in a receptor interaction surface. Preferably, the receptor interaction surface of IL-21 is a surface for interacting with an IL-21 receptor, more preferably IL-21 R / yc. There may be a defined group of amino acids which directly bind the receptor.Without wishing to be bound by theory, the amino acids which are present the “region adjacent” to the receptor interaction surface could play a role in mediating protein-protein interactions. In relation to IL-18, the region may be an exposed hydrophobic pocket and / or a surface region. In relation to IL-15 and IL-21, the region may be a surface region. Therefore, the term “region adjacent” may refer to surface amino acids which are in proximity to the receptor interaction site (see for example Figures 1A, 1B and 1 C). For IL-15 molecules, these amino acids may be proximal to the IL-15Ra interaction surface and may interact with IL-15Ra but do not interact with I L-15Rp or yc. Indeed, certain modifications may therefore act to enhance the interaction between the interleukin molecule and the receptor (e.g. , IL-15 and IL-15Ra) for example by increasing the affinity of binding. Further, modifications which alter the composition properties of amino acids on the surface of the interleukin molecule may impact the solubility of the interleukin molecule.

[0102] Given the mutation of select amino acids disclosed herein, the skilled person will readily appreciate that the term “region adjacent to the receptor interaction surface” also intends to cover amino acids in a region of IL-18 which may, or may not be, involved in coordinating, effecting or maintaining the interactions between IL-18 and IL-18R or IL-18BP. For instance, as described herein, the region adjacent to a receptor interaction surface of the IL-18 variant may be an exposed hydrophobic pocket and / or a surface region. The amino acids in the exposed hydrophobic pocket may not participate in interactions with IL-18R or IL-18BP, whereas the amino acids in the surface region may participate in interactions with IL-18R and / or IL-18BP. Therefore, the skilled person will readily understand that the term “region adjacent to a receptor interaction surface” in the context of IL-18 intends to cover any amino acids that are known to participate in interactions (Detry, 2022; Tsutsumi, 2014), but also those that may be a distance from the interaction surface and may not participate in interactions.

[0103] Certain amino acid modifications to the region may act to enhance an interaction between the interleukin variant and its receptor. By way of example, the interaction may be enhanced in that there is a lower rate constant of ligand dissociation (Kotf) (i.e. , high stability of receptor / interleukin variant complexes), ahigher rate constant of ligand association (Kon) (i.e. , fast recognition of the receptor by the interleukin variant and vice versa), and a lower equilibrium dissociation constant (Kd) (i.e., a high affinity interaction). Kdis the ratio between KOff and Kon(Kd= Koff / Kon). Other means for characterising binding kinetics will be apparent to those in the art. By way of example, in the context of IL-18, certain amino acid modifications to the region may act to disrupt the interaction between IL-18 and IL-18BP, for example by decreasing the affinity of binding or blocking binding all together. The amino acid modifications can block, prevent, or decrease the likelihood of an interaction between the IL-18 variant and IL-18BP. The modifications will preferably allow the IL-18 variant to be resistant to IL-18BP binding, and / or to preferably bind IL-18R over IL-18BP. The interaction between IL-18 and IL-18BP may be prevented in that there is a higher rate constant of ligand dissociation (Kotf) (i.e., low stability of IL-18 / IL-18BP complexes), a lower rate constant of ligand association (Kon) (i.e., slow or no recognition of IL-18BP by IL-18 and vice versa), and a higher equilibrium dissociation constant (Kd) (i.e., a low affinity interaction or no interaction).

[0104] In addition, amino acid modifications to the region which alter the composition properties of amino acids on the surface of the interleukin variant may impact the solubility of the interleukin variant, without impairing the biological activity of the interleukin variant.

[0105] The amino acid modifications which may be present within IL-15 to give rise to different IL-15 variants are described below. It will be understood that the nucleic acid construct may comprise a polynucleotide sequence which encodes said IL-15 variant. The skilled person will be aware of the methods and techniques used to encode amino acid modifications in a nucleic acid sequence.

[0106] In one embodiment, the IL-15 variant encoded by the nucleic acid construct may comprise an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1. In another embodiment, the IL-15 variant encoded by the nucleic acid construct 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 identityto 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.

[0107] In accordance with the invention, the IL-15 variant may be modified such that, in comparison to SEQ ID NO: 1 , the amino acid modifications occur at one or more of amino acid positions 45 and / or 49 and / or 52, or any combination thereof. These amino acid positions are located in a region adjacent to a receptor interaction surface of the IL-15 variant (preferably a surface region, even more preferably an interleukin 15 receptor alpha (IL-15Ra) interaction surface). The IL-15 variant may comprise a single amino acid modification at amino acid position 45; a single amino acid modification at amino acid position 49; a single amino acid modification at amino acid position 52; two amino acid modifications at amino acid positions 45 and 49; two amino acid modifications at amino acid positions 45 and 52; two amino acid modifications at amino acid positions 49 and 52; or three amino acid modifications at amino acid positions 45, 49, and 52. Any combination of these modifications is intended to be included. It will be understood that an IL-15 variant comprising any combination of amino acid mutations at amino acid positions 45 and / or 49 and / or 52 will retain at least 70% sequence identity to SEQ ID NO: 1.

[0108] The IL-15 variant encoded by the nucleic acid construct may comprise an amino acid modification at amino acid position 75. The amino acid modification may be present with any other modifications or combinations thereof described herein. For example, the IL-15 variant may comprise amino acid modifications at any of amino acid positions 45, 49, 52 and / or 75. For example, amino acid positions 75 and 45; amino acid positions 75 and 49; amino acid positions 75 and 52; amino acid positions 75, 45 and 49; amino acid positions 75, 45 and 52; amino acid positions 75, 49 and 52; or amino acid positions 75, 45, 49 and 52. Preferably, the IL-15 variant may comprise amino acid modifications at amino acid positions 75 and 45; amino acid positions 75, 45 and 49; amino acid positions 75, 45 and52; amino acid positions 75, 49 and 52. Preferably, in comparison to SEQ ID NO: 1 , the amino acid modifications occur at amino acid positions 45 and 75.

[0109] In some embodiments, the IL-15 variant encoded by the nucleic acid construct may comprise amino acid modifications whereby non-conservative amino acid substitutions are performed. In such embodiments, a hydrophobic amino acid naturally present in the wild-type sequence is replaced with a hydrophilic amino acid, or vice versa. In the case where a hydrophobic amino acid is in a solvent-exposed position on the three-dimensional protein structure, the protein may be destabilised. Therefore, mutation of such hydrophobic residues can serve to stabilise the protein.

[0110] In other embodiments, the IL-15 variant may comprise amino acid modifications whereby conservative amino acid substitutions are performed. In such embodiments, a hydrophobic amino acid naturally present in the wild-type sequence is replaced with another hydrophobic amino acid, or a hydrophilic amino acid naturally present in the wild-type sequence is replaced with another hydrophilic amino acid.

[0111] As used herein, the terms “hydrophobic” and “hydrophilic” are to be given their usual meanings in the art, that is having a weak affinity and strong affinity for water, respectively. “Hydrophobic amino acids” are amino acids which possess hydrophobic side chains, i.e. , side chains that do not interact with (repel) water. Hydrophobic side chains are typically or predominantly composed of carbon and hydrogen atoms. Hydrophobic amino acids are generally non-polar and therefore do not dissolve in polar solvents such as water. Hydrophobic amino acids that are naturally occurring may include glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (lie), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). However, any non-naturally occurring, synthetic, or modified amino acid with hydrophobic properties is also intended to be included by the term “hydrophobic amino acid”. “Hydrophilic amino acids” are amino acids which possess hydrophilic side chains, i.e., side chains that interact with (attract) water. Hydrophilic side chains are typically composed of oxygen, nitrogen, and sulphur atoms. Hydrophilic amino acids are generally polar and therefore can dissolve inpolar solvents such as water via the formation of hydrogen bonds. Hydrophilic amino acids that are naturally occurring may include serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), glutamine (Gin), and tyrosine (Tyr). Other naturally occurring hydrophilic amino acids may include arginine (Arg), histidine (His), lysine (Lys), aspartate (Asp), and glutamate (Glu), as these amino acids have electrically charged side chains. Arginine, histidine, and lysine have an overall positive charge, whereas aspartate and glutamate have an overall negative charge. However, any non-naturally occurring, synthetic, or modified amino acid with hydrophilic properties is also intended to be included by the term “hydrophilic amino acid”.

[0112] The IL-15 variant encoded by the nucleic acid construct may comprise amino acid modifications at one or more of amino acid positions 45 and / or 49 and / or 52 of SEQ ID NO: 1. In relation to SEQ ID NO: 1 , the wild-type amino acid at position 45 is leucine, the wild-type amino acid at position 49 is valine, and the wild-type amino acid at position 52 is leucine.

[0113] In a preferred embodiment, the amino acid modification at amino acid position 45 of SEQ ID NO: 1 is a hydrophobic-to-hydrophilic substitution. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of a hydrophobic amino acid for a hydrophilic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for any naturally occurring or non-naturally occurring hydrophilic amino acid. For example, any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan, may be substituted for any one of serine, threonine, cysteine, asparagine, glutamine, tyrosine, arginine, histidine, lysine, aspartate, or glutamate.

[0114] The wild-type amino acid at amino acid position 45 in relation to SEQ ID NO: 1 is leucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 45 of SEQ ID NO: 1 to be a leucine-to-serine substitution or a leucine-to-threonine substitution. Without wishing to be bound by theory, this non-conservative amino acid modification could act to alterthe physiochemical properties of the region in which the amino acid at position 45 resides. Specifically, the region may become more hydrophilic and therefore an IL-15 variant with such an amino acid modification may become more soluble. This alteration in physiochemical properties of the amino acid which is in a region adjacent to receptor interaction surface (e.g., an interleukin 15 receptor alpha (IL-15Ra) interaction surface) of the IL-15 variant may also serve to enhance an interaction between the IL-15 variant and a receptor. Therefore, IL-15 variants with such an amino acid modification may be more soluble in bacterial chassis than wild-type IL-15, while also exhibiting biological activity.

[0115] In another preferred embodiment, the amino acid modification at amino acid position 49 of SEQ ID NO: 1 is a hydrophobic-to-hydrophobic substitution. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of a hydrophobic amino acid for another hydrophobic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for another hydrophobic amino acid. For example, any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan, may be substituted for any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan. However, the amino acid modification at amino acid position 49 may alternatively be a hydrophobic-to-hydrophilic substitution, preferably a valine-to-serine substitution.

[0116] The wild-type amino acid at amino acid position 49 in relation to SEQ ID NO: 1 is valine. The preference of the present invention is for the hydrophobic-to-hydrophobic substitution at amino acid position 49 of SEQ ID NO: 1 to be a valine-to-alanine substitution. The inventors of the present invention have found that the hydrophobic-to-hydrophobic mutation of V49 enhances the properties of an IL-15 variant. Without wishing to be bound by theory, this relatively conservative amino acid modification could act to optimise the region in which the amino acid at position 49 resides. Specifically, alanine is a small amino acid with a molecular weight of 89.1 g / mol and a small methyl (-CH3) side chain, whereas valine is a larger amino acid with a molecular weight of 117.1 g / mol and a bulkier isopropylside chain (-CH(CH3)2). Such a substitution to a smaller / less bulky amino acid could serve to optimise the protein-protein interface for IL-15 / receptor interactions. Further, alanine is less hydrophobic than valine, therefore this alteration in the physiochemical properties of an amino acid which is in a surface region adjacent to receptor interaction surface of the IL-15 variant may also serve to increase the solubility of the IL-15 receptor. By removing the hydrophobicity of valine in such a substitution, the inventors of the present invention were able to assess the impact on solubility / stability of the IL-15 variant. Therefore, IL-15 variants with such an amino acid modification may have optimised solubility and functionality in comparison to wild-type IL-15.

[0117] In another preferred embodiment, the amino acid modification at amino acid position 52 of SEQ ID NO: 1 is a hydrophobic-to-hydrophilic substitution. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of a hydrophobic amino acid for a hydrophilic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for any occurring or non-naturally occurring hydrophilic amino acid. For example, any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan, may be substituted for any one of serine, threonine, cysteine, asparagine, glutamine, tyrosine, arginine, histidine, lysine, aspartate, or glutamate.

[0118] The wild-type amino acid at amino acid position 52 in relation to SEQ ID NO: 1 is leucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 52 of SEQ ID NO: 1 to be a leucine-to-lysine substitution or a leucine-to-arginine substitution. Without wishing to be bound by theory, this non-conservative amino acid modification could act to alter the physiochemical properties of the region in which the amino acid at position 52 resides. Specifically, the region may become more hydrophilic and therefore an IL-15 variant with such an amino acid modification may become more water-soluble. This alteration in physiochemical properties in of amino acid which is in a surface region adjacent to a receptor interaction surface (e.g., an interleukin 15receptor alpha (IL-15Ra) interaction surface) of the IL-15 variant may also serve to enhance an interaction between the IL-15 variant and a receptor. Therefore, IL-15 variants with such an amino acid modification may be more soluble than wild-type IL-15, while also preserving biological activity.

[0119] Any combination of amino acid modifications as described herein may be present in the IL-15 variant. There may be modifications present which give rise to an IL-15 variant according to any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10. However, in a preferred embodiment, there may be modifications present which give rise to an IL-15 variant according to SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 8. In preferred embodiments, where the nucleic acid construct encodes an IL-15 variant, the variant may have an amino acid sequence according to any of SEQ ID NOs: 4, 5 or 8.

[0120] Within IL-15, the amino acids at positions 45, 49 and 52 may, in some embodiments, not be substituted for bulky amino acids. Amino acids at positions 45, 49 and 52 are adjacent to the receptor binding surface, therefore substituting these residues for bulkier residues may cause steric hindrance that could interfere with receptor binding. Aspartate, glutamate, asparagine and glutamine are examples of bulky amino acids. Therefore, in some embodiments, the amino acid modifications at amino acid positions 45, 49 and 52 may not be substituted for any of aspartate, glutamate, asparagine or glutamine.

[0121] In a preferred embodiment, the IL-15 variant encoded by the nucleic acid construct may comprise an amino acid modification at amino acid position 75. The amino acid modification may be present with any other modification described herein. The preference of the present invention is for the amino acid modification at amino acid position 75 to be a serine-to-proline substitution (S75P). The serine-to-proline modification at amino acid position 75 has been described in the art (Behar, 2011). The inventors of the present invention selected the S75P IL-15 mutant (parental / M40 IL-15) as a starting sequence for generating IL-15 variants as the S75P IL-15 mutant was observed to be more soluble than unmodified IL-15 (Behar, 2011). An IL-15 variant comprising the S75P amino acid modification may have a sequence according to SEQ ID NO: 4, or a sequence comprising at least70% identity to SEQ ID NO: 4. In another embodiment, the IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 4; at least 80% sequence identity to SEQ ID NO: 4; at least 85% sequence identity to SEQ ID NO: 4; at least 90% sequence identity to SEQ ID NO: 4; at least 91% sequence identity to SEQ ID NO: 4; at least 92% sequence identity to SEQ ID NO: 4; at least 93% sequence identity to SEQ ID NO: 4; at least 94% sequence identity to SEQ ID NO: 4; at least 95% sequence identity to SEQ ID NO: 4; at least 96% sequence identity to SEQ ID NO: 4; at least 97% sequence identity to SEQ ID NO: 4; at least 98% sequence identity to SEQ ID NO: 4; at least 99% sequence identity to SEQ ID NO: 4; or at least 100% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid position 75. It will be understood that any of the amino acid modifications herein disclosed may be present in any combination with any of the other amino acid modifications herein disclosed.

[0122] In a preferred embodiment, the IL-15 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45 and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, the IL-15 variant may comprise (i) a leucine-to-serine substitution at amino acid position 45; and (ii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45 and 75. The IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 5; at least 80% sequence identity to SEQ ID NO: 5; at least 85% sequence identity to SEQ ID NO: 5; at least 90% sequence identity to SEQ ID NO: 5; at least 91% sequence identity to SEQ ID NO: 5; at least 92% sequence identity to SEQ ID NO: 5; at least 93% sequence identity to SEQ ID NO: 5; at least 94% sequence identity to SEQ ID NO: 5; at least 95% sequence identity to SEQ ID NO: 5; at least 96% sequence identity to SEQ ID NO: 5; at least 97% sequence identity to SEQ ID NO: 5; at least 98% sequence identity to SEQ ID NO: 5; at least 99% sequence identity to SEQID NO: 5; or at least 100% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45 and 75.

[0123] In one embodiment, the IL-15 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophobic substitution at amino acid position 49, and a serine-to-proline substitution at amino acid position 75. In a preferred embodiment, the IL-15 variant may comprise (i) a leucine-to-serine substitution at amino acid position 45, (ii) a valine-to-alanine substitution at amino acid position 49, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75. The IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 6; at least 80% sequence identity to SEQ ID NO: 6; at least 85% sequence identity to SEQ ID NO: 6; at least 90% sequence identity to SEQ ID NO: 6; at least 91% sequence identity to SEQ ID NO: 6; at least 92% sequence identity to SEQ ID NO: 6; at least 93% sequence identity to SEQ ID NO: 6; at least 94% sequence identity to SEQ ID NO: 6; at least 95% sequence identity to SEQ ID NO: 6; at least 96% sequence identity to SEQ ID NO: 6; at least 97% sequence identity to SEQ ID NO: 6; at least 98% sequence identity to SEQ ID NO: 6; at least 99% sequence identity to SEQ ID NO: 6; or at least 100% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

[0124] In one embodiment, the IL-15 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophilic substitution at amino acid position 52, and a serine-to-proline substitution at amino acid position 75. In a preferred embodiment, the IL-15 variant may comprise (i) a leucine-to-serine substitution at amino acid position 45, (ii) a leucine-to-lysine substitution at amino acid position 52, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 45, 52 and 75. The IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 7; at least 80% sequence identity to SEQ ID NO: 7; at least 85% sequence identity to SEQ ID NO: 7; at least 90% sequence identity to SEQ ID NO: 7; at least 91% sequence identity to SEQ ID NO: 7; at least 92% sequence identity to SEQ ID NO: 7; at least 93% sequence identity to SEQ ID NO: 7; at least 94% sequence identity to SEQ ID NO: 7; at least 95% sequence identity to SEQ ID NO: 7; at least 96% sequence identity to SEQ ID NO: 7; at least 97% sequence identity to SEQ ID NO: 7; at least 98% sequence identity to SEQ ID NO: 7; at least 99% sequence identity to SEQ ID NO: 7; or at least 100% sequence identity to SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 45, 52 and 75.

[0125] In preferred embodiment, the IL-15 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophilic substitution at amino acid position 52, and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, the IL-15 variant may comprise (i) a leucine-to-serine substitution at amino acid position 45, (ii) a leucine-to-arginine substitution at amino acid position 52, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise an amino acid sequence according to SEQ ID NO: 8, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 8, wherein the % sequence identity to SEQ ID NO: 8 retains the amino acid modifications at amino acid positions 45, 52 and 75. The IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 8; at least 80% sequence identity to SEQ ID NO: 8; at least 85% sequence identity to SEQ ID NO: 8; at least 90% sequence identity to SEQ ID NO: 8; at least 91% sequence identity to SEQ ID NO: 8; at least 92% sequence identity to SEQ ID NO: 8; at least 93% sequence identity to SEQ ID NO: 8; at least94% sequence identity to SEQ ID NO: 8; at least 95% sequence identity to SEQ ID NO: 8; at least 96% sequence identity to SEQ ID NO: 8; at least 97% sequence identity to SEQ ID NO: 8; at least 98% sequence identity to SEQ ID NO: 8; at least 99% sequence identity to SEQ ID NO: 8; or at least 100% sequence identity to SEQ ID NO: 8, wherein the % sequence identity to SEQ ID NO: 8 retains the amino acid modifications at amino acid positions 45, 52 and 75.

[0126] In one embodiment, the IL-15 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophobic substitution at amino acid position 49, and a serine-to-proline substitution at amino acid position 75. In a preferred embodiment, the IL-15 variant may comprise (i) a leucine-to-threonine substitution at amino acid position 45, (ii) a valine-to-alanine substitution at amino acid position 49, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 9, wherein the % sequence identity to SEQ ID NO: 9 retains the amino acid modifications at amino acid positions 45, 49 and 75. the IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 9; at least 80% sequence identity to SEQ ID NO: 9; at least 85% sequence identity to SEQ ID NO: 9; at least 90% sequence identity to SEQ ID NO: 9; at least 91% sequence identity to SEQ ID NO: 9; at least 92% sequence identity to SEQ ID NO: 9; at least 93% sequence identity to SEQ ID NO: 9; at least 94% sequence identity to SEQ ID NO: 9; at least 95% sequence identity to SEQ ID NO: 9; at least 96% sequence identity to SEQ ID NO: 9; at least 97% sequence identity to SEQ ID NO: 9; at least 98% sequence identity to SEQ ID NO: 9; at least 99% sequence identity to SEQ ID NO: 9; or at least 100% sequence identity to SEQ ID NO: 9, wherein the % sequence identity to SEQ ID NO: 9 retains the amino acid modifications at amino acid positions 45, 49 and 75.

[0127] In one embodiment, the IL-15 variant encoded by the nucleic acid construct may comprise a hydrophobic-to- hydrophobic substitution at amino acid position 49, a hydrophobic-to-hydrophilic substitution at amino acid position 52, and a serine-to-proline substitution at amino acid position 75. In a preferred embodiment, the IL-15 variant may comprise (i) a valine-to-alanine substitution at amino acid position 49, (ii) a leucine-to-arginine substitution at amino acid position 52, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may comprise an amino acid sequence according to SEQ ID NO: 10, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 10, wherein the % sequence identity to SEQ ID NO: 10 retains the amino acid modifications at amino acid positions 49, 52 and 75. The IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 10; at least 80% sequence identity to SEQ ID NO: 10; at least 85% sequence identity to SEQ ID NO: 10; at least 90% sequence identity to SEQ ID NO: 10; at least 91% sequence identity to SEQ ID NO: 10; at least 92% sequence identity to SEQ ID NO: 10; at least 93% sequence identity to SEQ ID NO: 10; at least 94% sequence identity to SEQ ID NO: 10; at least 95% sequence identity to SEQ ID NO: 10; at least 96% sequence identity to SEQ ID NO: 10; at least 97% sequence identity to SEQ ID NO: 10; at least 98% sequence identity to SEQ ID NO: 10; at least 99% sequence identity to SEQ ID NO: 10; or at least 100% sequence identity to SEQ ID NO: 10, wherein the % sequence identity to SEQ ID NO: 10 retains the amino acid modifications at amino acid positions 49, 52 and 75.

[0128] In addition to any amino acid modifications at any of amino acid positions 45, 49, and / or 52, and 75 of SEQ ID NO: 1, the nucleic acid construct may encode IL-15 variants with one or more amino acid modifications at amino acid positions 48, 60, and / or 71 of SEQ ID NO: 1. In one embodiment, the IL-15 variant may comprise a hydrophilic-to-hydrophobic substitution at amino acid position 48 of SEQ ID NO: 1, preferably a glutamine-to-isoleucine substitution or a glutamine-to-valine substitution at amino acid position 48 of SEQ ID NO: 1. In another embodiment, the IL-15 variant may comprise a hydrophilic-to-hydrophobic substitution at amino acid position 60 of SEQ ID NO: 1, preferably a histidine-to-valine substitution at amino acid position 60 of SEQ ID NO: 1. In another embodiment, the IL-15 variant may comprise a hydrophilic-to-hydrophobic substitution at amino acid position 70 of SEQ ID NO: 1, preferably an asparagine-to-isoleucine substitution at aminoacid position 70 of SEQ ID NO: 1. It will be understood that any of these modifications may be present in combination with any of the other amino acid modifications described herein, including those amino acid modifications at amino acid positions 45, 49 and / or 52 of SEQ ID NO: 1. In particular, there may be an IL-15 variant comprising amino acid modifications at amino acid positions 75 and 45; amino acid positions 75, 45 and 48; amino acid positions 75, 45 and 60; amino acid positions 75, 45 and 71; amino acid positions 75, 48 and 49; amino acid positions 75, 48 and 52; or any combinations thereof.

[0129] As detailed herein, there may be present in the nucleic acid construct an IL-15 variant comprising one or more amino acid modifications, wherein said amino acid modifications occur in a surface region adjacent to a receptor (e.g., IL-15Ra) interaction surface of the IL-15 variant (i.e., in comparison to SEQ ID NO: 1, at one or more of amino acid positions 45 and / or 49 and / or 52). The receptor interaction surface of the IL-15 variant may interact with or bind to IL-15 receptors. There exist three IL-15 receptors: IL-15Rp, yc, and IL-15Ra. IL-15Rp may also be known as I L-2Rp. These receptors form complexes with IL-15 via electrostatic interactions. In the sequence of parental IL-15 (SEQ ID NO: 4), the amino acids with the potential to be modified to enhance stability of the complex are shown in bold and underlined.

[0130] SEQ ID NO: 16

[0131] NWVNVI SDLKKIEDLI QSMH I DAT L YTE S DVHP SCKVTAMKC FLLE LQVI S LE S GDAS I HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0132] The inventors of the present invention have surprisingly found that the residues as shown in the sequence above are present in an exposed region of IL-15. Analysis of the sequence revealed in particular that lysine-41, leucine-45, glutamine-48, and leucine-52 are aggregation prone and therefore good candidates for amino acid modifications which enhance solubility and preserve function. As such, in a preferred embodiment, where the nucleic acid construct encodes an IL-15 variant, in comparison to SEQ ID NO: 1 the IL-15 variant may comprise one or more amino acid modifications in a surface region adjacent to areceptor interaction surface, the one or more amino acid modifications comprising any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophobic substitution at amino acid position 49; iii) a hydrophobic-to-hydrophilic substitution at amino acid position 52; and / or iv) a serine-to-proline substitution at amino acid position 75; preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface.

[0133] In a particularly preferred embodiment, the hydrophobic-to-hydrophilic substitution at amino acid position 45 is a leucine-to-serine or leucine-to-threonine substitution.

[0134] In another particularly preferred embodiment, the hydrophobic-to-hydrophilic substitution at amino acid position 52 is a leucine-to-lysine or leucine-to-arginine substitution.

[0135] In another particularly preferred embodiment, the hydrophobic-to-hydrophobic substitution at amino acid position 49 is a valine-to-alanine or valine-to-serine substitution.

[0136] Also encompassed by the term “IL-15 variant” are super agonists of the IL-15 variants. As detailed herein, where the nucleic acid construct encodes an IL-15 variant, the IL-15 variant may comprise one or more amino acid modifications, wherein said amino acid modifications occur in a surface region adjacent to a receptor (e.g., IL-15Ra) interaction surface of the IL-15 variant (i.e. , in comparison to SEQ ID NO: 1 , at one or more of amino acid positions 45 and / or 49 and / or 52). The receptor interaction surface of the IL-15 variant may interact with or bind to IL-15 receptors. There exist three IL-15 receptors: I L-15Rp, yc, and IL-15Ra. IL-15Rp may also be known as I L-2Rp. These receptors form complexes with IL-15 via electrostatic interactions. The IL-15Ra receptor contains protein-binding motifs known in the art as “sushi domains”. An IL-15 variant encoded by the nucleic acid construct defined herein may be fused to an IL-15 receptor molecule, in particular, an IL-15Ra molecule and / or the IL-15Ra sushi domain. The IL-15Ra molecule is a transmembrane protein, which has hydrophobic transmembranesegment, and can often be flexible and unstable. This makes expression of the entire IL-15Ra molecule challenging. Therefore, the IL-15 variant is preferably fused to the IL-15Ra sushi domain, as opposed to the entire IL-15Ra molecule. The IL-15Ra sushi domain is soluble and also the functional part of the receptor, meaning that only the sushi domain is required for functionality and is also easier to express alone. The fusion of the IL-15 variant and the sushi domain is termed a “super agonist” or “IL-15 variant super agonist” (“SAg”). As used herein, the term “fused” refers to any joining of two or more proteins. In the case of a fusion protein being encoded by the present nucleic acid construct, the appropriate sequences of DNA are to be arranged such that the resulting polypeptide transcribed from said DNA is a fusion protein. Proteins may be fused via covalent attachment, for example via a linker, and can be produced via the expression of a nucleic acid molecule comprising the nucleic acid sequences of the two or more proteins. In one embodiment, a fusion protein may comprise the sushi domain of an IL-15Ra molecule followed by a linker followed by any of the IL-15 variants described herein (IL-15Ra sushi:linker: IL-15 variant), i.e. , in some embodiments, the fusion is made to the amino (N) terminus of the IL-15 variant. The IL-15 variant may (or may not) be fused to a sushi domain of an IL-15Ra molecule. Any other suitable proteins or polypeptides may also be joined to the IL-15 variant and / or IL-15 / IL-15Ra sushi domain complex.

[0137] Any of the IL-15 variants detailed herein may be fused to an IL-15Ra molecule or sushi domain thereof and encoded by the nucleic acid construct of the present invention. As used herein, the term “super agonist” refers to a type of cytokine which is capable of inducing a maximal immune response greater than that of the agonist (i.e., the IL-15 variant) itself. The super agonists may be less soluble than their IL-15 variant counterparts.

[0138] Any of the IL-15 variants detailed herein may be fused to an IL-15Ra molecule or the sushi domain of an IL-15Ra molecule to form their super agonist counterpart. For example, in one embodiment, the nucleic acid construct may encode an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10, fused to an IL-15Ramolecule or the sushi domain of an IL-15Ra molecule via a GS linker such as that according to SEQ ID NO: 17 to form a super agonist. In another embodiment, the nucleic acid construct may encode an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10, or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 80% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 85% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 90% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 91% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 92% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 93% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 94% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 95% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 96% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 97% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 98% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; at least 99% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10; and / or at least 100% sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10, (wherein the % sequence identity to SEQ ID NOs: 4, 5, 6, 7, 8, 9, and / or 10 retains the amino acids at the relevant amino acid positions) fused to an IL-15Ra molecule or the sushi domain of an IL-15Ra molecule to form a super agonist. Preferably, the IL-15 variant is fused to the IL-15Ra sushi domain.

[0139] The term “relevant amino acid positions” is intended to encompass the amino acid modifications as described for each SEQ ID NO herein. For example, the amino acid modifications at the relevant amino acid positions for SEQ ID NO: 5 would be the amino acid modifications at amino acid positions 45 and 75.

[0140] In a preferred embodiment, the nucleic acid construct may encode an IL-15 super agonist comprising an amino acid sequence according to any one of SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16, or an amino acid sequence comprising at least 70% sequence identity to any one of SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16.SEQ ID NO: 11 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 5 (i.e. , the SAgO1 super agonist which comprises GBL01).

[0141] SEQ ID NO: 12 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 6 (i.e., the SAg15 super agonist which comprises GBL15).

[0142] SEQ ID NO: 13 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 7 (i.e., the SAg17 super agonist which comprises GBL17).

[0143] SEQ ID NO: 14 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 8 (i.e., the SAg18 super agonist which comprises GBL18).

[0144] SEQ ID NO: 15 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 9 (i.e., the SAg25 superagonist which comprises GBL25).

[0145] SEQ ID NO: 16 corresponds to an amino acid sequence comprising an IL-15Ra sushi domain and the IL-15 variant comprising an amino acid sequence according to SEQ ID NO: 10 (i.e., the SAg50 superagonist which comprises GBL50).

[0146] These super agonist sequences utilise the GS linker according to SEQ I D NO: 17, however, it will be appreciated by the skilled person that any suitable linker could be utilised in joining the IL-15 variant with an IL-15Ra molecule or the sushi domain of an IL-15Ra molecule.

[0147] Accordingly, with respect to the IL-15 variant, in another embodiment, the nucleic acid construct may encode an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16, or any combination thereof, or an amino acid sequence having or an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 80% sequence identity to SEQ I D NOs: 11, 12, 13, 14, 15, and / or 16; at least 85% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15,and / or 16; at least 90% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 91% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 92% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 93% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 94% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 95% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 96% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 97% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 98% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; at least 99% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16; and / or at least 100% sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16, (wherein the % sequence identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16 retains the amino acids at the relevant amino acid positions).

[0148] However, the preference is for the IL-15 super agonist to comprise an amino acid sequence according to any one of SEQ ID NOs: 12, 14, 15 and / or 16, or an amino acid sequence comprising at least 70% sequence identity with any one of SEQ ID NOs: 12, 14, 15 and / or 16 (wherein the % sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16 retains the amino acid modifications at the relevant amino acid positions), as it was found by the inventors of the present invention that these super agonists were more soluble than super agonists of wild-type IL-15. In another embodiment, the nucleic acid construct may encode an IL-15 super agonist comprising an amino acid sequence comprising at least 75% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 80% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 85% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 90% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 91% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 92% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 93% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 94% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 95% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 96% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 97% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; at least 98% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16;at least 99% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16; and / or at least 100% sequence identity to SEQ ID NOs: 12, 14, 15 and / or 16, wherein the % sequence identity retains the amino acid modifications at the relevant amino acid positions.

[0149] Such a fusion may be achieved by directly joining two or more proteins together (i.e. , a direct fusion protein), and / or indirectly joining two or more proteins via any suitable form of linkage. The linkage may be a covalent linkage. However, direct joining without a linker can lead to misfolding of the fusion protein, low solubility, or impaired bioactivity. Therefore, in a preferred embodiment, where the nucleic acid construct encodes an IL-15 variant, once transcribed and translated from said construct, the IL-15 variant is fused to the sushi domain of an IL-15Ra molecule via a linker, such as a flexible polypeptide linker. Flexible linkers, for example, those which make use of serine and glycine residues, allow the joined proteins to move relative to one another. The length of the linker may be increased to prevent steric interference between the proteins. A short linker may be approximately 4 to 6 amino acids in length, a medium length linker may be approximately 6 to 14 amino acids in length, and a long linker may be approximately 14 to 21 or more amino acids in length. The linker may be a naturally occurring linker or a synthetic linker. The linker may adopt secondary protein conformations such as alpha helices and / or beta strands / coils / bends. In a more preferred embodiment, the linker is a glycine-serine linker (GS linker). GS linkers primarily comprise stretches of glycine and serine residues, for example, (Ser-Gly-Gly-Gly-Gly)n, or (Ser-Gly-Gly-Gly)n. Any proportion of glycine and serine residues may be utilised, and other amino acids may be present, such as leucine or glutamine. The copy number ‘n’ may be adjusted to alter the length of the GS linker to achieve appropriate separation of the joined proteins, which can impact the functionality and interaction of said joined proteins. The copy number ‘n’ typically ranges between 1 and 6. However, the preferred GS linker of the present invention may comprise the sequence below:

[0150] SEQ ID NO: 17SGGGSGGGGSGGGGSGGGGSGGGSLQA

[0151] In a preferred embodiment, the linker utilised to fuse the IL-15 variant with an IL-15Ra molecule or the sushi domain of an IL-15Ra molecule comprises an amino acid sequence according to SEQ ID NO: 17, or amino acid sequence comprising at least 70%, at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 17. In otherwords, the nucleic acid construct may comprise a polynucleotide sequence which codes for the amino acid sequence as shown in SEQ ID NO: 17.

[0152] While the preference of the present invention is to use flexible GS linkers, other flexible linkers may be used, for example those which contain additional amino acids such as threonine, alanine, lysine and / or glutamate. Further, flexible linkers consisting purely of glycine or serine may also be appropriate, for example a (Gly)s linker.

[0153] In addition to the joining of an IL-15Ra sushi domain, there may be any other suitable protein or polypeptide joined to the IL-15 variant and / or the IL-15 super agonist. Such examples may include, but are not limited to, signal peptides, antibody fragments, antibodies, bi- or multi-specific antibodies, tissue-targeting sequences (such as the NGR (Asn-Gly-Arg) tripeptide), chemokines, interleukins (particularly those that stimulate cell mediated immunity (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21) or chemokines (e.g., CXCL9, CXCL10, CXCL11, CCL5, CCL2, CX3CL1).

[0154] As detailed herein, the nucleic acid construct may encode two or more of: an IL-15 molecule, an IL-18 molecule, an IL-21 molecule, and / or a CXCL9 molecule. Where the nucleic acid construct encodes an IL-18 variant, the amino acid modifications which may be present within IL-18 to give rise to different IL-18 variants are described below. It will be understood that the nucleic acid construct comprises a polynucleotide sequence which encodes said IL-18 variant. The skilled person will be aware of the methods and techniques used to encode amino acid modifications in a nucleic acid sequence.In one embodiment, the IL-18 variant encoded by the nucleic acid construct may comprise an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 2. The IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 2; at least 80% sequence identity to SEQ ID NO: 2; at least 85% sequence identity to SEQ ID NO: 2; at least 90% sequence identity to SEQ ID NO: 2; at least 91% sequence identity to SEQ ID NO: 2; at least 92% sequence identity to SEQ ID NO: 2; at least 93% sequence identity to SEQ ID NO: 2; at least 94% sequence identity to SEQ ID NO: 2; at least 95% sequence identity to SEQ ID NO: 2; at least 96% sequence identity to SEQ ID NO: 2; at least 97% sequence identity to SEQ ID NO: 2; at least 98% sequence identity to SEQ ID NO: 2; 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 the relevant amino acid positions.

[0155] In accordance with the invention, an IL-18 molecule may be modified such that, in comparison to SEQ ID NO: 2, the amino acid modifications occur at amino acid positions 48; and 51 and / or 60. For instance, the nucleic acid construct may encode an IL-18 variant comprising amino acid modifications at amino acid positions 48 and 60; 48 and 51; or 48, 51 and 60. The inventors of the present invention have found that amino acid modifications to the amino acids at positions 51 and 60 of IL-18 are all that is required to achieve decoy resistance in the IL-18 variant, and that an amino acid modification at amino acid position 48 allows for IL-18 variants comprising this modification to be solubilised (i.e., stabilised) while retaining similar biological activity levels to that of wild-type IL-18. Therefore, in one embodiment, the nucleic acid construct may encode an IL-18 variant comprising amino acid modifications at amino acid positions 51 and 60. However, the preference is for the IL-18 variant to comprise amino acid modifications at amino acid positions 48 and 60; 48 and 51; or 48, 51 and 60. An IL-18 molecule may be modified such that, in comparison to SEQ ID NO: 2, the amino acid modifications occur at amino acid positions 51 and 60 only.

[0156] In addition to the modifications at amino acid positions 48 and 51 and / or 60, the IL-18 variant may further comprise an amino acid modification at one or more ofamino acid positions 9, 46, 80, 105, 110, and / or 111. For example, a single amino acid modification at any one of amino acid positions 9, 46, 80, 105, 110, or 111; two amino acid modifications at any two of amino acid positions 9, 46, 80, 105, 110, and / or 111; three amino acid modifications at any three of amino acid positions 9, 46, 80, 105, 110, and / or 111; four amino acid modifications at any four of amino acid positions 9, 46, 80, 105, 110, and / or 111; five amino acid modifications at any five of amino acid positions 9, 46, 80, 105, 110, and / or 111; or six amino acid modifications at all of amino acid positions 9, 46, 80, 105, 110, and / or 111. Any combination of these modifications is intended to be included. The IL-18 variants comprising mutations at these combinations of amino acid positions were previously found by the inventors of the present invention to be soluble, active and decoy resistant. It will be understood that an IL-18 variant comprising any combination of these amino acid mutations at will retain at least 70% sequence identity to SEQ ID NO: 2.

[0157] In some embodiments, the IL-18 variant may comprise amino acid modifications whereby non-conservative amino acid substitutions are performed. In other embodiments, the IL-18 variant may comprise amino acid modifications whereby conservative amino acid substitutions are performed.

[0158] The IL-18 variant may comprise amino acid modifications at one or more of amino acid positions 48 and 51 and / or 60. The IL-18 variant may further comprise amino acid modifications at 9, 46, 80, 105, 110, and / or 111, or any combination thereof. In relation to SEQ ID NO: 2, the wild-type amino acid at position 9 is leucine, the wild-type amino acid at position 46 is isoleucine, the wild-type amino acid at position 48 is isoleucine, the wild-type amino acid at position 51 is methionine, the wild-type amino acid at position 60 is methionine, the wild-type amino acid at position 80 is isoleucine, the wild-type amino acid at position 110 is aspartate, and the wild-type amino acid at position 111 is asparagine.

[0159] Table 1 shows the wild-type amino acids of IL-18 (relative to SEQ ID NO: 2) and their preferred mutations.

[0160]

[0161]

[0162] able 1. Amino acids within IL-18 and their possible modifications.

[0163] In a preferred embodiment, the amino acid modification at amino acid position 9, 46, 48, and / or 80 of SEQ ID NO: 2 is a hydrophobic-to-hydrophilic substitution. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of a hydrophobic amino acid for a hydrophilic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for any naturally occurring or non-naturally occurring hydrophilic amino acid. For example, any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan, may be substituted for any one of serine, threonine, cysteine, asparagine, glutamine, tyrosine, arginine, histidine, lysine, aspartate, or glutamate.

[0164] The wild-type amino acid at amino acid position 9 in relation to SEQ ID NO: 2 is leucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 9 of SEQ ID NO: 2 to be a leucine-to-serine substitution or a leucine-to-aspartate substitution. In a more preferred embodiment, the substitution at amino acid position 9 of SEQ ID NO: 2 is leucine-to-serine substitution. The inventors of the present invention have previously found that the amino acid modification at amino acid position 9 of SEQ ID NO: 2 results in IL-18 variants which are more stable than wild-type IL-18, and consistently 25% more active than wild-type IL-18.The wild-type amino acid at position 46 in relation to SEQ ID NO: 2 is isoleucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 46 of SEQ ID NO: 2 to be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. In a more preferred embodiment, the substitution at amino acid position 46 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution.

[0165] The wild-type amino acid at position 48 in relation to SEQ ID NO: 2 is isoleucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 48 of SEQ ID NO: 2 to be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. In a more preferred embodiment, the substitution at amino acid position 48 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution. The inventors of the present invention have previously found that the amino acid modification at amino acid position 48 of SEQ ID NO: 2 allows for IL-18 variants comprising this modification to be solubilised (i.e., stabilised) while retaining similar biological activity levels to that of wild-type IL-18. IL-18 variants comprising an amino acid modification at amino acid position 48 were found to be approximately 15 times more stable than wild-type IL-18 and at least two-thirds as active as wild-type IL-18.

[0166] The wild-type amino acid at position 80 in relation to SEQ ID NO: 2 is isoleucine. The preference of the present invention is for the hydrophobic-to-hydrophilic substitution at amino acid position 80 of SEQ ID NO: 2 to be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. In a more preferred embodiment, the substitution at amino acid position 80 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution.

[0167] Without wishing to be bound by theory, these non-conservative amino acid modifications could act to alter the physiochemical properties of the region in which the amino acids at positions 9, 46, 48, and / or 80 of SEQ ID NO: 2 reside. Specifically, the region may become more hydrophilic which allows an IL-18 variant with such amino acid modifications may become more soluble / stable. These alterations may also serve to block or prevent the binding of IL-18BP. Therefore, IL-18 variants with such amino acid modifications may be more solublethan wild-type IL-18, while exhibiting biological activity and IL-18BP decoy resistance.

[0168] In another preferred embodiment, the amino acid modification at amino acid position 60 of SEQ ID NO: 2 involves the substitution of a large hydrophobic wildtype amino acid for another large amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for any large amino acid, such as leucine or lysine.

[0169] The wild-type amino acid at amino acid position 60 in relation to SEQ ID NO: 2 is methionine. The preference of the present invention is for the amino acid substitution at amino acid position 60 of SEQ ID NO: 2 to be a methionine-to-lysine substitution or a methionine-to-leucine substitution. In a more preferred embodiment, the substitution at amino acid position 60 of SEQ ID NO: 2 is a methionine-to-lysine substitution. Without wishing to be bound by theory, this amino acid modification could act to prevent or block an interaction with IL-18BP. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wild-type IL-18.

[0170] In another preferred embodiment, the amino acid modification at amino acid position 105 of SEQ ID NO: 2 involves the substitution of a wild-type amino acid for a polar amino acid, of a wild-type amino acid for a polar amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for a polar amino acid, including any one of lysine, threonine, cysteine, asparagine, glutamine, tyrosine, serine, arginine, histidine, aspartate, and glutamate.

[0171] The wild-type amino acid at amino acid position 105 in relation to SEQ ID NO: 2 is serine. The preference of the present invention is for the amino acid substitution at amino acid position 105 of SEQ ID NO: 2 to be a serine-to-aspartate substitution or a serine-to-asparagine substitution. In a more preferred embodiment, the substitution at amino acid position 105 of SEQ ID NO: 2 is a serine-to-aspartate substitution. Without wishing to be bound by theory, this amino acid modificationcould act to optimise the region in which the amino acid at position 105 resides such that an interaction with IL-18BP is prevented or blocked. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wild-type IL-18.

[0172] In another preferred embodiment, the amino acid modification at amino acid position 110 of SEQ ID NO: 2 involves the substitution of a wild-type amino acid for (a) a polar amino acid, or (b) a neutral amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for a polar amino acid, including any one of lysine, threonine, cysteine, asparagine, glutamine, tyrosine, serine, arginine, histidine, aspartate, and glutamate, or a neutral amino acid, such as glycine.

[0173] The wild-type amino acid at amino acid position 110 in relation to SEQ ID NO: 2 is aspartate. The preference of the present invention is for the amino acid substitution at amino acid position 110 of SEQ ID NO: 2 to be an aspartate-to-serine substitution or an aspartate-to-lysine substitution, or an aspartate-to-glycine substitution. In a more preferred embodiment, the substitution at amino acid position 110 of SEQ ID NO: 2 is an aspartate-to-lysine substitution. Without wishing to be bound by theory, this amino acid modification could act to optimise the region in which the amino acid at position 110 resides such that an interaction with IL-18BP is prevented or blocked. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wild-type IL-18.

[0174] In another preferred embodiment, the amino acid modification at amino acid position 111 of SEQ ID NO: 2 involves the substitution of a wild-type amino acid for (a) a positively charged amino acid, or (b) a neutral amino acid. This amino acid modification may be present with any other modification described herein. Such a substitution will involve the exchange of the wild-type amino acid for a positively charged amino acid, including any one of arginine, histidine or lysine, or a neutral amino acid, such as glycine.The wild-type amino acid at amino acid position 111 in relation to SEQ ID NO: 2 is asparagine. The preference of the present invention is for the amino acid substitution at amino acid position 111 of SEQ ID NO: 2 to be an asparagine-to-arginine substitution or an asparagine-to-histidine substitution, or an asparagine-to-glycine substitution. In a more preferred embodiment, the substitution at amino acid position 111 of SEQ ID NO: 2 is an asparagine-to-histidine substitution. Without wishing to be bound by theory, this amino acid modification could act to optimise the region in which the amino acid at position 111 resides such that an interaction with IL-18BP is prevented or blocked. Therefore, IL-18 variants with such an amino acid modification exhibit IL-18BP resistance in comparison to wildtype IL-18.

[0175] 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. As detailed herein, in a preferred embodiment, an IL-18 variant encoded by the nucleic acid construct may comprise amino acid modifications at amino acid positions 48 and 60 (V17); 48, 51, and 60 (V25); 48, 51, 60, and 111 (V27); or 9, 48, 51, 60, and 111 (V59). Activity assays have reproducibly demonstrated that the IL-18 variants denoted V17, V25, V27 and V59 are biologically active. Therefore, in a preferred embodiment, the nucleic acid construct may encode IL-18 variants with modifications present which give rise to an IL-18 variant according to any of SEQ ID NOs: 18, 19, 20, and / or 21.

[0176] In a preferred embodiment, the IL-18 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 48 and a substitution of a wild-type amino acid for a polar amino acid at amino acid position 60. In a more preferred embodiment, the IL-18 variant may comprise (i) the isoleucine-to-aspartate substitution at amino acid position 48; and (ii) the methionine-to-lysine substitution or a methionine-to-leucine substitution at amino acid position 60. In this embodiment, the IL-18 variant may comprise an amino acid sequence according to SEQ ID NO: 18, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 18, wherein the % sequence identity to SEQ ID NO: 18 retains the amino acid modifications at aminoacid positions 48 and 60. The IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 18; at least 80% sequence identity to SEQ ID NO: 18; at least 85% sequence identity to SEQ ID NO: 18; at least 90% sequence identity to SEQ ID NO: 18; at least 91% sequence identity to SEQ ID NO: 18; at least 92% sequence identity to SEQ ID NO: 18; at least 93% sequence identity to SEQ ID NO: 18; at least 94% sequence identity to SEQ ID NO: 18; at least 95% sequence identity to SEQ ID NO: 18; at least 96% sequence identity to SEQ ID NO: 18; at least 97% sequence identity to SEQ ID NO: 18; at least 98% sequence identity to SEQ ID NO: 18; at least 99% sequence identity to SEQ ID NO: 18; or at least 100% sequence identity to SEQ ID NO: 18, wherein the % sequence identity to SEQ ID NO: 18 retains the amino acid modifications at amino acid positions 48 and 60.

[0177] In another preferred embodiment, the IL-18 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 48, a substitution of a wild-type amino acid for a polar amino acid at amino acid position 51 , and a substitution of a wild-type amino acid for a large amino acid at amino acid position 60. In a more preferred embodiment, the IL-18 variant may comprise (i) the isoleucine-to-aspartate substitution at amino acid position 48; and (ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51; and (iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60. In this embodiment, the IL-18 variant may comprise an amino acid sequence according to SEQ ID NO: 19, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 19, wherein the % sequence identity to SEQ ID NO: 19 retains the amino acid modifications at amino acid positions 48, 51 , and 60. The IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 19; at least 80% sequence identity to SEQ ID NO: 19; at least 85% sequence identity to SEQ ID NO: 19; at least 90% sequence identity to SEQ ID NO: 19; at least 91% sequence identity to SEQ ID NO: 19; at least 92% sequence identity to SEQ ID NO: 19; at least 93% sequence identity to SEQ ID NO: 19; at least 94% sequence identity to SEQ ID NO: 19; at least 95% sequence identity to SEQ ID NO: 19; at least 96% sequence identity to SEQ IDNO: 19; at least 97% sequence identity to SEQ ID NO: 19; at least 98% sequence identity to SEQ ID NO: 19; at least 99% sequence identity to SEQ ID NO: 19; or at least 100% sequence identity to SEQ ID NO: 19, wherein the % sequence identity to SEQ ID NO: 19 retains the amino acid modifications at amino acid positions 48, 51, and 60.

[0178] In another preferred embodiment, the IL-18 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 48, a substitution of a wild-type amino acid for a polar amino acid at amino acid position 51, a substitution of a wild-type amino acid for a large amino acid at amino acid position 60, and a substitution of a wild-type amino acid for a positively charged amino acid ora neutral amino acid at amino acid position 111. In an even more preferred embodiment, the IL-18 variant may comprise (i) the isoleucine-to-aspartate substitution at amino acid position 48; and (ii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51; and (iii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and (iv) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111. In this embodiment, the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 20, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 20, wherein the % sequence identity to SEQ ID NO: 20 retains the amino acid modifications at amino acid positions 48, 51, 60, and 111. In another embodiment, the IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 20; at least 80% sequence identity to SEQ ID NO: 20; at least 85% sequence identity to SEQ ID NO: 20; at least 90% sequence identity to SEQ ID NO: 20; at least 91% sequence identity to SEQ ID NO: 20; at least 92% sequence identity to SEQ ID NO: 20; at least 93% sequence identity to SEQ ID NO: 20; at least 94% sequence identity to SEQ ID NO: 20; at least 95% sequence identity to SEQ ID NO: 20; at least 96% sequence identity to SEQ ID NO: 20; at least 97% sequence identity to SEQ ID NO: 20; at least 98% sequence identity to SEQ ID NO: 20; at least 99% sequence identity to SEQ ID NO: 20; or at least 100% sequence identity to SEQ ID NO: 20, wherein the % sequence identity to SEQ IDNO: 20 retains the amino acid modifications at amino acid positions 48, 51, 60, and 111.

[0179] In another preferred embodiment, the IL-18 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid position 9, a hydrophobic-to-hydrophilic substitution at amino acid position 48, a substitution of a wild-type amino acid for a polar amino acid at amino acid position 51, a substitution of a wild-type amino acid for a large amino acid at amino acid position 60, and a substitution of a wild-type amino acid for a positively charged amino acid or a neutral amino acid at amino acid position 111. In an even more preferred embodiment, the IL-18 variant may comprise (i) the leucine-to-serine substitution at amino acid position 9; and (ii) the isoleucine-to-aspartate substitution at amino acid position 48; and (iii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51; and (iv) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; and (v) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111. In this embodiment, the IL-18 variant may comprise an amino acid sequence according to SEQ ID NO: 21, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 21, wherein the % sequence identity to SEQ ID NO: 21 retains the amino acid modifications at amino acid positions 9, 48, 51, 60, and 111. In another embodiment, the IL-18 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 21; at least 80% sequence identity to SEQ ID NO: 21; at least 85% sequence identity to SEQ ID NO: 21; at least 90% sequence identity to SEQ ID NO: 21 ; at least 91 % sequence identity to SEQ ID NO: 21 ; at least 92% sequence identity to SEQ ID NO: 21; at least 93% sequence identity to SEQ ID NO: 21; at least 94% sequence identity to SEQ ID NO: 21; at least 95% sequence identity to SEQ ID NO: 21; at least 96% sequence identity to SEQ ID NO: 21; at least 97% sequence identity to SEQ ID NO: 21; at least 98% sequence identity to SEQ ID NO: 21; at least 99% sequence identity to SEQ ID NO: 21; or at least 100% sequence identity to SEQ ID NO: 21, wherein the % sequence identity to SEQ IDNO: 21 retains the amino acid modifications at amino acid positions 9, 48, 51, 60, and 111.

[0180] As discussed herein, in preferred embodiments the IL-18 variant may comprise amino acid modifications at amino acid positions 48 and 60; 48 and 51 ; or 48, 51 and 60. Decoy resistance is achieved via modifications to amino acid positions 51 or 60. Therefore, an IL-18 variant encoded by the nucleic acid construct may have amino acid mutations at amino acid positions 51 and 60 only. The amino acid modifications at amino acid positions 51 and 60 may as be described herein. For the avoidance of doubt, in a preferred embodiment, the amino acid modification at amino acid position 51 may involve a substitution of a wild-type amino acid for a polar amino acid. In a more preferred embodiment, the amino acid modification at amino acid position 51 may be a methionine-to-lysine substitution or a methionine-to-threonine substitution, preferably a methionine-to-threonine substitution. In another preferred embodiment, the amino acid modification at amino acid position 60 may involve substitution of a wild-type amino acid for a large amino acid. In a more preferred embodiment, the amino acid modification at amino acid position 60 may be a methionine-to-lysine substitution or a methionine-to-leucine substitution, preferably a methionine-to-lysine substitution. As such, a decoy resistant IL-18 variant may comprise the following decoy resistance mutations: M51K or M51T and M60L or M60K, preferably M51T and M60K.

[0181] Further, the inventors of the present invention have found the amino acid modification at amino acid position 48 allows for IL-18 variants comprising this modification to be solubilised (i.e., stabilised) while retaining similar biological activity levels to that of wild-type IL-18. Of the different residues tested for altering the solubility of IL-18 variants, mutation of amino acid 48 was surprisingly found to yield the greatest enhancement of solubility. Therefore, in a most preferred embodiment, where the nucleic acid construct encodes an IL-18 variant, the IL-18 variant may comprise amino acid modifications at amino acid positions 48, and 51 and / or 60. For instance, the IL-18 variant may comprise amino acid modifications at amino acid positions 48 and 51. The IL-18 variant may comprise amino acidmodifications at amino acid positions 48 and 60. The IL-18 variant may comprise amino acid modifications at amino acid positions 48, 51 and 60.

[0182] In this embodiment, the amino acid modification at amino acid position 48 may be an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution. Preferably, the amino acid modification at amino acid position 48 may be an isoleucine-to-aspartate substitution. The amino acid modification at amino acid position 51 may involve substitution of a wild-type amino acid for a polar amino acid. Preferably, the amino acid modification at amino acid position 51 may be a methionine-to-lysine substitution or a methionine-to-threonine substitution, more preferably a methionine-to-threonine substitution. The amino acid modification at amino acid position 60 may involve substitution of a wild-type amino acid for a large amino acid. Preferably, the amino acid modification at amino acid position 60 may be a methionine-to-lysine substitution or a methionine-to-leucine substitution, more preferably a methionine-to-lysine substitution. In a more preferred embodiment, the IL-18 variant according may comprise an I48D mutation and an M60K mutation. In another more preferred embodiment, the IL-18 variant may comprise an I48D mutation and an M51T mutation. In another more preferred embodiment, the IL-18 variant may comprise an I48D mutation and an M51T mutation and an M60K mutation.

[0183] As detailed herein, the nucleic acid construct may encode two or more of: an IL-15 molecule, an IL-18 molecule, an IL-21 molecule, and / or a CXCL9 molecule. When the nucleic acid construct encodes an IL-21 variant, the amino acid modifications which may be present within IL-21 to give rise to different IL-21 variants are described below. It will be understood that the nucleic acid construct comprises a polynucleotide sequence which encodes said IL-21 variant. The skilled person will be aware of the methods and techniques used to encode amino acid modifications in a nucleic acid sequence.

[0184] In one embodiment, the IL-21 variant encoded by the nucleic acid construct may comprise an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 3. The IL-21 variant may comprise an amino acid sequencecomprising 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; 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 the relevant amino acid positions.

[0185] In accordance with the invention, the IL-21 variant may be modified such that, in comparison to SEQ ID NO: 3, 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 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 some embodiments the IL-21 variant may comprise a single amino acid modification at any one of amino acid positions 13, 29, 33, 36, 71 or 74; two amino acid modifications at any two of amino acid positions 13, 29, 33, 36, 71 and / or 74; three amino acid modifications at any three of amino acid positions 13, 29, 33, 36, 71 and / or 74; four amino acid modifications at any four of amino acid positions 13, 29, 33, 36, 71 and / or 74; five amino acid modifications at any five of amino acid positions 13, 29, 33, 36, 71 and / or 74; or six amino acid modifications all of amino acid positions 13, 29, 33, 36, 71 and 74. In a preferred embodiment, the IL-21 variant may comprise two amino acid modifications at any two of amino acid positions 29, 33, 36, and / or 71. 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. lt 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: 3.In some embodiments, the IL-21 variant may comprise amino acid modifications whereby non-conservative amino acid substitutions are performed. In other embodiments, the IL-21 variant may comprise amino acid modifications whereby conservative amino acid substitutions are performed.

[0186] 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: 3, the wild-type amino acid at position 13 is isoleucine, the wildtype amino acid at position 29 is valine, the wild-type amino acid at position 33 is valine, the wild-type amino acid at position 36 is phenylalanine, the wild-type amino acid at position 71 is isoleucine, and the wild-type amino acid at position 74 is valine.

[0187] Table 2 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. In some instances, an additional methionine residue may be present at the beginning of the sequence which facilitates its translation in bacteria.

[0188]

[0189] able 2. Possible amino acid modifications within IL-21.

[0190] 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. Forexample, 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.

[0191] The wild-type amino acid at amino acid position 13 in relation to SEQ ID NO: 3 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.

[0192] The wild-type amino acid at position 29 in relation to SEQ ID NO: 3 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.

[0193] The wild-type amino acid at position 33 in relation to SEQ ID NO: 3 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.

[0194] The wild-type amino acid at position 36 in relation to SEQ ID NO: 3 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.

[0195] The wild-type amino acid at position 71 in relation to SEQ ID NO: 3 is isoleucine. The preference of the present invention is for the hydrophobic-to-hydrophilicsubstitution 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.

[0196] The wild-type amino acid at position 74 in relation to SEQ ID NO: 3 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.

[0197] 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.

[0198] 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.

[0199] Table 3 summarises the possible combinations of modifications / mutations present in each of the four IL-21 variants described above.

[0200]

[0201] Table 3. Four different IL-21 variants and their preferred mutations.As detailed herein, in a preferred embodiment, the IL-21 variant may comprise amino acid modifications at amino acid positions 29 and 36 (IL-21V36); 33 and 71 (IL-21 V52, IL-21V58); and / or 36 and 71 (IL-21V62). Four variants, referred to herein by SEQ ID NOs: 22, 23, 24 and 25 respectively (IL-21V36, IL-21V52, IL-21V58 and IL-21V62; Table 3) were found to be more soluble than wild-type IL-21, and have similar activity to wild-type IL-21. Therefore, in a preferred embodiment, the nucleic acid construct may encode IL-21 variants with modifications present which give rise to an IL-21 variant according to any of SEQ ID NOs: 22, 23, 24, and / or 25.

[0202] In a preferred embodiment, an IL-21 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid positions 29 and 36. In a more preferred embodiment, an IL-21 variant 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: 22, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 22, wherein the % sequence identity to SEQ ID NO: 22 retains the amino acid modifications at amino acid positions 29 and 36. The IL-21 variant encoded by the nucleic acid construct 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: 22; at least 85% sequence identity to SEQ ID NO: 22; at least 90% sequence identity to SEQ ID NO: 22; at least 91% sequence identity to SEQ ID NO: 22; at least 92% sequence identity to SEQ ID NO: 22; at least 93% sequence identity to SEQ ID NO: 22; at least 94% sequence identity to SEQ ID NO: 22; at least 95% sequence identity to SEQ ID NO: 22; at least 96% sequence identity to SEQ ID NO: 22; at least 97% sequence identity to SEQ ID NO:22; at least 98% sequence identity to SEQ ID NO: 22; at least 99% sequence identity to SEQ ID NO: 22; or at least 100% sequence identity to SEQID NO: 22, wherein the % sequence identity to SEQ ID NO: 22 retains the amino acid modifications at amino acid positions 29 and 36.

[0203] In another preferred embodiment, an IL-21 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid positions 33 and 71. In a more preferred embodiment, an IL-21 variant 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. In an 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: 23, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 23, wherein the % sequence identity to SEQ ID NO: 23 retains the amino acid modifications at amino acid positions 33 and 71. The IL-21 variant encoded by the nucleic acid construct may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 23; at least 80% sequence identity to SEQ ID NO: 23; at least 85% sequence identity to SEQ ID NO: 23; at least 90% sequence identity to SEQ ID NO: 23; at least 91% sequence identity to SEQ ID NO: 23; at least 92% sequence identity to SEQ ID NO: 23; at least 93% sequence identity to SEQ ID NO: 23; at least 94% sequence identity to SEQ ID NO: 23; at least 95% sequence identity to SEQ ID NO: 23; at least 96% sequence identity to SEQ ID NO: 23; at least 97% sequence identity to SEQ ID NO: 23; at least 98% sequence identity to SEQ ID NO: 23; at least 99% sequence identity to SEQ I D NO: 23; or at least 100% sequence identity to SEQ ID NO: 23, wherein the % sequence identity to SEQ ID NO: 23 retains the amino acid modifications at amino acid positions 33 and 71.

[0204] In another even more preferred embodiment, the IL-21 variant encoded by the nucleic acid construct 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: 24, or an amino acid sequence comprising at least 70%sequence identity to SEQ ID NO: 24, wherein the % sequence identity to SEQ ID NO: 24 retains the amino acid modifications at amino acid positions 33 and 71. The IL-21 variant encoded by the nucleic acid construct may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 24; at least 80% sequence identity to SEQ ID NO: 24; at least 85% sequence identity to SEQ ID NO: 24; at least 90% sequence identity to SEQ ID NO: 24; at least 91% sequence identity to SEQ ID NO: 24; at least 92% sequence identity to SEQ ID NO: 24; at least 93% sequence identity to SEQ ID NO: 24; at least 94% sequence identity to SEQ ID NO: 24; at least 95% sequence identity to SEQ ID NO: 24; at least 96% sequence identity to SEQ ID NO: 24; at least 97% sequence identity to SEQ ID NO: 24; at least 98% sequence identity to SEQ ID NO: 24; at least 99% sequence identity to SEQ ID NO: 24; or at least 100% sequence identity to SEQ ID NO: 24, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 33 and 71.

[0205] In another preferred embodiment, an IL-21 variant encoded by the nucleic acid construct may comprise a hydrophobic-to-hydrophilic substitution at amino acid positions 36 and 71. In a more preferred embodiment, the IL-21 variant 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: 25, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 25, wherein the % sequence identity to SEQ ID NO: 25 retains the amino acid modifications at amino acid positions 36 and 71. The IL-21 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 25; at least 80% sequence identity to SEQ ID NO: 25; at least 85% sequence identity to SEQ ID NO: 25; at least 90% sequence identity to SEQ ID NO: 25; at least 91% sequence identity to SEQ ID NO: 25; at least 92% sequence identity to SEQ ID NO: 25; at least 93% sequence identity to SEQ ID NO: 25; at least 94%sequence identity to SEQ ID NO: 25; at least 95% sequence identity to SEQ ID NO: 25; at least 96% sequence identity to SEQ ID NO: 25; at least 97% sequence identity to SEQ ID NO: 25; at least 98% sequence identity to SEQ ID NO: 25; at least 99% sequence identity to SEQ I D NO: 25; or at least 100% sequence identity to SEQ ID NO: 25, wherein the % sequence identity to SEQ ID NO: 25 retains the amino acid modifications at amino acid positions 36 and 71.

[0206] As detailed herein, the nucleic acid construct may encode two or more of: an IL-15 molecule, an IL-18 molecule, an IL-21 molecule, and / or a CXCL9 molecule. Where the nucleic acid construct encodes a CXCL9 molecule, it will be understood that the nucleic acid construct may comprise a polynucleotide sequence which encodes and therefore allows for expression of the CXCL9 protein. In some embodiments, the CXCL9 molecule is unmodified (wild-type), however in other embodiments, the CXCL9 molecule may comprise amino acid modifications.

[0207] In one embodiment, the CXCL9 molecule may comprise an amino acid sequence according to SEQ ID NO: 26, or a sequence comprising at least 70% sequence identity to SEQ ID NO: 26; at least 75% sequence identity to SEQ ID NO: 26; at least 80% sequence identity to SEQ ID NO: 26; at least 85% sequence identity to SEQ ID NO: 26; at least 90% sequence identity to SEQ ID NO: 26; at least 91% sequence identity to SEQ ID NO: 26; at least 92% sequence identity to SEQ ID NO: 26; at least 93% sequence identity to SEQ ID NO: 26; at least 94% sequence identity to SEQ ID NO: 26; at least 95% sequence identity to SEQ ID NO: 26; at least 96% sequence identity to SEQ ID NO: 26; at least 97% sequence identity to SEQ ID NO: 26; at least 98% sequence identity to SEQ ID NO: 26; or at least 99% sequence identity to SEQ ID NO: 26.

[0208] The nucleic acid construct may comprise a polynucleotide sequence according to SEQ ID NO: 27, ora sequence comprising at least 70% sequence identity to SEQ ID NO: 27; at least 75% sequence identity to SEQ ID NO: 27; at least 80% sequence identity to SEQ ID NO: 27; at least 85% sequence identity to SEQ ID NO: 27; at least 90% sequence identity to SEQ ID NO: 27; at least 91% sequence identity to SEQ ID NO: 27; at least 92% sequence identity to SEQ ID NO: 27; at least 93% sequence identity to SEQ ID NO: 27; at least 94% sequence identity toSEQ ID NO: 27; at least 95% sequence identity to SEQ ID NO: 27; at least 96% sequence identity to SEQ ID NO: 27; at least 97% sequence identity to SEQ ID NO: 27; at least 98% sequence identity to SEQ ID NO: 27; or at least 99% sequence identity to SEQ ID NO: 27. SEQ ID NO: 27 represents the DNA sequence of cxcl-9 (the gene encoding the CXCL9 protein).

[0209] In order to mount a strong immune response, for example, in the tumour microenvironment, the nucleic acid construct and the products transcribed and / or translated from it should be able to be produced and secreted. The cytokines are to be produced and secreted in situ, for example from therapeutic bacteria which possess the nucleic acid construct of the present invention. The nucleic acid construct may be encoded by a plasmid within the bacterium, or integrated into the bacterial chromosome. As such, the therapeutic bacteria must be able to produce and secrete immuno-stimulatory cytokines in situ. Therefore, in a preferred embodiment, the cytokines encoded by the nucleic acid construct (i.e. , IL-15 variant including IL-15 variant super agonists, IL-18 variant, IL-21 variant and / or CXCL9) are fused to a signal peptide (i.e., the nucleic acid construct may further comprise polynucleotide sequence(s) encoding signal peptides). As used herein, the term “signal peptide” refers to any signal sequence, localisation sequence or other polypeptide sequence which directs the cytokine to a particular intracellular or extracellular location. The addition of small (15 to 25 amino acid) sequences, known as signal peptides, to a protein of interest can be used to direct to a desired location.

[0210] 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 to the periplasm via signal peptides is beneficial to the production and correct folding of these proteins, andultimate secretion in situ. The 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 cytokines.

[0211] As an alternative to the use of signal peptides, cell lysis may be induced such as to release the cellular contents of said cell, including any cytokines expressed from the nucleic acid construct.

[0212] Where a signal peptide is utilised, the signal peptide may be a (bacterial) periplasmic signal peptide. The signal peptide directs the cytokine 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.

[0213] 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 exportation 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. The SEC pathway is shared by both Gramnegative and Gram-positive bacteria. In Gram-negative bacteria, as a result of the SEC pathway, proteins are secreted into the periplasm. In Gram-positive bacteria, as a result of the SEC pathway, proteins are secreted directly into the external environment. In some embodiments, the signal peptide used to direct proteins to the periplasm is N-terminally fused to the cytokine. However, thecytokine 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 cytokine.

[0214] From the periplasm, the cytokines can then be prompted into the supernatant or other extracellular environment. In fusing a periplasmic signal peptide to a cytokine, if expressed within a bacterial cell, the cytokine can be transported efficiently to the periplasm of the bacterial cell in order to achieve efficient secretion of the cytokines 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 cytokines. For example, upon infection and invasion of a host (e.g., eukaryotic) cell by Gram-negative bacteria expressing the nucleic acid construct of the present invention, the cytokines may be transferred into the cytoplasm of the host cell where they may achieve for example a therapeutic immunostimulatory effect.

[0215] The IL-15 variant, the IL-18 variant, the IL-21 variant, and / or CXCL9 is fused to a signal peptide. In a preferred embodiment of the invention, the signal peptide is a periplasmic signal peptide, such as MalE, PelB, DsbA, OmpA or PhoA. Signal peptides can vary in length and sequence; however, a common tripartite structure is found across signal peptides, which includes a positively charged N terminus, a hydrophobic core, and a C-terminal cleavage site that is polar and comprises the signal peptidase recognition site. Following secretion, the signal peptide may be cleaved by a signal peptidase enzyme at the Ala-X-Ala recognition sequence. Table 4 shows the sequences of the signal peptides that may be utilised herein.

[0216]

[0217]

[0218] Table 4. Signal peptides and sequences thereof.

[0219] In a more preferred embodiment, where the nucleic acid construct encodes an IL-15 variant, the IL-15 variant is fused to a MalE or PelB signal peptide.

[0220] In another more preferred embodiment, where the nucleic acid construct encodes an IL-18 variant, the IL-18 variant is fused to a MalE or PelB signal peptide.

[0221] In another more preferred embodiment, where the nucleic acid construct encodes an IL-21 variant, the IL-21 variant is fused to a DsbAor PhoA signal peptide.

[0222] In another more preferred embodiment, where the nucleic acid construct CXCL9, CXCL9 is fused to an OmpA or DsbA signal peptide.

[0223] In another embodiment, the signal peptide may be fused to the cytokine 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 (cytokine) that is to be secreted (i.e. , signal peptide:spacer:cytokine, i.e. , the spacer is located at the N terminus of the cytokine). 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.

[0224] The interleukin variants as described above, when arranged in specific combinations as part of a nucleic acid construct, have been found to provide improved stimulation of immune cells and trigger higher production of interferon-gamma, thereby having the capacity to promote an enhanced immune response. In accordance with a first aspect of the invention, there is provided a nucleic acid construct comprising polynucleotide sequences encoding two or more of: (i) an interleukin-15 (IL-15) molecule; (ii) an interleukin-18 (IL-18) molecule; (iii) an interleukin-21 (IL-21) molecule; and / or (iv) a CXCL9 molecule . Therefore, suitable combinations of these cytokines may be generated in order to achieve the desired immunostimulatory effect. A key advantage of the present invention is that the inventors have designed and created single transcriptional units that encode these combinations of cytokines (“multi-cytokine circuits”) and allow them to be produced from said single transcriptional unit. This means that space for other potentially therapeutic agents to be encoded by the nucleic acid construct is created while allowing for the efficient transcription and translation of the required cytokines to produce the immunostimulatory effect.

[0225] It can therefore be seen that the nucleic acid constructs according to the present invention confer distinct advantages in immunotherapy over what is known in the art. The effect of combining multiple interleukin variants (and / or CXCL9) which are individually designed (and, where the construct is for expression in bacteria, optimised for efficient expression, folding and bioactivity) and carefully arranged into a single unit is synergistic. For example, chemokines can attract appropriate immune cells to the tumour, where cytokines can also activate their proliferation and cytotoxicity. Further, the combination of cytokines can enhance the proliferation and cytotoxicity levels achieved. For example, Figure 4 shows how the combination of IL-15, IL-21, and IL-18 can boost the production of IFN-gamma, which plays a major role in activating anti-cancer immune responses.

[0226] Figure 10 shows the possible combinations of cytokines and the signal peptides which may be fused thereto. Figure 11 details the different nucleic acid constructs and the various cytokines comprised therein (“multi-cytokine circuits”).

[0227] The nucleic acid constructs of the present invention may be built using the techniques of nucleic acid digestion and ligation, with which the skilled person will be familiar. The ‘components’ of the construct, for example polynucleotide sequences encoding any of the interleukin variants and / or CXCL9, may beassembled into individual ‘modules’ via Bbsl Golden Gate reactions. Other suitable methods for assembly, such as Gibson assembly, will be known to those in the art. A module may contain a single interleukin variant or chemokine. Each module may also contain a ribozyme sequence and a strong ribosome binding site. The modules may then be combined as necessary into appropriate plasmid backbones, by digesting the vector and modules and ligating / assembling accordingly. Preferably, this assembly is carried out via a Bsal-dependent Golden Gate reaction. Digestion via the Bsal-dependent Golden Gate reaction generates compatible overhangs such that assembly may occur. For instance, the 3’ overhangs of a module left after digestion with Bsal are compatible with 5’ overhangs from the subsequent module or the recipient plasmid; and the 5’ overhang from the first module is compatible with the 3’ overhang of the recipient plasmid.

[0228] To build a nucleic acid construct according to the present invention, two or more of the interleukins described herein and CXCL9 may be present in any number and combination. In one embodiment, one or multiple copies of any particular interleukins and / or CXCL9 may be comprised within the nucleic acid construct. For example, there may be at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten copies of any particular interleukins and / or CXCL9 may be present in the nucleic acid construct of the present invention. In another embodiment, there may be one of each interleukins described herein and CXCL9 comprised within the nucleic acid construct.

[0229] The order, as well as number and combination, of the interleukin molecules described herein and CXCL9 has also been determined by the present inventors such as to achieve optimal expression of each cytokine. A number of suitable combinations and orders are described below.

[0230] In one embodiment, the nucleic acid construct may comprise an IL-15 molecule, an IL-18 molecule, an IL-21 molecule and / or a CXCL9 molecule. The nucleic acid construct may comprise any combination of cytokines, for example as shown in Figure 11. The nucleic acid construct may comprise a nucleic acid sequenceaccording to any of SEQ ID NOs: 33 to 48, or a sequence having at least 70% identity thereto, or at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0231] In another embodiment, the nucleic acid construct may comprise an IL-15 molecule and CXCL9. In a preferred embodiment, the nucleic acid construct may comprise an IL-15 variant and CXCL9.

[0232] In another embodiment, the nucleic acid construct may comprise an IL-15 molecule, an IL-18 molecule and CXCL9. In a preferred embodiment, the nucleic acid construct may comprise an IL-15 variant, an IL-18 variant and CXCL9.

[0233] In another embodiment, the nucleic acid construct may comprise an IL-15 molecule, an IL-18 molecule, an IL-21 molecule and CXCL9. In a preferred embodiment, the nucleic acid construct may comprise an IL-15 variant, an IL-18 variant, an IL-21 variant and CXCL9.

[0234] In another embodiment, the nucleic acid construct may comprise an IL-15 molecule, an IL-18 molecule and an IL-21 molecule. In a preferred embodiment, the nucleic acid construct may comprise an IL-15 variant, an IL-18 variant, and an IL-21 variant.

[0235] In another embodiment, the nucleic acid construct may comprise an IL-15 molecule and an IL-18 molecule. In a preferred embodiment, the nucleic acid construct may comprise an IL-15 variant and an IL-18 variant. Advantageously, the inventors of the present invention have found that multi-cytokine circuits encoding IL-15 and IL-18 are especially therapeutically relevant (se for example Figures 4, 16 and 17).

[0236] In another embodiment, the nucleic acid construct may comprise an IL-15 molecule, an IL-21 molecule, an IL-18 molecule and CXCL9. In a preferred embodiment, the nucleic acid construct may comprise an IL-15 variant, an IL-21 variant, an IL-18 variant and CXCL9.In another embodiment, the nucleic acid construct may comprise an IL-15 molecule, an IL-21 molecule and an IL-18 molecule. In a preferred embodiment, the nucleic acid construct may comprise an IL-15 variant, an IL-21 variant, and an IL-18 variant.

[0237] In another embodiment, the nucleic acid construct may comprise an IL-18 molecule, an IL-15 molecule and CXCL9. In a preferred embodiment, the nucleic acid construct may comprise an IL-18 variant, an IL-15 variant, and CXCL9.

[0238] In another embodiment, the nucleic acid construct may comprise an IL-18 molecule, an IL-15 molecule, an IL-21 molecule and CXCL9. In a preferred embodiment, the nucleic acid construct may comprise an IL-18 variant, an IL-15 variant, an IL-21 variant and CXCL9.

[0239] In another embodiment, the nucleic acid construct may comprise an IL-18 molecule, an IL-15 molecule and an IL-21 molecule. In a preferred embodiment, the nucleic acid construct may comprise an IL-18 variant, an IL-15 variant, and an IL-21 variant.

[0240] In another embodiment, the nucleic acid construct may comprise an IL-18 molecule and an IL-15 molecule. In a preferred embodiment, the nucleic acid construct may comprise an IL-18 variant, and an IL-15 variant.

[0241] In another embodiment, the nucleic acid construct may comprise an IL-18 molecule, an IL-21 molecule, an IL-15 molecule and CXCL9. In a preferred embodiment, the nucleic acid construct may comprise an IL-18 variant, an IL-21 variant, an IL-15 variant and CXCL9.

[0242] In another embodiment, the nucleic acid construct may comprise an IL-18 molecule, an IL-21 molecule and an IL-15 molecule. In a preferred embodiment, the nucleic acid construct may comprise an IL-18 variant, an IL-21 variant, and an IL-15 variant.

[0243] In another embodiment, the nucleic acid construct may comprise an IL-21 molecule, an IL-15 molecule, an IL-18 molecule and CXCL9. In a preferredembodiment, the nucleic acid construct may comprise an IL-21 variant, an IL-15 variant, an IL-18 variant and CXCL9.

[0244] In another embodiment, the nucleic acid construct may comprise an IL-21 molecule, an IL-15 molecule and an IL-18 molecule. In a preferred embodiment, the nucleic acid construct may comprise an IL-21 variant, an IL-15 variant, and an IL-18 variant.

[0245] In another embodiment, the nucleic acid construct may comprise an IL-21 molecule, an IL-18 molecule, an IL-15 molecule and CXCL9. In a preferred embodiment, the nucleic acid construct may comprise an IL-21 variant, an IL-18 variant, an IL-15 variant and CXCL9.

[0246] In another embodiment, the nucleic acid construct may comprise an IL-21 molecule, an IL-18 molecule and an IL-15 molecule. In a preferred embodiment, the nucleic acid construct may comprise an IL-21 variant, an IL-18 variant, and an IL-15 variant.

[0247] In a preferred embodiment, the nucleic acid construct may comprise an IL-15 variant comprising a sequence according to any of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and / or 16. In a more preferred embodiment, the IL-15 variant may comprise a sequence according to any of SEQ ID NOs: 4, 5 and / or 8. In a most preferred embodiment, the IL-15 variant may be SEQ ID NO: 8. Sequences having at least 70% identity to any of the aforementioned sequences are also intended to be covered.

[0248] In another preferred embodiment, the nucleic acid construct may comprise an IL-18 variant comprising a sequence according to any of SEQ ID NOs: 18, 19, 20 and / or 21. In a most preferred embodiment, the IL-18 variant may be SEQ ID NO: 19. Sequences having at least 70% identity to any of the aforementioned sequences are also intended to be covered.

[0249] In another preferred embodiment, the nucleic acid construct may comprise an IL-21 variant comprising a sequence according to any of SEQ ID NOs: 22, 23, 24 and / or 25. In a most preferred embodiment, the IL-21 variant may be SEQ ID NO:23. Sequences having at least 70% identity to any of the aforementioned sequences are also intended to be covered.

[0250] In another preferred embodiment, the nucleic acid construct may encode CXCL9 comprising a sequence according to SEQ ID NO: 26 (or the nucleic acid sequence of SEQ ID NO: 27). Sequences having at least 70% identity the aforementioned sequence are also intended to be covered.

[0251] The cytokines encoded by the nucleic acid construct may be fused to a signal peptide as described herein. The present inventors have identified the optimal signal peptides to be fused to each cytokine such as to achieve enhanced secretion from cells. In particular, for bacterial cells, the signal peptide may be any of MalE, PelB, DsbA, PhoA, or OmpA (given herein as SEQ ID NOs: 65, 66, 67, 68 and 69, respectively). For mammalian cells, the signal peptide may be a mammalian signal peptide, such as MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (SEQ ID NO77) for IL-15, MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESD (SEQ ID NO: 78) for IL-18, MKKSGVLFLLGIILLVLIGVQG (SEQ ID NO: 79) for CXCL-9, or MRSSPGNMERIVICLMVIFLGTLV (SEQ ID NO: 80) for IL-21.

[0252] In a preferred embodiment, CXCL9 is fused to an OmpA or DsbA signal peptide. However, the preference is for CXCL9 to be fused to an OmpA peptide. In a more preferred embodiment, the OmpA peptide is fused to a CXCL9 molecule comprising a sequence according to SEQ ID NO: 26, or a sequence having at least 70% identity thereto.

[0253] Any of the sequences described herein may also feature an ‘ATG’ sequence or ‘M’ residue at the N-terminus to facilitate cloning and translation.

[0254] In another preferred embodiment, the IL-15 molecule is fused to a MalE or PelB signal peptide. However, the preference is for the IL-15 molecule to be fused to a MalE signal peptide. In a more preferred embodiment, the MalE signal peptide is fused to an IL-15 variant comprising a sequence according to SEQ ID NO: 8, or a sequence having at least 70% identity thereto.In another embodiment, the IL-18 molecule is fused to a MalE or PelB signal peptide. However, the preference is for the IL-18 molecule to be fused to a PelB signal peptide. In a more preferred embodiment, the PelB signal peptide is fused to an IL-18 variant comprising a sequence according to SEQ ID NO: 19, or a sequence having at least 70% identity thereto.

[0255] In another embodiment, the IL-21 molecule is fused to a DsbA or PhoA signal peptide. However, the preference is for the IL-21 molecule to be fused to a DsbA signal peptide. In a more preferred embodiment, the DsbA signal peptide is fused to an IL-21 variant comprising a sequence according to SEQ ID NO: 23, or a sequence having at least 70% identity thereto.

[0256] The inventors of the present invention surprisingly found, in particular, that nucleic acid constructs encoding at least an IL-15 molecule demonstrated the most significant immuno-stimulatory capacity in comparison to those constructs not encoding an IL-15 variant. Therefore, in one embodiment, the nucleic acid construct may comprise polynucleotide sequences encoding three or more of an IL-15 molecule, an IL-18 molecule, an IL-21 molecule, and / or a CXCL9 molecule. In some embodiments, the nucleic acid construct may comprise polynucleotide sequences encoding IL-15 molecule and one or more polynucleotide sequences encoding an IL-18 molecule, an IL-21 molecule and / or a CXCL9 molecule, preferably wherein the interleukin molecules are interleukin variants.

[0257] In another embodiment, the nucleic acid construct may encode an IL-15 molecule and two or more of an IL-18 molecule, an IL-21 molecule and / or CXCL9, preferably wherein the interleukin molecules are interleukin variants.

[0258] In another embodiment, the nucleic acid construct may encode an IL-15 molecule and an IL-18 molecule, an IL-21 molecule and CXCL9, preferably wherein the interleukin molecules are interleukin variants.

[0259] For example, the nucleic acid construct may encode any of the following combinations of cytokines: an IL-15 variant and CXCL9; an IL-15 variant and IL-18 variant; an IL-15 variant and IL-21 variant; an IL-15 variant and CXCL9 and an IL-18 variant; an IL-15 variant and CXCL9 and an IL-21 variant; an IL-15 variantand CXCL9 and an IL-18 variant and an IL-21 variant; an IL-15 variant and an IL-18 variant and an IL-21 variant. Examples are demonstrated in Figures 10 and 11.

[0260] In a preferred embodiment, the nucleic acid construct may comprise two or more polynucleotide sequences encoding an amino acid sequence according to any of SEQ ID NOs: 8, 19, 23, and / or 26, or a sequence having at least 70% identity thereto. For example, a sequence having at least 70% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 75% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 80% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 85% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 90% identity to any of SEQ I D NOs: 8, 19, 23, and / or 26; at least 91 % identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 92% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 93% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 94% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 95% identity to any of SEQ I D NOs: 8, 19, 23, and / or 26; at least 96% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 97% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 98% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; at least 99% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26; or 100% identity to any of SEQ ID NOs: 8, 19, 23, and / or 26.

[0261] In one embodiment, the nucleic acid construct may comprise a polynucleotide sequence encoding a MalE signal peptide upstream of the polynucleotide sequence encoding the amino acid sequence according to SEQ ID NO: 8. In one embodiment, the nucleic acid construct may comprise a polynucleotide sequence encoding a PelB signal peptide upstream of the polynucleotide sequence encoding the amino acid sequence according to SEQ ID NO: 19. In one embodiment, the nucleic acid construct may comprise a polynucleotide sequence encoding a DsbA signal peptide upstream of the polynucleotide sequence encoding the amino acid sequence according to SEQ ID NO: 23. In one embodiment, the nucleic acid construct may comprise a polynucleotide sequence encoding a OmpA signal peptide upstream of the polynucleotide sequence encoding the amino acid sequence according to SEQ ID NO: 26.It is also possible that the nucleic acid construct comprises different IL-15 variants, IL-18 variants, and / or IL-21 variants. It is also possible that the nucleic acid construct comprises one or more copies of the cytokines, for example two, three or four copies of a cytokine.

[0262] In one embodiment, the nucleic acid molecule is a DNA molecule. In another embodiment, the nucleic acid molecule is an RNA molecule. Preferably, the nucleic acid construct comprises an DNA molecule. Ribozyme sequences may be present with any combination of cytokines, as described herein.

[0263] The nucleic acid may be expressed in a host cell, such that the cytokine molecules are expressed within and secreted from the host cell. The host cell may be a bacterial cell (i.e., a bacterial cell which has been engineered or modified to express said nucleic acid construct). Alternatively, the host cell may be a eukaryotic cell, such as a mammalian cell.

[0264] Exemplary mammalian cells that the nucleic acid construct may be expressed in include human cells, such as stem cells, bone cells, blood cells, muscle cells, nerve cells, epithelial cells, adipocytes, endothelial cells, cartilage cells, glandular cells, and hepatocytes.

[0265] However, it is a preference of the present invention that the nucleic acid construct may be comprised within a bacterium. The nucleic acid construct may be comprised within a live attenuated bacterium. The bacterium may be Gramnegative or Gram-positive. Any suitable bacterium capable of comprising a nucleic acid construct according to the present invention may be utilised. However, in a particularly preferred embodiment, there is provided a Gram-negative bacterium, comprising the nucleic acid construct according to the present invention.

[0266] Any Gram-negative bacterium capable of expressing and secreting heterologous cytokines as disclosed herein may be utilised. Examples of Gram-negative bacteria for use in the present invention include, but are not limited to, Salmonella, Escherichia (e.g., E. coli), Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia and Yersinia.Preferably, the Gram-negative bacteria 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.

[0267] As described herein, the bacteria may alternatively be Gram-positive bacteria. Any Gram-positive 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.

[0268] Preferably, the bacterium will be a live attenuated 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.

[0269] As used herein, the term “non-natural bacterium or bacteria” refers to bacterial (prokaryotic) cells that have been genetically modified or “engineered” such that itis 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.

[0270] 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 asM01ZH09). Derivatives or variants of the Salmonella enterica Typhi ZH9 strain are also intended to be included, including genetically modified variants.

[0271] Such a bacterium may be engineered to express the any of the nucleic acid constructs 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.

[0272] Expression of a nucleic acid construct according to the present invention by bacteria 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.

[0273] The immune response may be, amongst others, a systemic immune response, a local immune response, an innate immune response, an adaptive immune response, a memory immune response, a primary and / or secondary immune response, a specific and / or non-specific immune response, immune cell activation, proliferation, and / or differentiation or the like, or any combinations thereof. As used herein, the terms “systemic immune response” and “systemic immunity” are used interchangeably and refers to a widespread immune response throughout the body of a subject directed against the eliciting agent, as well as widespread non-specific immune activation, as opposed to a local, spatially restricted response. Such a response involves a complex interaction between the different cells of the immune response, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by the above cells or the liver, and in the context of the present invention is thought to prime the immune system of the subject, such that the subject is more responsive to another therapy such as an anti-cancer therapy. A “systemic immune response” may therefore be measured and quantified via the analysis of various different immune cell types, including, but not limited to, neutrophils, monocytes, dendritic cells, T cells (e.g., CD4+ and / or CD8+ T cells) and natural killer cells. The methods by which these effects can be measured are well known to those skilled in the art, for example, flow cytometry. A “systemic immune response” may also be measured and quantified by the presence of antibodies, including but not limited to IgG and IgA isotype antibodies. The methods by which these antibodies can be measured are well known to those skilled in the art, for example, ELISA. Accordingly, the invention may act to prime (used interchangeably with “condition”, “boost”, “amplify”, “enhance”, “improve”, “augment” or “promote”) the immune response of a subject following administration.Accordingly, the combination of the nucleic acid construct comprising the cytokines and the bacterium, wherein the cytokines are expressed and secreted by the bacteria, offers another level of synergy, and will provide an enhanced immune response in a subject thereby enabling 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 may be of a therapeutic level in itself or be of a sub-therapeutic level requiring the subsequent administration of further dose of to exert a therapeutic effect.

[0274] Cells engineered to express a nucleic acid construct according to the invention would act as therapeutic strains. Therefore, such cells may be used in therapy.

[0275] Due to the wide-spread involvement of cytokines in several different signalling pathways (including both pro-inflammatory and anti-inflammatory signalling), it will be understood that the present invention has therapeutic implications. Therefore, in accordance with a third aspect of the invention, there is provided a nucleic acid of the first aspect of the invention, or a cell of the second aspect of the invention, for use as a therapy.

[0276] In particular, 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 a fourth aspect of the invention, there is provided a nucleic acid construct of the first aspect or the cell of the second aspect for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

[0277] 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.

[0278] 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, IVorV) 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.

[0279] 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; non-encapsulating 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.

[0280] 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.

[0281] In some embodiments, the neoplastic disease is a malignant neoplastic disease.

[0282] In some embodiments the neoplastic disease is not a benign neoplastic disease.

[0283] In one preferred embodiment, the malignant tumour may be associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer,hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial carcinoma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

[0284] In another preferred embodiment of the invention, the nucleic acid construct or cell is to be administered intratumourally, intravesically, intravaginally, intravenously, intraperitoneally, or orally administered. In a most preferred embodiment, nucleic acid construct is administered intratumourally. However, it is also contemplated that other methods of administration may be used in some cases. Therefore, in certain instances the nucleic acid constructs 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, 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, intraperitoneally, intrapleurally, intravesically, intravaginally, peritumoral injection, or intratumoural injection.

[0285] As used herein, the terms “oral administration” or “orally administered” refer to the route of administration in which a 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 thevagina. 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 nucleic acid construct directly into the neoplastic disease of the subject.

[0286] 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.

[0287] Therapeutic cells in accordance with the second aspect of the invention 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, Gram-negative bacteria expressing the present nucleic acid constructs are particularly suitable for the treatment of cancer in vivo.The amount of the cell, for example a bacterium, administered to the subject is sufficient to deliver a therapeutically effective amount of the nucleic acid construct 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, and the medical history of the subject to be treated.

[0288] 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.

[0289] 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.

[0290] 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 thesubject's size (e.g., weight), the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0291] Where the cell described herein is a bacterium, the bacterium may be administered at a dose of between 105and 1012CFU, where CFU is a colonyforming unit. For example, suitable doses may be between 105and 106CFU, 105and 107CFU, 105and 108CFU, 105and 109CFU, 105and 101° CFU, 105and 1011CFU, 106and 107CFU, 106and 108CFU, 106and 109CFU, 106, and 101° CFU, 106and 1011CFU, 106and 1012CFU, 107and 108CFU, 107and 109CFU, 107and 1O10CFU, 107and 1011CFU, 107and 1012CFU, 108and 109CFU, 108and 1O10CFU, 108and 1011CFU, 108and 1012CFU, 109and 101°CFU, 109and 1011CFU, 109and 1012CFU, 1O10and 1011CFU, 1O10and 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 cytokines to be delivered, as well as the specific indication to be treated.

[0292] The nucleic acid construct or cell, for example 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 nucleic acid construct or cell 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 nucleic acid construct or cell may be administered “in” or “on” the tissue or surrounding tissue. As such, the term “local administration” refers to any context in which the nucleic acid construct or cell 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 nucleic acid construct or cell is administered to a subject. As such, the resulting immune responseproduced 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.

[0293] In a preferred embodiment of the present invention, the nucleic acid construct or cell, for example bacterium, described herein are administered intratumourally, intravesically, intravaginally, intravenously, intraperitoneally, or orally administered. In a most preferred embodiment, the cell is administered intratumourally. However, it is also contemplated that other methods of administration may be used in some cases. Therefore, in certain instances the cell 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. Where the cell is a mammalian cell, it may be derived from the subject in need of treatment. Such a cell may then be engineered to express the nucleic acid construct, and delivered back into the subject. Where the nucleic acid construct is to be delivered directly to the subject, it may be formulated as a nucleic acid vaccine, for example a DNA vaccine. The nucleic acid construct may be expressed by a bacterium, and said bacterium be administered to the subject.

[0294] It is understood that the specific method of administration of the nucleic acid construct or cell 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 beprevented 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.

[0295] 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 a fourth aspect of the invention, there is provided a nucleic acid construct according to the first aspect of the invention or cell according to the second aspect of the invention for use in the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject. Further, in a fifth aspect of the invention, there is provided a use of the nucleic acid construct according to the first aspect of the invention or cell according to the second aspect of the invention in the manufacture of a medicament for a therapy. 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 a nucleic acid construct according to the first aspect of the invention or a cell according to the second aspect of the invention.

[0296] Where the disease to be treated is a neoplastic disease, the nucleic acid construct or cell, such as a bacterium, 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 nucleic acid construct or cell with other known cancer therapies. Preferably, the cancer therapy may be an immunotherapy, radiotherapy, chemotherapy, or another 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 nucleic acid construct or cell 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 nucleic acid construct or cell may be administered separately from, simultaneously with or sequentially to (prior to and / or following) the other known cancer therapy.

[0297] 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.

[0298] 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 celladenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating 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 neurogenictumour; 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.

[0299] 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, 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.

[0300] In some embodiments, the neoplastic disease is a malignant neoplastic disease.

[0301] In some embodiments the neoplastic disease is not a benign neoplastic disease.In one preferred embodiment, the malignant tumour may be associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial carcinoma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

[0302] 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 a nucleic acid construct according to the first aspect of the invention or a cell according to the second aspect of the invention. The present invention therefore also provides for a method treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject. As such, the method of the present invention may be used to reduce or inhibit metastasis of a primary tumour or cancer to other sites, or the formation or establishment of metastatic tumours or cancers at other sites distal from the primary tumour or cancer thereby inhibiting or reducing tumour or cancer relapse or tumour or cancer progression. Accordingly, the present invention provides a detectable or measurable improvement in a condition of a given subject, such as alleviating or ameliorating one or more adverse (physical) symptoms or consequences associated with the presence of a cell proliferative or cellular hyperprol iterative disorder, neoplasia, tumour or cancer, or metastasis, i.e., a therapeutic benefit or a beneficial effect. A therapeutic benefit or beneficial effect is any objective or subjective, transient, temporary, or long-term improvement in the condition or pathology, or a reduction in onset, severity, duration or frequency of an adverse symptom associated with or caused by cell proliferation ora cellular hyperprol iterative disorder such as a neoplasia, tumour or cancer, or metastasis. It may lead to improved survival. A satisfactory clinical endpoint of a treatment method in accordance with the invention is achieved, for example, when there is an incremental or a partial reduction in severity, duration or frequency of one or more associated pathologies, adverse symptoms or complications, or inhibition or reversal of one or more of the physiological, biochemical or cellular manifestationsor characteristics of cell proliferation or a cellular hyperprol iterative disorder such as a neoplasia, tumour or cancer, or metastasis. A therapeutic benefit or improvement therefore may include, but is not limited to, destruction of target proliferating cells (e.g., neoplasia, tumour or cancer, or metastasis) or ablation of one or more, most or all pathologies, adverse symptoms or complications associated with or caused by cell proliferation or the cellular hyperprol iterative disorder such as a neoplasia, tumour or cancer, or metastasis. However, a therapeutic benefit or improvement need not be a cure or complete destruction of all target proliferating cells (e.g., neoplasia, tumour or cancer, or metastasis) or ablation of all pathologies, adverse symptoms or complications associated with or caused by cell proliferation or the cellular hyperprol iterative disorder such as a neoplasia, tumour or cancer, or metastasis. For example, partial destruction of a tumour or cancer cell mass, or a stabilization of the tumour or cancer mass, size, or cell numbers by inhibiting progression or worsening of the tumour or cancer, can reduce mortality and prolong lifespan even if only for a few days, weeks or months, even though a portion or the bulk of the tumour or cancer mass, size or cells remain.

[0303] 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.

[0304] 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.

[0305] 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, painand discomfort. Thus, a partial or complete decrease or reduction in the severity, duration or frequency of an adverse symptom or complication associated with or caused by a cellular hyperproliferative disorder, an improvement in the subject’s quality of life and / or well-being, such as increased energy, appetite, psychological well-being, are all particular non-limiting examples of therapeutic benefit.

[0306] 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.

[0307] 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.

[0308] The nucleic acid construct or cell may also be delivered together with a pharmaceutically acceptable carrier / adjuvant / diluent or excipient. The phrases "pharmaceutically” and “pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Such preparations will be known to those skilled in the art. Moreover, for animal (e.g., human or any other member of the animal kingdom) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards, as applicable.

[0309] Such administration may be intended to enhance an immune response of a subject. Accordingly, the nucleic acid construct or cell 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, whichis 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.

[0310] 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.

[0311] 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 coatingmaterials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0312] 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.

[0313] 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 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).

[0314] 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.

[0315] 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.

[0316] The invention is further described with reference to the following non-limiting examples:

[0317] EXAMPLES

[0318] Example 1 - Construction of informed multicytokine circuits and strains

[0319] Figure 3 shows a schematic representation of the development of bacterial strains expressing the multicytokine circuits. Loading vectors pSEC_F1_Pro1, pSEC_F1_ProA, pSEC_F1_ProB, pSEC_F1_ProC, pSEC_F1_Pro1, pSEC_F2_riboJ_End, pSEC_F2_riboJ_Link, pSEC_F3_VtmoJ_End, pSEC_F3_VtmoJ_Link, pSEC_F4_SccJ_End, pSEC_F4_SccJ_Link, pSEC_F5_PlmJ_End, pSEC_F5_PlmJ_Link, and pSEC_F6_LtsvJ_End (given herein as SEQ ID NOs: 49-61) were built by ligating the eBlock (synthesized through IDT DNA) encoding for each sub-module encoding the cytokine molecules (wild-type and / or variant) into pMiniT 2.0 (New England Biolabs) using the reagents from the PCR Cloning Kit (New England Biolabs) and following the manufacturer recommendations. Reactions were transformed into DH10p cellsand grown overnight in ampicillin-selective LB media. Two growing clones per construct were grown overnight in ampicillin-supplemented LB broth and miniprepped. Recovered DNA was validated by sequencing.

[0320] Gene blocks encoding for cytokines (ordered as either gBIocks or eBlocks from IDT DNA) and bearing compatible Bbsl restrictions sites were loaded onto each plasmid via Bbsl-dependent Golden Gate. Briefly, the gene block (30 fmol) was assembled into the recipient vector (15 fmol, carbenicilin-resistant, bla) into a reaction volume, 5 pL with mixture DNA volume brought to 3 pL and topped up with 2 pL of NEB Bridge + Bbsl (1.667 pL NEB Bridge and 0.333 pL of Bbsl). To ensure a successful reaction, after the mixture samples were mixed properly, centrifuged, and cycled at 37°C, 4 min and 16°C, 2 min for 30 cycles (~4h run). The resulting assemblies were transformed into DH10p (2 pL from the 5 pL reaction). Plasmid assembly was validated by sequencing as previously described. Plasmid library was stored into 96-well plates in 30 fmol / pL dilutions.

[0321] Multi-cytokine circuits were assembled by combining appropriate cytokine-loaded plasmids together with a recipient vector from the SEVA collection (e.g., pSEVA-37-SEQ ID NO: 62. Other variants of this plasmid may be utilised) and processed with the Golden Gate protocol previously described, using Bsal instead of Bbsl as restriction enzyme. Assemblies were validated as described, with the added advantage that negative clones were red-coloured. Final plasmids were stored at -20°C.

[0322] Example 2 - Stimulation of NK cells

[0323] Primary NK cells were isolated from single, healthy donor PBMCs by magnetic bead selection (NK Cell Isolation Kit, human; Miltenyi Biotech) and checked for purity by flow cytometry. Resulting cells were aliquoted and stored at -80 °C until required.

[0324] Cells were thawed at 37 °C, counted and assessed for viability with Nucleocounter (97.1 % viability), and seeded in a 96-well plate at 105cells / well in a final 25 pL / well volume in LGM-3 Growth Medium (Lonza). Cells were stimulated by adding 25 pL of media supplemented with recombinant cytokines at appropriate concentrations(0-100 ng / mL). Final wells were brought up to 100 pil_ and grown for 24 h at 37 °C and 5 % CO2. The final plate was spun down at 300xg for 5 min to collect the supernatant, which was transferred to a fresh plate and stored at -20 °C until required.

[0325] Stimuli response was measured through IFNy ELISA by thawing the pre-collected supernatants on ice and using the IFN-gamma duoset ELISA (R&D Systems) as indicated by the manufacturer. Concentrations were determined from a standard curve.

[0326] Figure 4 shows a heatmap of NK cell stimulation using different cytokine combinations. Interestingly, combination of IL-15 with IL-18 was shown to have high synergistic effects in NK cell activation at circa 1 ng / mL each protein.

[0327]

[0328] Reporter plasmid pSPEBIa (Figure 14) was built via Gibson Assembly by combining a vector backbone bearing pSC101 and the chloramphenicol acetyltransferase (cat) gene, generated via PCR from the NL plasmid (SEQ ID NO: 63) using CloneAmp mastermix (Takara) and primers vector_F (actagtcttggactcctgttgatagatcc — SEQ ID NO: 70) and vector_R (CAGCAGATAGGGACGACGTGGTGTTAG - SEQ ID NO: 71), following manufacturer recommendations, and an eBlock (SEQ ID NO: 64) with proC controlling transcription of a fusion reporter gene of mature beta-lactamase (mbla) with two Golden gate sites at its 5’ end, replacing the signal peptide (b / asp): a Bsal- dependent multi-cloning site containing a replaceable strong RBS and ATG- StrepTagll sequence and a Bbsl-dependent multi-cloning site containing a replaceable FLAG tag sequence. The former can be used to load signal peptides and the latter can be used to load the relevant payload (e.g., cytokines). Gibson assembly was performed with the NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) following manufacturer’s recommendations and validated via sequencing as previously described.

[0329] pSPEBIa was loaded with eBlocks encoding cytokines via Bbsl-dependent Golden Gate, as previously described. These express the cytokine-b / a fusion into thecytosol. To direct protein fusions into the periplasm, signal peptide sequences with strong ribosome binding sites were loaded via Bsal-dependent Golden Gate, using conditions previously described.

[0330] Signal peptides MalE, PelB, DsbA, PhoA, and OmpA, amino acid sequences given herein as SEQ ID NOs: 28-32, nucleic acid sequences given here as SEQ ID NOs: 65-69). Various combinations of cytokines and signal peptides are given in Figure 6.

[0331] Final plasmids were transformed into S. enterica Typhi ZH9 via electroporation and recovered in antibiotic-supplemented media overnight. Three independent single colonies per evaluated construct were grown overnight in a 1 mL LB broth supplemented with 25 pig / mL of Chloramphenicol, L-Tyrosine, and aromatic amino acid mix in a 96-deep well plate at 37°C and 700 rpm. Cultures were diluted 1 :500 into fresh media containing appropriate concentrations of carbenicillin (0 - 4 mg / mL) in 100 piL of LB broth in a 96-well plate. Plates were grown for 16-18 hours at 37 °C and 700 rpm. Protein export into the periplasm is directly proportional to the minimum inhibitory concentration of carbenicillin (Figure 5).

[0332] Example 4 - Analysis of transcriptional activity impact on strain growth

[0333] Single and multi-cytokine circuits in plasmids were transformed into the ZH9 strain via electroporation and recovered in LB Agar media supplemented with 25 pig / mL of Chloramphenicol, L-Tyrosine, and aromatic amino acid mix. Single clones were selected and grown overnight in LB Broth supplemented with 25 pig / mL of Chloramphenicol, L-Tyrosine, and aromatic amino acid mix in a 1 mL of broth in a 96-deep well plate. Overnight pre-cultures were diluted 1:500 in fresh LB media and grown for 24 hours at 37 °C and 700 rpm in a BMC ClarioStar plate reader monitoring bacterial density as Optical Density at 600 nm (OD6oo) every 30 min. Final growth between constructs was compared, showing no visible growth defects. Figure 12 shows that the expression of different MCCs in ZH9 did not impact ZH9 growth, therefore confirming that the MCCs do not inflict excessive cell burden.

[0334]

[0335] Circuits containing CXCL-9 were transformed into ZH9, as previously described. Single colonies were grown in 96-deep well plates using previously defined overnight protocol. Cultures were diluted in 5 mL of fresh media in a 50 mL Falcon T ube and grown for 6 h (OD6oo ~ 1.0) at 37 °C and 250 rpm. Bacteria were pelleted by centrifugation at 20,000xg for 5 min and the supernatant filter-sterilized and transferred to a fresh recipient. Proteins in the supernatant were precipitated by adding 20 mL of ice-cold acetone, followed by incubation at -20 °C overnight. Final pellet was separated from supernatant and re-suspended in 500 piL of Phosphate Buffered Saline (PBS). Cell pellets were stored overnight at -20°C and lysed by sonication in 100 piL of PBS. Insoluble aggregates were removed by centrifugation at 20,000xg for 20 min and 4 °C.

[0336] Protein content in each sample was determined via HPLC-MS and quantified through a titration curve calculated with purified CXCL-9 peptide.

[0337] The production of interleukin variants was assessed in the same way as described above for CXCL9.

[0338] Figure 7 shows a schematic representation of the analysis of the impact the strength of the promoter has on single cytokine production. Figure 8 shows the analysis of the impact of the signal peptides, promoters, and cargoes on bacterial growth. Figure 9 shows that CXCL9 production increased linearly with promoter activity.

[0339] Figure 13 shows CXCL9 production in bacteria with multi-cytokine circuits (x axis identifier codes correspond to those given in Figure 11). Note that addition of multiple cytokines and that positional effects have an impact on the overall yield of CXCL-9 either on periplasm or whole cell. Intriguingly, periplasmic protein accounts for 1-10% of total protein produced.

[0340] Further examples of expression from the multicytokine circuits are given in Figures 16, 17 and 18. Figure 16 shows relative production of one, two or three cytokines in bacteria. In Figure 16, the especially therapeutically relevant multi-cytokinecircuits encoding IL15+IL18 and IL-15+IL18+CXCL9 show better-than-average results for all three cytokines.

[0341] Figures 17 and 18 show the expression of cytokines in multicytokine circuits using constitutive systems or quorum sensing-based expression systems. The skilled person will be aware of such quorum sensing systems.

[0342] Example 6 - Construction of multicytokine circuits with wild type cytokines

[0343] The inventors of the present invention have found that the principle of designing and constructing multicytokine circuits as described herein can be applied to both wild-type and variant forms of the cytokine molecules, including combinations of wild-type and variant cytokines. As such, the skilled person will readily understand that the data and disclosures of the preceding examples may also apply to wildtype cytokines.

[0344] Figures 10 shows possible combinations of cytokines and signal peptides, and Figure 11 shows a number of multi-cytokine circuits which were constructed.

[0345] Construction of circuits comprising variant (‘evolved’) and wild-type cytokine sequences was achieved using the molecular tools previously described. Briefly, DNA blocks encoding for each of the cytokines, for example GBL01 and I L-18^, were designed to contain Bbsl-dependent restriction sites compatible with library storage vectors described in Example 1 and later used in combinatorial assembly, also as described in Example 1. The skilled person would understand that such DNA blocks can be encoded to contain 15-20 bp overhangs compatible with Gibson Assembly and assemble libraries using a NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) following manufacturer’s recommendations and onto previously prepared vector backbones.

[0346] Mammalian expression vectors with the cytokine circuits can be built through either the described Golden Gate or Gibson Assembly methodologies described above with the following design changes: a functional Kozak sequence (e.g., GCCGCCACCATGG, where ATG is the start codon) instead of a Shine-Dalgarno or Ribosome Binding Site sequence, addition of a poly-A tail upstream of eachribozyme sequence or at the end of the mRNA sequence, exchange of the prokaryotic (SEC-dependent) signal peptide for the natural signal peptide for each of the cytokines (i.e., SEQ ID NO: 77 for IL-15, SEQ ID NO: 78 for IL-18, SEQ ID NO: 79 for CXCL-9, or SEQ ID NO: 80 for IL-21), and addition of maintenance and expression sequences (e.g., expression promoters such as CMV or SV40; selection markers such as Neomycin or Hygromycin genes, or viral-derived origins of replication such as EBV).REFERENCES

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[0402] Zhou, T., Damsky, W, Weizman, OE. et al (2020) IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature. 583:609-614.SEQUENCES FORMING PART OF THE DESCRIPTION

[0403] SEQ ID NO: 1 (mature IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0404] SEQ ID NO: 2 (mature IL-18) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRG MAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0405] SEQ ID NO: 3 (mature IL-21) HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS LLQKMIHQHLSSRTHGSEDS

[0406] SEQ ID NO: 4 (parental / M40 IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0407] SEQ ID NO: 5 (GBL01) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISLESGDASI HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0408] SEQ ID NO: 6 (GBL15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQAISLESGDASI HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0409] SEQ ID NO: 7 (GBL17) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISKESGDASI HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0410] SEQ ID NO: 8 (IL-15 GBL18)NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISRESGDASI HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0411] SEQ ID NO: 9 (IL-15 GBL25) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLTELQAISLESGDASI HDTVENLI ILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0412] SEQ ID NO: 10 (IL-15 GBL50) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQAISRESGDASI HDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0413] SEQ ID NO: 11 (IL-15 SAg01) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLSELQVISLESGDASIHDTVENLIILANNSLPSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0414] SEQ ID NO: 12 (IL-15 SAg15) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLSELQAISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0415] SEQ ID NO: 13 (IL-15 SAg17) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLSELQVISKESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0416] SEQ ID NO: 14 (IL-15 SAg18) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISRESGDASIHDTVENLIILANNSLPSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0417] SEQ ID NO: 15 (IL-15 SAg25) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLTELQAISLESGDASIHDTVENLIILANNSLPSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0418] SEQ ID NO: 16 (IL-15 SAg50) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATL YTESDVHPSCKVTAMKCFLLELQAISRESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS

[0419] SEQ ID NO: 17 (exemplary GS linker) SGGGSGGGGSGGGGSGGGGSGGGSLQA SEQ ID NO: 18 (IL-18 V17) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFDISMYKDSQPRG KAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0420] SEQ ID NO: 19 (IL-18 V25) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFDISTYKDSQPRG KAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0421] SEQ ID NO: 20 (IL-18 V27) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFDISTYKDSQPRG KAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDHKMQFESS SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0422] SEQ ID NO: 21 (IL-18 V59)YFGKLESKSSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFDISTYKDSQPRG KAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDHKMQFESS SYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0423] SEQ ID NO: 22 (IL-21 V36) HKSSSQGQDRHMIRMRQLIDIVDQLKNYTNDLVPEKLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS LLQKMIHQHLSSRTHGSEDS

[0424] SEQ ID NO: 23 (IL-21 V52) HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLTPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERKINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS LLQKMIHQHLSSRTHGSEDS

[0425] SEQ ID NO: 24 (IL-21 V58) HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLDPEFLPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERKINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS LLQKMIHQHLSSRTHGSEDS

[0426] SEQ ID NO: 25 (IL-21 V62) HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEELPAPEDVETNCEWSAFSCFQKAQ LKSANTGNNERKINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKS LLQKMIHQHLSSRTHGSEDS

[0427] SEQ ID NO: 26 (CXCL9) TPWRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSAD VKEL I KKWEKQVS QKKKQKNGKKHQKKKVLKVRKS QRSRQKKTT

[0428] SEQ ID NO: 27 (cxcl-9 nucleic acid sequence) ACCCCAGTAGTGAGAAAGGGTCGCTGTTCCTGCATCAGCACCAACCAAGGGACTATCCA CCTACAATCCTTGAAAGACCTTAAACAATTTGCCCCAAGCCCTTCCTGCGAGAAAATTG AAATCATTGCTACACTGAAGAATGGAGTTCAAACATGTCTAAACCCAGATTCAGCAGAT GTGAAGGAACTGATTAAAAAGTGGGAGAAACAGGTCAGCCAAAAGAAAAAGCAAAAGAAT G GGAAAAAACAT C AAAAAAAGAAAG T T C T GAAAGT T C GAAAAT C T CAAC GT T C T C G T C AAAAGAAAAC TACA

[0429] SEQ ID NO: 28 (MalE)

[0430] MK I KT GARI LAL SAL T TMMF S AS ALA

[0431] SEQ ID NO: 29 (PelB)

[0432] MKYLLPTAAAGLLLLAAQPAMA

[0433] SEQ ID NO: 30 (DsbA)

[0434] MKKIWLALAGLVLAFSASA

[0435] SEQ ID NO: 31 (PhoA)

[0436] MKQSTIALALLPLLFTPVTKA

[0437] SEQ ID NO: 32 (OmpA)

[0438] MKKTAIAIAVALAGFATVAQA

[0439] SEQ ID NO: 33 (MC1000) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTATTATGAAAATTAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACG ACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGTGAACGTTATTAGCGACCTGAA AAAAATCGAAGATCTGATCCAGAGCATGCATATTGATGCAACCCTGTATACCGAAAGTG ATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGTTTTCTGAGCGAACTGCAGGTG ATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATACCGTTGAAAACCTGATTATTCT GGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCGAAAGCGGTTGTAAAGAATGTG AAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAGAGCTTCGTGCATATCGTGCAG ATGTTTATTAACACCAGCTAAGAGCCGTATAGTcctgagacggtccactagtcttg

[0440] SEQ ID NO: 34 (MC1200)GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTATTATGAAAATTAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACG ACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGTGAACGTTATTAGCGACCTGAA AAAAATCGAAGATCTGATCCAGAGCATGCATATTGATGCAACCCTGTATACCGAAAGTG ATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGTTTTCTGAGCGAACTGCAGGTG ATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATACCGTTGAAAACCTGATTATTCT GGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCGAAAGCGGTTGTAAAGAATGTG AAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAGAGCTTCGTGCATATCGTGCAG ATGTTTATTAACACCAGCTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGC TGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAAT C T AAAT TAT C AG T C AT AAGAAAT T T GAAT GAC C AAG T T C T C T T CAT T GAC CAAG GAAAT CGGCCTCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAATGCACCCCGGACCAT ATTTGATATAAGTACCTATAAAGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTG T GAAGT GT GAGAAAAT T T CAAC T C T C T C C T GT GAGAACAAAAT T AT T T C C T T T AAGGAA AT GAAT C C T C C T GAT AAC AT CAAG GAT AC AAAAAG T GAC AT CAT AT T C T T T CAGAGAAG TGTCCCAGGACATGATAATAAGATGCAATTTGAATCTTCATCATACGAAGGATACTTTC T AGC T T GT GAAAAAGAGAGG GAC C T T T T T AAAC T CAT T T T GAAAAAAGAG GAT GAAT T G GGGGATAGATCTATAATGTTCACTGTTCAAAACGAGGACTAAGAGCCGTATAGTcctga gacggtccactagtcttg

[0441] SEQ ID NO: 35 (MC1230) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTATTATGAAAATTAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACG ACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGTGAACGTTATTAGCGACCTGAA AAAAATCGAAGATCTGATCCAGAGCATGCATATTGATGCAACCCTGTATACCGAAAGTGATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGTTTTCTGAGCGAACTGCAGGTG ATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATACCGTTGAAAACCTGATTATTCT GGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCGAAAGCGGTTGTAAAGAATGTG AAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAGAGCTTCGTGCATATCGTGCAG ATGTTTATTAACACCAGCTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGC TGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAAT C T AAAT TAT C AG T C AT AAGAAAT T T GAAT GAC C AAG T T C T C T T CAT T GAC CAAG GAAAT CGGCCTCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAATGCACCCCGGACCAT ATTTGATATAAGTACCTATAAAGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTG T GAAGT GT GAGAAAAT T T CAAC T C T C T C C T GT GAGAACAAAAT T AT T T C C T T T AAGGAA AT GAAT C C T C C T GAT AAC AT CAAG GAT AC AAAAAGT GACAT CAT AT T C T T T C AGAGAAG TGTCCCAGGACATGATAATAAGATGCAATTTGAATCTTCATCATACGAAGGATACTTTC T AGC T T GT GAAAAAGAGAG G GAC C T T T T T AAAC T CAT T T T GAAAAAAGAG GAT GAAT T G GGGGATAGATCTATAATGTTCACTGTTCAAAACGAGGACTAAGAGCGTTGCCAAGACGC AGC

[0442] SEQ ID NO: 36 (MC1240) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTATTATGAAAATTAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACG ACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGTGAACGTTATTAGCGACCTGAA AAAAATCGAAGATCTGATCCAGAGCATGCATATTGATGCAACCCTGTATACCGAAAGTG ATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGTTTTCTGAGCGAACTGCAGGTG ATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATACCGTTGAAAACCTGATTATTCT GGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCGAAAGCGGTTGTAAAGAATGTG AAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAGAGCTTCGTGCATATCGTGCAG ATGTTTATTAACACCAGCTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAAT C T AAAT TAT C AG T C AT AAGAAAT T T GAAT GAC C AAG T T C T C T T CAT T GAC CAAG GAAAT CGGCCTCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAATGCACCCCGGACCAT ATTTGATATAAGTACCTATAAAGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTG T GAAG T GT GAGAAAAT T T C AAC T C T C T C C T G T GAGAAC AAAAT T AT T T C C T T T AAGGAA AT GAAT C C T C C T GAT AAC AT CAAG GAT AC AAAAAGT GACAT CAT AT T C T T T C AGAGAAG TGTCCCAGGACATGATAATAAGATGCAATTTGAATCTTCATCATACGAAGGATACTTTC T AGC T T GT GAAAAAGAGAGG GAC C T T T T T AAAC T CAT T T T GAAAAAAGAG GAT GAAT T G GGGGATAGATCTATAATGTTCACTGTTCAAAACGAGGACTAAGAGCGTTGCCAAGACGC AGC

[0443] SEQ ID NO: 37 (MC1320) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTATTATGAAAATTAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACG ACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGTGAACGTTATTAGCGACCTGAA AAAAATCGAAGATCTGATCCAGAGCATGCATATTGATGCAACCCTGTATACCGAAAGTG ATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGTTTTCTGAGCGAACTGCAGGTG ATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATACCGTTGAAAACCTGATTATTCT GGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCGAAAGCGGTTGTAAAGAATGTG AAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAGAGCTTCGTGCATATCGTGCAG ATGTTTATTAACACCAGCTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAGATCGTAGGATGAAAAAGATTTGGCTGGCGCTGGCT GGTTTAGTTTTAGCGTTTAGCGCATCGGCGATGCACAAATCAAGCTCCCAAGGTCAAGA T C GC C ACAT GAT T AGAAT GC G T CAAC T T AT AGAT AT T G T T GAT C AG C T GAAAAAT T AT G TGAATGACTTGACCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAG TGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAA T GAAAGGAAAAT CAAT GTAT CAAT TAAAAAGCT GAAGAGGAAACCACCT T CCACAAAT G CAGGGAGGAGACAGAAACACAGAC T AACAT GC C C T T CAT GT GAT T C T TAT GAGAAAAAA C C AC C C AAAGAAT T C C T AGAAAGAT T CAAAT C AC T T C T C C AAAAGAT GAT T CAT CAG C ATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTAAGAGCGTTGCCAAGACGCAGCAG ATGCTGTAGTGGGATGTGTGTCTCACCTGAAGAGTACAAAAGTCCGAAACGGTATCCTC TACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAAATAGTCCATGAAATACCTGCT GCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCGATGTACT TTGGCAAGCTTGAATCTAAATTATCAGTCATAAGAAATTTGAATGACCAAGTTCTCTTC ATTGACCAAGGAAATCGGCCTCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAA TGCACCCCGGACCATATTTGATATAAGTACCTATAAAGATAGCCAGCCTAGAGGTAAAG CTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAACTCTCTCCTGTGAGAACAAAATT AT T T C C T T T AAG GAAAT GAAT C C T C C T GAT AAC AT C AAGGAT AC AAAAAG T GAC AT CAT AT T C T T T C AGAGAAG T GT C C C AG GACAT GAT AAT AAGAT G CAAT T T GAAT C T T CAT CAT ACGAAGGATACTTTCTAGCTTGTGAAAAAGAGAGGGACCTTTTTAAACTCATTTTGAAA AAAGAGGATGAATTGGGGGATAGATCTATAATGTTCACTGTTCAAAACGAGGACTAAGA GCCGTATAGTcctgagacggtccactagtcttg

[0444] SEQ ID NO: 38 (MC1324) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTATTATGAAAATTAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACG ACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGTGAACGTTATTAGCGACCTGAA AAAAATCGAAGATCTGATCCAGAGCATGCATATTGATGCAACCCTGTATACCGAAAGTG ATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGTTTTCTGAGCGAACTGCAGGTG ATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATACCGTTGAAAACCTGATTATTCT GGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCGAAAGCGGTTGTAAAGAATGTG AAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAGAGCTTCGTGCATATCGTGCAG ATGTTTATTAACACCAGCTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAGATCGTAGGATGAAAAAGATTTGGCTGGCGCTGGCT GGTTTAGTTTTAGCGTTTAGCGCATCGGCGATGCACAAATCAAGCTCCCAAGGTCAAGA T C GC C ACAT GAT T AGAAT GC G T CAAC T T AT AGAT AT T G T T GAT C AG C T GAAAAAT T AT G TGAATGACTTGACCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAG TGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAAT GAAAGGAAAAT CAAT GTAT CAAT TAAAAAGCT GAAGAGGAAACCACCT T CCACAAAT G CAGGGAGGAGACAGAAACACAGAC T AACAT GC C C T T CAT GT GAT T C T TAT GAGAAAAAA C C AC C C AAAGAAT T C C T AGAAAGAT T CAAAT C AC T T C T C C AAAAGAT GAT T CAT CAG C A TCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTAAGAGCGTTGCCAAGACGCAGCAG ATGCTGTAGTGGGATGTGTGTCTCACCTGAAGAGTACAAAAGTCCGAAACGGTATCCTC TACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAAATAGTCCATGAAATACCTGCT GCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCGATGTACT T T GG C AAG C T T GAAT C T AAAT T AT C AGT C AT AAGAAAT T T GAAT GAC C AAGT T C T C T T C ATTGACCAAGGAAATCGGCCTCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAA TGCACCCCGGACCATATTTGATATAAGTACCTATAAAGATAGCCAGCCTAGAGGTAAAG CTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAACTCTCTCCTGTGAGAACAAAATT AT T T C C T T T AAG GAAAT GAAT C C T C C T GAT AACAT C AAGGAT AC AAAAAG T GAC AT CAT AT T C T T T C AGAGAAGT G T C C C AGGAC AT GAT AAT AAGAT G CAAT T T GAAT C T T CAT CAT ACGAAGGATACTTTCTAGCTTGTGAAAAAGAGAGGGACCTTTTTAAACTCATTTTGAAA AAAGAGGAT GAAT T GGGGGATAGAT CTATAAT GT T CACT GTT CAAAACGAGGACTAAGA GCCACTTCATTGCCGGCAAGTCATAAGTCTGGGCTAAGCCCACTGATGAGTCGCTGAAA TGCGACGAAACTTATGACCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAA GATGTTCGTATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTAC CGTAGCGCAGGCCATGACCCCAGTAGTGAGAAAGGGTCGCTGTTCCTGCATCAGCACCA ACCAAGGGACTATCCACCTACAATCCTTGAAAGACCTTAAACAATTTGCCCCAAGCCCT TCCTGCGAGAAAATTGAAATCATTGCTACACTGAAGAATGGAGTTCAAACATGTCTAAA CCCAGATTCAGCAGATGTGAAGGAACTGATTAAAAAGTGGGAGAAACAGGTCAGCCAAA AGAAAAAG C AAAAGAAT G GGAAAAAACAT C AAAAAAAGAAAG T T C T GAAAGT T C GAAAA TCTCAACGTTCTCGTCAAAAGAAAACTACATAAGAGCCGTATAGTcctgagacggtcca ctagtcttg

[0445] SEQ ID NO: 39 (MC1400) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTATTATGAAAATTAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACG ACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGTGAACGTTATTAGCGACCTGAAAAAAATCGAAGATCTGATCCAGAGCATGCATATTGATGCAACCCTGTATACCGAAAGTG ATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGTTTTCTGAGCGAACTGCAGGTG ATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATACCGTTGAAAACCTGATTATTCT GGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCGAAAGCGGTTGTAAAGAATGTG AAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAGAGCTTCGTGCATATCGTGCAG ATGTTTATTAACACCAGCTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT T T AAG C GAC T T T AAAGAG GAGAAGAT GT T C GT AT GAAAAAGACAG C TAT C GC GAT T G C A GTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCATGACCCCAGTAGTGAGAAAGGG TCGCTGTTCCTGCATCAGCACCAACCAAGGGACTATCCACCTACAATCCTTGAAAGACC T T AAAC AAT T T G C C C C AAGC C C T T C C T G C GAGAAAAT T GAAAT C AT T G C T AC AC T GAAG AATGGAGTTCAAACATGTCTAAACCCAGATTCAGCAGATGTGAAGGAACTGATTAAAAA GT GGGAGAAACAGGTCAGCCAAAAGAAAAAGCAAAAGAAT GGGAAAAAACAT CAAAAAA AGAAAGT T C T GAAAGT T C GAAAAT C T CAAC GT T C T C GT CAAAAGAAAAC T ACAT AAGAG CCGTATAGTcctgagacggtccactagtcttg

[0446] SEQ ID NO: 40 (MC2000) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGC CCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAATCTAAATTATCAGTCATAAGAA ATTTGAATGACCAAGTTCTCTTCATTGACCAAGGAAATCGGCCTCTATTTGAAGATATG ACTGATTCTGACTGTAGAGATAATGCACCCCGGACCATATTTGATATAAGTACCTATAA AGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAA CTCTCTCCTGTGAGAACAAAATTATTTCCTTTAAGGAAATGAATCCTCCTGATAACATC AAGGATACAAAAAGTGACAT CATAT T CT T T CAGAGAAGTGT CCCAGGACATGATAATAA GATGCAATTTGAATCTTCATCATACGAAGGATACTTTCTAGCTTGTGAAAAAGAGAGGG ACCTTTTTAAACTCATTTTGAAAAAAGAGGATGAATTGGGGGATAGATCTATAATGTTC ACTGTTCAAAACGAGGACTAAGAGCCGTATAGTcctgagacggtccactagtcttg

[0447] SEQ ID NO: 41 (MC2100)GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGC CCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAATCTAAATTATCAGTCATAAGAA ATTTGAATGACCAAGTTCTCTTCATTGACCAAGGAAATCGGCCTCTATTTGAAGATATG ACTGATTCTGACTGTAGAGATAATGCACCCCGGACCATATTTGATATAAGTACCTATAA AGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAA CTCTCTCCTGTGAGAACAAAATTATTTCCTTTAAGGAAATGAATCCTCCTGATAACATC AAGGATACAAAAAGTGACAT CATAT T CT T T CAGAGAAGTGT CCCAGGACATGATAATAA GATGCAATTTGAATCTTCATCATACGAAGGATACTTTCTAGCTTGTGAAAAAGAGAGGG ACCTTTTTAAACTCATTTTGAAAAAAGAGGATGAATTGGGGGATAGATCTATAATGTTC ACTGTTCAAAACGAGGACTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAGATCGTATTATGAAAATTAAAACAGGTGCACGCATC CTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGT GAACGTTATTAGCGACCTGAAAAAAATCGAAGATCTGATCCAGAGCATGCATATTGATG CAACCCTGTATACCGAAAGTGATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGT TTTCTGAGCGAACTGCAGGTGATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATAC CGTTGAAAACCTGATTATTCTGGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCG AAAGCGGTTGTAAAGAATGTGAAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAG AGCTTCGTGCATATCGTGCAGATGTTTATTAACACCAGCTAAGAGCCGTATAGTcctga gacggtccactagtcttg

[0448] SEQ ID NO: 42 (MC2130) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGC CCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAATCTAAATTATCAGTCATAAGAA ATTTGAATGACCAAGTTCTCTTCATTGACCAAGGAAATCGGCCTCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAATGCACCCCGGACCATATTTGATATAAGTACCTATAA AGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAA CTCTCTCCTGTGAGAACAAAATTATTTCCTTTAAGGAAATGAATCCTCCTGATAACATC AAGGATACAAAAAGTGACAT CATAT T CT T T CAGAGAAGTGT CCCAGGACATGATAATAA GAT G C AAT T T GAAT C T T CAT CAT AC GAAG GAT AC TTTCTAGCTTGT GAAAAAGAGAG G G ACCTTTTTAAACTCATTTTGAAAAAAGAGGATGAATTGGGGGATAGATCTATAATGTTC ACTGTTCAAAACGAGGACTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAGATCGTATTATGAAAATTAAAACAGGTGCACGCATC CTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGT GAACGTTATTAGCGACCTGAAAAAAATCGAAGATCTGATCCAGAGCATGCATATTGATG CAACCCTGTATACCGAAAGTGATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGT TTTCTGAGCGAACTGCAGGTGATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATAC CGTTGAAAACCTGATTATTCTGGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCG AAAGCGGT T GTAAAGAAT GT GAAGAACT GGAAGAGAAAAACAT CAAAGAAT TT CT GCAG AGCTTCGTGCATATCGTGCAGATGTTTATTAACACCAGCTAAGAGCGTTGCCAAGACGC AGCAGATGCTGTAGTGGGATGTGTGTCTCACCTGAAGAGTACAAAAGTCCGAAACGGTA TCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAAGATCGTAGGATGAAAA AGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGATGCACAAA TCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGT TGATCAGCTGAAAAATTATGTGAATGACTTGACCCCTGAATTTCTGCCAGCTCCAGAAG ATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAG T CAGCAAATACAGGAAACAAT GAAAGGAAAAT CAAT GTAT CAAT TAAAAAGCT GAAGAG GAAAC CAC C T T C CACAAAT GCAGGGAGGAGACAGAAACACAGAC T AACAT GC C C T T CAT G T GAT T C T T AT GAGAAAAAAC CAC C CAAAGAAT T C C T AGAAAGAT T C AAAT CAC T T C T C CAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTAAGA GCCGTATAGTcctgagacggtccactagtcttg

[0449] SEQ ID NO: 43 (MC2134) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGC CCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAATCTAAATTATCAGTCATAAGAA ATTTGAATGACCAAGTTCTCTTCATTGACCAAGGAAATCGGCCTCTATTTGAAGATATG ACTGATTCTGACTGTAGAGATAATGCACCCCGGACCATATTTGATATAAGTACCTATAA AGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAA CTCTCTCCTGTGAGAACAAAATTATTTCCTTTAAGGAAATGAATCCTCCTGATAACATC AAGGATACAAAAAGTGACAT CATAT T CT T T CAGAGAAGTGT CCCAGGACATGATAATAA GATGCAATTTGAATCTTCATCATACGAAGGATACTTTCTAGCTTGTGAAAAAGAGAGGG ACCTTTTTAAACTCATTTTGAAAAAAGAGGATGAATTGGGGGATAGATCTATAATGTTC ACTGTTCAAAACGAGGACTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAGATCGTATTATGAAAATTAAAACAGGTGCACGCATC CTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGT GAACGTTATTAGCGACCTGAAAAAAATCGAAGATCTGATCCAGAGCATGCATATTGATG CAACCCTGTATACCGAAAGTGATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGT TTTCTGAGCGAACTGCAGGTGATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATAC CGTTGAAAACCTGATTATTCTGGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCG AAAGCGGTTGTAAAGAATGTGAAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAG AGCTTCGTGCATATCGTGCAGATGTTTATTAACACCAGCTAAGAGCGTTGCCAAGACGC AGCAGATGCTGTAGTGGGATGTGTGTCTCACCTGAAGAGTACAAAAGTCCGAAACGGTA TCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAAGATCGTAGGATGAAAA AGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGATGCACAAA TCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGT TGATCAGCTGAAAAATTATGTGAATGACTTGACCCCTGAATTTCTGCCAGCTCCAGAAG ATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAG T CAGCAAATACAGGAAACAAT GAAAGGAAAAT CAAT GTAT CAAT TAAAAAGCT GAAGAG GAAAC CAC C T T C CACAAAT GCAGGGAGGAGACAGAAACACAGAC T AACAT GC C C T T CAT G T GAT T C T T AT GAGAAAAAAC CAC C C AAAGAAT T C C T AGAAAGAT T CAAAT CAC T T C T C CAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTAAGA GCCACTTCATTGCCGGCAAGTCATAAGTCTGGGCTAAGCCCACTGATGAGTCGCTGAAA TGCGACGAAACTTATGACCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAA GATGTTCGTATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTAC CGTAGCGCAGGCCATGACCCCAGTAGTGAGAAAGGGTCGCTGTTCCTGCATCAGCACCAAC CAAG GGAC TAT C CAC C T AC AAT C C T T GAAAGAC C T T AAAC AAT T T G C C C C AAGC C C T TCCTGCGAGAAAATTGAAATCATTGCTACACTGAAGAATGGAGTTCAAACATGTCTAAA CCCAGATTCAGCAGATGTGAAGGAACTGATTAAAAAGTGGGAGAAACAGGTCAGCCAAA AGAAAAAG C AAAAGAAT G GGAAAAAACAT C AAAAAAAGAAAG T T C T GAAAGT T C GAAAA TCTCAACGTTCTCGTCAAAAGAAAACTACATAAGAGCCGTATAGTcctgagacggtcca ctagtcttg

[0450] SEQ ID NO: 44 (MC2140) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGC CCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAATCTAAATTATCAGTCATAAGAA AT T T GAAT GAC CAAG T T C T C T T C AT T GAC CAAGGAAAT C G GC C T C T AT T T GAAGAT AT G ACTGATTCTGACTGTAGAGATAATGCACCCCGGACCATATTTGATATAAGTACCTATAA AGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAA CTCTCTCCTGTGAGAACAAAATTATTTCCTTTAAGGAAATGAATCCTCCTGATAACATC AAGGATACAAAAAGTGACAT CATAT T CT T T CAGAGAAGTGT CCCAGGACATGATAATAA GAT G C AAT T T GAAT C T T CAT CAT AC GAAG GAT AC TTTCTAGCTTGT GAAAAAGAGAG G G ACCTTTTTAAACTCATTTTGAAAAAAGAGGATGAATTGGGGGATAGATCTATAATGTTC ACTGTTCAAAACGAGGACTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAGATCGTATTATGAAAATTAAAACAGGTGCACGCATC CTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGT GAACGTTATTAGCGACCTGAAAAAAATCGAAGATCTGATCCAGAGCATGCATATTGATG CAACCCTGTATACCGAAAGTGATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGT TTTCTGAGCGAACTGCAGGTGATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATAC CGTTGAAAACCTGATTATTCTGGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCG AAAGCGGT T GTAAAGAAT GT GAAGAACT GGAAGAGAAAAACAT CAAAGAAT TT CT GCAG AGCTTCGTGCATATCGTGCAGATGTTTATTAACACCAGCTAAGAGCGTTGCCAAGACGC AGCAGATGCTGTAGTGGGATGTGTGTCTCACCTGAAGAGTACAAAAGTCCGAAACGGTA T C C T C T AC AAAT AAT T T T GT T T AAG C GAC T T T AAAGAG GAGAAGAT GT T C GT AT GAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCATG ACCCCAGTAGTGAGAAAGGGTCGCTGTTCCTGCATCAGCACCAACCAAGGGACTATCCA CCTACAATCCTTGAAAGACCTTAAACAATTTGCCCCAAGCCCTTCCTGCGAGAAAATTG AAATCATTGCTACACTGAAGAATGGAGTTCAAACATGTCTAAACCCAGATTCAGCAGAT GT GAAGGAACT GAT TAAAAAGTGGGAGAAACAGGT CAGCCAAAAGAAAAAGCAAAAGAA T G GGAAAAAACAT C AAAAAAAGAAAG T T C T GAAAGT T C GAAAAT C T CAAC GT T C T C G T C AAAAGAAAACTACATAAGAGCCGTATAGTcctgagacggtccactagtcttg

[0451] SEQ ID NO: 45 (MC2314) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGC CCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAATCTAAATTATCAGTCATAAGAA ATTTGAATGACCAAGTTCTCTTCATTGACCAAGGAAATCGGCCTCTATTTGAAGATATG ACTGATTCTGACTGTAGAGATAATGCACCCCGGACCATATTTGATATAAGTACCTATAA AGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAA CTCTCTCCTGTGAGAACAAAATTATTTCCTTTAAGGAAATGAATCCTCCTGATAACATC AAGGAT AC AAAAAG T GAC AT CAT AT T C T T T C AGAGAAG T G T C C C AG GAC AT GAT AAT AA GATGCAATTTGAATCTTCATCATACGAAGGATACTTTCTAGCTTGTGAAAAAGAGAGGG ACCTTTTTAAACTCATTTTGAAAAAAGAGGATGAATTGGGGGATAGATCTATAATGTTC ACTGTTCAAAACGAGGACTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGG ATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGT TTAAGCGACTTTAAAGAGGAGAAGATCGTAGGATGAAAAAGATTTGGCTGGCGCTGGCT GGTTTAGTTTTAGCGTTTAGCGCATCGGCGATGCACAAATCAAGCTCCCAAGGTCAAGA T C GC C ACAT GAT T AGAAT GC G T CAAC T T AT AGAT AT T G T T GAT C AG C T GAAAAAT T AT G TGAATGACTTGACCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAG TGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAA T GAAAG GAAAAT C AAT GT AT C AAT T AAAAAG C T GAAGAG GAAAC CAC C T T C CAC AAAT G CAGGGAGGAGACAGAAACACAGAC T AACAT GC C C T T CAT GT GAT T C T TAT GAGAAAAAA C CAC C C AAAGAAT T C C T AGAAAGAT T CAAAT CAC T T C T C C AAAAGAT GAT T CAT CAG C A TCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTAAGAGCGTTGCCAAGACGCAGCAGATGCTGTAGTGGGATGTGTGTCTCACCTGAAGAGTACAAAAGTCCGAAACGGTATCCTC T ACAAAT AAT T T T G T T T AAG C GAC T T T AAAGAG GAGAAGAT C GT AT TAT GAAAAT T AAA ACAGGTGCACGCATCCTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGC TCTCGCCAATTGGGTGAACGTTATTAGCGACCTGAAAAAAATCGAAGATCTGATCCAGA GCATGCATATTGATGCAACCCTGTATACCGAAAGTGATGTTCATCCGAGCTGTAAAGTT ACCGCCATGAAATGTTTTCTGAGCGAACTGCAGGTGATTAGCCGTGAAAGCGGTGATGC AAGCATTCATGATACCGTTGAAAACCTGATTATTCTGGCCAACAATTCCCTGCCGAGCA ATGGTAATGTTACCGAAAGCGGTTGTAAAGAATGTGAAGAACTGGAAGAGAAAAACATC AAAGAATTTCTGCAGAGCTTCGTGCATATCGTGCAGATGTTTATTAACACCAGCTAAGA GCCACTTCATTGCCGGCAAGTCATAAGTCTGGGCTAAGCCCACTGATGAGTCGCTGAAA TGCGACGAAACTTATGACCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAA GATGTTCGTATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTAC CGTAGCGCAGGCCATGACCCCAGTAGTGAGAAAGGGTCGCTGTTCCTGCATCAGCACCA AC CAAG GGAC TAT C CAC C T AC AAT C C T T GAAAGAC C T T AAAC AAT T T G C C C C AAGC C C T TCCTGCGAGAAAATTGAAATCATTGCTACACTGAAGAATGGAGTTCAAACATGTCTAAA CCCAGATTCAGCAGATGTGAAGGAACTGATTAAAAAGTGGGAGAAACAGGTCAGCCAAA AGAAAAAG C AAAAGAAT G GGAAAAAACAT C AAAAAAAGAAAG T T C T GAAAGT T C GAAAA TCTCAACGTTCTCGTCAAAAGAAAACTACATAAGAGCCGTATAGTcctgagacggtcca ctagtcttg

[0452] SEQ ID NO: 46 (MC3120) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTAGGATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGC GCATCGGCGATGCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCG T C AAC T T AT AGAT AT T G T T GAT CAG C T GAAAAAT TAT G T GAAT GAC T T GAC C C C T GAAT TTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTT CAGAAGGCCCAACTAAAGT CAGCAAATACAGGAAACAAT GAAAGGAAAAT CAAT GTAT C AATTAAAAAGCT GAAGAGGAAACCACCT T CCACAAAT GCAGGGAGGAGACAGAAACACA GACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAA AGAT T C AAAT CAC T T C T C CAAAAGAT GAT T CAT CAG CAT CTGTCCTC T AGAAC ACAC G GAAGTGAAGATTCCTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGGATGTG TATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGTTTAAG CGACTTTAAAGAGGAGAAGATCGTATTATGAAAATTAAAACAGGTGCACGCATCCTCGC ATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAATTGGGTGAACG TTATTAGCGACCTGAAAAAAATCGAAGATCTGATCCAGAGCATGCATATTGATGCAACC CTGTATACCGAAAGTGATGTTCATCCGAGCTGTAAAGTTACCGCCATGAAATGTTTTCT GAGCGAACTGCAGGTGATTAGCCGTGAAAGCGGTGATGCAAGCATTCATGATACCGTTG AAAACCTGATTATTCTGGCCAACAATTCCCTGCCGAGCAATGGTAATGTTACCGAAAGC GGTTGTAAAGAATGTGAAGAACTGGAAGAGAAAAACATCAAAGAATTTCTGCAGAGCTT CGTGCATATCGTGCAGATGTTTATTAACACCAGCTAAGAGCGTTGCCAAGACGCAGCAG ATGCTGTAGTGGGATGTGTGTCTCACCTGAAGAGTACAAAAGTCCGAAACGGTATCCTC TACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAGAAATAGTCCATGAAATACCTGCT GCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCGATGTACT T T GG C AAG C T T GAAT C T AAAT T AT C AGT C AT AAGAAAT T T GAAT GAC C AAGT T C T C T T C ATTGACCAAGGAAATCGGCCTCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAA TGCACCCCGGACCATATTTGATATAAGTACCTATAAAGATAGCCAGCCTAGAGGTAAAG CTGTAACTATCTCTGTGAAGTGTGAGAAAATTTCAACTCTCTCCTGTGAGAACAAAATT AT T T C C T T T AAG GAAAT GAAT C C T C C T GAT AAC AT C AAGGAT AC AAAAAG T GAC AT CAT AT T C T T T C AGAGAAGT G T C C C AGGAC AT GAT AAT AAGAT G C AAT T T GAAT C T T CAT CAT ACGAAGGATACTTTCTAGCTTGTGAAAAAGAGAGGGACCTTTTTAAACTCATTTTGAAA AAAGAGGATGAATTGGGGGATAGATCTATAATGTTCACTGTTCAAAACGAGGACTAAGA GCCGTATAGTcctgagacggtccactagtcttg

[0453] SEQ ID NO: 47 (MC3124) GCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGA TAACTTTACGGGCATGCATAAGGCTCGTAAGAtATATTCAGGCACAGCACAACGGTTTC CTTTtcgtctcactcgAATAGTATTCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGCGACTTTAAAGAGGAG AAGATCGTAGGATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGC GCATCGGCGATGCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCG T C AAC T T AT AGAT AT T G T T GAT CAG C T GAAAAAT TAT G T GAAT GAC T T GAC C C C T GAAT TTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTT C AGAAG GC C C AAC T AAAG T CAG CAAAT AC AGGAAAC AAT GAAAG GAAAAT CAAT GT AT CAATTAAAAAGCT GAAGAGGAAACCACCT T CCACAAAT GCAGGGAGGAGACAGAAACACA GACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAA AGAT T C AAAT CAC T T C T C CAAAAGAT GAT T CAT C AG CAT CTGTCCTC T AGAAC ACAC G G AAGTGAAGATTCCTAAGAGCCCCGTCACGCTCCTACGCCAGCAGTCCGTAGTGGATGTG TATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGTTTAAG CGACTTTAAAGAGGAGAAATAGTCCATGAAATACCTGCTGCCGACCGCTGCTGCTGGTC TGCTGCTCCTCGCTGCCCAGCCGGCGATGGCGATGTACTTTGGCAAGCTTGAATCTAAA TTATCAGTCATAAGAAATTTGAATGACCAAGTTCTCTTCATTGACCAAGGAAATCGGCC TCTATTTGAAGATATGACTGATTCTGACTGTAGAGATAATGCACCCCGGACCATATTTG ATATAAGTACCTATAAAGATAGCCAGCCTAGAGGTAAAGCTGTAACTATCTCTGTGAAG T G T GAGAAAAT T T C AAC T C T C T C C T G T GAGAAC AAAAT T AT T T C C T T T AAGGAAAT GAA TCCTCCTGATAACATCAAGGATACAAAAAGTGACATCATATTCTTTCAGAGAAGTGTCC C AGGAC AT GAT AAT AAGAT G C AAT T T GAAT C T T CAT CAT AC GAAG GAT AC T T T C TAG C T TGTGAAAAAGAGAGGGACCTTTTTAAACTCATTTTGAAAAAAGAGGATGAAT...

Claims

CLAIMS1. A nucleic acid construct comprising polynucleotide sequences encoding two or more of:(i) an interleukin-15 (IL-15) molecule;(ii) an interleukin-18 (IL-18) molecule;(iii) an interleukin-21 (IL-21) molecule; and / or(iv) a CXCL9 molecule.

2. The nucleic acid construct according to claim 1 , wherein the nucleic acid construct comprises polynucleotide sequences encoding three or more of:(i) an interleukin-15 (IL-15) molecule;(ii) an interleukin-18 (IL-18) molecule;(iii) an interleukin-21 (IL-21) molecule; and / or(iv) a CXCL9 molecule3. The nucleic acid construct according to claim 1 or 2, wherein the nucleic acid construct comprises polynucleotide sequences encoding an IL-15 molecule and any of: an IL-18 molecule, an IL-21 molecule and a CXCL9 molecule.

4. The nucleic acid construct according to any of claims 1 to 3, wherein the interleukin molecules are wild-type or interleukin variants, and wherein the nucleic acid construct comprises at least one interleukin variant.

5. The nucleic acid construct according to claim 4, wherein in comparison to wild-type interleukins, the interleukin variants comprise one or more amino acid modifications in a region adjacent to a receptor interaction surface of the interleukin variants.

6. The nucleic acid construct according to claim 5, wherein in comparison to wild-type IL-15, an IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to an interleukin-15 receptor alpha (IL-15Ra) interaction surface.

7. The nucleic acid construct according to any of claims 4 to 6, wherein in comparison to wild-type IL-18, an IL-18 variant preferentially binds an IL-18 receptor over an IL-18 binding protein (IL-18BP).

8. The nucleic acid construct according to any of claims 4 to 7 , wherein wild-type IL-15 comprises an amino acid sequence according to SEQ ID NO: 1;wherein wild-type IL-18 comprises an amino acid sequence according to SEQ ID NO: 2; orwherein wild-type IL-21 comprises an amino acid sequence according to SEQ ID NO: 3.

9. The nucleic acid construct according to claim 8,wherein, when the nucleic acid construct comprises a polynucleotide sequence encoding an IL-15 variant, in comparison to SEQ ID NO: 1, the amino acid modifications occur at one or more of amino acid positions 45 and / or 49 and / or 52;wherein, when the nucleic acid construct comprises a polynucleotide sequence encoding an IL-18 variant, in comparison to SEQ ID NO: 2, the amino acid modifications occur at one or more of amino acid positions (i) 48; and (ii) 51 and / or 60; orwherein, when the nucleic acid construct comprises a polynucleotide sequence encoding an IL-21 variant, in comparison to SEQ ID NO: 3, the amino acid modifications occur at one or more of amino acid positions 29, 33, 36 and / or 71.

10. The nucleic acid construct according to claim 9, wherein in comparison to SEQ ID NO: 1, the IL-15 variant further comprises an amino acid modification atamino acid position 75, preferably wherein the amino acid modification at amino acid position 75 is a serine-to-proline substitution.

11. The nucleic acid construct according to claim 9 or 10, wherein in comparison to SEQ ID NO: 2, the IL-18 variant further comprises an amino acid modification at one or more of amino acid positions 9, 46, 80, 105, 110, and / or 111.

12. The nucleic acid construct according to any of claims 9 to 11 , wherein in comparison to SEQ ID NO: 3, the IL-21 variant further comprises amino acid modifications at amino acid positions 13 and / or 74, preferably wherein the IL-21 variant comprises an amino acid modification at amino acid positions 29 and 36; 33 and 71; or 36 and 71.

13. The nucleic acid construct according to any of claims 10 to 12 , wherein the amino acid modification at amino acid position 45 of SEQ ID NO: 1 is a hydrophobic-to-hydrophilic substitution.

14. The nucleic acid construct according to claim 13, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 45 of SEQ ID NO: 1 is a leucine-to-serine substitution or a leucine-to-threonine substitution.

15. The nucleic acid construct according to any of claims 9 to 14, wherein the amino acid modification at amino acid position 49 of SEQ ID NO: 1 is a hydrophobic-to-hydrophobic substitution.

16. The nucleic acid construct according to claim 15, wherein the hydrophobic-to-hydrophobic substitution at amino acid position 49 of SEQ ID NO: 1 is a valine-to-alanine substitution.

17. The nucleic acid construct according to any of claims 9 to 16, wherein the amino acid modification at amino acid position 52 of SEQ ID NO: 1 is a hydrophobic-to-hydrophilic substitution.

18. The nucleic acid construct according to claim 17, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 52 of SEQ ID NO: 1 is a leucine-to-lysine substitution or a leucine-to-arginine substitution.

19. The nucleic acid construct according to claim 10, wherein the IL-15 variant comprises a 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 modification at amino acid position 75.

20. The nucleic acid construct according to claim 19, wherein the IL-15 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, wherein the % sequence identity to SEQ ID NO: 4 retains the amino acid modification at amino acid position 75.

21. The nucleic acid construct according to any of claims 9 to 20, wherein the IL-15 variant comprisesi) the leucine-to-serine substitution at amino acid position 45; andii) the serine-to-proline substitution at amino acid position 75.

22. The nucleic acid construct according to claim 21, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45 and 75.

23. The nucleic acid construct according to claim 22, wherein the IL-15 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, wherein the % sequence identity to SEQ ID NO: 5 retains the amino acid modification at amino acid position 45 and 75.

24. The nucleic acid construct according to any of claims 9 to 23, wherein the IL-15 variant comprises:i) the leucine-to-serine substitution at amino acid position 45; andii) the valine-to-alanine substitution at amino acid position 49; andiii) the serine-to-proline substitution at amino acid position 75.

25. The nucleic acid construct according to claim 24, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

26. The nucleic acid construct according to claim 25, wherein the IL-15 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49 and 75.

27. The nucleic acid construct according to any of claims 9 to 26, wherein the IL-15 variant comprises:i) the leucine-to-serine substitution at amino acid position 45; andii) the leucine-to-lysine substitution at amino acid position 52; andiii) the serine-to-proline substitution at amino acid position 75.

28. The nucleic acid construct according to claim 27, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 45, 52 and 75.

29. The nucleic acid construct according to claim 28, wherein the IL-15 variant comprises an amino acid sequence comprising at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 7, wherein the % sequence identity to SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 45, 52 and 75.

30. The nucleic acid construct according to any of claims 9 to29, wherein the IL-15 variant comprisesi) the leucine-to-serine substitution at amino acid position 45; andii) the leucine-to-arginine substitution at amino acid position 52; andiii) the serine-to-proline substitution at amino acid position 75.

31. The nucleic acid construct according to claim 30, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 8, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 8, wherein the % sequence identity to SEQ ID NO: 8 retains the amino acid modifications at amino acid positions 45, 52 and 75.

32. The nucleic acid construct according to claim 31, wherein the IL-15 variant comprises an amino acid sequence comprising at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 8, wherein the % sequence identity to SEQ ID NO: 8 retains the amino acid modifications at amino acid positions 45, 52 and 75.

33. The nucleic acid construct according to any of claims 9 to 32, wherein the IL-15 variant comprises:i) the leucine-to-threonine substitution at amino acid position 45; andii) the valine-to-alanine substitution at amino acid position 49; andiii) the serine-to-proline substitution at amino acid position 75.

34. The nucleic acid construct according to claim 33, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 9, wherein the % sequence identity to SEQ ID NO: 9 retains the amino acid modifications at amino acid positions 45, 49 and 75.

35. The nucleic acid construct according to claim 34, wherein the IL-15 variant comprises an amino acid sequence comprising at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 29, wherein the % sequence identity to SEQ ID NO: 29 retains the amino acid modifications at amino acid positions 45, 49 and 75.

36. The nucleic acid construct according to any of claims 9 to 35, wherein the IL-15 variant comprises:i) the valine-to-alanine substitution at amino acid position 49; andii) the leucine-to-arginine substitution at amino acid position 52; andiii) the serine-to-proline substitution at amino acid position 75.

37. The nucleic acid construct according to claim 36, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 10, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 10, wherein the % sequence identity to SEQ ID NO: 10 retains the amino acid modifications at amino acid positions 49, 52 and 75.

38. The nucleic acid construct according to claim 37, wherein the IL-15 variant comprises an amino acid sequence comprising at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 10, wherein the % sequence identity to SEQ ID NO: 10 retains the amino acid modifications at amino acid positions 49, 52 and 75.

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

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

41. The nucleic acid construct according to claim 39 or 40, wherein the IL-15 variant is fused to the IL-15Ra molecule or the sushi domain of the IL-15Ra molecule via a linker, preferably wherein the linker is a glycine-serine linker.

42. The nucleic acid construct according to claim 41, wherein the linker comprises a sequence according to SEQ ID NO: 17, or a sequence comprising at least 70% identity to SEQ ID NO: 17.

43. The nucleic acid construct according to any of claims 39 to 42, wherein the IL-15 variant comprises a sequence according to any of SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16, or a sequence comprising at least 70% identity to SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16, retains the amino acid modifications at the relevant amino acid positions.

44. The nucleic acid construct according to claim 43, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 11, 12, 13, 14, 15, and / or 16, wherein the % sequence identity to any one of SEQ I D NOs: 11, 12, 13, 14, 15, and / or 16, retains the amino acid modifications at the relevant amino acid positions.

45. The nucleic acid construct according to any of claims 9 to 44, wherein the amino acid modification at amino acid position 48 of SEQ ID NO: 2 is a hydrophobic-to-hydrophilic substitution.

46. The nucleic acid construct according to claim 45, wherein the amino acid modification at amino acid position 48 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

47. The nucleic acid construct according to claim 46, wherein the amino acid modification at amino acid position 48 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution.

48. The nucleic acid construct according to any of claims 9 to 47, wherein the amino acid modification at amino acid position 51 of SEQ ID NO: 2 involves substitution of a wild-type amino acid for a polar amino acid.

49. The nucleic acid construct according to claim 48, wherein the amino acid modification at amino acid position 51 of SEQ ID NO: 2 is a methionine-to-lysine substitution or a methionine-to-threonine substitution.

50. The nucleic acid construct according to claim 49, wherein the amino acid modification at amino acid position 51 of SEQ ID NO: 2 is a methionine-to-threonine substitution.

51. The nucleic acid construct according to any of claims 9 to 50, wherein the amino acid modification at amino acid position 60 of SEQ ID NO: 2 involves substitution of a wild-type amino acid for a large amino acid.

52. The nucleic acid construct according to claim 51 , wherein the amino acid modification at amino acid position 60 of SEQ ID NO: 2 is a methionine-to-lysine substitution or a methionine-to-leucine substitution.

53. The nucleic acid construct according to claim 52, wherein the amino acid modification at amino acid position 60 of SEQ ID NO: 2 is a methionine-to-lysine substitution.

54. The nucleic acid construct according to any of claims 12 to 54, wherein the amino acid modification at amino acid positions 9, 46, and / or 80 of SEQ ID NO: 2 is a hydrophobic-to-hydrophilic substitution.

55. The nucleic acid construct according to claim 54, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 9 of SEQ ID NO: 2 is a leucine-to-serine substitution or a leucine-to-aspartate substitution.

56. The nucleic acid construct according to claim 55, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 9 of SEQ ID NO: 2 is a leucine-to-serine substitution.

57. The nucleic acid construct according to any of claims 54 to 56, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 46 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

58. The nucleic acid construct according to claim 57, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 46 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution.

59. The nucleic acid construct according to any of claims 54 to 58, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 80 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

60. The nucleic acid construct according to claim 59, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 80 of SEQ ID NO: 2 is an isoleucine-to-aspartate substitution.

61. The nucleic acid construct according to any of claims 11 to 60, wherein the amino acid modification at amino acid position 105 of SEQ ID NO: 2 involves substitution of a wild-type amino acid for a polar amino acid.

62. The nucleic acid construct according to claim 61 , wherein the amino acid modification at amino acid position 105 of SEQ ID NO: 2 is a serine-to-aspartate substitution or a serine-to-asparagine substitution.

63. The nucleic acid construct according to claim 62, wherein the amino acid modification at amino acid position 105 of SEQ ID NO: 2 is a serine-to-aspartate substitution.

64. The nucleic acid construct according to any of claims 11 to 63, wherein the amino acid modification at amino acid position 110 of SEQ ID NO: 2 involves substitution of a wild-type amino acid for (a) a polar amino acid, or (b) a neutral amino acid.

65. The nucleic acid construct according to claim 64, wherein the amino acid modification at amino acid position 110 of SEQ ID NO: 2 is (a) an aspartate-to-serine substitution or an aspartate-to-lysine substitution, or (b) an aspartate-to-glycine substitution.

66. The nucleic acid construct according to claim 65, wherein the amino acid modification at amino acid position 110 of SEQ ID NO: 2 is an aspartate-to-lysine substitution.

67. The nucleic acid construct according to any of claims 11 to 66, wherein the amino acid modification at amino acid position 111 of SEQ ID NO: 2 involves substitution of a wild-type amino acid for (a) a positively charged amino acid, or (b) a neutral amino acid.

68. The nucleic acid construct according to claim 67, wherein the amino acid modification at amino acid position 111 of SEQ ID NO: 2 is (a) an asparagine-to-arginine substitution or an asparagine-to-histidine substitution, or (b) an asparagine-to-glycine substitution.

69. The nucleic acid construct according to claim 68, wherein the amino acid modification at amino acid position 111 of SEQ ID NO: 2 is an asparagine-to-histidine substitution.

70. The nucleic acid construct according to any of claims 9 to 69, wherein the IL-18 variant comprisesi) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; andii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60,preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48 and the methionine-to-lysine substitution at amino acid position 60.

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

72. The nucleic acid construct according to claim 71, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 18, wherein the % sequence identity to SEQ ID NO: 18 retains the amino acid modifications at amino acid positions 48 and 51.

73. The nucleic acid construct according to any of claims 9 to 72, wherein the IL-18 variant comprises an amino acid mutation at amino acid positions 48, 51 and 60.

74. The nucleic acid construct according to claim 73, wherein the IL-18 variant comprises:i) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; andii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51; andiii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60,preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51, and the methionine-to-lysine substitution at amino acid position 60.

75. The nucleic acid construct according to claim 74, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 19, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 19, wherein the % sequence identity to SEQ ID NO: 19 retains the amino acid modifications at amino acid positions 48, 51, and 60.

76. The nucleic acid construct according to claim 75, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 19, wherein the % sequence identity to SEQ ID NO: 19 retains the amino acid modifications at amino acid positions 48, 51, and 60.

77. The nucleic acid construct according to any of claims 9 or 11 to 76, wherein the IL-18 variant comprises an amino acid mutation at amino acid positions 48, 51 , 60 and 111.

78. The nucleic acid construct according to claim 77, wherein the IL-18 variant comprises:i) the isoleucine-to-aspartate or isoleucine-to-lysine substitution at amino acid position 48; andii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51; andiii) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; andiv) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111,preferably wherein the IL-18 variant comprises the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , the methionine-to-lysine substitution at amino acid position 60, and the asparagine-to-histidine substitution at amino acid position 111.

79. The nucleic acid construct according to claim 78, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 20, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 20, wherein the % sequence identity to SEQ ID NO: 20 retains the amino acid modifications at amino acid positions 48, 51, 60, and 111.

80. The nucleic acid construct according to claim 79, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 20, wherein the % sequence identity to SEQ ID NO: 20 retains the amino acid modifications at amino acid positions 48, 51, 60, and 111.

81. The nucleic acid construct according to any of claims 9 or 11 to 80, wherein the IL-18 variant comprises:i) the leucine-to-serine substitution or leucine-to-aspartate substitution at amino acid position 9; andii) the isoleucine-to-aspartate substitution or isoleucine-to-lysine substitution at amino acid position 48; andiii) the methionine-to-lysine substitution or methionine-to-threonine substitution at amino acid position 51; andiv) the methionine-to-lysine substitution or methionine-to-leucine substitution at amino acid position 60; andv) the asparagine-to-arginine substitution or asparagine-to-histidine substitution, or asparagine-to-glycine substitution at amino acid position 111,preferably wherein the IL-18 variant comprises the leucine-to-serine substitution at amino acid position 9, the isoleucine-to-aspartate substitution at amino acid position 48, the methionine-to-threonine substitution at amino acid position 51 , the methionine-to-lysine substitution at amino acid position 60, and the asparagine-to-histidine substitution at amino acid position 111.

82. The nucleic acid construct according to claim 81, wherein the IL-18 variant comprises an amino acid sequence according to SEQ ID NO: 21, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 21, wherein the % sequence identity to SEQ ID NO: 21 retains the amino acid modifications at amino acid positions 9, 48, 51, 60, and 111.

83. The nucleic acid construct according to claim 82, wherein the IL-18 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 21, wherein the % sequence identity to SEQ ID NO: 21 retains the amino acid modifications at amino acid positions 9, 48, 51, 60, and 111.

84. The nucleic acid construct according to claims 9 or 12, wherein the amino acid modification at amino acid positions 13, 29, 33, 36, 71, and / or 74 of SEQ ID NO: 3 is a hydrophobic-to-hydrophilic substitution.

85. The nucleic acid construct according to claim 84, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 29 of SEQ ID NO: 3 is a valine-to-threonine substitution.

86. The nucleic acid construct according to any of claims 84 to 85, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 33 of SEQ ID NO: 3 is valine-to-threonine substitution or a valine-to-aspartate substitution.

87. The nucleic acid construct according to any of claims 84 to 86, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 36 of SEQ IDNO: 3 is a phenylalanine-to-glutamine substitution or a phenylalanine-to-lysine substitution.

88. The nucleic acid construct according to claim 87, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 36 of SEQ ID NO: 3 is a phenylalanine-to-lysine substitution.

89. The nucleic acid construct according to any of claims 84 to 88, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 71 of SEQ ID NO: 3 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

90. The nucleic acid construct according to claim 89, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 71 of SEQ ID NO: 3 is an isoleucine-to-lysine substitution.

91. The nucleic acid construct according to any of claims 84 to 90, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 13 of SEQ ID NO: 3 is an isoleucine-to-aspartate substitution or an isoleucine-to-lysine substitution.

92. The nucleic acid construct according to claim 91, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 13 of SEQ ID NO: 3 is an isoleucine-to-aspartate substitution.

93. The nucleic acid construct according to any of claims 84 to 92, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 29 of SEQ ID NO: 3 is a valine-to-threonine substitution or a valine-to-aspartate substitution.

94. The nucleic acid construct according to any of claims 84 to 93, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 74 of SEQ ID NO: 3 is a valine-to-threonine substitution or a valine-to-aspartate substitution.

95. The nucleic acid construct according to claim 94, wherein the hydrophobic-to-hydrophilic substitution at amino acid position 74 is a valine-to-threonine substitution.

96. The nucleic acid construct according to any of claims 9 or 12 to 95, wherein the IL-21 variant comprises:i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 29; andii) 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.

97. The nucleic acid construct according to claim 96, wherein the IL-21 variant comprises an amino acid sequence according to SEQ ID NO: 22, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 22, wherein the % sequence identity to SEQ ID NO: 22 retains the amino acid modifications at amino acid positions 29 and 36.

98. The nucleic acid construct according to claim 97, 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: 22, wherein the % sequence identity to SEQ ID NO: 22 retains the amino acid modifications at amino acid positions 29 and 36.

99. The nucleic acid construct according to any of claims 9 or 12 to 98, wherein the IL-21 variant comprises:i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33; andii) 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.

100. The nucleic acid construct according to claim 99, wherein the IL-21 variant comprises an amino acid sequence according to SEQ ID NO: 23, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 23, wherein the % sequence identity to SEQ ID NO: 23 retains the amino acid modifications at amino acid positions 33 and 71.

101. The nucleic acid construct according to claim 100, 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: 23, wherein the % sequence identity to SEQ ID NO: 23 retains the amino acid modifications at amino acid positions 33 and 71.

102. The nucleic acid construct according to any of claims 9 or 12 to 101, wherein the IL-21 variant comprises:i) the valine-to-threonine substitution or valine-to-aspartate substitution at amino acid position 33; andii) 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.

103. The nucleic acid construct according to claim 102, wherein the IL-21 variant comprises an amino acid sequence according to SEQ ID NO: 24, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 24, wherein the % sequence identity to SEQ ID NO: 24 retains the amino acid modifications at amino acid positions 33 and 71.

104. The nucleic acid construct according to claim 103, 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: 24, wherein the % sequence identity to SEQ ID NO: 24 retains the amino acid modifications at amino acid positions 33 and 71.

105. The nucleic acid construct according to any of claims 9 or 12 to 104, wherein the IL-21 variant comprises:i) the phenylalanine-to-lysine substitution or phenylalanine-to- glutamine substitution at amino acid position 36; andii) 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.

106. The nucleic acid construct according to claim 105, wherein the IL-21 variant comprises an amino acid sequence according to SEQ ID NO: 25, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 25, wherein the % sequence identity to SEQ ID NO: 25 retains the amino acid modifications at amino acid positions 36 and 71.

107. The nucleic acid construct according to claim 106, 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: 25, wherein the % sequence identity to SEQ ID NO: 25 retains the amino acid modifications at amino acid positions 36 and 71.

108. The nucleic acid construct according to any preceding claim, wherein the two or more interleukin molecules are fused to a signal peptide.

109. The nucleic acid construct according to claim 108, wherein the signal peptide is a periplasmic signal peptide or a mammalian signal peptide.

110. The nucleic acid construct according to any preceding claim, wherein, when the nucleic acid construct comprises a polynucleotide sequence encoding an IL-15 variant, the IL-15 variant is fused to a MalE or PelB signal peptide.

111. The nucleic acid construct according to any preceding claim, wherein, when the nucleic acid construct comprises a polynucleotide sequence encoding an IL-18 variant, the IL-18 variant is fused to a MalE or PelB signal peptide.

112. The nucleic acid construct according to any preceding claim, wherein, when the nucleic acid construct comprises a polynucleotide sequence encoding an IL-21 variant, the IL-21 variant is fused to a DsbAor PhoA signal peptide.

113. The nucleic acid construct according to any preceding claim, wherein, when the nucleic acid construct comprises a polynucleotide sequence encoding a CXCL9 molecule, the CXCL9 molecule is fused to an OmpA or DsbA signal peptide.

114. The nucleic acid construct according to any preceding claim, wherein the construct encodes an IL-15 molecule and one or of an IL-18 molecule, an IL-21 molecule, and / or a CXCL9 molecule, preferably wherein the interleukin molecules are interleukin variants.

115. The nucleic acid construct according to any preceding claim, wherein the nucleic acid construct comprises polynucleotide sequences encoding an IL-15 molecule and a CXCL9 molecule, preferably wherein the IL-15 molecule is an IL-15 variant.

116. The nucleic acid construct according to any preceding claim, wherein the nucleic acid construct comprises polynucleotide sequences encoding an IL-15 molecule, an IL-18 molecule, and a CXCL9 molecule, preferably wherein the interleukin molecules are interleukin variants.

117. The nucleic acid construct according to any preceding claim, wherein the nucleic acid construct comprises polynucleotide sequences encoding an IL-15 molecule, an IL-18 molecule, an IL-21 molecule, and a CXCL9 molecule, preferably wherein the interleukin molecules are interleukin variants.

118. The nucleic acid construct according to any preceding claim, wherein the nucleic acid construct comprises polynucleotide sequences encoding an IL-15 molecule, an IL-18 molecule, and an IL-21 molecule, preferably wherein the interleukin molecules are interleukin variants.

119. The nucleic acid construct according to any preceding claim, wherein the construct comprises polynucleotide sequences encoding an IL-15 molecule and an IL-18 molecule, preferably wherein the interleukin molecules are interleukin variants.

120. The nucleic acid construct according to any preceding claim, wherein the construct comprises polynucleotide sequences encoding two or more an amino acid sequence according to SEQ ID NOs: 8, 19, 23, and / or 26.

121. The nucleic acid construct according to claim 120, wherein the construct comprises a polynucleotide sequence encoding a MalE signal peptide upstream of the polynucleotide sequence encoding the amino acid sequence according to SEQ ID NO: 8.

122. The nucleic acid construct according to claim 120 or 121, wherein the construct comprises a polynucleotide sequence encoding a PelB signal peptide upstream of the polynucleotide sequence encoding the amino acid sequence according to SEQ ID NO: 19.

123. The nucleic acid construct according to any of claims 120 to 122, wherein the construct comprises a polynucleotide sequence encoding a DsbA signal peptide upstream of the polynucleotide sequence encoding the amino acid sequence according to SEQ ID NO: 23.

124. The nucleic acid construct according to any of claims 120 to 123, wherein the construct comprises a polynucleotide sequence encoding a OmpA signal peptide upstream of the polynucleotide sequence encoding the amino acid sequence according to SEQ ID NO: 26.

125. The nucleic acid construct according to any preceding claim, wherein the nucleic acid molecule comprises DNA.

126. The nucleic acid construct according to any preceding claim, wherein the nucleic acid construct comprises RNA.

127. The nucleic acid construct according to claim 126, wherein the nucleic acid construct comprises mRNA.

128. The nucleic acid construct according to any preceding claim, further comprising a ribozyme sequence.

129. The nucleic acid construct according to claim 128, wherein the nucleic acid construct comprises a ribozyme sequence upstream of each polynucleotide sequence encoding a cytokine molecule.

130. The nucleic acid construct according to any preceding claim, wherein nucleic acid construct is controlled by a single promoter.

131. A cell comprising the nucleic acid construct according to any preceding claim.

132. The cell according to claim 131, wherein the cell is a bacterium or a mammalian cell.

133. The cell according to claim 132, wherein the bacterium is a live attenuated bacterium.

134. The cell according to claim 133, wherein the bacterium is a live attenuated Gram-negative bacterium.

135. The cell according to claim 134, wherein the bacterium is a Salmonella spp.

136. The cell according to claim 135, wherein the bacterium is Salmonella enterica, preferably wherein the bacterium is Salmonella enterica serovarTyphi.

137. The cell according to claim 136, wherein the bacterium is Salmonella enterica serovarTyphi ZH9.

138. The nucleic acid construct according to any of claims 1 to 130, or the cell according to any of claims 131 to 137, for use as a therapy.

139. The nucleic acid construct or cell for use according to claim 135, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

140. The nucleic acid construct or cell for use according to claim 139, wherein the neoplastic disease is a solid cancer and / or a haematological malignancy.

141. The nucleic acid construct or cell for use according to claim 140, wherein the solid cancer and / or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, endometrial cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

142. The nucleic acid construct or cell for use according to any of claims 138 to 141, wherein the nucleic acid construct or cell is administered to the subject orally, or via local instillation, subcutaneously, intramuscularly, intraperitoneally, intrapleurally, intravesically, peritumoral injection or intratumoural injection.

143. Use of the nucleic acid construct according to any of claims 1 to 130, or the cell according to any of claims 131 to 137, in the manufacture of a medicament for a therapy.

144. The use according to claim 143, wherein the therapy is the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease in a subject.

145. A method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject the nucleic acid construct according to any of claims 1 to 130, or the cell according to any of claims 131 to 137.

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