Interleukin-15 variant

Abstract

A bacterium containing an interleukin-15 (IL-15) variant is provided, wherein, compared to wild-type IL-15, the IL-15 variant contains one or more amino acid modifications in a surface region adjacent to the receptor-interacting surface, preferably wherein the receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface.

Description

Technical Field

[0001] This invention relates to the interleukin-15 (IL-15) molecule and its variants. Background Technology

[0002] Microbial immunotherapy has proven to be an effective postoperative treatment to reduce the incidence of tumor recurrence, and BCG therapy is the current standard of care for certain cancer types, particularly for patients with non-MIBC (Alhunaidi, 2019). BCG therapy has limitations, including efficacy range (40-60% of patients will experience tumor recurrence within 5 years post-surgery), adverse reactions and safety issues, complex manufacturing requirements, and a long treatment regimen (patients undergo 27 intravesical, transurethral infusions over 36 months). To improve efficacy, microbial immunotherapy in conjunction with co-immunostimulatory molecules has been proposed.

[0003] In recent years, there has been a significant increase in evidence supporting the use of immunostimulatory molecules such as cytokines in cancer treatment. Interleukin-15 (IL-15) and IL-15 agonist molecules are considered promising immunotherapeutic agents due to their roles in CD8 T cell and NK cell activation and proliferation (Guo, 2017; Robinson, 2017). Clinical trials are currently underway to test the combination therapy of intravesical BCG plus ANKTIVA® (formerly ALT-803, N803), an IL-15 super-agonist molecule (developed by ImmunityBio (formerly Altor BioScience)).

[0004] However, a challenge associated with using IL-15 as an immunostimulatory anticancer therapy is its expression in bacteria, particularly in the context of in vivo microbial therapy. Many existing technologies utilize IL-15 from *Escherichia coli* (…). E. coli Examples of expressed and purified cytokines exist, but a common problem with expressing eukaryotic cytokines using bacterial systems is inclusion body formation. Bacterial expression of cytokines often leads to the formation of nonfunctional inclusion bodies. Therefore, the deposition of cytokines in bacteria imposes an additional and laborious requirement for the dissolution and refolding of proteins from inclusion bodies, which often results in low yields of proteins unsuitable for in vivo therapy (Ferrer-Miralles, 2009; de Marco, 2009). High protein expression rates or unfavorable reducing conditions in the cytoplasm can impair the folding of nascent peptides, leading to the formation of insoluble protein aggregates. That is, the highly reducing environment of the bacterial cytoplasm hinders the formation of disulfide bonds between cysteine ​​residues during protein folding—which can be problematic for the expression of cytokines with disulfide bonds in their protein structure, including IL-15.

[0005] Attempts to improve cytokine expression levels and solubility in bacteria such as E. coli include lowering culture temperatures, co-expression of chaperone proteins, in vitro rehydration and refolding protocols, using weak promoters, fusion to solubility enhancer tags, adding signal peptides for periplasmic localization, or N-terminal fusion to vectors for extracellular export (Behar 2011, Devi 2016, Nausch 2013).

[0006] The same limitations on cytokine expression efficiency observed in Escherichia coli are also seen in Salmonella ( Salmonella ) strains were observed. In fact, the inventors of this invention had previously observed that Salmonella typhi from temperature-inducible LPR promoters ( ) in cultures grown at a suboptimal induction temperature of 37°C Salmonella The expression of human IL-15 in Typhi ZH9 leads to the accumulation of IL-15 in insoluble aggregates.

[0007] Another challenge posed by protein expression in Gram-negative bacterial strains such as Escherichia coli and Salmonella is that even after successful expression of cytokines, proteins must be properly folded and then transported across the two cell membranes, which are separated by the bacterial periplasmic space, in order to be secreted from the cell.

[0008] Therefore, it can be seen that there is a need to develop cytokines that can be optimally expressed in bacteria and effectively secreted by bacteria. Summary of the Invention

[0009] This invention provides IL-15 variants with optimized solubility and retained biological activity. The inventors of this invention unexpectedly discovered that combinations of certain mutations in IL-15 produce IL-15 variants that are more soluble and retain functionality than wild-type IL-15. Therefore, the variants provided herein are ideal candidates for expression and secretion in efficient expression systems such as bacteria (e.g., Gram-negative and / or Gram-positive bacteria).

[0010] Therefore, in a first aspect of the invention, bacteria comprising an interleukin-15 (IL-15) variant are provided, wherein, compared to wild-type IL-15, the IL-15 variant contains one or more amino acid modifications in a surface region adjacent to the receptor-interacting surface, preferably wherein the receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface.

[0011] In a second aspect of the invention, an interleukin-15 (IL-15) variant is provided, wherein the wild-type IL-15 comprises the amino acid sequence according to SEQ ID NO: 1, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, and wherein, compared with SEQ ID NO: 1, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor-interacting surface, and wherein said one or more amino acid modifications comprise any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophilic 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 said receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface.

[0012] In a third aspect of the invention, a nucleic acid molecule encoding an IL-15 variant as defined in the second aspect of the invention is provided.

[0013] In a fourth aspect of the invention, bacteria comprising an IL-15 variant according to a second aspect of the invention or a nucleic acid molecule according to a third aspect of the invention are provided.

[0014] In a fifth aspect of the invention, bacteria according to the first or fourth aspect of the invention, or IL-15 variants according to the second aspect of the invention, or nucleic acid molecules according to the third aspect of the invention, are provided for therapeutic use.

[0015] In a sixth aspect of the invention, the use of bacteria according to the first or fourth aspect of the invention, or IL-15 variants according to the second aspect of the invention, or nucleic acid molecules according to the third aspect of the invention, in the preparation of a medicament for treatment is provided.

[0016] In a seventh aspect of the invention, a method is provided for treating, inhibiting, preventing recurrence or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject bacteria according to a first or fourth aspect of the invention, or an IL-15 variant according to a second aspect of the invention, or a nucleic acid molecule according to a third aspect of the invention. Attached Figure Description

[0017] The present invention is described with reference to the accompanying drawings, wherein: Figure 1 The structure of a variant of interleukin-15 (IL-15, PDB ID: 4GS7) is shown, demonstrating amino acid modifications in a surface region adjacent to the IL-15Rα interacting surface. IL-15Rα, IL-2Rβ (IL-15Rβ), and γC are also shown.

[0018] Figure 2 A schematic diagram of IL-15 trans-presentation and biological function is shown.

[0019] Figure 3 The sequence-structure relationship of wild-type IL-15 and its interaction with homologous ligands are shown. Short and long (mammalian) signal peptide sequences of precursor IL-15 (A), mature IL-15 (B), and IL-15 tertiary structure (C) are shown.

[0020] Figure 4 The aggregation tendency and hydrophobicity score of IL-15 are shown using AggreScan and AggreScan 3D.

[0021] Figure 5 An overview of IL-15 and IL-15 agonists for use in immunotherapy is shown.

[0022] Figure 6 The structures of IL-15 variants are shown, with amino acids at positions 45 and / or 49 and / or 52 marked. IL-15Rα, IL-2Rβ, and γC are also shown.

[0023] Figures 7A-7C The structure of the quaternary complex of IL-15 with IL-15Rα, IL-15Rβ and γC (PDB: 4GS7) is shown.

[0024] Figures 8A-8D The solubility and functionality of different IL-15 variants are shown. (A) The total number of soluble variants obtained through different mutagenesis strategies is shown. (B) The solubility score distribution for each mutagenesis strategy is shown, with computer simulation design showing a higher overall success rate and a wider distribution of error-prone PCR (epPCR) variants. (C) The total number of active variants obtained through different mutagenesis strategies is shown. (D) Activity measurements (expressed in relative luminescent units (RLU)) based on the distribution of IL-15 bioassay cells (Promega) reporter cell lines under each mutagenesis strategy are shown.

[0025] Figures 9A-9C The bioactivity of the IL-15 / SAg variants was demonstrated using three independent assays (IL-15 bioactivity assay, NK proliferation assay, and IFNγ production assay).

[0026] Figures 10A-10B Protein purification data confirming the isolation of different IL-15 variants and IL-15 SAg variants are shown.

[0027] Figure 11A and 11BPreclinical candidate screening of IL-15 variants is shown. Results of incubating gradient concentrations of purified IL-15 variants with reporter cell lines for 6 hours are shown.

[0028] Figure 12 In vitro human cell assays are shown. Results of incubating primary NK cells with IL-15 variants at 71 nM (Experiment 1) and 58 nM (Experiment 2) for 24 hours are shown.

[0029] Figure 13 The results show the activation of NK cells in mouse spleen cells mediated by the IL-15 variant after 72 hours of incubation with the IL-15 variant. Detailed Implementation

[0030] The present invention provides optimized IL-15 variants (and / or their super-agonists, i.e., SAg variants), which are both soluble and functional, and are therefore the best candidates for expression in efficient secretion systems that allow the production and secretion of said IL-15 variants and SAg variants.

[0031] Therefore, in a first aspect, the present invention provides bacteria comprising an interleukin-15 (IL-15) variant, wherein, compared to wild-type IL-15, the IL-15 variant includes one or more amino acid modifications in a surface region adjacent to the receptor-interacting surface, preferably wherein the receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface. Figure 1 ).

[0032] As used herein, the term "interleukin" (IL) refers to any member of a family of glycoproteins expressed by leukocytes (white blood cells) and involved in regulating immune responses. Interleukins are also known as cytokines. Interleukins play multiple roles in the production of immune cells, including immune cell activation and differentiation, as well as proliferation, maturation, migration, and adhesion. Interleukins can possess both pro-inflammatory and anti-inflammatory properties. Interleukins constitute a large group of proteins that can trigger several responses in cells and tissues by binding to receptors called interleukin receptors to initiate downstream signaling from the receptors.

[0033] Interleukin-15 (IL-15) is a tetraalpha-helical 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 variety of cellular organisms, including dendritic cells, monocytes, macrophages, bone marrow stromal cells, and intestinal epithelial cells. Cells expressing IL-15 also express its high-affinity receptor-α, IL-15Rα. Unlike most cytokines that are secreted in a soluble form, IL-15 has a unique expression form, which binds to its high-affinity receptor IL-15Rα and shuttles to the surface of IL-15-producing cells as a heterodimeric complex. IL-15 can also be secreted independently of IL-15Rα as a soluble IL-15. The IL-15 / IL-15Rα complex is a cell surface complex that effectively stimulates neighboring cells via a trans-presentation mechanism through the IL-2 / IL-15Rβ and γc complex (IL-15Rβγ). Figure 2 IL-15 preferentially stimulates the activation, proliferation, and cytolysis of NK (natural killer) and CD8+ T cells (Guo, 2007; Santana-Carrero, 2019).

[0034] Trans-presentation of IL-15 mediates cellular responses during homeostasis. Evidence suggests that the IL-15Rα / IL-15 complex cleaves from the surface of presenting cells in response to various types of immunostimuli, such as whole-body irradiation, TLR stimulation, viral infection, CD40 stimulation, activation of type I IFN (IFN-I) and the STING pathway, thereby releasing the soluble heterodimeric IL-15 / IL-15Rα complex (Bergamaschi, 2012; Anthony, 2016).

[0035] Human IL-15 is a protein of 162 amino acids (18,086 kDa), containing 4 helices, 2 disulfide bonds, and 1 glycosylation site. Figure 3It has two isoforms produced by alternatively spliced ​​transcripts: one with a short signal peptide (SSP), containing a 21-amino acid leader peptide that is retained in the cytosol, and the other with a long signal peptide (LSP), containing a 48-amino acid signal peptide that allows the protein to be secreted into the extracellular environment (Saeed and Revell, 2001; Duitman, 2008). This secretion occurs via independent export of the signal and receptor α to the Golgi apparatus, followed by a high-affinity interaction between the signal and receptor, and export of the protein into the complex environment via the receptor α. The signal peptide is always cleaved at the same location, producing the same mature form of IL-15, possibly via the highly conserved sequence N-GLPKTEA / NW-C, although SSP IL-15 has shown biological activity, indicating some flexibility in the presence of the N-terminal peptide (Bergamaschi, 2009). Mature IL-15 then folds into a four-helix bundle motif (four α-helices arranged longitudinally in an antiparallel direction), each α-chain containing 16-21 amino acids, where α1 interacts with α3 and α2 interacts with α4 (the interactions between the helices are...). Figure 3 The key residues involved in protein-protein interactions are highlighted by dashed lines. A stable disulfide bond exists between Cys42 and Cys85 for the second type of interaction, and a second disulfide bond exists between Cys35 and Cys88. Two highly disordered regions exist between α1 and α2 (rings 1-2) and α3 and α4 (rings 3-4).

[0036] The crystal structure of IL-15 co-coordinated with its receptors IL-2Rβ, γc, and IL-15Rα has been resolved (Ring, 2012). This indicates that the signaling protein interacts with IL-2Rβ through polar interactions with helical α1 and α3, where the interactions IL15_D8:IL-2Rβ {H133, Y134}, IL15_D61{IL2Rβ_{K71}, and IL15_N65:IL-2Rβ {R42, Q70, Y134} are essential for stabilizing this interaction, consistent with previously published mutagenesis studies (Eisenman, 2002; Pettit 1997). Other amino acids involved in stabilizing the interaction are I68, L69, K10, K11, and S7.

[0037] On the other hand, the interaction with γc exhibits a more generalized interaction, lacking a highly defined chemical signature, highlighting the heterogeneity of γc interactions with multiple cytokines. The interaction with γc occurs through chains α1 and α4, with Q108, M109, and N112 being key amino acids involved in this interaction. Additional contact sites are formed with the extended α chain to compensate for the smaller size of IL-15.

[0038] Finally, the interaction with IL-15Rα has been found to occur via α2. Chirifu et al. (Chirifu, 2007) described the interaction between IL-15 and IL-15Rα as a weak electrostatic (van der Waals) interaction and identified the following amino acids in the protein-protein interaction: D22, T24, Y26, E46, Q48, E53, E87, and E90. The disulfide bond between C42 and C88 is thought to stabilize the contact with IL-15Rα and impart molecular rigidity.

[0039] The inventors of this invention have analyzed the amino acid sequence of IL-15 using AggreScan, ProtScale, and AggreScan3D to identify potential aggregation sites responsible for protein aggregation and inclusion body formation during recombinant overexpression in *E. coli* or *Salmonella typhi*. Regions with a higher tendency to aggregate are those that form α-helices (…). Figure 4 These regions involve protein-protein interactions. The rational design and directed evolution approaches discussed below aim to minimize these hydrophobic interactions without altering the binding properties of IL-15 to its receptor.

[0040] Given its role in stimulating the proliferation and cytotoxicity of CD8 T cells and NK cells, IL-15 has been extensively tested as a cancer immunotherapy agent. The efficacy of IL-15 is limited by the short in vivo half-life of the protein. Many IL-15 variants have been developed to improve protein stability and efficacy.

[0041] Figure 5Five (1-5) different IL-15-based agents for stimulating cytotoxic T-cell and NK-cell responses were described. In order of increasing IL-15 signal intensity / duration, (1) rIL-15 is the first form of IL-15 detected in vivo. When administered, rIL-15 is thought to bind primarily to IL-15Rα on the cell surface, where it is trans-presented to IL-15-responsive cells. (2) Heterodimeric IL-15 is the natural form of IL-15 that is cleaved from cells and can stimulate responses independent of cell-cell interactions. Novartis is producing heterodimeric hIL-15 as a therapeutic agent. (3) RLI (Cytune Pharmaceuticals) is a fusion protein consisting of IL-15 linked to the sushi (cytokine-binding) domain of IL-15Rα, which can act as a soluble IL-15 agonist. (4) The IL-15 / IL-15Rα-Fc complex is produced by mixing commercially available IL-15Rα-Fc chimeric fusion protein with rIL-15 and has been widely used in preclinical studies. (5) ANKTIVA® (formerly ALT-803, N-803) (ImmunityBio, formerly Altor Pharmaceutical) is an IL-15 super agonist composed of mutated IL-15 (the asparagine at position 72 of the mature IL-15 amino acid sequence is replaced by an aspartic acid residue), which has increased affinity for CD122-expressing immune cells and is linked to the sushi domain of IL-15Rα fused with the Fc fragment. The signal intensity and duration of these IL-15-based agents increase with increasing agonist abundance, increased in vivo half-life (conferred by IL-15 binding to IL-15Rα and the presence of the Fc fragment), agonist dimerization, and increased affinity for the IL-2Rβ / γC complex (conferred by IL-15 binding to the sushi domain and mutations present in ALT-803).

[0042] There is a clear need to develop soluble IL-15 variants that exhibit biological activity. As used herein, the term “wild-type” (“WT”) should be given its usual meaning in the art, referring to a protein or gene encoding a protein found in its native, non-mutated, or unaltered form. In one embodiment, wild-type IL-15 is mature (i.e., unmodified) IL-15. As used herein, the term “parental IL-15” refers to the baseline sequence derived from all IL-15 variants according to the invention. Parental IL-15, also known as IL-15M40 or M40 (SEQ ID NO: 16), is derived from wild-type IL-15 and comprises the amino acid sequence according to SEQ ID NO: 1. The difference between wild-type IL-15 and M40 IL-15 lies in the presence of an amino acid modification in which the serine of amino acid 75 of SEQ ID NO: 1 is replaced by a proline (S75P). Thus, the S75P mutation from SEQ ID NO: 1 (wild-type) produces SEQ ID NO: 16 (parental). Therefore, in some embodiments, the IL-15 variant contains an amino acid modification at position 75. In a preferred embodiment, the IL-15 variant contains a serine-to-proline substitution (S75P) at position 75. Any IL-15 variant described herein may be characterized by an S75P mutation. The S75P mutation can improve the solubility of the IL-15 variant to further stabilize the IL-15 variant and thus optimize its expression in bacteria.

[0043] The term "interleukin variant" refers to an IL-15 molecule that contains one or more amino acid modifications compared to wild-type IL-15. As used herein, the term "amino acid modification" refers to any alteration or mutation in the wild-type amino acid sequence. Amino acid modifications may or may not alter the structure, function, and / or physicochemical properties of a 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 reduction 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 replacing one amino acid with another. Substitution mutations can be conserved or non-conserved.

[0044] As those skilled in the art will understand, amino acids can be broadly grouped according to their properties (i.e., size, volume, charge, hydrophobicity, polarity, etc.). The similarity or dissimilarity between amino acids can also be calculated using substitution matrices and physicochemical distances. Conservative amino acid substitutions involve replacing or exchanging one amino acid with another that has similar properties. For example, replacing one hydrophobic amino acid with another (e.g., valine with alanine) constitutes a conserved amino acid substitution. Non-conservative or extreme amino acid substitutions refer to replacing or exchanging one amino acid with another that has different or dissimilar properties. For example, replacing a hydrophobic amino acid with a hydrophilic amino acid (e.g., leucine with serine) constitutes a non-conservative amino acid substitution. Compared to conserved amino acid substitutions, non-conservative amino acid substitutions are more likely to be associated with changes in protein structure and function.

[0045] Several methods for inducing amino acid modifications are well known in the art. As will be readily understood by those skilled in the art, nucleic acid sequences encoding the target modified or mutated amino acid sequence can be generated by several methods known in the art (i.e., de novo synthesis). Such genes can be introduced into expression vectors or expression systems that support gene expression and messenger RNA (mRNA) translation. The gene introduced into the expression system can be a heterologous gene (i.e., a foreign gene), meaning that the gene originates from a cell type derived from an organism different from the recipient expression system. Methods for heterologous expression of recombinant proteins are well known to those skilled in the art. The expression system of the present invention is a bacterial expression system, preferably bacterial cells, such as Salmonella strains.

[0046] The IL-15 variants according to the invention may contain one or more, two or more, three or more, four or more, five or more, or more than five amino acid modifications. However, the IL-15 variants may preferably contain one, two, or three amino acid modifications.

[0047] According to a first aspect of the invention, bacteria comprising an IL-15 variant are provided, wherein, compared to wild-type IL-15, the IL-15 variant contains one or more amino acid modifications in a surface region adjacent to the receptor-interacting surface, preferably wherein the receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface.

[0048] Amino acid modifications will preferably be present in the surface regions of the IL-15 variant. As used herein, the term "surface region" refers to the portion of a protein exposed to a solvent. In the overall three-dimensional tertiary structure of a protein, the protein surface is generally referred to as the solvent-accessible region of the protein. For soluble proteins, the protein surface is primarily composed of hydrophilic amino acid residues, while the protein core (i.e., the interior) will consist of hydrophobic residues stacked together away from the solvent. Amino acid modifications on the IL-15 variant of the present invention will preferably involve those amino acids exposed to the solvent and located on the outer / external surface of the protein. Modification of amino acids on the protein surface generally involves protein-protein interactions, as the protein surface typically contains protein-protein interfaces (i.e., molecular surfaces through which proteins contact and interact with each other). The protein surface may contain large, flat interfaces and / or smaller depressions or even single residues, which can form anchoring sites for one or more interacting proteins. The protein surfaces of the interacting proteins can be complementary in shape and / or chemical properties.

[0049] As those skilled in the art will understand, modifications to the surface of or around a protein involving a protein-protein interface can affect protein-protein interactions. In the context of IL-15, protein-protein interactions can include interactions between IL-15 and its receptors. For example, IL-15 can interact with any of its receptors IL-15Rα, IL-15Rβ, and γc. Such interactions occur on the surface of IL-15 at receptor-interacting surfaces.

[0050] Therefore, the surface region is the region adjacent to the receptor-interacting surface of the IL-15 variant.

[0051] As used herein, the terms “receptor-interacting surface,” “receptor-binding surface,” “interacting surface,” “binding surface,” and “ligand-binding surface” are used interchangeably and refer to the surface of IL-15 involved in coordinating receptor interactions and binding to receptors (i.e., protein-protein interactions). There may be a precise set of amino acids that directly bind to the receptor. In a preferred embodiment, the receptor-interacting surface is the IL-15 receptor-interacting surface. In a more preferred embodiment, the receptor-interacting surface is the IL-15 receptor α (IL-15Rα)-interacting surface. The interacting surface is a surface region, not a domain. A protein domain is a unit within a protein that has a unique function and / or structure, is self-stable, and independent of the rest of the protein. An example of a protein domain is the sushi domain of the IL-15Rα receptor. The interacting surface is a surface region, not a domain. A protein domain is a unit within a protein that has a unique function and / or structure, is self-stable, and independent of the rest of the protein. An example of a protein domain is the sushi domain of the IL-15Rα receptor.

[0052] Regarding SEQ ID NO: 1, the “receptor interaction surface” is defined as those amino acids at the following positions: D8, D61, and N65 of IL-15Rβ (i.e., IL-15 α1 and α3) (and other amino acids including I68, L69, K10, K11, and S7); Q108, M109, and N112 of γC (i.e., IL-15 α1 and α4); and D22, T24, Y26, E46, Q48, E53, E87, and E90 of IL-15Rα (i.e., IL-15 α2). Therefore, the “receptor interaction surface” is well characterized in the art.

[0053] The inventors of this invention unexpectedly discovered regions of IL-15 that can be modified with amino acids, thereby producing IL-15 variants with increased solubility and maintained biological activity. The amino acids to be modified are located in surface regions adjacent to the receptor-interacting surface of IL-15. These amino acids were chosen for modification because they form a portion of the available space in the IL-15 structure that does not require interaction with IL-15Rβ or γC.

[0054] In particular, the inventors of this invention have unexpectedly identified the following amino acid positions that can 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 IL-15Rβ or γc. IL-15Rβ or γc is expressed on the surface of target cells to be activated (NK cells and CD8+ T cells). Therefore, the “receptor-interacting surface-adjacent region” can be described as any region in IL-15 where the amino acid does not interact with IL-15Rβ or γc. Such interactions include any covalent and electrostatic interactions, including van der Waals interactions and hydrogen bonds. However, the modified amino acids described herein can interact with IL-15Rα. For example, within IL-15 (i.e., SEQ ID NO: 1), L45, V49, and L52 have been described as van der Waals contacts of IL-15Rα, and Q48 has been described as forming hydrogen bonds with IL-15Rα (Chirifu et al., 2007). Furthermore, it is known in the art that N71 mutations within IL-15 (N71D in the ALT-803 IL-15 superagonist) contribute to IL-15 activity. Therefore, as used herein, the “region adjacent to the receptor-interacting surface” may comprise amino acids within IL-15 α2.

[0055] Beyond theoretical constraints, amino acids present in the "adjacent region" of the receptor-interacting surface can play a role in mediating protein-protein interactions. Therefore, the term "adjacent region" can refer to surface amino acids that are close to the receptor (e.g., IL-15Rα) and can interact with IL-15Rα but not with IL-15Rβ or γC. In practice, certain modifications can therefore be used to enhance the interaction between IL-15 and its receptor (e.g., IL-15Rα), for example, by increasing binding affinity. Furthermore, modifications altering the amino acid composition of the IL-15 surface can affect the solubility of IL-15.

[0056] Therefore, in a preferred embodiment, the amino acid modification will be present near the surface region of the IL-15 variant adjacent to the surface of the receptor (e.g., IL-15Rα) that interacts with the IL-15 variant, thereby enhancing the interaction between the IL-15 variant and the receptor. For example, the interaction between IL-15 and IL-15Rα can be enhanced because of the presence of a lower ligand dissociation rate constant (K). off (i.e., the high stability of the IL-15 / IL-15Rα complex), and a high ligand binding rate constant (K). on (i.e., IL-15 rapidly recognizes IL-15Rα, and vice versa) and has a lower equilibrium dissociation constant (K). d (i.e., high-affinity interactions). Kd It is K off and K on The ratio between (K) d =K off / K on Other methods for characterizing binding dynamics are well known to those skilled in the art.

[0057] According to a first aspect of the invention, bacteria comprising an IL-15 variant are provided, wherein the IL-15 variant contains one or more amino acid modifications in a surface region adjacent to a receptor-interacting surface, compared to wild-type IL-15, preferably wherein said receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface. As detailed herein, wild-type IL-15 may comprise mature IL-15, or the amino acid sequence according to SEQ ID NO: 1, or comprise an amino acid sequence having at least 70% identity with SEQ ID NO: 1. The IL-15 variant may also be present with other protein sequences such as signaling sequences, localization sequences, peptide tags, peptide linkers, other interleukins or their receptors, other immunostimulatory proteins, or any combination thereof.

[0058] According to a second aspect of the invention, a variant of interleukin-15 (IL-15) is provided, wherein the wild-type IL-15 comprises the amino acid sequence according to SEQ ID NO: 1, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, and wherein, compared to SEQ ID NO: 1, the IL-15 variant includes one or more amino acid modifications in a surface region adjacent to the receptor-interacting surface, and wherein said one or more amino acid modifications comprise any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophilic 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 said receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface. The IL-15 variant includes amino acid modifications at one or more of amino acids 45 and / or 49 and / or 52, or any combination thereof. Figure 6 ).

[0059] The IL-15 variant may contain an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1. As used herein, the terms “sequence identity” and “sequence homology” are interchangeable and refer to the number of identical residues within a specified length range in a given alignment. To calculate the percentage of sequence identity for any sequence disclosed herein, sequence comparison software can be used, for example, using the default settings on the BLAST software package (V2.10.1).

[0060] In another embodiment, the IL-15 variant may comprise the following amino acid sequences: said amino acid sequences having at least 75% sequence identity with SEQ ID NO: 1; having at least 80% sequence identity with SEQ ID NO: 1; having at least 85% sequence identity with SEQ ID NO: 1; having at least 90% sequence identity with SEQ ID NO: 1; having at least 91% sequence identity with SEQ ID NO: 1; having at least 92% sequence identity with SEQ ID NO: 1; having at least 93% sequence identity with SEQ ID NO: 1; having at least 94% sequence identity with SEQ ID NO: 1; having at least 95% sequence identity with SEQ ID NO: 1; having at least 96% sequence identity with SEQ ID NO: 1; having at least 97% sequence identity with SEQ ID NO: 1; having at least 98% sequence identity with SEQ ID NO: 1; or having at least 99% sequence identity with SEQ ID NO: 1.

[0061] According to the present invention, the IL-15 variant can be modified such that, compared with SEQ ID NO: 1, the amino acid modification occurs at one or more of amino acids at positions 45 and / or 49 and / or 52. These amino acid positions are located in a surface region adjacent to the receptor-interacting surface of the IL-15 variant (preferably the interleukin-15 receptor α (IL-15Rα) interacting surface). The IL-15 variant may comprise: a single amino acid modification at position 45; a single amino acid modification at position 49; a single amino acid modification at position 52; two amino acid modifications at positions 45 and 49; two amino acid modifications at positions 45 and 52; two amino acid modifications at positions 49 and 52; or three amino acid modifications at positions 45, 49, and 52. Any combination of these modifications is considered included. It should be understood that any combination of amino acid mutations at positions 45 and / or 49 and / or 52 will retain at least 70% sequence identity with SEQ ID NO: 1.

[0062] According to any aspect of the invention, the IL-15 variant may contain an amino acid modification at amino acid position 75. The amino acid modification may be present together with any other modification or combination thereof described herein. For example, the IL-15 variant may contain an amino acid modification at any of the following positions: amino acids 45, 49, 52, and / or 75. For example, amino acids 75 and 45; amino acids 75 and 49; amino acids 75 and 52; amino acids 75, 45, and 49; amino acids 75, 45, and 52; amino acids 75, 49, and 52; or amino acids 75, 45, 49, and 52. Preferably, the IL-15 variant may contain an amino acid modification at the following positions: amino acids 75 and 45; amino acids 75, 45, and 49; amino acids 75, 45, and 52; amino acids 75, 49, and 52.

[0063] In some embodiments, the IL-15 variant may include amino acid modifications, thereby making non-conserved amino acid substitutions. In such embodiments, hydrophobic amino acids naturally present in the wild-type sequence are replaced with hydrophilic amino acids, or vice versa. Proteins may be unstable when hydrophobic amino acids are located at solvent-exposed positions on a three-dimensional protein structure. Therefore, mutations in such hydrophobic residues can be used to stabilize proteins.

[0064] In other embodiments, the IL-15 variant may include amino acid modifications, thereby performing conserved amino acid substitutions. In such embodiments, a hydrophobic amino acid naturally present in the wild-type sequence is replaced by another hydrophobic amino acid, or a hydrophilic amino acid naturally present in the wild-type sequence is replaced by another hydrophilic amino acid.

[0065] As used herein, the terms “hydrophobic” and “hydrophilic” should be given their common meaning in the art, namely, weak affinity and strong affinity for water, respectively. A “hydrophobic amino acid” is an amino acid with a hydrophobic side chain, i.e., a side chain that does not interact (repel) water. The hydrophobic side chain typically consists of carbon and hydrogen atoms, or is primarily composed of carbon and hydrogen atoms. Hydrophobic amino acids are generally nonpolar and therefore insoluble in polar solvents such as water. Naturally occurring hydrophobic amino acids can include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). However, the term “hydrophobic amino acid” is also intended to include any non-naturally occurring, synthetic, or modified amino acid that is hydrophobic. A “hydrophilic amino acid” is an amino acid with a hydrophilic side chain, i.e., a side chain that interacts (attracts) water. The hydrophilic side chain typically consists of oxygen, nitrogen, and sulfur atoms. Hydrophilic amino acids are typically polar and therefore can dissolve in polar solvents such as water by forming hydrogen bonds. Naturally occurring hydrophilic amino acids can include serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), and tyrosine (Tyr). Other naturally occurring hydrophilic amino acids can include arginine (Arg), histidine (His), lysine (Lys), aspartic acid (Asp), and glutamic acid (Glu) because these amino acids have charged side chains. Arginine, histidine, and lysine are generally positively charged, while aspartic acid and glutamic acid are generally negatively charged. However, the term "hydrophilic amino acid" is also intended to include any non-naturally occurring, synthetic, or modified amino acid that is hydrophilic.

[0066] According to the present invention, the IL-15 variant may contain amino acid modifications at one or more of the 45th and / or 49th and / or 52nd amino acid positions. Regarding SEQ ID NO: 1, the wild-type amino acid at position 45 is leucine, the wild-type amino acid at position 49 is valine, and the wild-type amino acid at position 52 is leucine.

[0067] In a preferred embodiment of any aspect of the invention, the amino acid modification at position 45 is a hydrophobic-to-hydrophilic substitution. This amino acid modification may be present in conjunction with any other modifications described herein. This substitution involves exchanging a hydrophobic amino acid for a hydrophilic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or replaced by any naturally occurring or non-naturally occurring hydrophilic amino acid. For example, any of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan may be replaced by any of serine, threonine, cysteine, asparagine, glutamine, tyrosine, arginine, histidine, lysine, aspartic acid, or glutamic acid.

[0068] In SEQ ID NO: 1, the wild-type amino acid at amino acid position 45 is leucine. Preferably, the hydrophobic-to-hydrophilic substitution at amino acid position 45 is leucine replaced by serine or leucine replaced by threonine. Not bound by theory, this non-conservative amino acid modification can alter the physicochemical properties of the region containing amino acid position 45. Specifically, this region can become more hydrophilic, thus making the IL-15 variant with this amino acid modification more soluble. This alteration of the physicochemical properties of amino acids in surface regions adjacent to the receptor-interacting surface of the IL-15 variant (e.g., the interleukin-15 receptor α (IL-15Rα) interaction surface) can also enhance the interaction between the IL-15 variant and the receptor. Therefore, the IL-15 variant with this amino acid modification may be more soluble in bacterial chassis than wild-type IL-15 while still exhibiting biological activity.

[0069] In another preferred embodiment, the amino acid modification at position 49 is a hydrophobic-to-hydrophobic substitution. This amino acid modification may be present along with any other modifications described herein. This substitution will involve exchanging a hydrophobic amino acid for another hydrophobic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or replaced by another hydrophobic amino acid. For example, any of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan may be replaced by any of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan. However, the amino acid modification at position 49 may alternatively be a hydrophobic-to-hydrophilic substitution, preferably replacing valine with serine.

[0070] In SEQ ID NO: 1, the wild-type amino acid at amino acid position 49 is valine. Preferably, the hydrophobic-to-hydrophobic substitution at amino acid position 49 is the replacement of valine with alanine. The inventors of this invention unexpectedly discovered that the hydrophobic-to-hydrophobic mutation at V49 enhances the properties of the IL-15 variant. Without being bound by theory, this relatively conserved amino acid modification can optimize the region containing amino acid position 49. Specifically, alanine is a small amino acid with a molecular weight of 89.1 g / mol and a small methyl (-CH3) side chain, while valine is a larger amino acid with a molecular weight of 117.1 g / mol and a larger isopropyl side chain (-CH(CH3)2). This substitution with a smaller / smaller amino acid can be used to optimize the protein-protein interface of the IL-15 / receptor interaction. Furthermore, alanine is less hydrophobic than valine; therefore, this physicochemical modification of the amino acids in the adjacent surface region of the receptor-interacting surface of the IL-15 variant also helps to improve the solubility of the IL-15 receptor. By removing the hydrophobicity of valine through this substitution, the inventors were able to evaluate the effect on the solubility / stability of the IL-15 variant. Therefore, the IL-15 variant with this amino acid modification exhibits optimized solubility and functionality compared to wild-type IL-15.

[0071] In another preferred embodiment, the amino acid modification at position 52 is a hydrophobic-to-hydrophilic substitution. This amino acid modification may be present along with any other modifications described herein. This substitution involves exchanging a hydrophobic amino acid for a hydrophilic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or replaced by any naturally occurring or non-naturally occurring hydrophilic amino acid. For example, any of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan may be replaced by any of serine, threonine, cysteine, asparagine, glutamine, tyrosine, arginine, histidine, lysine, aspartic acid, or glutamic acid.

[0072] In SEQ ID NO: 1, the wild-type amino acid at amino acid position 52 is leucine. Preferably, the hydrophobic-to-hydrophilic substitution at amino acid position 52 is leucine replaced with lysine or leucine replaced with arginine. Not bound by theory, this non-conservative amino acid modification can alter the physicochemical properties of the region containing amino acid position 52. Specifically, this region can become more hydrophilic, thus making the IL-15 variant with this amino acid modification more water-soluble. Such physicochemical property alteration of amino acids in surface regions adjacent to the receptor-interacting surface of the IL-15 variant (e.g., the interleukin-15 receptor α (IL-15Rα) interaction surface) also contributes to enhanced interaction between the IL-15 variant and its receptor. Therefore, the IL-15 variant with this amino acid modification may be more soluble than wild-type IL-15 while retaining its biological activity.

[0073] In some embodiments, amino acids at positions 45, 49, and 52 may not be replaced by bulkier amino acids. These amino acids are adjacent to the receptor-binding surface, and replacing them with bulkier residues could lead to steric hindrance that could interfere with receptor binding. Aspartic acid, glutamic acid, asparagine, and glutamine are examples of bulkier amino acids. Therefore, in some embodiments, the amino acid modification at positions 45, 49, and 52 may not be replaced by any one of aspartic acid, glutamic acid, asparagine, or glutamine.

[0074] As described herein, the IL-15 variant according to the invention may contain an amino acid modification at amino acid position 75. This amino acid modification may be present along with any other modifications described herein. Preferably, the amino acid modification at amino acid position 75 is a serine-to-proline substitution (S75P). Serine-to-proline modifications at amino acid position 75 have been described in the art (Behar, 2011). The inventors of the invention chose the S75P IL-15 mutant (parental / M40IL-15) as the starting sequence for generating the IL-15 variant because the S75P IL-15 mutant was observed to be more soluble than unmodified IL-15 (Behar, 2011). The IL-15 variant containing the S75P amino acid modification may have the sequence according to SEQ ID NO: 16, or a sequence having at least 70% identity with SEQ ID NO: 16. In another embodiment, the IL-15 variant may comprise the following amino acid sequences: said amino acid sequence having at least 75% sequence identity with SEQ ID NO: 16; having at least 80% sequence identity with SEQ ID NO: 16; having at least 85% sequence identity with SEQ ID NO: 16; having at least 90% sequence identity with SEQ ID NO: 16; having at least 91% sequence identity with SEQ ID NO: 16; having at least 92% sequence identity with SEQ ID NO: 16; having at least 93% sequence identity with SEQ ID NO: 16; having at least 94% sequence identity with SEQ ID NO: 16; having at least 95% sequence identity with SEQ ID NO: 16; having at least 96% sequence identity with SEQ ID NO: 16; having at least 97% sequence identity with SEQ ID NO: 16; having at least 98% sequence identity with SEQ ID NO: 16; having at least 99% sequence identity with SEQ ID NO: 16; or having the same sequence identity as SEQ ID NO: 16. NO: 16 has at least 100% sequence identity with respect to the amino acid modification at amino acid position 75. It should be understood that any amino acid modification disclosed herein may exist in any combination with any other amino acid modification disclosed herein.

[0075] Therefore, in a preferred embodiment, the IL-15 variant according to any aspect of the invention may include 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 according to any aspect of the invention may include: (i) a leucine substitution at amino acid position 45 for serine; and (ii) a serine substitution at amino acid position 75 for proline. In this embodiment, the IL-15 variant may comprise the amino acid sequence according to SEQ ID NO: 2, or comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2, wherein the percentage of sequence identity with SEQ ID NO: 2 retains the amino acid modifications at amino acids positions 45 and 75.

[0076] In another embodiment, the IL-15 variant may comprise the following amino acid sequences: said amino acid sequences having at least 75% sequence identity with SEQ ID NO: 2; having at least 80% sequence identity with SEQ ID NO: 2; having at least 85% sequence identity with SEQ ID NO: 2; having at least 90% sequence identity with SEQ ID NO: 2; having at least 91% sequence identity with SEQ ID NO: 2; having at least 92% sequence identity with SEQ ID NO: 2; having at least 93% sequence identity with SEQ ID NO: 2; having at least 94% sequence identity with SEQ ID NO: 2; having at least 95% sequence identity with SEQ ID NO: 2; having at least 96% sequence identity with SEQ ID NO: 2; having at least 97% sequence identity with SEQ ID NO: 2; having at least 98% sequence identity with SEQ ID NO: 2; having at least 99% sequence identity with SEQ ID NO: 2; or having at least 100% sequence identity with SEQ ID NO: 2, wherein said amino acid sequences have at least 75% sequence identity with SEQ ID NO: 2; having at least 80% sequence identity with SEQ ID NO: 2; having at least 85% sequence identity with SEQ ID NO: 2; having at least 91% sequence identity with SEQ ID NO: 2; having at least 92% sequence identity with SEQ ID NO: 2; having at least 93% sequence identity with SEQ ID NO: 2; having at least 94% sequence identity with SEQ ID NO: 2; having at least 95% sequence identity with SEQ ID NO: 2; having at least 96% sequence identity with SEQ ID NO: 2; having at least 97% sequence identity with SEQ ID NO: 2; having at least 98% sequence identity with SEQ ID NO: 2; having at least 99% sequence identity with SEQ ID NO: 2; or having at least 100% sequence identity with The sequence identity percentage of NO:2 retains the amino acid modifications at positions 45 and 75.

[0077] In another preferred embodiment, the IL-15 variant according to any aspect of the invention may include a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophobic substitution at amino acid position 49, and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, the IL-15 variant according to any aspect of the invention may include (i) a leucine substitution at amino acid position 45 for serine, (ii) a valine substitution at amino acid position 49 for alanine, and (iii) a serine substitution at amino acid position 75 for proline. In this embodiment, the IL-15 variant may comprise the amino acid sequence according to SEQ ID NO: 3, or a sequence having at least 70% sequence identity with SEQ ID NO: 3, wherein the percentage of sequence identity with SEQ ID NO: 3 retains the amino acid modifications at amino acids positions 45, 49, and 75.

[0078] In another embodiment, the IL-15 variant may comprise the following amino acid sequences: said amino acid sequences having at least 75% sequence identity with SEQ ID NO: 3; having at least 80% sequence identity with SEQ ID NO: 3; having at least 85% sequence identity with SEQ ID NO: 3; having at least 90% sequence identity with SEQ ID NO: 3; having at least 91% sequence identity with SEQ ID NO: 3; having at least 92% sequence identity with SEQ ID NO: 3; having at least 93% sequence identity with SEQ ID NO: 3; having at least 94% sequence identity with SEQ ID NO: 3; having at least 95% sequence identity with SEQ ID NO: 3; having at least 96% sequence identity with SEQ ID NO: 3; having at least 97% sequence identity with SEQ ID NO: 3; having at least 98% sequence identity with SEQ ID NO: 3; having at least 99% sequence identity with SEQ ID NO: 3; or having at least 100% sequence identity with SEQ ID NO: 3, wherein said amino acid sequences have at least 75% sequence identity with SEQ ID NO: 3; having at least 80% sequence identity with SEQ ID NO: 3; having at least 85% sequence identity with SEQ ID NO: 3; having at least 91% sequence identity with SEQ ID NO: 3; having at least 92% sequence identity with SEQ ID NO: 3; having at least 93% sequence identity with SEQ ID NO: 3; having at least 94% sequence identity with SEQ ID NO: 3; having at least 95% sequence identity with SEQ ID NO: 3; having at least 96% sequence identity with SEQ ID NO: 3; having at least 97% sequence identity with SEQ ID NO: 3; having at least 98% sequence identity with SEQ ID NO: 3; having at least 99% sequence identity with SEQ ID NO: 3; or having at least 100% sequence identity with The sequence identity percentage of NO:3 retains the amino acid modifications at positions 45, 49, and 75.

[0079] In another preferred embodiment, the IL-15 variant according to any aspect of the invention may include 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 according to any aspect of the invention may include (i) a leucine substitution at amino acid position 45 for serine, (ii) a leucine substitution at amino acid position 52 for lysine, and (iii) a serine substitution at amino acid position 75 for proline. In this embodiment, the IL-15 variant may comprise the amino acid sequence according to SEQ ID NO: 4, or a sequence having at least 70% sequence identity with SEQ ID NO: 4, wherein the percentage of sequence identity with SEQ ID NO: 4 retains the amino acid modifications at amino acids positions 45, 52, and 75.

[0080] In another embodiment, the IL-15 variant may comprise the following amino acid sequences: said amino acid sequences having at least 75% sequence identity with SEQ ID NO: 4; having at least 80% sequence identity with SEQ ID NO: 4; having at least 85% sequence identity with SEQ ID NO: 4; having at least 90% sequence identity with SEQ ID NO: 4; having at least 91% sequence identity with SEQ ID NO: 4; having at least 92% sequence identity with SEQ ID NO: 4; having at least 93% sequence identity with SEQ ID NO: 4; having at least 94% sequence identity with SEQ ID NO: 4; having at least 95% sequence identity with SEQ ID NO: 4; having at least 96% sequence identity with SEQ ID NO: 4; having at least 97% sequence identity with SEQ ID NO: 4; having at least 98% sequence identity with SEQ ID NO: 4; having at least 99% sequence identity with SEQ ID NO: 4; or having at least 100% sequence identity with SEQ ID NO: 4, wherein said amino acid sequences have at least 75% sequence identity with SEQ ID NO: 4; having at least 80% sequence identity with SEQ ID NO: 4; having at least 85% sequence identity with SEQ ID NO: 4; having at least 91% sequence identity with SEQ ID NO: 4; having at least 92% sequence identity with SEQ ID NO: 4; having at least 93% sequence identity with SEQ ID NO: 4; having at least 94% sequence identity with SEQ ID NO: 4; having at least 95% sequence identity with SEQ ID NO: 4; having at least 96% sequence identity with SEQ ID NO: 4; having at least 97% sequence identity with SEQ ID NO: 4; having at least 98% sequence identity with SEQ ID NO: 4; having at least 99% sequence identity with SEQ ID NO: 4; or having at least 100% sequence identity with The sequence identity percentage of NO:4 retains the amino acid modifications at positions 45, 52, and 75.

[0081] In another preferred embodiment, the IL-15 variant according to any aspect of the invention may include 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 according to any aspect of the invention may include (i) a leucine substitution at amino acid position 45 for serine, (ii) a leucine substitution at amino acid position 52 for arginine, and (iii) a serine substitution at amino acid position 75 for proline. In this embodiment, the IL-15 variant may comprise the amino acid sequence according to SEQ ID NO: 5, or comprise an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 5, wherein the percentage of sequence identity with SEQ ID NO: 5 retains the amino acid modifications at amino acids positions 45, 52, and 75.

[0082] In another embodiment, the IL-15 variant may comprise the following amino acid sequences: said amino acid sequences having at least 75% sequence identity with SEQ ID NO: 5; having at least 80% sequence identity with SEQ ID NO: 5; having at least 85% sequence identity with SEQ ID NO: 5; having at least 90% sequence identity with SEQ ID NO: 5; having at least 91% sequence identity with SEQ ID NO: 5; having at least 92% sequence identity with SEQ ID NO: 5; having at least 93% sequence identity with SEQ ID NO: 5; having at least 94% sequence identity with SEQ ID NO: 5; having at least 95% sequence identity with SEQ ID NO: 5; having at least 96% sequence identity with SEQ ID NO: 5; having at least 97% sequence identity with SEQ ID NO: 5; having at least 98% sequence identity with SEQ ID NO: 5; having at least 99% sequence identity with SEQ ID NO: 5; or having at least 100% sequence identity with SEQ ID NO: 5, wherein said amino acid sequences have at least 75% sequence identity with SEQ ID NO: 5; having at least 80% sequence identity with SEQ ID NO: 5; having at least 85% sequence identity with SEQ ID NO: 5; having at least 90% sequence identity with SEQ ID NO: 5; having at least 91% sequence identity with SEQ ID NO: 5; having at least 92% sequence identity with SEQ ID NO: 5; having at least 93% sequence identity with SEQ ID NO: 5; having at least 94% sequence identity with SEQ ID NO: 5; having at least 95% sequence identity with SEQ ID NO: 5; having at least 96% sequence identity with SEQ ID NO: 5; having at least 97% sequence identity with SEQ ID NO: 5; having at least 98% sequence identity with SEQ ID NO: 5; having at least 99% sequence identity with SEQ ID The sequence identity percentage of NO:5 retains the amino acid modifications at positions 45, 52, and 75.

[0083] In another preferred embodiment, the IL-15 variant according to any aspect of the invention may include a hydrophobic-to-hydrophilic substitution at amino acid position 45, a hydrophobic-to-hydrophobic substitution at amino acid position 49, and a serine-to-proline substitution at amino acid position 75. In an even more preferred embodiment, the IL-15 variant according to any aspect of the invention may include (i) a leucine substitution at amino acid position 45 for threonine, (ii) a valine substitution at amino acid position 49 for alanine, and (iii) a serine substitution at amino acid position 75 for proline. In this embodiment, the IL-15 variant may comprise the amino acid sequence according to SEQ ID NO: 6, or a sequence having at least 70% sequence identity with SEQ ID NO: 6, wherein the percentage of sequence identity with SEQ ID NO: 6 retains the amino acid modifications at amino acids positions 45, 49, and 75.

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

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

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

[0087] In addition to any amino acid modification at any of the amino acid positions 45, 49, and / or 52 and 75, IL-15 variants having one or more amino acid modifications at the amino acid positions 48, 60, and / or 71 are provided. In one embodiment, an IL-15 variant is provided comprising a hydrophilic-to-hydrophobic substitution at the amino acid position 48, preferably a glutamine-to-isoleucine substitution or a glutamine-to-valine substitution at the amino acid position 48. In another embodiment, an IL-15 variant may be present comprising a hydrophilic-to-hydrophobic substitution at the amino acid position 60, preferably a histidine-to-valine substitution at the amino acid position 60. In yet another embodiment, an IL-15 variant may be present comprising a hydrophilic-to-hydrophobic substitution at the amino acid position 70, preferably an asparagine-to-isoleucine substitution at the amino acid position 70. It should be understood that any of these modifications may be present in combination with any other amino acid modifications described herein, including those amino acid modifications at the amino acid positions 45, 49, and / or 52. In particular, an IL-15 variant may exist which contains amino acid modifications at the following amino acid positions: amino acids at positions 75 and 45; amino acids at positions 75, 45 and 48; amino acids at positions 75, 45 and 60; amino acids at positions 75, 45 and 71; amino acids at positions 75, 48 and 49; amino acids at positions 75, 48 and 52; or any combination thereof.

[0088] As described herein, IL-15 variants comprising one or more amino acid modifications are provided, wherein the amino acid modifications occur in a surface region adjacent to the receptor (e.g., IL-15Rα) interacting surface of the IL-15 variant (i.e., at one or more of amino acids at positions 45 and / or 49 and / or 52, compared to SEQ ID NO: 1). The receptor-interacting surface of the IL-15 variant can interact with or bind to an IL-15 receptor. Three IL-15 receptors exist: IL-15Rβ, γC, and IL-15Rα. IL-15Rβ may also be referred to as IL-2Rβ. These receptors form complexes with IL-15 via electrostatic interactions (Figure 7). In the sequence of the parental IL-15 (SEQ ID NO: 16), amino acids with the potential to be modified to enhance the stability of the complex are shown in bold and underlined.

[0089] SEQ ID NO: 16 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPS C KV T AM K CFL L EL Q VIS LESGDASIHDTVENLIILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS The inventors of this invention have surprisingly discovered that residues as shown in the sequence above are present in the exposed region of IL-15. Sequence analysis, in particular, reveals that lysine-41, leucine-45, glutamine-48, and leucine-52 readily aggregate, and are therefore promising candidates for amino acid modifications that improve solubility while preserving functionality.

[0090] Therefore, according to a second aspect of the invention, an interleukin-15 (IL-15) variant is provided, wherein the wild-type IL-15 comprises the amino acid sequence according to SEQ ID NO: 1, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, and wherein, compared with SEQ ID NO: 1, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor-interacting surface, and wherein said one or more amino acid modifications comprise any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophilic 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 said receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface.

[0091] In a particularly preferred embodiment, the hydrophobic-to-hydrophilic substitution at the 45th amino acid position is a substitution of leucine to serine or leucine to threonine.

[0092] In another particularly preferred embodiment, the hydrophobic-to-hydrophilic substitution at the 52nd amino acid position is a substitution of leucine to lysine or a substitution of leucine to arginine.

[0093] In another particularly preferred embodiment, the hydrophobic-to-hydrophobic substitution at the 49th amino acid position is a substitution of valine to alanine or a substitution of valine to serine.

[0094] The IL-15Rα receptor contains a protein-binding motif known in the art as the “sushi domain.” According to the invention, any IL-15 variant of the invention detailed herein can be fused to an IL-15R molecule, particularly an IL-15Rα molecule and / or the IL-15Rα sushi domain. The IL-15Rα molecule is a transmembrane protein with a hydrophobic transmembrane segment and is generally flexible and unstable. This makes the expression of the whole IL-15Rα molecule challenging. Therefore, the invention preferably fuses the IL-15 variant with the IL-15Rα sushi domain, rather than the whole IL-15Rα molecule. The IL-15Rα sushi domain is soluble and is the functional part of the receptor, meaning that only the sushi domain is functionally necessary and is also easier to express alone. As used herein, the term “fusion” refers to any connection of two or more proteins. Proteins can be fused via covalent linkage (e.g., via a linker) and can be produced via expression of a nucleic acid molecule containing the nucleic acid sequences of two or more proteins. In one embodiment, the fusion protein may comprise the sushi domain of the IL-15Rα molecule, followed by a linker, followed by any IL-15 variant described herein (IL-15Rα sushi: linker: IL-15 variant), i.e., in some embodiments, fused to the amino (N) terminus of said IL-15 variant.

[0095] The IL-15 variant may (or may not) fuse with the sushi domain of the IL-15Rα molecule. The fusion of the sushi domain with the IL-15 variant produces a super agonist (SAg) molecule. Any other suitable protein or peptide may also be linked to the IL-15 variant and / or the IL-15 / IL-15Rα sushi domain complex.

[0096] According to any aspect of the invention, any IL-15 variant detailed herein may be fused with the IL-15Rα molecule or its sushi domain. Such a fusion is referred to herein as a “super agonist,” “SAg,” “SAg variant,” or “IL-15 super agonist.” As used herein, the term “super agonist” refers to a cytokine capable of inducing a maximal immune response stronger than that of the agonist (i.e., the IL-15 variant) itself. The solubility of said super agonist may be lower than that of its corresponding IL-15 variant.

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

[0098] The term "related amino acid position" is intended to encompass the amino acid modifications described herein for each SEQ ID NO. For example, the amino acid modification at the related amino acid position of SEQ ID NO: 2 refers to the amino acid modification at positions 45 and 75.

[0099] In a preferred embodiment, the IL-15 super agonist may comprise an amino acid sequence according to any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, or an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13.

[0100] SEQ ID NO: 8 corresponds to an amino acid sequence containing an IL-15Rα sushi domain and an IL-15 variant containing the amino acid sequence according to SEQ ID NO: 2 (i.e., an SAg01 super agonist containing GBL01). SEQ ID NO: 9 corresponds to an amino acid sequence containing an IL-15Rα sushi domain and an IL-15 variant containing the amino acid sequence according to SEQ ID NO: 3 (i.e., an SAg15 super agonist containing GBL15). SEQ ID NO: 10 corresponds to an amino acid sequence containing an IL-15Rα sushi domain and an IL-15 variant containing the amino acid sequence according to SEQ ID NO: 4 (i.e., an SAg17 super agonist containing GBL17). SEQ ID NO: 11 corresponds to an amino acid sequence containing an IL-15Rα sushi domain and an IL-15 variant containing the amino acid sequence according to SEQ ID NO: 5 (i.e., an SAg18 super agonist containing GBL18). SEQ ID NO: 12 corresponds to an amino acid sequence containing the IL-15Rα sushi domain and an IL-15 variant containing the amino acid sequence according to SEQ ID NO: 6 (i.e., an SAg25 super agonist containing GB125). SEQ ID NO: 13 corresponds to an amino acid sequence containing the IL-15Rα sushi domain and an IL-15 variant containing the amino acid sequence according to SEQ ID NO: 7 (i.e., an SAg50 super agonist containing GBL50). These super agonist sequences utilize the GS linker according to SEQ ID NO: 17; however, those skilled in the art will understand that any suitable linker can be used to connect the IL-15 variant to the IL-15Rα molecule or the sushi domain of the IL-15Rα molecule.

[0101] Therefore, in another embodiment, an IL-15 variant is provided comprising an amino acid sequence according to any one or any combination of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 80% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 85% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 90% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 91% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 92% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 92% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 95 ...2% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 95% sequence identity with SEQ ID NO: 8, 9 SEQ ID NOs 8, 9, 10, 11, 12 and / or 13 have at least 93% sequence identity; have at least 94% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 95% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 96% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 97% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 98% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 99% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; and / or have SEQ ID NOs 8, 9, 10, 11, 12 and / or 13. SEQ ID NO: 8, 9, 10, 11, 12 and / or 13 have at least 100% sequence identity (wherein the percentage of sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13 preserves the amino acids at the relevant amino acid positions).

[0102] However, the preferred IL-15 super agonist may comprise an amino acid sequence according to any one of SEQ ID NO: 9, 11, 12 and / or 13, or comprise an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NO: 9, 11, 12 and / or 13 (wherein the percentage of sequence identity with SEQ ID NO: 9, 11, 12 and / or 13 preserves the amino acid modification at the relevant amino acid position), because the inventors of the present invention have surprisingly found that these super agonists are more soluble than the super agonist of wild-type IL-15. In another embodiment, the IL-15 superagonist may comprise the following amino acid sequences: said amino acid sequences having at least 75% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 80% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 85% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 90% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 91% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 92% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 93% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 94% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 94% sequence identity with SEQ ID NO: 9, 11, 12 and / or 13; having at least 95 ... SEQ ID NOs 9, 11, 12 and / or 13 have at least 95% sequence identity; at least 96% sequence identity with SEQ ID NOs 9, 11, 12 and / or 13; at least 97% sequence identity with SEQ ID NOs 9, 11, 12 and / or 13; at least 98% sequence identity with SEQ ID NOs 9, 11, 12 and / or 13; at least 99% sequence identity with SEQ ID NOs 9, 11, 12 and / or 13; and / or at least 100% sequence identity with SEQ ID NOs 9, 11, 12 and / or 13, wherein the percentage of sequence identity preserves the amino acid modifications at the relevant amino acid positions.

[0103] Such fusion can be achieved by directly linking two or more proteins together (i.e., direct fusion) and / or indirectly linking two or more proteins via any suitable linkage mechanism. The linkage can be covalent. However, direct linking without a linker can lead to folding errors, low solubility, or impaired biological activity of the fusion protein. Therefore, in a preferred embodiment, the IL-15 variant is fused to the sushi domain of the IL-15Rα molecule via a linker (such as a flexible peptide linker). Flexible linkers (e.g., linkers utilizing serine and glycine residues) allow the linked proteins to move relative to each other. The length of the linker can be increased to prevent steric interference between proteins. Short linkers can be approximately 4 to 6 amino acids long, medium-length linkers can be approximately 6 to 14 amino acids long, and long linkers can be approximately 14 to 21 or more amino acids long. The linker can be naturally occurring or synthetic. The linker can adopt protein secondary conformations such as α-helices and / or β-strands / coils / bends. In a more preferred embodiment, the linker is a glycine-serine linker (GS linker). The GS linker mainly consists of segments containing glycine and serine residues, such as (Ser-Gly-Gly-Gly-Gly). n Or (Ser-Gly-Gly-Gly) n Any ratio of glycine and serine residues can be used, and other amino acids, such as leucine or glutamine, may be present. The copy number 'n' can be adjusted to change the length of the GS linker to achieve proper dissociation of the linker protein, which can affect the function and interactions of the linker protein. The copy number 'n' is typically between 1 and 6. However, the preferred GS linker of the present invention may comprise the following sequence: SEQ ID NO: 17 SGGGSGGGGSGGGGSGGGGSGGSLQA In a preferred embodiment, the connector for fusing the IL-15 variant with the IL-15Rα molecule or the sushi domain of the IL-15Rα molecule comprises the amino acid sequence according to SEQ ID NO: 17, or comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 17.

[0104] While the present invention preferably uses a flexible GS connector, other flexible connectors may also be used, such as those containing additional amino acids such as threonine, alanine, lysine, and / or glutamic acid. Furthermore, flexible connectors consisting solely of glycine or serine may also be suitable, such as (Gly)8 connectors.

[0105] In addition to linking the IL-15Rα sushi domain, any other suitable protein or polypeptide linked to the IL-15 variant and / or IL-15 superagonist may be present. Such examples may include, but are not limited to, signal peptides, antibody fragments, antibodies, bispecific or multispecific antibodies, tissue-targeting sequences (such as NGR (Asn-Gly-Arg) tripeptides), chemokines, interleukins (especially those that stimulate cell-mediated immunity (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, 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).

[0106] In particular, to generate a strong immune response, for example, in the tumor microenvironment, therapeutic bacteria must be able to produce and secrete immunostimulatory cytokines in situ. Therefore, in a preferred embodiment, the IL-15 variant and / or IL-15 superagonist is fused with a signal peptide. As used herein, the term "signal peptide" refers to any signaling sequence, localizing sequence, or other polypeptide sequence that directs the IL-15 variant and / or IL-15 superagonist to a specific intracellular or extracellular location. Adding a small (15 to 25 amino acid) sequence, called a signal peptide, to a target protein can be used to guide it to the desired location.

[0107] Human IL-15 is produced in two isoforms via alternative splicing of its mRNA transcript (which occurs only in mammalian cells and not in bacterial cells). In humans, one isoform is a short signal peptide (SSP) (SEQ ID NO: 15) containing 21 amino acids and retained in the cytoplasm. The other isoform is a long signal peptide (LSP) (SEQ ID NO: 14) containing 48 amino acids, which allows it to be secreted into the extracellular environment.

[0108] For expression in bacteria, a bacterial signal peptide is required to secrete the IL-15 variant from the bacterial cytosol and into, for example, the periplasm. In a preferred embodiment, the signal peptide guides the IL-15 variant into the bacterial periplasm in a rapid and robust manner. Therefore, the preferred signal peptide of the present invention is a (bacterial) periplasmic signal peptide. A periplasmic signal peptide is a polypeptide sequence that allows proteins linked to the signal peptide to be transported, guided, localized, or targeted to the periplasm of bacterial cells. No universal signal peptide exists that can effectively translocate any protein; therefore, the inventors of the present invention have also screened for optimal signal peptides for each IL-15 variant.

[0109] As those skilled in the art will understand, the periplasm is a concentrated matrix within the space defined between the endoplasmic membrane and the outer membrane of Gram-negative bacteria. The periplasm is an oxidizing compartment, ideal for the formation of disulfide bonds, which are ubiquitous in human cytokines. Chaperone proteins are also present in the periplasm, which contribute to and promote disulfide bond formation. Conversely, the cytosol is a reducing environment unfavorable to disulfide bond formation. Therefore, it can be seen that directing cytokines (IL-15 variants and their superagonists) to the periplasm via signal peptides facilitates the production, proper folding, and eventual in situ secretion of these proteins.

[0110] In bacteria, there are two main pathways for protein export across the bacterial plasma membrane: the secretion (SEC) pathway or the diarginine translocation (TAT) pathway. The SEC pathway allows unfolded proteins to cross the inner membrane (IM), while the TAT pathway allows proteins folded in the cytoplasm to cross. Therefore, the signal peptide used for exporting any IL-15 variant or its superagonist according to the invention can be an SEC or TAT signal peptide. However, the SEC pathway allows proteins to fold in a suitable environment, so in a preferred embodiment, the signal peptide is an SEC signal peptide. The SEC pathway is common to both Gram-negative and Gram-positive bacteria. In Gram-negative bacteria, as a result of the SEC pathway, proteins are secreted into the periplasm. In Gram-positive bacteria, as a result of the SEC pathway, proteins are secreted directly into the external environment. In some embodiments, the N-terminus of the signal peptide used to guide the protein to the periplasm is fused to an IL-15 variant or IL-15 superagonist. However, the IL-15 variant or IL-15 superagonist can be exported extracellularly via other signal peptides, such as type I signal peptides. Therefore, in other embodiments, the C-terminus of the signal peptide is fused to an IL-15 variant or an IL-15 superagonist.

[0111] The IL-15 variant or superagonist can then be transferred from the periplasm into the supernatant or other extracellular environment. When the periplasmic signal peptide is fused with any of the IL-15 variants and / or IL-15 superagonists described herein, if expressed intracellularly in bacterial cells, the IL-15 variant and / or IL-15 superagonist can be efficiently transported into the periplasm of the bacterial cells to achieve proper folding and subsequent secretion from the bacterial cells. This secretion will enable the delivery of these molecules to target sites where they are needed, including to lesion sites in subjects who may benefit from such enhanced immune responses induced by the IL-15 variant and / or IL-15 superagonist. For example, when Gram-negative bacteria expressing the IL-15 variant or superagonist of the present invention infect and invade host (e.g., eukaryotic) cells, the IL-15 variant or superagonist can be transferred into the cytoplasm of the host cells, where it can achieve, for example, therapeutic immunostimulatory effects.

[0112] In one embodiment, the signal peptide is fused to the IL-15 variant and / or superagonist via a spacer sequence. As used herein, the terms "spacer region" and "spacer sequence" are used interchangeably and refer to any amino acid sequence located between the signal peptide and the carrier protein to be secreted (the IL-15 variant or its superagonist) (i.e., signal peptide:spacer region:sushi:linker:IL-15 variant, and / or signal peptide:spacer region:IL-15 variant). Preferably, the spacer region is a very short amino acid sequence, typically 1 to 5 amino acids in length (e.g., 1, 2, 3, 4, 4, or 5 amino acids). Any suitable amino acid sequence can be used as the spacer region. Even more preferably, the spacer region is present immediately following the AXA domain of the signal peptide. The AXA domain of the signal peptide is recognized by a signal peptidase that cleaves the signal peptide from the mature protein. The purpose of the spacer sequence is to increase the rate of unfolded protein export to the periplasm and to improve the cleavage of the signal peptide, thereby releasing the mature protein.

[0113] According to a third aspect of the invention, a nucleic acid molecule encoding an interleukin-15 (IL-15) variant according to a second aspect of the invention is provided. The invention also provides a nucleic acid molecule encoding an IL-15 variant contained in bacteria according to a first aspect of the invention. As used herein, the term "nucleic acid molecule" refers to a recombinant or synthetically produced nucleic acid molecule, including DNA and / or RNA, which contains a series of specific nucleic acid elements that allow a specific nucleic acid sequence to be expressed in a host cell. Specifically, the nucleic acid molecule can be expressed in bacterial cells, preferably in Gram-negative bacterial cells. Nucleic acid molecules may contain sequences encoding the following amino acid sequences: amino acid sequences according to any one or any combination of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 (preferably any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13), or amino acid sequences having at least 70% identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 preserves the amino acid modifications at the relevant amino acid positions. In some implementations, the expression of the IL-15 variant can be controlled by modulating the spatiotemporal activation and intensity of the promoter, i.e., by using a promoter activated at a specific location (e.g., intracellular or extracellular), at a specific time, or in response to a specific chemical substance.

[0114] In one embodiment, the nucleic acid molecule is a DNA molecule. As used herein, the terms “DNA” and “deoxyribonucleic acid” are given their common meaning in the art and are used interchangeably to refer to nucleic acids composed of thymine, adenine, guanine, and cytosine deoxyribonucleic acid bases. These terms and concepts are well known to those skilled in the art. In another embodiment, the nucleic acid molecule is an RNA molecule. As used herein, the terms “RNA” and “ribonucleic acid” are given their common meaning in the art and are used interchangeably to refer to nucleic acids composed of uracil, adenine, guanine, and cytosine ribonucleic acid bases. These terms and concepts are well known to those skilled in the art. Types of RNA molecules include, for example, mRNA, siRNA, shRNA, miRNA, tRNA, and rRNA. In a preferred embodiment, the RNA molecule is an mRNA molecule. As used herein, the terms “mRNA” and “messenger RNA” are used interchangeably to refer to a single-stranded RNA molecule involved in protein synthesis, which is transcribed from DNA and translated into an amino acid sequence. Bacterial mRNA molecules are transcribed from non-compartmentalized DNA, and transcription and translation are coupled in the cytosol. These terms and concepts are well known to those skilled in the art. Eukaryotic mRNA molecules are transcribed from DNA in the nucleus of eukaryotic cells and subsequently exported from the nucleus to the cytoplasm of the eukaryotic cell, where the mRNA molecules are translated into proteins.

[0115] Nucleic acids can be expressed in host cells, such as bacterial cells that have been engineered or modified to express the nucleic acids. Alternatively, nucleic acids can be expressed in eukaryotic cells. In this embodiment, the nucleic acid (e.g., RNA) can be contained within the bacterial cell and then delivered to the eukaryotic cell. The RNA molecule has a nucleotide-coding structure that, in the case of mRNA, allows it to be transcribed within the bacteria themselves and then translated and expressed as a protein within the eukaryotic cell as the bacteria invade and replicate in the target eukaryotic cell.

[0116] The nucleic acid molecule may comprise a sequence encoding an amino acid sequence according to any one or any combination of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16. In a preferred embodiment, the nucleic acid molecule encodes any one or any combination of SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 11, 12, 13 and / or 16. In a more preferred embodiment, the nucleic acid molecule encodes any one or any combination of SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 11, 12 and / or 13. In another embodiment, the nucleic acid molecule encodes an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, or a sequence having at least 70% identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 preserves amino acid modifications at the relevant amino acid positions.

[0117] In another embodiment, a nucleic acid molecule may be provided comprising a sequence encoding the following amino acid sequences: said amino acid sequences having at least 75% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; having at least 80% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; having at least 85% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; having at least 90% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; and having at least 90% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16. SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 have at least 91% sequence identity; have at least 92% sequence identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 93% sequence identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 94% sequence identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 95 ...5% sequence identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 have at least 96% sequence identity; have at least 97% sequence identity; have at least 98% sequence identity; have at least 99% sequence identity; and / or have at least 100% sequence identity, wherein the sequence identity is with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16. The percentage of sequence identity of any one of NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 preserves the amino acid modifications at the relevant amino acid positions.

[0118] In the most preferred embodiment, the nucleic acid molecule may comprise a sequence encoding the following amino acid sequence: said amino acid sequence is any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; or said amino acid sequence has at least 70% identity with any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; has at least 75% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; has at least 80% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; has at least 85% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; has at least 85% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; has at least 7 ... SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13 have at least 90% sequence identity; have at least 91% sequence identity with SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; have at least 92% sequence identity with SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; have at least 93% sequence identity with SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; have at least 94 ...2% sequence identity with SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; have at least 93% sequence identity with SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13; have at least 94% sequence identity with SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13 SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13 have at least 95% sequence identity; have at least 96% sequence identity; have at least 96% sequence identity; have at least 97 ...8% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 98% sequence identity; have at least 99% sequence identity; have at least 9 NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13 have at least 100% sequence identity, wherein the percentage of sequence identity preserves the amino acid modifications at the relevant amino acid positions.

[0119] In a fourth aspect of the invention, bacteria comprising an IL-15 variant according to a second aspect of the invention or a nucleic acid molecule according to a third aspect of the invention are provided. As those skilled in the art will understand, interleukins play multiple roles in inducing immune responses. Given the role of IL-15 in stimulating the proliferation and cytotoxic function of CD8 T cells and NK cells, the therapeutic potential of IL-15 is evident. The inventors of the present invention have surprisingly discovered that certain mutations in the amino acid structure of IL-15 can produce IL-15 variants optimized in terms of solubility without affecting biological activity. Furthermore, the corresponding super agonists of these IL-15 variants may have the potential to induce maximal and durable immune responses against a variety of diseases in subjects. Therefore, in a fifth aspect of the invention, bacteria according to a first or fourth aspect of the invention, or an IL-15 variant according to a second aspect of the invention, or a nucleic acid molecule according to a third aspect of the invention, are provided for therapeutic use. Any IL-15 variant and / or IL-15 super agonist disclosed herein may be used for treatment in any combination.

[0120] Because interleukins are widely involved in several different signaling pathways (including both pro-inflammatory and anti-inflammatory signaling), it should be understood that this invention relates to the treatment, prevention, mitigation, suppression, 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, infectious diseases, cardiovascular diseases, neurodegenerative diseases, gastrointestinal diseases, respiratory diseases, kidney diseases, liver diseases, autoimmune diseases, inflammatory diseases, or genetic disorders. In a preferred embodiment, the disease may be a neoplastic disease. Therefore, this invention provides bacteria according to the first or fourth aspect of the invention, or IL-15 variants according to the second aspect of the invention, or nucleic acid molecules according to the third aspect of the invention, for the treatment, prevention, mitigation, suppression, prevention of recurrence, or control of neoplastic diseases in subjects.

[0121] The terms “tumor,” “cancer,” “malignant tumor,” and “tumor formation” are used interchangeably to refer to cells or groups of cells that grow, proliferate, or survive in greater numbers than their normal counterparts, such as in proliferative or differentiation disorders. Typically, this growth is uncontrolled. The term “malignant tumor” refers to invasion of nearby tissues. The term “metastasis” refers to the spread or dissemination of a tumor, cancer, or tumor formation to other sites, locations, or regions within the subject that are distinct from the primary tumor or cancer.

[0122] In one implementation, the neoplastic disease can be a solid carcinoma and / or a hematologic malignancy. Tumor formation, tumor, and cancer 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 tumor formation, tumor, or cancer, or tumor formation, tumor, cancer, or metastasis that is progressing, worsening, stable, or in remission.

[0123] The cancers treatable according to the present invention include, but are not limited to, cells or tumors of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, cancer can specifically have the following histological types, but is not limited to: tumor formation; malignant tumor; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial adenomatous polyposis; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic cell carcinoma; eosinophilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell adenocarcinoma. Cancer; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Uncapsulated sclerosing carcinoma; Adrenocortical carcinoma; Endometrial cancer; Skin adnexal cancer; Apocrine gland cancer; Sebaceous gland cancer; Cerumen gland cancer; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous gland carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant androblastoma; Sertoli cell carcinoma; Malignant testicular stromal cell tumor; Malignant lipocyte tumor; Malignant paraganglioma; Malignant breast Extraglandular paraganglioma; Pheochromocytoma; Angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial diffuse melanoma; Malignant melanoma in giant nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Acinar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant stromal tumor; Malignant Brenner's tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonic carcinoma; Malignant teratoma; Malignant ovarian goiter; Choriocarcinoma Malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma;Retinoblastoma; olfactory neurogenic tumors; malignant meningiomas; neurofibrosarcomas; malignant schwannomas; malignant granular cell tumors; malignant lymphomas; Hodgkin's disease; Hodgkin's lymphoma; paragranulomas; small lymphocytic malignant lymphomas; diffuse large cell malignant lymphomas; follicular malignant lymphomas; mycosis fungoides; other specific non-Hodgkin's lymphomas; malignant histiocytic proliferative disorders; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel diseases; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0124] Preferably, the solid tumor and / or hematologic malignancy can be a cancer selected from prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, malignant epithelial tumors, head and neck cancer, endometrial cancer, skin cancer, or sarcoma. More preferably, the neoplastic disease can be associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular carcinoma, melanoma, esophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer, or breast cancer.

[0125] In some implementation schemes, neoplastic disease is defined as malignant neoplastic disease.

[0126] In some implementations, neoplastic diseases are not benign neoplastic diseases.

[0127] In a preferred embodiment, the malignant tumor may be associated with cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial cancer, thyroid cancer, melanoma, malignant epithelial tumor, head and neck cancer, skin cancer, or sarcoma.

[0128] In another preferred embodiment of the invention, the IL-15 variant or IL-15 super agonist will be administered intratumorally, intravesically, intravenously, intraperitoneally, or orally. In the most preferred embodiment, the IL-15 variant or IL-15 super agonist is administered intratumorally. However, other methods of administration are also contemplated in some cases. Thus, in certain circumstances, the IL-15 variant or IL-15 super agonist of the present invention may be administered by injection, infusion, continuous infusion, intradermal, intraarterial, intralesional, intravaginal, intrarectal, intramuscular, subcutaneous, subconjunctival mucosa, intraperitoneal, intraumbilical, intraocular, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intranasal, inhalation (e.g., aerosol inhalation), via catheter, via irrigation, or by other methods known to those skilled in the art, or any combination of the foregoing methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition. Mack Printing Company, 1990). However, the present invention is preferably administered by local infusion, intraperitoneal, intrapleural, intravesical, peritumoral injection, intratumoral injection or oral administration.

[0129] As used herein, the term "infusion" refers to the introduction of the composition into the relevant anatomical site and its residence there for a specified duration, followed by drainage, emptying, or aspiration. As used herein, the term "intraperitoneal" refers to the injection of the composition into the peritoneum of a subject. As used herein, the term "intrapleural" refers to the injection of the composition into the pleura or pleural cavity of a subject. As used herein, the term "intravesical" refers to the injection or infusion of the composition into the bladder of a subject via a catheter. As used herein, the term "peritumoral injection" refers to the injection of the composition around the lesion of a neoplastic disease, in cases where the disease under discussion is neoplastic. As used herein, the term "intratumoral injection" refers to the direct injection of an IL-15 variant or an IL-15 superagonist into the lesion of a subject's neoplastic disease, in cases where the disease under discussion is neoplastic.

[0130] It should be understood that the specific method of administration of the composition may depend on the disease to be treated, such as the location and type of the disease. For example, if a large surface area of ​​a body cavity needs to be treated, such as the subject's pleural cavity, administration by infusion may be most appropriate. Alternatively, if the disease is located in the peritoneal cavity, administration via intraperitoneal injection may be most appropriate. Furthermore, if the disease to be prevented and / or treated is, for example, a hematologic malignancy, it should be noted that intratumoral injection may not be the preferred method of administration.

[0131] In addition to direct administration of any IL-15 variant and / or its superagonist disclosed herein, for optimal and efficient expression, folding, and secretion of said IL-15 variant or its superagonist, this document also provides a bacterium containing any IL-15 variant and / or IL-15 variant superagonist or any combination thereof disclosed herein, or containing a nucleic acid molecule encoding said variant. The IL-15 variants and / or their superagonists disclosed herein can be expressed and secreted by a suitable expression system and isolated for direct administration to a subject in need. However, in a preferred embodiment, the IL-15 variant and / or its superagonist can be administered co-administered with bacteria, thereby administering bacteria containing the IL-15 variant and / or its superagonist or a nucleic acid molecule encoding the IL-15 variant and / or its superagonist and expressing and secreting the IL-15 variant and / or its superagonist. In some embodiments, the bacteria may be Gram-negative bacteria. In other embodiments, the bacteria may be Gram-positive bacteria.

[0132] Any suitable bacteria can be used that are capable of containing an IL-15 variant and / or its superagonist according to the invention, or containing a nucleic acid molecule encoding an IL-15 variant and / or its superagonist. However, in a particularly preferred embodiment, Gram-negative bacteria comprising a nucleic acid construct according to the invention are provided.

[0133] For example, a bacterium, such as a Gram-negative bacterium, may be provided comprising a nucleic acid molecule, said nucleic acid molecule comprising a sequence encoding the following amino acid sequence: said amino acid sequence is any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, or has at least 70% identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; has at least 75% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; has at least 80% sequence identity with ...75% sequence identity with SEQ ID NO SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 have at least 85% sequence identity; have at least 90% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 91% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 92% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 93% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 93% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 95 ... SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 have at least 94% sequence identity; have at least 95% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 96% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 97% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; have at least 98 ...4% sequence identity with SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 have at least 99% sequence identity;And / or having at least 100% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 preserves the amino acid modifications at the relevant amino acid positions. Preferably, the bacteria may comprise a nucleic acid molecule comprising a sequence encoding an amino acid sequence according to any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and / or 13.

[0134] According to the present invention, any suitable bacteria capable of containing IL-15 variants can be used. However, in a particularly preferred embodiment, the bacteria are Gram-negative bacteria.

[0135] Any Gram-negative bacteria capable of expressing and secreting heterologous interleukins as described herein. Examples of Gram-negative bacteria used in this invention include, but are not limited to, Salmonella, Escherichia coli, and Shigella. Shigella ), Pseudomonas ( Pseudomonas Moraxella ( ) Moraxella ), Helicobacter pylori ( Helicobacter ), Oligotrophomonas ( Stenotrophomonas ), Bdellovibrio ( Bdellovibrio Legionella ( Legionella ), Chlamydia ( Chlamydia ) and Yersinia ( Yersinia Preferably, the Gram-negative bacteria are live, attenuated Gram-negative bacteria. As used herein, in the context of this invention, the term "attenuated" refers to altering a microorganism to reduce its pathogenicity, rendering it harmless to the host while maintaining its viability. This approach is commonly used in vaccine development because it can elicit a highly specific immune response while maintaining acceptable safety. The development of such vaccines can involve a variety of methods, examples including, but not limited to, passage of pathogens under in vitro conditions until loss of virulence, chemical mutagenesis, and genetic engineering techniques. Such attenuated microorganisms are preferably live, although non-live attenuated microorganisms are also disclosed. As used herein, the term "inactivating mutation" refers to a modification of the natural genetic code of a particular gene or a gene promoter associated with that gene, such as by altering the nucleotide code or deleting nucleotide segments or adding non-coding nucleotides or non-natural nucleotides, such that the particular gene cannot be properly transcribed or translated or expressed as an inactive protein, resulting in the complete elimination or reduction of the gene's natural function to an unmeasurable degree. Thus, a mutation of a gene inactivates the function of that gene or the protein encoded by that gene.

[0136] In other embodiments, the bacteria may be Gram-positive bacteria. Any Gram-positive bacteria capable of expressing and secreting the heterologous cytokines disclosed herein may be used. Examples of Gram-positive bacteria used in this invention include, but are not limited to, Bacillus spp. Bacillus Clostridium ( Clostridium Corynebacterium spp. Corynebacterium Listeria ( ) Listeria ) and Gardnerella spp. Gardnerella Similarly, live attenuated bacteria are preferred.

[0137] As used herein, the term "non-natural bacteria" refers to bacterial (prokaryotic) cells that have been genetically modified or "engineered" to alter their structure relative to naturally occurring cells. Such genetic modification can be, for example, incorporating additional genetic information into the cell, modifying existing genetic information, or effectively deleting existing genetic information. This can be achieved, for example, by transfecting recombinant plasmids into the cell or by directly modifying the bacterial genome. Alternatively, bacterial cells can be genetically modified by chemical mutagenesis, for example, to achieve attenuation, methods well known to those skilled in the art. Therefore, the term "non-natural bacteria" can refer to both recombinantly modified and non-recombinantly modified bacterial strains. As used herein, the terms "recombinant," "recombinant strain," or "recombinant bacteria" are used interchangeably, and in the context of this invention, refer to bacterial strains that have been genetically engineered so that their DNA has been altered by the introduction of new DNA. Recombinant DNA methods typically involve the introduction of new DNA via a vector (e.g., plasmid). These methods are well known to those skilled in the art. Using recombinant bacterial strains can confer advantageous properties on bacterial strains, such as prolonged activity, eliciting a stronger immune response in a subject, or introducing desired molecules.

[0138] Preferably, the Gram-negative bacteria of the present invention can be intestinal bacteria. Examples of intestinal bacteria include, but are not limited to, Enterobacteriaceae (such as Escherichia coli, Klebsiella pneumoniae, etc.). Klebsiella ), Proteus ( Proteus ) and Enterobacteriaceae ( Enterobacter Salmonella, Shigella, Yersinia, and Campylobacter jejuni ( Campylobacter jejuni ).

[0139] Preferably, the Gram-negative bacteria are species of the genus *Salmonella*. An example of a *Salmonella* species used in this invention is *Salmonella enterica* (…). Salmonella enterica ) and Salmonella Bongomery ( Salmonella bongori Enteric Salmonella can be further subdivided into different serotypes or serovariates. An example of a serotype or serovariate used in this invention is *Salmonella typhi* (…). Salmonella enterica Typhi), Salmonella paratyphi A ( Salmonella enterica Paratyphi A), Salmonella paratyphi B ( Salmonella enterica Paratyphi B), Salmonella paratyphi C ( Salmonella enterica Paratyphi C), Salmonella typhimurium ( Salmonella enterica Typhimurium and Salmonella enteritidis Salmonella enterica (Enteritidis). In a preferred embodiment, the live attenuated Gram-negative bacteria is any attenuated, non-pathogenic Salmonella typhi or Salmonella typhimurium serotype strain. In the most preferred embodiment, the live attenuated Gram-negative bacteria is Salmonella typhi. In an even more preferred embodiment, the live attenuated bacteria is Salmonella typhi ZH9 (also known as M01ZH09). Derivatives or variants of Salmonella typhi ZH9 strains are also intended to be included, including genetically modified variants.

[0140] This bacterium will be engineered to express any IL-15 variant and / or its super agonist disclosed herein. Many methods and techniques for genetically engineering bacterial strains will be well known to those skilled in the art. These techniques include those required to introduce heterologous genes into bacteria via chromosomal integration or by introducing stable autosomal self-replicating genetic elements. Exemplary methods for genetically modifying (also known as “transforming” or “engineering”) bacterial cells include phage infection, transduction, conjugation, lipid transfection, or electroporation. Other techniques are also intended to be included, including CRISPR and CRISPR-related proteins such as Cas9, CasCLOVER, TALEN, retrotranscriptans, homing endonucleases or meganucleases, zinc finger nucleases, and transposon-based methods. An overview of genetic engineering methods in molecular and cellular biochemistry can be found in the following standard textbooks, such as *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook et al., HaRBor Laboratory Press 2001); *Short Protocols in Molecular Biology*, 4th edition (edited by Ausubel et al., John Wiley & Sons 1999); and *Protein Methods* (Bollag et al., John Wiley & Sons 1996). These references are cited here.

[0141] For example, a bacterium, such as a Gram-negative bacterium, is provided that comprises an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and / or 16, more preferably any one of SEQ ID NO: 2, 3, 4, 5, 6, 7 and / or 16, even more preferably any one of SEQ ID NO: 2, 3, 4, 5, 6 and / or 7, or comprising an amino acid sequence having at least 70% sequence identity with any of these sequences, wherein the percentage of sequence identity preserves the amino acid modifications at the relevant amino acid positions. In another embodiment, a bacterium, such as a Gram-negative bacterium, may be provided comprising an IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NO: 2, 3, 4, 5, 6, 7 and / or 16, or comprising the following amino acid sequences having at least 75% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7 and / or 16; having at least 80% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7 and / or 16; having at least 85% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7 and / or 16; having at least 90% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7 and / or 16; having at least 91% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7 and / or 16; and having at least 91% sequence identity with SEQ ID NO: 2, 3, 4, 5, 6, 7 and / or 16. SEQ ID NOs 2, 3, 4, 5, 6, 7 and / or 16 have at least 92% sequence identity; have at least 93% sequence identity; have at least 94% sequence identity; have at least 95% sequence identity; have at least 96% sequence identity; have at least 97% sequence identity; have at least 98% sequence identity; have at least 99% sequence identity; and / or have at least 99% sequence identity; and / or have at least 98% sequence identity; and / or have at least 99% sequence identity; and / or have at least 99% sequence identity. NO: 2, 3, 4, 5, 6, 7 and / or 16 have at least 100% sequence identity, wherein the percentage of sequence identity preserves the amino acid modifications at the relevant amino acid positions.

[0142] In another preferred embodiment, bacteria comprising an IL-15 superagonist, such as Gram-negative bacteria, may be provided, the IL-15 superagonist comprising an amino acid sequence according to any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, or comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, wherein the sequence identity percentage preserves amino acid modifications at the relevant amino acid positions. In an even more preferred embodiment, the bacteria may comprise an IL-15 superagonist comprising an amino acid sequence according to any one of SEQ ID NO: 9, 11, 12 and / or 13.

[0143] In another embodiment, bacteria comprising an IL-15 superagonist, such as Gram-negative bacteria, may be provided, wherein the IL-15 superagonist comprises an amino acid sequence according to any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, or comprises the following amino acid sequences having at least 75% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 80% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 85% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 90% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 91 ...75% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 80% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 91% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 90% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13; having at least 91% sequence identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or SEQ ID NOs 8, 9, 10, 11, 12 and / or 13 have at least 92% sequence identity; have at least 93% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 94% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 95% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 96% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 97% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 98 ...4% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 95% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 96% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 97% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 98% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12 and / or 13; have at least 9 SEQ ID NOs 8, 9, 10, 11, 12, and / or 13 have at least 99% sequence identity; and / or have at least 100% sequence identity with SEQ ID NOs 8, 9, 10, 11, 12, and / or 13, wherein said sequence identity percentage preserves amino acid modifications at the relevant amino acid positions. As those skilled in the art will understand, this is also intended to include bacteria, such as Gram-negative bacteria, comprising nucleic acid molecules encoding any of the amino acid sequences described herein.

[0144] Unless otherwise stated, bacteria containing IL-15 variants and / or their IL-15 superagonists (SAg variants) are referred to herein as compositions. The IL-15 variants and / or their SAg variants will promote an enhanced immune response, and the bacteria themselves 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, such as lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, which result in the selective damage, destruction, or elimination of cancer cells from the body. As used herein, the term "cellular components of the immune system" refers to immune cells, such as lymphocytes, including T and B lymphocytes, γ-δ T cells, and NK cells, which can recognize specific antigens, such as prions, viruses, bacteria, yeast, fungi, parasites, tumor-associated or tumor-specific antigens, or other antigens associated with a particular disease, symptom, or condition. Other immune cells we refer to include blood cells, which may be granulocytes or agranulocytes. Examples of immune cells include neutrophils, eosinophils, basophils, lymphocytes, monocytes, and macrophages. Dendritic cells, microglia, and other antigen-presenting cells are also included in this definition.

[0145] An immune response can be 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 any combination thereof. As used herein, the terms “systemic immune response” and “systemic immunity” are used interchangeably and refer to a broad immune response against the inducer throughout the subject’s body, as well as broad non-specific immune activation, unlike a local, spatially confined response. Such a response involves complex interactions between different cells of the immune response, such as lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by these cells or the liver, which, in the context of this invention, are considered to prime the subject’s immune system so that when the second composition is administered locally to the lesion site of a neoplastic disease, the subject is more responsive to the second composition containing live attenuated bacteria. Therefore, the “systemic immune response” can be measured and quantified by analyzing 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. Methods for measuring such effects are well known to those skilled in the art, such as flow cytometry. The “systemic immune response” can also be measured and quantified by the presence of antibodies (including but not limited to IgG and IgA subtype antibodies). Methods for measuring these antibodies are well known to those skilled in the art, such as ELISA. Therefore, the first composition can induce (and be used interchangeably with “modulate,” “enhance,” “amplify,” “strengthen,” “improve,” “enhance,” or “promote”) the immune response of a subject after administration of the second composition.

[0146] Therefore, the combination of the IL-15 variant and / or IL-15 superagonist with bacteria (wherein the IL-15 variant and / or IL-15 superagonist is expressed and secreted by the bacteria) can produce a synergistic effect and provide an enhanced immune response in the subject. The IL-15 variant or IL-15 superagonist of the present invention (i.e., the composition) delivered by bacteria (such as Gram-negative bacteria as defined herein) will enable the subject's immune system to generate a potent and durable effective immune response against disease, and may lead to enhanced therapeutic benefit. The immune response induced by the administration of the composition may be therapeutically adequate or subtherapeutic, requiring subsequent administration of additional doses of the composition to achieve a therapeutic effect.

[0147] Bacteria engineered to express any IL-15 variant and / or its superagonist according to any aspect of the invention (e.g., Gram-negative bacteria) will function as therapeutic strains. Therefore, the bacteria disclosed herein can be used for treatment. These therapeutic strains can be administered to subjects requiring such therapy. The term “treatment” or “therapy” refers to the administration of an active agent to a subject with the aim of curing, healing, alleviating, resolving, altering, remedying, improving, ameliorating, or influencing a condition (e.g., a disease), symptoms of a condition, or preventing or delaying the onset of symptoms, complications, biochemical markers of a disease, or statistically significantly halting or inhibiting the further development of a disease, condition, or symptom. As used herein, the term “subject” is intended to include humans and non-human animals (i.e., any member of the animal kingdom). Preferred subjects include humans (i.e., human patients requiring enhanced immune responses). The methods are particularly suitable for treating patients (preferably human patients) suffering from conditions that can be treated by enhancing immune responses. In one specific embodiment, the IL-15 variant and / or its superagonist is particularly suitable for in vivo treatment of cancer.

[0148] The amount of bacteria administered to the subject is sufficient to deliver a therapeutically effective amount of the IL-15 variant and / or IL-15 superagonist to the subject. Those skilled in the art will readily understand that the specific dosage will depend on many factors, such as the disease to be treated, the IL-15 variant and / or its IL-15 superagonist to be expressed and secreted, and the medical history of the subject to be treated.

[0149] The terms "therapeutic effective amount" and "effective amount" refer to an amount of a pharmaceutical agent sufficient to provide a desired biological or therapeutic outcome. This outcome can be a reduction, improvement, mitigation, alleviation, delay, and / or relief of signs, symptoms, or causes of one or more diseases, or any other desired alteration of a biological system. Regarding cancer, an effective amount can include an amount sufficient to cause tumor shrinkage and / or a decrease in the rate of tumor growth (e.g., inhibition of tumor growth) or to prevent or delay the proliferation of other unwanted cells. In some embodiments, an effective amount is an amount sufficient to delay the development of cancer or tumor or prolong survival or induce cancer or tumor stabilization. In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay recurrence. A therapeutically effective amount can be administered by a single or multiple doses. A therapeutically effective amount of a drug or combination thereof may result in one or more of the following outcomes: (i) a reduction in the number of cancer cells; (ii) a reduction in tumor size; (iii) to some extent an inhibition, delay, slowing, and preferably terminating of the invasion of cancer cells into peripheral organs; (iv) an inhibition (i.e., to some extent a slowing and preferably cessation) of tumor metastasis; (v) an inhibition of tumor growth; (vi) prevention or delay of tumor occurrence and / or recurrence; and / or (vii) to some extent an relief of one or more cancer-related symptoms.

[0150] For example, in the treatment of tumors, a “therapeutic effective dose” can induce a reduction in tumor size by at least about 5% relative to baseline measurements, such as at least about 10%, or about 20%, or about 60% or more. Baseline measurements can be obtained from untreated subjects.

[0151] A therapeutically effective amount of the therapeutic compound can reduce tumor size or otherwise improve symptoms in a subject. Those skilled in the art will be able to determine this amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration chosen.

[0152] The bacteria can reach 10 5 Up to 10 12 CFU dosage, where CFU stands for colony-forming unit. For example, a suitable dosage could be 10... 5 Up to 10 6 CFU, 10 5 Up to 10 7 CFU, 10 5 Up to 10 8 CFU, 10 5 Up to 10 9 CFU, 10 5 Up to 10 10 CFU, 10 5 Up to 10 11 CFU, 10 6 Up to 10 7 CFU, 10 6 Up to 10 8 CFU, 10 6 Up to 10 9 CFU, 10 6 Up to 10 10 CFU, 10 6 Up to 10 11 CFU, 10 6 Up to 10 12 CFU, 10 7 Up to 10 8 CFU, 10 7 Up to 10 9 CFU, 10 7 Up to 10 10 CFU, 10 7 Up to 10 11 CFU, 10 7 Up to 10 12 CFU, 10 8 Up to 10 9 CFU, 10 8 Up to 10 10 CFU, 10 8 Up to 1011 CFU, 10 8 Up to 10 12 CFU, 10 9 Up to 10 10 CFU, 10 9 Up to 10 11 CFU, 10 9 Up to 10 12 CFU, 10 10 Up to 10 11 CFU, 10 10 Up to 10 12 CFU, or 10 11 Up to 10 12 CFU. The composition can be administered as a single dose or in multiple doses. The specific number of doses to be administered should be understood to depend on the IL-15 variant to be delivered and the specific indication to be treated.

[0153] The bacteria can be administered in any manner that places them in the vicinity of the lesion. As used herein, the term "vicinity" means an area or range extending a certain distance around the lesion. For example, in one embodiment, the term "vicinity" may refer to an area / range extending, for example, from the disease boundary to a maximum of 10 mm, 5 mm, or 2.5 mm. Administration of the composition at a distance from the lesion site can exert biological effects (e.g., recruitment and activation of various immune cell types) on the local environment of the relevant lesion while having minimal / no effect on tissues located in unrelated areas of the body. Additionally, the composition can be administered into or onto the surface of tissue or surrounding tissue. Therefore, the term "local administration" refers to any situation in which the bacteria can come into contact with the lesion or with immediately surrounding tissue to have the desired effect. As used herein, the terms "local" and "local administration" are used interchangeably and, in the context of this invention, refer to the manner in which a variant of IL-15 or its superagonist and / or bacteria are administered to a subject. Therefore, the resulting immune response in the subject is also considered to be local to the lesion site, i.e., not a broad systemic immune response.

[0154] In a preferred embodiment of the invention, the bacteria described herein are administered intratumorally, intrabladderally, intravenously, intraperitoneally, or orally. In the most preferred embodiment, the bacteria are administered intratumorally or orally. However, it is also contemplated that other methods of administration may be used in some cases. Thus, in certain circumstances, the bacteria of the invention may be administered by injection, infusion, continuous infusion, intradermal, intraarterial, intralesional, intravaginal, intrarectal, intramuscular, subcutaneous, subconjunctival mucosa, pericardial, umbilical, intraocular, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, inhalation (e.g., aerosol inhalation), via catheter, via irrigation, or by other methods known to those skilled in the art, or any combination of the foregoing methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition. Mack Printing Company, 1990).

[0155] It should be understood that the specific method of administration of the bacteria described herein may depend on the disease to be treated, such as its location and type. For example, if a large surface area of ​​a body cavity needs to be treated, such as the subject's pleural cavity, administration by infusion may be most appropriate. Alternatively, if the disease is located in the peritoneal cavity, administration via intraperitoneal injection may be most appropriate. Furthermore, if the disease to be prevented and / or treated is, for example, a hematologic malignancy, it should be noted that intratumoral injection may not be the preferred method of administration.

[0156] Because interleukins are widely involved in several different signal transduction pathways (including pro-inflammatory and anti-inflammatory signal transduction), it should be understood that this invention relates to the treatment, prevention, mitigation, inhibition, prevention of recurrence, or control of a variety of diseases. In a preferred embodiment, the disease is a human disease. Diseases may include, but are not limited to, infectious diseases, cardiovascular diseases, neurodegenerative diseases, gastrointestinal diseases, respiratory diseases, kidney diseases, liver diseases, autoimmune diseases, inflammatory diseases, or genetic disorders. In a preferred embodiment, the disease may be a neoplastic disease. Therefore, according to the invention, bacteria according to the first or fourth aspect of the invention, or IL-15 variants according to the second aspect of the invention, or nucleic acid molecules according to the third aspect of the invention, are provided for the treatment, prevention, mitigation, inhibition, prevention of recurrence, or control of neoplastic diseases in subjects. Furthermore, in a sixth aspect of the invention, the use of bacteria according to the first or fourth aspect of the invention, or IL-15 variants according to the second aspect of the invention, or nucleic acid molecules according to the third aspect of the invention, is provided in the preparation of a medicament for treatment.

[0157] When the disease to be treated is a neoplastic disease, the bacteria and their IL-15 variants and / or SAg variants can be administered in combination with another therapy. Neoplastic diseases are known to have multifaceted and diverse causes, often leading to prevention and treatment strategies involving multiple therapies to achieve optimal outcomes. Therefore, the present invention may relate to combining bacteria expressing the aforementioned IL-15 variants and / or IL-15 superagonists with other known cancer therapies. Preferably, the bacteria expressing the aforementioned IL-15 variants and / or IL-15 superagonists can be administered in combination with immunotherapy, radiotherapy, chemotherapy, or anticancer agents. As used herein, the term "anticancer agent" refers to any agent that effectively kills cancer cells, stops cancer cell division, or helps prevent cancer cell recurrence, but is not considered immunotherapy, radiotherapy, or chemotherapy. In a preferred embodiment, the bacteria expressing the aforementioned IL-15 variants and / or their SAg variants can be administered in combination with immunotherapy. Preferably, the immunotherapy may include checkpoint inhibitors, antigen-specific T cells, adoptive T cell therapy, therapeutic antibodies, cancer vaccines, or any other engineered cell immunotherapy. Bacteria expressing the IL-15 variant and / or IL-15 superagonist can be administered separately, simultaneously, or sequentially (before and / or after) other known cancer therapies.

[0158] In one implementation, the neoplastic disease can be a solid carcinoma and / or a hematologic malignancy. Tumor formation, tumor, and cancer 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 tumor formation, tumor, or cancer, or tumor formation, tumor, cancer, or metastasis that is progressing, worsening, stable, or in remission.

[0159] The cancers treatable according to the present invention include, but are not limited to, cells or tumors of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, cancer can specifically have the following histological types, but is not limited to: malignant tumors; malignant epithelial tumors; undifferentiated malignant epithelial tumors; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial adenomatous polyposis; solid carcinoma; malignant carcinoid tumors; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic cell carcinoma; eosinophilic adenocarcinoma; basophilic cell carcinoma; clear cell carcinoma. Cellular adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Non-capsulated sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Skin adnexal carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous gland carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant androblastoma; Sertoli cell carcinoma; Malignant testicular stromal cell tumor; Malignant lipocyte tumor Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial diffuse melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Acinar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant stromal tumor; Malignant Brenner's tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonic carcinoma; Malignant teratoma Tumors; Malignant ovarian goiter; Choriocarcinoma; Malignant mesonephroma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Cortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing sarcoma; Malignant odontogenic tumors; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pineal tumors; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibroblastic astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumors; Cerebellar sarcoma;Ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specific non-Hodgkin's lymphomas; malignant histiocytic proliferative disorders; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and piloblastic leukemia.

[0160] Preferably, the solid tumor and / or hematologic malignancy can be a cancer selected from prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, mesothelioma, thyroid cancer, melanoma, malignant epithelial tumor, head and neck cancer, skin cancer, or sarcoma. Even more preferably, the neoplastic disease can be associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular carcinoma, melanoma, esophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer, or breast cancer.

[0161] In some implementation schemes, neoplastic disease is defined as malignant neoplastic disease.

[0162] In some implementations, neoplastic diseases are not benign neoplastic diseases.

[0163] In a preferred embodiment, the malignant tumor may be associated with cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial cancer, thyroid cancer, melanoma, malignant epithelial tumor, head and neck cancer, skin cancer, or sarcoma.

[0164] In a seventh aspect of the invention, a method is provided for treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject a bacterium according to a first or fourth aspect of the invention, or an IL-15 variant according to a second aspect of the invention, or a nucleic acid molecule according to a third aspect of the invention.

[0165] Therefore, the present invention also provides methods for treating, inhibiting, preventing recurrence, or controlling neoplastic diseases in subjects. Thus, the methods of the present invention can be used to reduce or inhibit the metastasis of a primary tumor or cancer to other sites, or to reduce or inhibit the formation or establishment of metastatic tumors or cancers in sites other than the primary tumor or cancer, thereby inhibiting or reducing tumor or cancer recurrence or progression. Therefore, the present invention provides a detectable or measurable improvement in the condition of a given subject, such as relief or improvement of one or more adverse (physical) symptoms or consequences associated with the presence of a proliferative or hyperproliferative condition, tumor formation, tumor or cancer, or metastasis, i.e., a therapeutic benefit or 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 the onset, severity, duration, or frequency of adverse symptoms associated with or caused by a proliferative or hyperproliferative condition (such as tumor formation, tumor or cancer, or metastasis). It may improve survival. According to the treatment methods of the present invention, a satisfactory clinical endpoint is considered to have been reached when, for example, the severity, duration, or frequency of one or more related pathologies, adverse symptoms, or complications progressively decreases or partially decreases, or one or more physiological, biochemical, or cellular manifestations or characteristics of cell proliferation or cell hyperproliferative disorders (such as tumor formation, tumors, or cancer or metastasis) are inhibited or reversed. Therefore, treatment benefits or improvements may include, but are not limited to: destruction of target proliferating cells (e.g., tumor formation, tumors, or cancer or metastasis), or elimination of one or more, most or all, pathologies, adverse symptoms, or complications associated with or caused by cell proliferation or cell hyperproliferative disorders (such as tumor formation, tumors, or cancer, or metastasis). However, treatment benefits or improvements need not be a cure or complete destruction of all target proliferating cells (e.g., tumor formation, tumors, or cancer or metastasis) or elimination of all pathologies, adverse symptoms, or complications associated with or caused by cell proliferation or cell hyperproliferative disorders (e.g., tumor formation, tumors, or cancer, or metastasis). For example, by inhibiting the progression or worsening of a tumor or cancer, partially destroying a cluster of tumors or cancer cells, or stabilizing the size or number of cells in a cluster of tumors or cancer cells, mortality can be reduced and lifespan extended, even if only for a few days, weeks, or months, even if part or most of the cluster of tumors or cancer cells, size, or cells still remain.

[0166] Specific, non-limiting examples of therapeutic benefits include reducing tumor formation, tumor or cancer volume (size or cell cluster) or cell number; inhibiting or preventing the increase of tumor formation, tumor or cancer volume (e.g., stabilization); slowing or inhibiting tumor formation, tumor or cancer progression, worsening or metastasis; or inhibiting tumor formation, tumor or cancer proliferation, growth or metastasis.

[0167] The method of the present invention may not take effect immediately. For example, tumor formation, an increase in the number or clusters of tumors or cancer cells may occur after treatment, but over time, the eventual stabilization or reduction of tumor cell clusters, size, or number of cells in a given subject may subsequently occur.

[0168] Additional adverse symptoms and complications associated with tumor formation, tumors, cancer, and metastasis that can be suppressed, alleviated, reduced, delayed, or prevented include, for example, nausea, loss of appetite, somnolence, pain, and malaise. Therefore, partial or complete reduction or alleviation of the severity, duration, or frequency of adverse symptoms or complications associated with or caused by cell hyperproliferative disorders, and improvements in the subject's quality of life and / or health, such as increased energy, appetite, and mental well-being, are specific, non-limiting examples of therapeutic benefits.

[0169] Therefore, treatment benefits or improvements may also include a subjective improvement in the subject's quality of life. In another embodiment, a method prolongs or extends the subject's lifespan (survival). In yet another embodiment, a method improves the subject's quality of life.

[0170] Treatment benefits may also include prevention of recurrence of tumor formation, tumors, cancer, and metastases, for example, where the tumor formation, tumors, cancer, and metastases have been surgically or chemically removed.

[0171] Bacteria expressing the IL-15 variants and / or IL-15 superagonists of the present invention are typically administered to subjects in compositions comprising an effective amount of bacteria (e.g., Gram-negative bacteria, such as Salmonella Typhi ZH9 serotype) and also comprising a pharmaceutically acceptable carrier / adjuvant / diluent or excipient. The terms “pharmaceuticalally” and “pharmaceuticalally acceptable” mean molecular entities and compositions that, when properly administered to animals (e.g., humans), do not produce adverse, allergic, or other adverse reactions. Such formulations are known to those skilled in the art. Furthermore, for administration to animals (e.g., humans or any other member of the animal kingdom), it should be understood that the formulation should meet standards of sterility, pyrogenicity, general safety, and purity (if applicable).

[0172] The composition is designed to enhance the immune response of the subject. Therefore, bacteria expressing IL-15 variants and / or IL-15 superagonists can be provided as vaccines or vaccine compositions. These terms are used interchangeably and refer to biological agents to which the subject develops an immune response, thus providing active acquired immunity against a specific infectious disease, such as one caused by Salmonella. In the context of this invention, the vaccine may contain an agent or “exotic” agent similar to the bacteria causing the infection, which is a weakened or inactivated form of the bacteria, or any part or fragment of bacterial protein, capsule, DNA, or RNA. Such an exotic agent will be recognized by the vaccine recipient’s immune system, which in turn will destroy the agent and develop a “memory” against the bacteria, inducing a durable level of protection against future bacterial infections from the same or similar viruses. Through a vaccination route including the vaccine composition of this invention, it is envisioned that once a vaccinated subject encounters again the same bacteria or bacterial isolate targeted by the vaccine, the individual’s immune system can thereby recognize the bacteria or bacterial isolate and trigger a more effective defense against the infection. The active acquired immunity induced in the subject as a result of the vaccine can be humoral and / or cellular in nature. The vaccine composition may further include adjuvants, pharmaceutically acceptable carriers, or excipients.

[0173] As used herein, “pharmaceuticalally 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 retardants, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and other materials and combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329). Examples include, but are not limited to, disodium hydrogen phosphate, soybean peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, calcium chloride, sucrose, borate buffer, sterile physiological saline (0.9% NaCl), and sterile water.

[0174] Suitable aqueous and non-aqueous carriers that can be used in the vaccine compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). For example, appropriate flowability can be maintained by using coating materials such as lecithin, maintaining the desired particle size in the dispersion system, and using surfactants.

[0175] The vaccine compositions disclosed herein may further contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of unwanted microorganisms can be ensured by the sterilization procedures described above and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. Furthermore, delayed absorption in the form of injectable drugs can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin). The vaccine compositions may also optionally include additional therapeutic agents known to be effective in, for example, infectious or neoplastic diseases. Therefore, the vaccine compositions disclosed herein may also contain antiretroviral drugs, antibiotics, antifungal agents, antiparasitic agents, and anticancer agents.

[0176] The vaccine composition may also contain other components designed to enhance the immune response. Examples of such other components include, but are not limited to: aluminum salts (such as aluminum hydroxide, aluminum oxide, and aluminum phosphate), oil-based adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), mycoester adjuvants (such as trehalose dimethicone), bacterial lipopolysaccharides (LPS), peptidoglycans (such as muramin, peptidoglycans, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (such as those extracted from Klebsiella pneumoniae), streptococcal preparations (such as OK432), muramyl dipeptides, immunostimulatory complexes (such as "Iscoms" disclosed in EP 109 942, EP 180 564, and EP 231 039), saponins, DEAE-glucan, neutral oils (such as miglyol), vegetable oils (such as peanut oil), liposomes, polyols, and Ribi adjuvant systems (see, for example, GB-A-2189). 141) Vitamin E, carbomer, interferons (e.g., IFN-α, IFN-γ, or IFN-β), or other (unmodified) interleukins, especially those that can 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).

[0177] The bacteria in the vaccine compositions disclosed herein may include any characteristic of the bacteria disclosed herein or any combination thereof.

[0178] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any of the stated or enumerated components.

[0179] As used herein, “about” means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” may mean within a standard deviation of 1 or greater than 1. Alternatively, “about” may mean a range up to 20%. When a particular value is given in this application and claims, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable range of error for that particular value.

[0180] The invention is further described with reference to the following non-limiting embodiments: Example Example 1: Identification and bioactivity evaluation of soluble IL-15 sequence variants.

[0181] Variant Design Cytokine variants were designed using three methods: • Targeted computer simulation screening: Cargo modeling and computational analysis were performed on the reported crystal structure of the key receptor complex (PDB ID: 4GS7). The analysis considered improvements in solubility and stability without loss of activity. This yielded five identified variants containing multiple mutations synthesized from the IDT gene.

[0182] • Semi-rational candidate design: Computational tools (e.g., Aggrescan3D) are used to identify amino acids that readily aggregate, and then only these positions are mutated. This produces two libraries, depending on the method used to generate the mutants: a library of 72 members, where each variant contains 1 or 2 defined amino acid changes relative to IL-15M40; and a variant pool of 729 variants, each with 0–6 mutations (randomized). Over 85% of the variants in the pool have 3–6 mutations. The first library was synthesized via the IDT gene, and the second via the TWIST gene.

[0183] • Random candidates: Random mutants were generated by error-prone PCR using the Genemorph II kit (Agilent), and two pools were generated according to the manufacturer’s instructions, one with a fragment expected to have 0-2 mutations and the other with a fragment expected to have 2-4 mutations.

[0184] Solubility screening Each fragment generated during the design phase was cloned into the solubility reporter plasmid pBRED, derived from a system reported by Foit et al. (2009 Molecular Cell 36 861-871), which is based on the survival ability of bacterial cells in penicillin antibiotics (such as carbenicillin, ampicillin, and penicillin V) depending on the stability of the target protein. The β-lactamase gene contained in pBRED is divided into two independently foldable domains (α and ω). Two flexible linkers between these two domains allow fusion with any protein carrier protein, resulting in the final protein conformation: Bla α -Connector-Carrier Protein-Connector-Bla ω If the carrier protein is inherently stable / soluble, the α and ω subdomains work synergistically to degrade penicillin antibiotics. Conversely, if the carrier protein is unstable / insoluble and degrades, the two elements separate, reporting that the bacteria cannot survive the antibiotics. Survival of each experimental strain was tested in either liquid or solid media. Both methods produced consistent and reproducible results, with liquid assays providing faster and more easily interpreted readings.

[0185] The first iteration of screening of the parental sequence IL-15M40 revealed that it produced cells sensitive to carbenicillin at a concentration of 1 mg / mL. The known stable protein mScarlet was used as a positive control or baseline.

[0186] Four out of five sequences in the target library (1) were found to be more stable than the parental control. In the semi-rationally designed library, from 72 variants designed to contain 1–2 amino acid mutations, most single amino acid substitutions (75%) and double amino acid substitutions (72%) improved stability. In the pool where 85% of the variants carried 3–6 mutations, 94 / 176 positive sequences were generated after screening (53%) and sequenced. The number of mutations in the samples was normally distributed with a mean of 4, and mutations at positions 45 and 49 were found to be the most common. Finally, a total of 42 / 176 (24%) variants generated by error-prone PCR were positive in the assay. The final pool of positive candidates contained 169 sequences.

[0187] Activity screening The activity of cytokine variants that were found to be more stable than the parental sequence (IL-15M40) was assessed using three assays: • Promega IL-15 Kit: Rapid assay using the IL-15 Bioassay Cell (Promega) Kit. Allows for S-curve evaluation of activity and is suitable for high-throughput assays. Performs initial screening assays for soluble candidates. In this assay, reporter cells are added to wells containing immobilized cytokine proteins at the bottom and incubated for 6 hours as per manufacturer's instructions. After incubation, substrate is added to the plate as per manufacturer's instructions, and luminescence is measured on a Tecan plate reader.

[0188] • NK Cell Amplification Assay: NK cells isolated from Prokarium were induced with candidate reagents and incubated at 37°C and 5% CO2 for 4 days. Customized assays were then performed based on this incubation (see below). Positive activity resulted in NK cell proliferation, as measured by increased optical density (OD). This provides a more accurate, albeit narrower, output of candidate activity. Positive soluble candidates were then screened using a bioactivity assay kit (Promega).

[0189] • IFN-γ (IFN-γ) Production Assay: A customized assay was performed based on the incubation of NK cells isolated from Prokarium for 1 day after candidate induction (see below). Positive activity resulted in the production of interferon-γ by NK cells, which was quantified by ELISA. Similar to the NK amplification assay, this provides a more accurate output of candidate activity. A third and final screening assay was performed on the soluble candidates that showed positive results using the Bioactivity Assay Cell (Promega) kit and the NK amplification assay.

[0190] The 169 variants obtained through solubility screening were transferred into protein expression plasmids that constitutively express C-terminal His 10 Each target delivery protein was tagged. Cells containing 4 mL of each plasmid were cultured to OD200. 600 Centrifuge at approximately 1.0 μL. Discard the culture supernatant and resuspend the cell pellet in 200 μL NPI buffer (50 mM NaH2PO4, 300 mM NaCl) supplemented with 20 mM imidazole. Then lyse the cells using a PIXUL® multisample sonicator. Separate the insoluble fraction from the soluble protein by centrifugation at 12000 xg for 20 min and load the fractions into Ni-NTA HisSorb (QIAGEN) or HisPur (ThermoFisher) plates. Allow the proteins to bind at room temperature for 1 h. Then wash the plate three times with NPI supplemented with 50 mM imidazole and twice with PBS (37 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4) to remove excess imidazole. Then perform the activity assay as described above using the plate.

[0191] Cell screening using IL-15 bioactivity assays revealed that only variants from sequence libraries containing 1 to 2 amino acid mutations exhibited bioactivity.

[0192] A total of 438 IL-15 variants were screened. Of these, 169 were found to be 2-40 times more soluble than the reference sequence. Figure 8A and 8B Of these soluble variants, 13 were found to also possess biological activity using a high-throughput screening bioactivity assay (Promega). Figure 8C and 8D (Table 1). Seven variants listed in Table 1 were selected for further experiments (GBL01, GBL15, GBL17, GBL18, GBL25, GBL50). Table 1. Solubility of different IL-15 variants. All IL-15 variants listed above have the S75P mutation. The parental IL-15M40 (IL-15S75P) has a solubility score of 1.0, and the mScarlet has a solubility score of 19.7 ± 0.8.

[0193] Example 2: Generation of IL-15 super agonist (SAg) using IL-15Rα sushi domain.

[0194] Super agonists corresponding to 13 soluble and functional variants of IL-15 were generated by fusing with IL-15Rα (sushi domain) via the Gly-Ser flexible linker (ILR) (Table 2). Table 2. Solubility of different IL-15 variants fused with the IL-15Rα sushi domain.

[0195] As previously described, after cloning the superagonist into the reporter plasmid pBRED, an antibiotic challenge assay was performed using the parental IL-15M40 superagonist as a reference and the fusion of mScarlet with the IL-15Rα-ILR construct as a positive control.

[0196] Superagonists were found to be less soluble than their corresponding IL-15 counterparts, requiring moderate to low assay conditions to identify soluble chaperones. Five of the 13 superagonists (38%) were found to be more soluble than parental IL-15-based superagonists. These soluble superagonists are SAg15, SAg17, SAg18, SAg25, and SAg50.

[0197] Example 3: Validation of the biological activity of the IL-15 / SAg variant Cell bioactivity assay using IL-15 ( Figure 9A Primary NK cell proliferation assay Figure 9B ) and interferon-γ release assay ( Figure 9C Further validation of 13 candidate IL-15 variants and super agonists SAg15, SAg18, SAg25 and SAg50 was conducted.

[0198] All IL-15 variants except GBL34 and GBL43 produced reproducible measurements in the Promega Bioactivity Assay Kit. This assay suggests that SAg15, SAg18, and SAg25 may possess bioactivity. Figure 9A ).

[0199] For proliferation assays, the same sample extracts were used as in the bioassay. To show differences, sample results were normalized to values ​​from the mScarlet negative control. Results of NK cell induction with different IL-15 / Sag carrier proteins reflect the results obtained in the bioassay cells (Promega) assay. Figure 9B ).

[0200] The IFN-γ production assay results were consistent with other experiments and showed better quantification of activity than the proliferation assay. Figure 9C ).

[0201] Example 4: Protein purification of IL-15 variants and IL-15 / Sag variants.

[0202] Variants GBL01, GBL18, GBL25, SAg18, and SAg25 were selected for more thorough characterization. The genes encoding these variants were placed in positions compatible with His... 10 The C-terminal tag fused to the expression plasmid was expressed in *E. coli*. A total of 1 L of culture for each variant was centrifuged, and the resulting precipitate was lysed by sonication. Soluble fractions were purified by Ni-NTA chromatography to yield purified His-tagged IL-15, IL-15 variants, and a superagonist (…). Figure 10B The protein was then quantified by HPLC / MS, using commercially available IL-15 as a sample to calculate a standard curve. The standard curve was calculated using IL-15 concentration versus MS peak area. Figure 10A Calculate the concentration of purified protein (Table 3). Table 3. Concentrations of purified proteins obtained by Ni-NTA affinity chromatography.

[0203] Example 5: In vitro and ex vivo characterization of recombinant IL-15 variants Using the Promega bioactivity assay, different concentration gradients (from 10) were analyzed. -2The activity of the recombinant purified IL-15 variant (-2.5 nM) was determined. The IL-15 variant was incubated with a reporter cell line that expresses luc2 in response to IL-15 signaling for 6 hours. Figure 11A The results are shown as the average fold induction (samples / blank) for n=3 independent replicates (+ / -SD). Figure 11B This indicates INF-γ release. From the lowest EC 50 It is evident that the wild-type IL-15 protein exhibits the greatest activity; among the candidate variants, GBL18 and GBL01 show the highest activity; while SAg18 and SAg25 fail to induce activation in bioactivity assay reporter cell lines. Figure 11A (Table 4). While this indicates that the wild-type IL-15 is the most active protein, the activities of GBL1 and GBL18 are comparable. The variant GBL25 was found to require approximately twice the concentration to activate luminescence. Table 4. EC50 for each IL-15 variant 50 The result is calculated as the average of the three independent experiments.

[0204] The results in Figure 11 were validated by incubating the purified variant at a known concentration with NK cells for 24 hours and measuring the concentration of IFN-γ produced in the supernatant. Figure 12 ).exist Figure 12 In this study, primary human NK cells were isolated from peripheral blood mononuclear cells (PBMCs) using negative sorting with magnetic microbeads. The purified IL-15 variant was incubated with the primary NK cells for 24 hours at 71 nM (Experiment 1) and 58 nM (Experiment 2). Figure 12 The results are shown as the mean + / - standard error (SEM) of stimulation replicates from a single experiment performed in three trials using two different cell donors. The presence of IFN-γ in the supernatant was then detected using a human two-component IFN-γ ELISA. Peprotech is a commercially available recombinant IL-15. All candidates induced IFN-γ production in primary NK cells. Cells stimulated with wild-type IL-15 produced the highest concentrations of IFN-γ, followed by NK cells stimulated with SAg25, and then those stimulated with GBL01 and GBL18.

[0205] Finally, NK cells in mouse (initial balb / c) spleen cells were stimulated with a variant for 72 hours. The spleen cells were then stained, and NK cells and NK activation markers were assessed by flow cytometry (Table 5). Figure 13 GBL01 and GBL18 exhibited similar activity and were the most effective variants. In some assays, they even outperformed the wild type. Table 5. Biomarkers used in in vitro assays to analyze mouse cell survival, proliferation, activation, and effector molecule expression.

[0206] Example 6: Long-term expression of IL-15 in ZH9 Salmonella strain in tumors.

[0207] Due to the difficulty in detecting IL-15 carrier proteins, pharmacokinetics were assessed using IL-15 gene expression from Salmonella.

[0208] Mice with subcutaneous MC38 tumors were intratumorally injected with PBS control, ZH9 Salmonella strain, or a candidate ZH9 variant expressing IL-15. Tissue samples were collected at designated time points after treatment, RNA was extracted, and the copy number of the Salmonella-derived IL-15 gene was counted by TaqMan quantitative PCR. Gene copy numbers were normalized to tumor volume.

[0209] The results of this experiment indicate that, following intratumoral administration, Salmonella bacteria can be detected in tumor tissue for an extended period (≥7 days). il-15 mRNA.

[0210] Furthermore, wild-type human IL-15 is expressed in Salmonella ZH9. Western blot analysis showed that wild-type human IL-15 was mainly detected in the bacterial insoluble fraction.

[0211] Example 7: In vivo bladder tumor model study shows the improved efficacy of IL-15 variants compared to chassis strains. MB49-luc tumor cells were injected intravesically (IVES) into female C57BL / 6 mice and treated with various Salmonella strains (PBS control, ZH9 Salmonella strain, or candidate ZH9 variant strains expressing the IL-15 variant). Survival of these mice was monitored for 60 days. In all cases, mice injected with candidate ZH9 variant strains expressing the IL-15 variant showed a longer median survival compared to mice injected with the ZH9 Salmonella strain (i.e., strains lacking the IL-15 variant).

[0212] The present invention can be described in any of the following embodiments.

[0213] Implementation Scheme 1. An interleukin-15 (IL-15) variant, wherein, compared to wild-type IL-15, the IL-15 variant contains one or more amino acid modifications in a surface region adjacent to a receptor-interacting surface, preferably wherein the receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface.

[0214] Implementation Scheme 2. The IL-15 variant according to Implementation Scheme 1, wherein the wild-type IL-15 comprises the amino acid sequence according to SEQ ID NO: 1, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1.

[0215] Implementation Scheme 3. The IL-15 variant according to Implementation Scheme 2, wherein, compared to SEQ ID NO: 1, the amino acid modification occurs in one or more of the 45th and / or 49th and / or 52nd amino acids or any combination thereof.

[0216] Implementation Scheme 4. An interleukin-15 (IL-15) variant, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, and wherein, compared with SEQ ID NO: 1, the IL-15 variant contains an amino acid modification at one or more of the 45th and / or 49th and / or 52nd amino acids, or any combination thereof.

[0217] Implementation Scheme 5. An IL-15 variant according to any one of Implementation Schemes 2 to 4, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 1.

[0218] Implementation Scheme 6. An IL-15 variant according to any one of Implementation Schemes 2 to 5, wherein the IL-15 variant contains an amino acid modification at the 75th amino acid position.

[0219] Implementation Scheme 7. An IL-15 variant according to Implementation Scheme 3 or 4 or any of the preceding schemes, wherein the amino acid modification at position 45 is a hydrophobic-to-hydrophilic substitution.

[0220] Implementation Scheme 8. The IL-15 variant according to Implementation Scheme 7, wherein the hydrophobic-to-hydrophilic substitution is a substitution of leucine to serine or a substitution of leucine to threonine.

[0221] Implementation Scheme 9. The IL-15 variant according to Implementation Scheme 3 or 4 or any of the preceding schemes, wherein the amino acid modification at amino acid position 49 is a hydrophobic-to-hydrophobic substitution.

[0222] Implementation Scheme 10. The IL-15 variant according to Implementation Scheme 9, wherein the hydrophobic-to-hydrophobic substitution is a valine-to-alanine substitution.

[0223] Implementation Scheme 11. The IL-15 variant according to Implementation Scheme 3 or 4 or any one thereof, wherein the amino acid modification at amino acid position 52 is a hydrophobic to hydrophilic substitution.

[0224] Implementation Scheme 12. The IL-15 variant according to Implementation Scheme 11, wherein the hydrophobic-to-hydrophilic substitution is a substitution of leucine to lysine or a substitution of leucine to arginine.

[0225] Implementation Scheme 13. An IL-15 variant according to Implementation Scheme 3 or 4 or any of the preceding schemes, wherein the amino acid modification at position 75 is a substitution of serine for proline.

[0226] Implementation Scheme 14. The IL-15 variant according to Implementation Scheme 13, wherein the IL-15 variant comprising a serine-to-proline substitution at amino acid position 75 comprises the sequence according to SEQ ID NO: 16, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 16, wherein the percentage of sequence identity with SEQ ID NO: 16 is retained by the amino acid modification at amino acid position 75.

[0227] Implementation Scheme 15. A variant of IL-15 according to Implementation Scheme 3 or 4 or any of the preceding embodiments, wherein the IL-15 variant comprises: i) The substitution of leucine at amino acid position 45 with serine; and ii) Replacement of serine with proline at amino acid position 75.

[0228] Implementation Scheme 16. The IL-15 variant according to Implementation Scheme 15, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 2 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2, wherein the percentage of sequence identity with SEQ ID NO: 2 preserves the amino acid modifications at amino acid positions 45 and 75.

[0229] Implementation Scheme 17. The IL-15 variant according to Implementation Scheme 16, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 2, wherein the percentage of sequence identity with SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 45 and 75.

[0230] Implementation Scheme 18. A variant of IL-15 according to Implementation Scheme 3 or 4 or any of the preceding embodiments, wherein the IL-15 variant comprises: i) The substitution of leucine at amino acid position 45 with serine; and ii) The substitution of valine for alanine at amino acid position 49; and iii) Replacement of serine with proline at amino acid position 75.

[0231] Implementation Scheme 19. The IL-15 variant according to Implementation Scheme 18, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 3 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 3, wherein the percentage of sequence identity with SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 45, 49 and 75.

[0232] Implementation Scheme 20. The IL-15 variant according to Implementation Scheme 19, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 3, wherein the percentage of sequence identity with SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 45, 49, and 75.

[0233] Implementation Scheme 21. A variant of IL-15 according to Implementation Scheme 3 or 4 or any of the preceding embodiments, wherein the IL-15 variant comprises: i) The substitution of leucine at amino acid position 45 with serine; and ii) The substitution of leucine for lysine at amino acid position 52; and iii) Replacement of serine with proline at amino acid position 75.

[0234] Implementation Scheme 22. The IL-15 variant according to Implementation Scheme 21, wherein the IL-15 variant comprises the amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4, wherein the percentage of sequence identity with SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 45, 52 and 75.

[0235] Implementation Scheme 23. The IL-15 variant according to Implementation Scheme 22, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 4, wherein the percentage of sequence identity with SEQ ID NO: 4 retains the amino acid modifications at amino acid positions 45, 52, and 75.

[0236] Implementation Scheme 24. A variant of IL-15 according to Implementation Scheme 3 or 4 or any of the preceding embodiments, wherein the IL-15 variant comprises: i) The substitution of leucine at amino acid position 45 with serine; and ii) The substitution of leucine for arginine at amino acid position 52; and iii) Replacement of serine with proline at amino acid position 75.

[0237] Implementation Scheme 25. The IL-15 variant according to Implementation Scheme 24, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 5 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 5, wherein the percentage of sequence identity with SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45, 52 and 75.

[0238] Implementation Scheme 26. The IL-15 variant according to Implementation Scheme 25, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 5, wherein the percentage of sequence identity with SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45, 52, and 75.

[0239] Implementation Scheme 27. A variant of IL-15 according to Implementation Scheme 3 or 4 or any of the preceding embodiments, wherein the IL-15 variant comprises: i) The substitution of leucine for threonine at amino acid position 45; and ii) The substitution of valine for alanine at amino acid position 49; and iii) Replacement of serine with proline at amino acid position 75.

[0240] Implementation Scheme 28. The IL-15 variant according to Implementation Scheme 27, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 6 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 6, wherein the percentage of sequence identity with SEQ ID NO: 6 preserves the amino acid modifications at amino acid positions 45, 49 and 75.

[0241] Implementation Scheme 29. The IL-15 variant according to Implementation Scheme 28, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 6, wherein the percentage of sequence identity with SEQ ID NO: 6 retains the amino acid modifications at amino acid positions 45, 49, and 75.

[0242] Implementation Scheme 30. A variant of IL-15 according to Implementation Scheme 3 or 4 or any of the preceding embodiments, wherein the IL-15 variant comprises: i) The substitution of valine for alanine at amino acid position 49; and ii) The substitution of leucine for arginine at amino acid position 52; and iii) Replacement of serine with proline at amino acid position 75.

[0243] Implementation Scheme 31. The IL-15 variant according to Implementation Scheme 30, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 7 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 7, wherein the percentage of sequence identity with SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52 and 75.

[0244] Implementation Scheme 32. The IL-15 variant according to Implementation Scheme 31, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 7, wherein the percentage of sequence identity with SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52, and 75.

[0245] Implementation Scheme 33. An IL-15 variant according to any one of the above embodiments, wherein the IL-15 variant is fused with an interleukin-15 receptor α (IL-15Rα) molecule.

[0246] Implementation Scheme 34. An IL-15 variant according to any one of the above embodiments, wherein the IL-15 variant is fused to the sushi domain of the interleukin-15 receptor α (IL-15Rα) molecule.

[0247] Implementation Scheme 35. The IL-15 variant according to Implementation Scheme 33 or 34, wherein the IL-15 variant is fused via a linker, preferably wherein the linker is a glycine-serine linker.

[0248] Implementation Scheme 36. The IL-15 variant according to Implementation Scheme 35, wherein the connector comprises a sequence according to SEQ ID NO: 17 or a sequence having at least 70% identity with SEQ ID NO: 17.

[0249] Implementation Scheme 37. An IL-15 variant according to any one of Implementation Schemes 33 to 36, wherein the IL-15 variant comprises a sequence according to any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, or a sequence having at least 70% identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, wherein the percentage of sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13 is retained at the amino acid modification at the relevant amino acid position.

[0250] Implementation Scheme 38. The IL-15 variant according to Implementation Scheme 37, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12, and / or 13, wherein the percentage of sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12, and / or 13 is retained at the amino acid modification at the relevant amino acid position.

[0251] Implementation Scheme 39. An IL-15 variant according to any one of the above implementation schemes, wherein the IL-15 variant is fused with a signal peptide.

[0252] Implementation Scheme 40. The IL-15 variant according to Implementation Scheme 39, wherein the signal peptide is a periplasmic signal peptide.

[0253] Implementation Scheme 41. The IL-15 variant according to Implementation Scheme 39 or 40, wherein the signal peptide is fused to the IL-15 variant via a spacer sequence, preferably wherein the spacer sequence is 1 to 5 amino acids in length.

[0254] Implementation Scheme 42. The IL-15 variant according to any one of the above implementation schemes, for therapeutic use.

[0255] Implementation Scheme 43. An IL-15 variant according to any one of the above implementation schemes, used for the treatment, prevention, reduction, inhibition, prevention of recurrence, or control of neoplastic diseases in a subject.

[0256] Implementation Scheme 44. An IL-15 variant for the stated purpose according to Implementation Scheme 43, wherein the neoplastic disease is solid carcinoma and / or hematologic malignancy.

[0257] Implementation Scheme 45. The IL-15 variant for the stated purpose according to Implementation Scheme 44, wherein the solid cancer and / or hematologic malignancy is selected from cancers such as prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, malignant epithelial tumors, head and neck cancer, endometrial cancer, skin cancer, or sarcoma.

[0258] Implementation Scheme 46. An IL-15 variant for the purpose according to any one of Implementation Schemes 42 to 45, wherein the IL-15 variant is administered to the subject via local infusion, intraperitoneal, intrapleural, intravesical, intravaginal, peritumoral, intratumoral injection or oral administration.

[0259] Implementation Scheme 47. A nucleic acid molecule encoding an IL-15 variant as defined in any one of Implementation Schemes 1 to 41.

[0260] Implementation Scheme 48. The nucleic acid molecule according to Implementation Scheme 47, wherein the nucleic acid molecule encodes an IL-15 variant, the IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, or a sequence having at least 70% identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 preserves amino acid modifications at the relevant amino acid positions.

[0261] Implementation Scheme 49. The nucleic acid molecule according to Implementation Scheme 48, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 preserves the amino acid modifications at the relevant amino acid positions.

[0262] Implementation Scheme 50. A nucleic acid molecule according to any one of Implementation Schemes 47 to 49, wherein the nucleic acid molecule comprises a DNA molecule.

[0263] Implementation Scheme 51. A nucleic acid molecule according to any one of Implementation Schemes 47 to 50, wherein the nucleic acid molecule comprises an RNA molecule.

[0264] Implementation Scheme 52. The nucleic acid molecule according to Implementation Scheme 51, wherein the RNA molecule comprises an mRNA molecule.

[0265] Implementation Scheme 53. A Gram-negative bacterium comprising any one of the IL-15 variants of Implementation Scheme 1 to 41 or any one of the nucleic acid molecules of Implementation Scheme 47 to 52.

[0266] Implementation Scheme 54. Gram-negative bacteria according to Implementation Scheme 53, wherein the Gram-negative bacteria are live attenuated Gram-negative bacteria.

[0267] Implementation Scheme 55. Gram-negative bacteria according to Implementation Scheme 54, wherein the live attenuated Gram-negative bacteria is Salmonella.

[0268] Implementation Scheme 56. The Gram-negative bacteria according to Implementation Scheme 55, wherein the live attenuated Gram-negative bacteria is Salmonella enterica, preferably wherein the live attenuated Gram-negative bacteria is Salmonella typhi serotype.

[0269] Implementation Scheme 57. The Gram-negative bacteria according to Implementation Scheme 56, wherein the live attenuated Gram-negative bacteria is Salmonella typhi serotype ZH9.

[0270] Implementation Scheme 58. Gram-negative bacteria according to any one of Implementation Schemes 53 to 57, for therapeutic use.

[0271] Implementation Scheme 59. Gram-negative bacteria according to any one of Implementation Schemes 53 to 58, used for the treatment, prevention, mitigation, inhibition, prevention of recurrence, or control of neoplastic diseases in the subject.

[0272] Implementation Scheme 60. Gram-negative bacteria for the purpose as described in Implementation Scheme 59, wherein the neoplastic disease is solid carcinoma and / or hematologic malignancy.

[0273] Implementation Scheme 61. Gram-negative bacteria for the stated purpose as described in Implementation Scheme 60, wherein the solid carcinoma and / or hematologic malignancy is selected from the following cancers: prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, malignant epithelial tumors, head and neck cancer, skin cancer, or sarcoma.

[0274] Implementation Scheme 62. Gram-negative bacteria for the purpose according to any one of Implementation Schemes 53 to 61, wherein the Gram-negative bacteria are administered to the subject by local infusion, intraperitoneal, intrapleural, intravenous, intravesical, peritumoral injection, intratumoral injection or oral administration.

[0275] Implementation Scheme 63. Use of an IL-15 variant as defined in any of Implementation Schemes 1 to 41, or a nucleic acid molecule as defined in any of Implementation Schemes 47 to 52, or a Gram-negative bacterium as defined in any of Implementation Schemes 53 to 62, in the preparation of a medicament for therapeutic purposes.

[0276] Implementation Scheme 64. The use according to Implementation Scheme 63, wherein the treatment is the treatment, prevention, reduction, suppression, prevention of recurrence, or control of a neoplastic disease in the subject.

[0277] Implementation Scheme 65. A method for treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject an IL-15 variant as defined in any one of Implementation Schemes 1 to 41, or a nucleic acid molecule as defined in any one of Implementation Schemes 47 to 52, or a Gram-negative bacterium as defined in any one of Implementation Schemes 53 to 62.

[0278] The sequence that constitutes the instruction manual SEQ ID NO: 1 (Wild type / unmodified IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 2 (GBL01) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISLESGDASIHDTVENLIILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 3 (GBL15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQAISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 4(GBL17) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISKESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 5(GBL18) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISRESGDASIHDTVENLIILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 6(GBL25) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLTELQAISLESGDASIHDTVENLIILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 7(GBL50) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQAISRESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 8(SAg01) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISLESGDASIHDTVENLIILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 9(SAg15) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQAISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 10(SAg17) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISKESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 11(SAg18) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLSELQVISRESGDASIHDTVENLIILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 12(SAg25) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLTELQAISLESGDASIHDTVENLIILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 13(SAg50) MITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQAISRESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 14 (LSP) MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA SEQ ID NO: 15 (SSP) MVLGTIDLCSCFSAGLPKTEA SEQ ID NO: 16 (Parent / M40 IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLPSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 17 (Exemplary GS Linker) SGGGSGGGGSGGGGSGGGGSGGGSLQA References Alhunaidi, O. & A. 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Kai, Y.Yamagata, S. J. Davis & S. Ikemizu (2007) Crystal structure of the IL-15-IL-15Ralpha complex, a cytokine-receptor unit presented in trans. Nat Immunol . 8(9): 1001-1007. de Marco, A. (2009) Strategies for successful recombinant expressionof disulfide bond-dependent proteins in Escherichia coli. Microbial Cell Factories . 8. Devi, N., Adivitiya & Y. P. Khasa (2016) A combinatorial approach ofN-terminus blocking and codon optimization strategies to enhance the solubleexpression of recombinant hIL-7 in E. coli fed-batch culture. Appl Microbiol Biotechnol . 100(23): 9979-9994. Duitman, E. H., Z. Orinska, E. Bulanova, R. Paus & S. Bulfone-Paus(2008) How a cytokine is chaperoned through the secretory pathway bycomplexing with its own receptor: lessons from interleukin-15 (IL-15) / IL-15receptor alpha. Mol Cell Biol . 28(15): 4851-4861. Eisenman, J., M. Ahdieh, C. Beers, K. Brasel, M. K. Kennedy, T. Le,T. P. Bonnert, R. J. Paxton & L. S. Park (2002) Interleukin-15 interactionswith interleukin-15 receptor complexes: characterization and speciesspecificity. Cytokine . 20(3): 121-129. Ferrer-Miralles N., Domingo-Espin J., Corchero JL, Vazquez E., &Villaverde A. (2009) Microbial factories for recombinant pharmaceuticals. Microbial Cell Factories . . . . 8. Guo, Y., L. Luan, NK Patil & ER Sherwood (2017) Immunobiologyof the IL-15 / IL-15Rα complex as an antitumor and antiviral agent. Cytokine Growth Factor Rev 38: 10-2 Kuhlman , TE & EC Cox ( 2010 ) Site-specific chromosome correction of large synthetic constructs . Nucleic Acids . . . . Res 38(6): e92. Nausch , H. , J. Huckauf , R. Koslowski , U. Meyer , I. Broer & H. Mikschofsky ( 2013 ) Recombinant production of human interleukin 6 in Escherichia coli . PLoS One 8(1):e54933. Park, Y., A. Espah Borujeni, TE Gorochowski, J. Shin & CA Voigt(2020) Precision design of stable genetic circuits carried in highly-insulated E. coli genomic landing pads. 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Gangadharan, et al (2019) IL-15 is a component of the inflammatory milieuin the tumor microenvironment promoting antitumor responses. P roc Natl Acad Sci USA 116(2): 599-608.

Claims

1. A bacterium containing an interleukin-15 (IL-15) variant, wherein, compared to wild-type IL-15, the IL-15 variant contains one or more amino acid modifications in a surface region adjacent to a receptor-interacting surface, preferably wherein the receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα) interacting surface.

2. The bacteria according to claim 1, wherein the bacteria are live attenuated bacteria.

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

4. The bacteria according to claim 3, wherein the live attenuated Gram-negative bacteria is Salmonella; preferably, wherein the live attenuated Gram-negative bacteria is Salmonella enterica; more preferably, wherein the live attenuated Gram-negative bacteria is Salmonella typhi serotype; and even more preferably, wherein the live attenuated Gram-negative bacteria is Salmonella typhi serotype ZH9.

5. The bacteria according to any one of the preceding claims, wherein wild-type IL-15 comprises the amino acid sequence according to SEQ ID NO: 1, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

16.

6. The bacteria of claim 5, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:

1.

7. The bacteria according to claim 5 or 6, wherein, compared to SEQ ID NO: 1, the amino acid modification occurs in one or more of the 45th and / or 49th and / or 52nd amino acids or any combination thereof.

8. The bacteria according to any one of the preceding claims, wherein the IL-15 variant comprises an amino acid modification at the 75th amino acid position, preferably wherein the amino acid modification at the 75th amino acid position is a substitution of serine for proline.

9. The bacteria of claim 8, wherein the IL-15 variant comprising a serine-to-proline substitution at amino acid position 75 comprises the sequence according to SEQ ID NO: 16, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 16, wherein the percentage of sequence identity with SEQ ID NO: 16 preserves the amino acid modification at amino acid position 75.

10. The bacteria according to any one of claims 7 to 9, wherein the amino acid modification at the 45th amino acid position is a hydrophobic-to-hydrophilic substitution, preferably wherein the hydrophobic-to-hydrophilic substitution is a substitution of leucine to serine or a substitution of leucine to threonine.

11. The bacteria according to any one of claims 7 to 10, wherein the amino acid modification at the 49th amino acid position is a hydrophobic-to-hydrophobic substitution, preferably wherein the hydrophobic-to-hydrophobic substitution is a valine-to-alanine substitution.

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

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

14. The bacteria of claim 13, wherein the IL-15 variant comprises the amino acid sequence according to SEQ ID NO: 2, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2, wherein the percentage of sequence identity with SEQ ID NO: 2 preserves the amino acid modifications at amino acid positions 45 and 75.

15. The bacteria of claim 14, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 2, wherein the percentage of sequence identity with SEQ ID NO: 2 preserves the amino acid modifications at amino acid positions 45 and 75.

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

17. The bacteria of claim 16, wherein the IL-15 variant comprises the amino acid sequence according to SEQ ID NO: 3, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 3, wherein the percentage of sequence identity with SEQ ID NO: 3 retains the amino acid modifications at amino acids 45, 49 and 75.

18. The bacteria of claim 17, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 3, wherein the percentage of sequence identity with SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 45, 49, and 75.

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

20. The bacteria of claim 19, wherein the IL-15 variant comprises the amino acid sequence according to SEQ ID NO: 4, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4, wherein the percentage of sequence identity with SEQ ID NO: 4 preserves the amino acid modifications at amino acids 45, 52 and 75.

21. The bacteria of claim 20, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 4, wherein the percentage of sequence identity with SEQ ID NO: 4 preserves the amino acid modifications at amino acid positions 45, 52, and 75.

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

23. The bacteria of claim 22, wherein the IL-15 variant comprises the amino acid sequence according to SEQ ID NO: 5, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 5, wherein the percentage of sequence identity with SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45, 52, and 75.

24. The bacteria of claim 23, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 5, wherein the percentage of sequence identity with SEQ ID NO: 5 retains the amino acid modifications at amino acid positions 45, 52, and 75.

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

26. The bacteria of claim 25, wherein the IL-15 variant comprises the amino acid sequence according to SEQ ID NO: 6, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 6, wherein the percentage of sequence identity with SEQ ID NO: 6 preserves the amino acid modifications at amino acids 45, 49 and 75.

27. The bacteria of claim 26, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 6, wherein the percentage of sequence identity with SEQ ID NO: 6 preserves the amino acid modifications at amino acids 45, 49, and 75.

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

29. The bacteria of claim 28, wherein the IL-15 variant comprises the amino acid sequence according to SEQ ID NO: 7, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 7, wherein the percentage of sequence identity with SEQ ID NO: 7 preserves the amino acid modifications at amino acids 49, 52 and 75.

30. The bacteria of claim 29, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 7, wherein the percentage of sequence identity with SEQ ID NO: 7 retains the amino acid modifications at amino acid positions 49, 52, and 75.

31. The bacteria according to any one of the preceding claims, wherein the IL-15 variant is fused with an interleukin-15 receptor α (IL-15Rα) molecule.

32. The bacteria according to any one of the preceding claims, wherein the IL-15 variant is fused to the sushi domain of the interleukin-15 receptor α (IL-15Rα) molecule.

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

34. The bacteria of claim 33, wherein the adapter comprises a sequence according to SEQ ID NO: 17 or a sequence having at least 70% identity with SEQ ID NO:

17.

35. The bacteria according to any one of claims 31 to 34, wherein the IL-15 variant comprises a sequence according to any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, or a sequence having at least 70% identity with SEQ ID NO: 8, 9, 10, 11, 12 and / or 13, wherein the percentage of sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12 and / or 13 is preserved in the amino acid modifications at the relevant amino acid positions.

36. The bacteria of claim 35, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12, and / or 13, wherein the percentage of sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12, and / or 13 is preserved in the amino acid modifications at the relevant amino acid positions.

37. The bacteria according to any one of the preceding claims, wherein the IL-15 variant is fused with a signal peptide.

38. The bacteria of claim 37, wherein the signal peptide is a periplasmic signal peptide.

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

40. The bacteria according to any one of the preceding claims, wherein the IL-15 variant is encoded by a nucleic acid molecule.

41. The bacteria of claim 40, wherein the nucleic acid molecule encodes an IL-15 variant, the IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, or a sequence having at least 70% identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 is preserved in the amino acid modifications at the relevant amino acid positions.

42. The bacterium of claim 41, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 is preserved in the amino acid modifications at the relevant amino acid positions.

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

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

45. A variant of interleukin-15 (IL-15), wherein the wild-type IL-15 comprises the amino acid sequence according to SEQ ID NO: 1, or comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, and wherein, compared with SEQ ID NO: 1, the IL-15 variant includes one or more amino acid modifications in a surface region adjacent to the receptor-interacting surface, and wherein said one or more amino acid modifications comprise any of the following: i) The hydrophobic to hydrophilic substitution at the 45th amino acid position; ii) Hydrophobic-to-hydrophobic substitution at amino acid position 49; iii) A hydrophobic-to-hydrophilic substitution at amino acid position 52; and / or iv) Replacement of serine with proline at amino acid position 75. Preferably, the receptor-interacting surface is an interleukin-15 receptor α (IL-15Rα)-interacting surface.

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

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

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

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

50. The nucleic acid molecule of claim 49, wherein the nucleic acid molecule encodes an IL-15 variant, the IL-15 variant comprising an amino acid sequence according to any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, or a sequence having at least 70% identity with any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 is preserved in the amino acid modifications at the relevant amino acid positions.

51. The nucleic acid molecule of claim 50, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the percentage of sequence identity with any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 is retained at the amino acid modifications at the relevant amino acid positions.

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

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

54. A bacterium comprising an IL-15 variant as claimed in any one of claims 45 to 48, or a nucleic acid molecule as claimed in any one of claims 49 to 53.

55. The bacteria according to claim 54, wherein the bacteria are live attenuated bacteria.

56. The bacteria of claim 55, wherein the bacteria is a live, attenuated Gram-negative bacterium.

57. The bacteria of claim 56, wherein the live attenuated Gram-negative bacteria is Salmonella; preferably, wherein the live attenuated Gram-negative bacteria is Salmonella enterica; more preferably, wherein the live attenuated Gram-negative bacteria is Salmonella typhi serotype; and even more preferably, wherein the live attenuated Gram-negative bacteria is Salmonella typhi serotype ZH9.

58. The bacteria of any one of claims 1 to 44 or 54 to 57, the IL-15 variant of any one of claims 45 to 48, or the nucleic acid molecule of any one of claims 49 to 53, for therapeutic use.

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

60. The bacteria, IL-15 variant, or nucleic acid molecule for the purpose according to claim 59, wherein the neoplastic disease is solid carcinoma and / or hematologic malignancy.

61. The bacteria, IL-15 variant, or nucleic acid molecule for the stated purpose according to claim 60, wherein the solid tumor and / or hematologic malignancy is selected from the following cancers: prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, malignant epithelial tumor, head and neck cancer, skin cancer, or sarcoma.

62. The bacteria, IL-15 variant, or nucleic acid molecule for the stated purpose according to claim 61, wherein the bacteria, IL-15 variant, or nucleic acid molecule is administered to the subject by local infusion, intraperitoneal, intrapleural, intravenous, intravesical, peritumoral injection, intratumoral injection, or oral administration.

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

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

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

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