Interleukin-15 variant

Abstract

The present invention provides a bacterium containing an interleukin-15 (IL-15) variant, wherein, compared to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, preferably the receptor interaction surface being an interleukin-15 receptor alpha (IL-15Rα) interaction surface.

Description

[Technical Field]

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

[0002] Microbiotalysis has been shown to be effective as a postoperative treatment to reduce the incidence of tumor recurrence, and BCG treatment is the current standard of care for certain cancer types, particularly for NMIBC patients (Alhunaidi, 2019). BCG treatment has limitations, including its efficacy range (40-60% of patients experience tumor recurrence within 5 years of surgery), concerns about adverse effects and safety, complex production requirements, and a long treatment regimen (patients receive 27 transurethral intravesical infusions over 36 months). To improve efficacy, it has been proposed to use microbiotalysis in combination with co-immunostimulatory molecules.

[0003] Evidence supporting the use of immunostimulatory molecules such as cytokines in cancer treatment has increased significantly in recent years. Interleukin-15 (IL-15) and IL-15 agonist molecules are considered promising immunotherapies 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 combination therapy with intrabladder BCG and the IL-15 superagonist molecule ANKTIVA® (formerly ALT-803, N803) (developed by ImmunityBio, formerly Altor BioScience).

[0004] However, a challenge associated with the use of IL-15 as an immunostimulatory anti-cancer treatment, particularly in the context of microorganisms, is its expression in bacteria. While there are many examples in the prior art of cytokines expressed and purified from E. coli, a common problem associated with the use of bacterial systems to express eukaryotic cytokines is the formation of inclusion bodies. Bacterial expression of cytokines generally results in the formation of non-functional inclusion bodies. Therefore, the accumulation of cytokines in bacteria imposes additional and labor-intensive requirements of solubilizing and refolding the protein from the inclusion bodies, which often leads to low protein yields and is not suitable for live therapeutic agents (Ferrer-Miralles, 2009; de Marco, 2009). High protein expression rates or unfavorable reducing conditions in the cytoplasm can inhibit the folding of nascent peptides, resulting in the formation of insoluble protein aggregates. That is, the highly reducing environment of the bacterial cytoplasm interferes with the formation of disulfide linkages between cysteine residues during protein folding, which can be problematic for the expression of cytokines (including IL-15) that have disulfide bonds in their protein structure.

[0005] Attempts to improve cytokine expression levels and solubility in bacteria, such as E. coli, include lowering culture temperature, co-expression of chaperones, in vitro resolubilization and refolding protocols, use of weak promoters, fusion to solubility-enhancing tags, addition of signal peptides for periplasmic localization, or N-terminal fusion to carriers for extracellular transport (Behar 2011, Devi 2016, Nausch 2013).

[0006] The same limitation on cytokine expression efficiency observed in Escherichia coli has also been observed in strains of Salmonella. In fact, the inventors of the present invention have previously observed that expression of human IL-15 in Salmonella Typhi ZH9 from a temperature-inducible LPR promoter in cultures grown at a sub-optimal induction temperature of 37°C results in the accumulation of IL-15 in insoluble aggregates.

[0007] A further challenge presented by protein expression in Gram-negative bacterial strains such as Escherichia coli and Salmonella is that even after successful expression of the cytokine, the protein must be correctly folded and then transported across the two cell membranes with the bacterial periplasm in between in order to be secreted from the cell.

[0008] Accordingly, it can be seen that there is a need to develop cytokines that can be optimally expressed and efficiently secreted in bacteria. SUMMARY OF THE INVENTION

[0009] The present invention provides IL-15 variants with both optimized solubility and retained biological activity. The inventors of the present invention have surprisingly identified certain combinations of mutations in IL-15 that result in IL-15 variants that are more soluble than wild-type IL-15 and retain functionality. The variants provided herein are therefore ideal candidates for efficient expression systems, for example expression in and secretion from bacteria, such as Gram-negative bacteria and / or Gram-positive bacteria.

[0010] Therefore, in a first aspect of the present invention, a bacterium is provided that contains an interleukin-15 (IL-15) variant, wherein, compared to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, and preferably, the receptor interaction surface is an interleukin-15 receptor alpha (IL-15Rα) interaction surface.

[0011] In a second aspect of the present invention, an interleukin-15 (IL-15) variant is provided, wherein wild-type IL-15 comprises the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 1, and, compared to SEQ ID NO: 1, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, and the one or more amino acid modifications comprise any of i) a hydrophobic to hydrophilic substitution at amino acid position 45; ii) a hydrophobic to hydrophobic substitution at amino acid position 49; iii) a hydrophobic to hydrophilic substitution at amino acid position 52; and / or iv) a serine to proline substitution at amino acid position 75; preferably, the receptor interaction surface is the interleukin-15 receptor alpha (IL-15Rα) interaction surface.

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

[0013] A fourth embodiment of the present invention provides a bacterium comprising an IL-15 variant according to a second embodiment of the present invention, or a nucleic acid molecule according to a third embodiment of the present invention.

[0014] In a fifth aspect of the present invention, a bacterium according to the first or fourth aspect of the present invention, an IL-15 variant according to the second aspect of the present invention, or a nucleic acid molecule according to the third aspect of the present invention is provided for therapeutic use.

[0015] A sixth aspect of the present invention provides the use of a bacterium according to the first or fourth aspect of the present invention, an IL-15 variant according to the second aspect of the present invention, or a nucleic acid molecule according to the third aspect of the present invention in the production of a therapeutic pharmaceutical.

[0016] A seventh aspect of the present invention provides a method for treating, suppressing, preventing or controlling a neoplastic disease in a subject, comprising administering to the subject a bacterium according to the first or fourth aspect of the present invention, an IL-15 variant according to the second aspect of the present invention, or a nucleic acid molecule according to the third aspect of the present invention.

[0017] The present invention is described with reference to the accompanying drawings. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 shows the structure of an interleukin-15 (IL-15, PDB ID: 4GS7) variant, pointing to amino acid modifications in the surface region adjacent to the IL-15Rα interaction surface. IL-15Rα, IL-2Rβ (IL-15Rβ), and GammaC (γc) are also shown. [Figure 2] Figure 2 shows a schematic representation of IL-15 trans presentation and biological function. [Figure 3] Figure 3 shows the sequence-structure relationship of wild-type IL-15 and its interaction with cognitive ligands. It shows the short and long (mammalian) signal peptide sequences of pre-IL-15 (A), mature IL-15 (B), and the tertiary structure of IL-15 (C). [Figure 4-1] Figure 4-1 shows the aggregation tendency and hydrophobicity score of IL-15 calculated using AggreScan and AggreScan 3D. [Figure 4-2] Figure 4-2 shows the aggregation tendency and hydrophobicity score of IL-15 calculated using AggreScan and AggreScan 3D. [Figure 5]Figure 5 shows a summary of IL-15 and IL-15 agonists used in immunotherapy. [Figure 6] Figure 6 shows the structure of the IL-15 variant, pointing to amino acid positions 45 and / or 49 and / or 52. IL-15Rα, IL-2Rβ, and GammaC(γc) are also shown. [Figure 7A] Figures 7A-7C show the structures of IL-15 in a quaternary complex with IL-15Rα, IL-15Rβ, and GammaC(γc) (PDB:4GS7). [Figure 7B] Figures 7A-7C show the structures of IL-15 in a quaternary complex with IL-15Rα, IL-15Rβ, and GammaC(γc) (PDB:4GS7). [Figure 7C] Figures 7A-7C show the structures of IL-15 in a quaternary complex with IL-15Rα, IL-15Rβ, and GammaC(γc) (PDB:4GS7). [Figure 8A] Figures 8A–8D demonstrate the solubility and functionality of different IL-15 variants. (A) shows the total number of soluble variants obtained through different mutagenesis strategies. (B) shows the solubility score distribution for each mutagenesis strategy, with in silico designs showing a higher overall success rate and epPCR variants showing greater variability. (C) shows the total number of active variants obtained through different mutagenesis strategies. (D) shows the distribution of activity measurements as relative luminescence units (RLU) from IL-15 Bioassay Cells (Promega) reporter cell lines for each mutagenesis strategy. [Figure 8B]Figures 8A–8D demonstrate the solubility and functionality of different IL-15 variants. (A) shows the total number of soluble variants obtained through different mutagenesis strategies. (B) shows the solubility score distribution for each mutagenesis strategy, with in silico designs showing a higher overall success rate and epPCR variants showing greater variability. (C) shows the total number of active variants obtained through different mutagenesis strategies. (D) shows the distribution of activity measurements as relative luminescence units (RLU) from IL-15 Bioassay Cells (Promega) reporter cell lines for each mutagenesis strategy. [Figure 8C] Figures 8A–8D demonstrate the solubility and functionality of different IL-15 variants. (A) shows the total number of soluble variants obtained through different mutagenesis strategies. (B) shows the solubility score distribution for each mutagenesis strategy, with in silico designs showing a higher overall success rate and epPCR variants showing greater variability. (C) shows the total number of active variants obtained through different mutagenesis strategies. (D) shows the distribution of activity measurements as relative luminescence units (RLU) from IL-15 Bioassay Cells (Promega) reporter cell lines for each mutagenesis strategy. [Figure 8D] Figures 8A–8D demonstrate the solubility and functionality of different IL-15 variants. (A) shows the total number of soluble variants obtained through different mutagenesis strategies. (B) shows the solubility score distribution for each mutagenesis strategy, with in silico designs showing a higher overall success rate and epPCR variants showing greater variability. (C) shows the total number of active variants obtained through different mutagenesis strategies. (D) shows the distribution of activity measurements as relative luminescence units (RLU) from IL-15 Bioassay Cells (Promega) reporter cell lines for each mutagenesis strategy. [Figure 9A]Figures 9A–9C demonstrate the biological activity of the IL-15 / SAg variant using three independent assays (IL-15 Bioassay Cells, NK proliferation assay, and IFNγ production assay). [Figure 9B] Figures 9A–9C demonstrate the biological activity of the IL-15 / SAg variant using three independent assays (IL-15 Bioassay Cells, NK proliferation assay, and IFNγ production assay). [Figure 9C] Figures 9A–9C demonstrate the biological activity of the IL-15 / SAg variant using three independent assays (IL-15 Bioassay Cells, NK proliferation assay, and IFNγ production assay). [Figure 10A] Figures 10A-10B show protein purification data supporting the isolation of different IL-15 variants and IL-15 SAg variants. [Figure 10B] Figures 10A-10B show protein purification data supporting the isolation of different IL-15 variants and IL-15 SAg variants. [Figure 11A] Figures 11A and 11B show the preclinical candidate selection of IL-15 variants. The results of incubating purified IL-15 variants at stepwise concentrations with reporter cell lines for 6 hours are shown. [Figure 11B] Figures 11A and 11B show the preclinical candidate selection of IL-15 variants. The results of incubating purified IL-15 variants at stepwise concentrations with reporter cell lines for 6 hours are shown. [Figure 12] Figure 12 shows the in vitro human cell assay. It shows the results of incubating IL-15 variants with primary NK cells for 24 hours at 71 nM (Experiment 1) and 58 nM (Experiment 2). [Figure 13] Figure 13 shows the results of IL-15 variant-mediated NK activation in mouse spleen cells after 72 hours of incubation with an IL-15 variant. [Modes for carrying out the invention]

[0019] Detailed explanation The present invention provides an optimized IL-15 variant (and / or its superagonist, i.e., SAg variant) which is both soluble and functional, and is therefore an optimal candidate for expression in an efficient secretion system that enables the production and secretion of the IL-15 variant and SAg variant.

[0020] Therefore, in the first embodiment, the present invention provides an interleukin-15 (IL-15) barrier. The present invention provides a bacterium containing an IL-15 variant, wherein, compared to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, preferably the receptor interaction surface being an interleukin-15 receptor alpha (IL-15Rα) interaction surface (Figure 1).

[0021] As used herein, the term “interleukin” (IL) refers to any member of the glycoprotein family that is expressed by leukocytes (white blood cells) and is involved in regulating the immune response. Interleukins are sometimes referred to as cytokines. Interleukins have diverse roles in the proliferation, maturation, migration, and adhesion of immune cells, as well as in the generation of immune cells, including immune cell activation and differentiation. Interleukins can have pro-inflammatory and anti-inflammatory properties. Interleukins constitute a large group of proteins that have the ability to induce several responses in cells and tissues by binding to receptors known as interleukin receptors and triggering downstream signaling from those receptors.

[0022] Interleukin-15 (IL-15) is a four-alpha-helix bundle cytokine belonging to the same family as IL-2, IL-4, IL-7, IL-9, and IL-21. IL-15 is constitutively expressed by a diverse range of cells, including dendritic cells, monocytes, macrophages, bone marrow stromal cells, and intestinal epithelial cells. Cells expressing IL-15 also express its high-affinity receptor, IL-15Rα. Unlike most cytokines secreted in soluble form, IL-15 has a unique expression morphology, being expressed in association with its high-affinity receptor, IL-15Rα, and transported to the surface of IL-15-producing cells as a heterodimer complex. IL-15 can also be secreted as soluble IL-15 independently of IL-15Rα. The IL-15 / IL-15Rα complex is a cell surface complex that efficiently stimulates adjacent cells via the IL-2 / IL-15Rβ and GammaC(γc) complex (IL-15Rβγ) through the trans-presentation mechanism (Figure 2). IL-15 preferentially stimulates the activation, proliferation, and cytolytic activity of NK (natural killer) and CD8+ T cells (Guo, 2007; Santana-Carrero, 2019).

[0023] Trans-presentation of IL-15 mediates cellular responses during homeostasis. There is evidence that the IL-15Rα / IL-15 complex is cleaved from the surface of presenting cells in response to numerous types of immune stimuli, such as total body irradiation, TLR stimulation, viral infection, CD40 stimulation, type I IFN (IFN-I), and activation of the interferon gene-stimulating (STING) pathway, releasing a soluble heterodimer IL-15 / IL-15Rα complex (Bergamaschi, 2012; Anthony, 2016).

[0024] Human IL-15 is a 162-amino acid (18,086 kDa) protein containing four helices, two disulfide bonds, and one glycosylation site (Figure 3). It has two isoforms produced from alternatively spliced ​​transcripts: one form has a short-chain signal peptide (SSP) containing a 21-amino acid leader peptide and remaining in the cellular cytosol, while the other form has a long-chain signal peptide (LSP) containing a 48-amino acid signal peptide that enables the secretion of the protein into the extracellular environment (Saeed and Revell, 2001; Duitman, 2008). This secretion occurs through the independent transport of the signal and receptor alpha into the Golgi apparatus, followed by high-affinity interactions between the signal and receptor, and then the transport of the complex into the external environment via receptor alpha. The signal peptide is always cleaved at the same site, presumably via the highly conserved sequence N-GLPKTEA / NW-C, resulting in the same mature form of IL-15, although SSP IL-15 has already been shown to be bioactive, pointing to a certain flexibility in the presence of the N-terminal peptide (Bergamaschi, 2009). Mature IL-15 then folds into a 4-helix bundle motif (the four α-helices are constructed longitudinally in antiparallel orientation), with 16-21 amino acids per α-chain, where α1 interacts with α3 and α2 with α4 (inter-helix interactions are shown as dashed lines in Figure 3, with key residues involved in protein-protein interactions highlighted). There are disulfide bonds between Cys42-Cys85 that stabilize this latter interaction, and between Cys35-Cys88 that stabilize the second interaction. There are two highly structurally undefined regions that form loops between α1 and α2 (loop 1-2) and between α3 and α4 (loop 3-4).

[0025] The crystal structures of IL-15 co-coordinated with receptors IL-2Rβ, γc, and IL-15Rα have been elucidated (Ring, 2012). This indicates that this signaling protein interacts with IL-2Rβ via polar interactions with helices α1 and α3, and that the interactions of 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.

[0026] On the other hand, the interaction with γc exhibits a more general interaction that lacks a highly defined chemical signature, highlighting the promiscuous nature of γc, which interacts with multiple cytokines. The interaction with γc occurs via the α1 and α4 chains, with Q108, M109, and N112 being key amino acids involved in this interaction. To compensate for the smaller size of IL-15, an additional contact site is created with the elongated alpha chain.

[0027] Finally, it has been found that the interaction with IL-15Rα occurs 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 contact with IL-15Rα and confer rigidity to the molecule.

[0028] The amino acid sequence of IL-15 was analyzed by the inventors of this invention using AggreScan, ProtScale, and AggreScan3D to identify potential aggregation sites that cause protein aggregation and inclusion formation during recombinant overexpression in E. coli or Salmonella cifida. Regions with a higher tendency to aggregate are those that form alpha helices (Figures 4-1 and 4-2), as these are regions involved in protein-protein interactions. Both the rational design and directional evolutionary approaches discussed below aim to minimize these hydrophobic interactions without altering IL-15's receptor binding properties.

[0029] Given its role in stimulating the proliferation and cytotoxicity of CD8 T cells and NK cells, IL-15 has been widely tested as a cancer immunotherapy. The efficacy of IL-15 is limited by the short half-life of the protein in vivo. Numerous IL-15 variants have been generated to improve the stability and efficacy of the protein.

[0030] Figure 5 shows five (1-) used to stimulate cytotoxic T cell and NK cell responses. 5) Describe different IL-15-based agents. In order of increasing IL-15 signal intensity / duration, (1) rIL-15 was the first form of IL-15 to investigate in vivo. When administered, rIL-15 is thought to primarily bind to cell surface IL-15Rα, where it is trans-presented to IL-15-responsive cells. (2) Heterodimeric IL-15 is the native form of IL-15 that can be cleaved from cells and stimulate a response independently of cell interactions. Heterodimeric hIL-15 is manufactured as a therapeutic agent by Novartis. (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 generated by mixing commercially available IL-15Rα-Fc chimeric fusion proteins with rIL-15 and has been used extensively in preclinical studies. (5) ANKTIVA® (formerly ALT-803, N-803) (ImmunityBio, formerly Altor Pharmaceutical) is an IL-15 superagonist consisting of a mutant IL-15 with increased affinity for CD122-expressing immune cells (in which the asparagine at position 72 in the mature IL-15 amino acid sequence is replaced with an aspartic acid residue) linked to the sushi domain of IL-15Rα fused to the Fc fragment. The signal intensity and duration of these IL-15-based agents increase with increasing agonist abundance, increased in vivo half-life (contributed 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 (contributed by IL-15 binding to the sushi domain and mutations present in ALT-803).

[0031] There is a clear need to develop IL-15 variants that are soluble and exhibit biological activity. As used herein, the term “wild-type” (“WT”) is given its usual meaning in the art and refers to a protein or the gene encoding such protein found in its natural, unmutated, or unmodified 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 from which all IL-15 variants according to the present invention are derived. Parental IL-15 is also referred to as IL-15M40 or M40 (SEQ ID NO: 16) and is derived from wild-type IL-15 containing the amino acid sequence described in SEQ ID NO: 1. The difference between wild-type IL-15 and M40 IL-15 is the presence of an amino acid modification (S75P) in which serine at amino acid position 75 of SEQ ID NO: 1 is replaced with proline. Thus, the S75P mutation gives rise to SEQ ID NO: 16 (parent) from SEQ ID NO: 1 (wild-type). Therefore, in some embodiments, the IL-15 variant includes an amino acid modification at position 75. In preferred embodiments, the IL-15 variant includes a serine-to-proline substitution (S75P) at position 75. Any of the IL-15 variants described herein may feature the S75P mutation. The S75P mutation can provide increased solubility of the IL-15 variant, further stabilizing it and thus optimizing its expression in bacteria.

[0032] Compared to "wild-type" IL-15, the term "interleukin variant" refers to an IL-15 molecule containing one or more amino acid modifications compared to wild-type IL-15. As used herein, the term "amino acid modification" refers to any change or mutation to the wild-type amino acid sequence. Amino acid modifications may or may not alter the structure, function, and / or physiological and chemical 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 and result in a frameshift or a decrease in the length of the amino acid sequence. Insertion mutations involve the addition of an amino acid and result in a frameshift or an increase in the length of the amino acid sequence. Substitution mutations involve the replacement of one amino acid with another amino acid. Substitution mutations may be conservative or non-conservative.

[0033] As will be understood by those skilled in the art, amino acids can be broadly grouped according to their properties (i.e., size, bulkiness, charge, hydrophobicity, polarity, etc.). Similarity or dissimilarity between amino acids can also be calculated using substitution matrices and physicochemical distances. Conservative amino acid substitutions involve the replacement or exchange of one amino acid for another amino acid having similar properties. For example, the substitution of a hydrophobic amino acid for another hydrophobic amino acid (e.g., valine to alanine) constitutes a conservative amino acid substitution. Non-conservative or radical amino acid substitutions involve the replacement or exchange of one amino acid for another amino acid having different or dissimilar properties. For example, the substitution of a hydrophobic amino acid for a hydrophilic amino acid (e.g., leucine to serine) constitutes a non-conservative amino acid substitution. Non-conservative amino acid substitutions may be more likely to be associated with changes in protein structure and function compared to conservative amino acid substitutions.

[0034] Several methods for inducing amino acid modification are well known in the art. As will be readily apparent to those skilled in the art, nucleic acid sequences encoding a modified or mutated amino acid sequence of interest may be generated by several means known in the art (i.e., de novo synthesis). Such genes may be introduced into an expression vector or expression system that supports gene expression and messenger RNA (mRNA) translation. The gene introduced into the expression system may be a heterologous gene (i.e., an exogenous gene), meaning that the gene originates from a cell type of 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 a bacterial cell, such as a Salmonella strain.

[0035] The IL-15 variant according to the present invention may contain one or more, two or more, three or more, four or more, five or more, or five or more amino acid modifications. However, the IL-15 variant may preferably contain one, two, or three amino acid modifications.

[0036] According to a first aspect of the present invention, a bacterium comprising an IL-15 variant is provided, wherein, compared to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, preferably the receptor interaction surface being an interleukin-15 receptor alpha (IL-15Rα) interaction surface.

[0037] Amino acid modifications are preferably located in the surface region of the IL-15 variant. As used herein, the term “surface region” refers to the portion of the protein exposed to the 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 mainly composed of hydrophilic amino acid residues, while the protein core (i.e., interior) is composed of hydrophobic residues packed together away from the solvent. The amino acid modifications on the IL-15 variant of the present invention preferably involve amino acids that are exposed to the solvent and are located on the outer / external surface of the protein. Modifications to amino acids on the protein surface often affect protein-protein interactions, as the protein surface typically contains protein-protein interfaces (i.e., molecular surfaces on which proteins come into contact and interact with each other). The protein surface may contain large, flat interfaces and / or smaller depressions or single residues, which may form anchor sites for one or more interacting proteins. The protein surfaces of interacting proteins may be morphologically and / or chemically complementary.

[0038] As those skilled in the art will understand, modifications to or around the protein surface involved in the protein-protein interface can affect the protein-protein interaction. In the context of IL-15, the protein-protein interaction may include the interaction between IL-15 and its receptor. For example, IL-15 may interact with any of its receptors, IL-15Rα, IL-15Rβ, and γc. Such interactions occur at the receptor interaction surface on the surface of IL-15.

[0039] Therefore, the surface region is the region adjacent to the receptor interaction surface of the IL-15 variant.

[0040] As used herein, the terms “receptor interaction surface,” “receptor binding surface,” “interaction surface,” “binding surface,” and “ligand binding surface” are interchangeable and refer to the surface of IL-15 that coordinates to, interacts with, and binds to the receptor (i.e., protein-protein interaction). There may be a precise group of amino acids that directly bind to the receptor. In a preferred embodiment, the receptor interaction surface is the IL-15 receptor interaction surface. In a more preferred embodiment, the receptor interaction surface is the IL-15 receptor alpha (IL-15Rα) interaction surface. An interaction surface is a surface region, as opposed to a domain. A protein domain is a distinct functional and / or structural unit within a protein, possessing self-stability and independent of the rest of the protein. An example of a protein domain is the sushi domain of the IL-15Rα receptor. An interaction surface is a surface region, as opposed to a domain. A protein domain is a distinct functional and / or structural unit within a protein, possessing self-stability and independent of the rest of the protein. An example of a protein domain is the sushi domain of the IL-15Rα receptor.

[0041] With respect to Sequence ID No. 1, the “receptor interaction surface” is defined as the amino acids at amino acid positions D8, D61 and N65 (with other amino acids involved at positions I68, L69, K10, K11 and S7) for IL-15Rβ (i.e., IL-15 α1 and α3); Q108, M109 and N112 for γc (i.e., IL-15 α1 and α4); and D22, T24, Y26, E46, Q48, E53, E87 and E90 for IL-15Rα (i.e., IL-15 α2). The “receptor interaction surface” is therefore well characterized in the art.

[0042] The inventors of this invention have remarkably identified a region of IL-15 in which amino acids may be modified to produce IL-15 variants with increased solubility and maintained biological activity. The amino acids to be modified are located in the surface region adjacent to the receptor interaction surface of IL-15. These amino acids were selected for modification because they form a portion of the available space in the IL-15 structure that is not required for interaction with IL-15Rβ or γc.

[0043] In particular, the inventors of this invention have surprisingly identified that the following amino acid positions: L45, Q48, V49, L52, H60, and N71 may be modified within this region of IL-15 (i.e., Sequence ID No. 1). None of these amino acids interact with IL-15Rβ or γc. IL-15Rβ or γc are expressed on the surface of the target cells to be activated (NK cells and CD8+ T cells). Thus, the “region adjacent to the receptor interaction surface” can be described as any region of IL-15 in which the amino acids do 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 amino acids modified herein may interact with IL-15Rα. For example, within IL-15 (i.e., Sequence ID No. 1), L45, V49, and L52 are described as making van der Waals contacts with IL-15Rα, and Q48 is described as forming a hydrogen bond with IL-15Rα (Chirifu et al, 2007). Furthermore, it is known in the art that mutations in N71 within IL-15 (N71D; in ALT-803 IL-15 superagonist) contribute to the activity of IL-15. Therefore, as used herein, the “region adjacent to the receptor interaction surface” may include amino acids within IL-15α2.

[0044] While not wishing to be bound by theory, amino acids present in the "adjacent region" to the receptor interaction surface may play a role in mediating protein-protein interactions. Accordingly, the term "adjacent region" may refer to surface amino acids that are in the vicinity of the interaction surface of a receptor (e.g., IL-15Rα) and are capable of interacting with IL-15Rα but do not interact with IL-15Rβ or γc. Indeed, certain modifications may thus act to enhance the interaction between IL-15 and a receptor (e.g., IL-15Rα), for example by increasing binding affinity. Furthermore, modifications that alter the compositional properties of amino acids on the surface of IL-15 can affect the solubility of IL-15.

[0045] Thus, in a preferred embodiment, the amino acid modification is present adjacent to the surface region of the IL-15 variant that is adjacent to the receptor (e.g., IL-15Rα) interaction surface of the IL-15 variant, whereby the amino acid modification enhances the interaction between the IL-15 variant and the receptor. For example, the interaction between IL-15 and IL-15Rα can be defined by the rate constant for ligand dissociation (K off ) is lower (i.e., higher stability of the IL-15 / IL-15Rα complex), the rate constant for ligand association (K on ) is higher (i.e., recognition of IL-15Rα by IL-15 and vice versa is more rapid), as well as the equilibrium dissociation constant (K d ) is lower (i.e., a high-affinity interaction), the interaction may be enhanced. K d is K off and K on (K d =K off / K on ). Other means for characterizing binding kinetics are apparent to those skilled in the art.

[0046] According to a first aspect of the present invention, a bacterium comprising an IL-15 variant is provided, wherein, compared to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, preferably the receptor interaction surface being an interleukin-15 receptor alpha (IL-15Rα) interaction surface. As detailed herein, wild-type IL-15 may comprise mature IL-15, or the amino acid sequence described in SEQ ID NO: 1, or 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 signal sequences, localization sequences, peptide tags, peptide linkers, other interleukins or their receptors, other immunostimulatory proteins, or any combination thereof.

[0047] According to a second aspect of the present invention, an interleukin-15 (IL-15) variant is provided, wherein wild-type IL-15 comprises the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 1, and, compared to SEQ ID NO: 1, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, and the one or more amino acid modifications comprise any of i) a hydrophobic to hydrophilic substitution at amino acid position 45; ii) a hydrophobic to hydrophobic substitution at amino acid position 49; iii) a hydrophobic to hydrophilic substitution at amino acid position 52; and / or iv) a serine to proline substitution at amino acid position 75; preferably, the receptor interaction surface is the interleukin-15 receptor alpha (IL-15Rα) interaction surface. This includes amino acid modifications at one or more amino acid positions 45 and / or 49 and / or 52, or any combination thereof (Figure 6).

[0048] An IL-15 variant may contain an amino acid sequence that has at least 70% sequence identity with respect to SEQ ID NO: 1. As used herein, the terms “sequence identity” and “sequence homology” refer to the number of interchangeable and identical residues over a defined length in a given alignment. To calculate the sequence identity percentage of any sequence disclosed herein, sequence comparison software may be used, for example, with default settings on the BLAST software package (V2.10.1).

[0049] In another embodiment, the IL-15 variant may include an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1; at least 80% sequence identity to SEQ ID NO: 1; at least 85% sequence identity to SEQ ID NO: 1; at least 90% sequence identity to SEQ ID NO: 1; at least 91% sequence identity to SEQ ID NO: 1; at least 92% sequence identity to SEQ ID NO: 1; at least 93% sequence identity to SEQ ID NO: 1; at least 94% sequence identity to SEQ ID NO: 1; at least 95% sequence identity to SEQ ID NO: 1; at least 96% sequence identity to SEQ ID NO: 1; at least 97% sequence identity to SEQ ID NO: 1; at least 98% sequence identity to SEQ ID NO: 1; or at least 99% sequence identity to SEQ ID NO: 1.

[0050] According to the present invention, an IL-15 variant may be modified such that, compared to SEQ ID NO: 1, an amino acid modification is present at one or more amino acid positions 45 and / or 49 and / or 52. These amino acid positions are located in the surface region adjacent to the receptor interaction surface of the IL-15 variant (preferably the interleukin-15 receptor alpha (IL-15Rα) interaction surface). An IL-15 variant may include a single amino acid modification at amino acid position 45; a single amino acid modification at amino acid position 49; a single amino acid modification at amino acid position 52; two amino acid modifications at amino acid positions 45 and 49; two amino acid modifications at amino acid positions 45 and 52; two amino acid modifications at amino acid positions 49 and 52; or three amino acid modifications at amino acid positions 45, 49, and 52. Any combination of these modifications is intended to be included. It is understood that an IL-15 variant including any combination of amino acid mutations at amino acid positions 45 and / or 49 and / or 52 will retain at least 70% sequence identity with respect to SEQ ID NO: 1.

[0051] In any aspect of the present invention, the IL-15 variant may include an amino acid modification at amino acid position 75. The amino acid modification may be present in combination with any other modifications or combinations thereof as described herein. For example, the IL-15 variant may include an amino acid modification at any of amino acid positions 45, 49, 52 and / or 75. For example, amino acid positions 75 and 45; amino acid positions 75 and 49; amino acid positions 75 and 52; amino acid positions 75, 45 and 49; amino acid positions 75, 45 and 52; amino acid positions 75, 49 and 52; or amino acid positions 75, 45, 49 and 52. Preferably, the IL-15 variant may include an amino acid modification at amino acid positions 75 and 45; amino acid positions 75, 45 and 49; amino acid positions 75, 45 and 52; amino acid positions 75, 49 and 52.

[0052] In some embodiments, the IL-15 variant may include amino acid modifications in which non-conservative amino acid substitutions are made. In such embodiments, hydrophobic amino acids naturally present in the wild-type sequence are replaced with hydrophilic amino acids, or vice versa. If hydrophobic amino acids are located in positions on the three-dimensional protein structure that are exposed to the solvent, the protein may be destabilized. Therefore, mutations in such hydrophobic residues can destabilize the protein. It can be useful for standardizing the process.

[0053] In other embodiments, the IL-15 variant may include amino acid modifications in which conservative amino acid substitutions are performed. In such embodiments, hydrophobic amino acids naturally present in the wild-type sequence are replaced with other hydrophobic amino acids, or hydrophilic amino acids naturally present in the wild-type sequence are replaced with other hydrophilic amino acids.

[0054] As used herein, the terms “hydrophobic” and “hydrophilic” are given their usual meanings in the art, i.e., having a weak affinity and a strong affinity for water, respectively. “Hydrophobic amino acids” are amino acids that have hydrophobic side chains, i.e., side chains that do not interact with water (repell water). Hydrophobic side chains are typically or 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 may include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). However, any naturally occurring, synthetic, or modified amino acids possessing hydrophobic properties are also intended to be included in the term “hydrophobic amino acids.” “Hydrophilic amino acids” are amino acids that have hydrophilic side chains, i.e., side chains that interact with water (attract water). Hydrophilic side chains are typically composed of oxygen, nitrogen, and sulfur atoms. Hydrophilic amino acids are generally polar and can therefore dissolve in polar solvents, such as water, via the formation of hydrogen bonds. Naturally occurring hydrophilic amino acids may include serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), and tyrosine (Tyr). Other naturally occurring hydrophilic amino acids may include arginine (Arg), histidine (His), lysine (Lys), aspartic acid (Asp), and glutamic acid (Glu), which have electrically charged side chains. Arginine, histidine, and lysine have an overall positive charge, while aspartic acid and glutamic acid have an overall negative charge. However, any naturally occurring, synthetic, or modified amino acids with hydrophilic properties are also intended to be included in the term “hydrophilic amino acids.”

[0055] According to the present invention, an IL-15 variant may include an amino acid modification at one or more amino acid positions 45 and / or 49 and / or 52. With respect to SEQ ID NO: 1, the wild-type amino acid at position 45 is leucine, the wild-type amino acid at position 49 is valine, and the wild-type amino acid at position 52 is leucine.

[0056] In preferred embodiments of any aspect of the present invention, the amino acid modification at amino acid position 45 is a hydrophobic to hydrophilic substitution. This amino acid modification may be present in conjunction with any other modifications described herein. Such substitutions involve the exchange of a hydrophobic amino acid for a hydrophilic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for any naturally occurring or non-naturally occurring hydrophilic amino acid. For example, any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan may be substituted for serine, threonine, cysteine, asparagine, glutamine, tyrosine, arginine, histidine, lysine, aspartic acid, or glutamic acid.

[0057] The wild-type amino acid at amino acid position 45 in relation to Sequence ID No. 1 is leucine. In the present invention, it is preferable that the hydrophobic-to-hydrophilic substitution at amino acid position 45 is a substitution from leucine to serine or a substitution from leucine to threonine. Although we do not wish to be bound by theory, this non-conservative amino acid modification is the amino acid at position 45. It may be possible to act to alter the physiological and chemical properties of the region where the amino acid is present. In particular, the region may become more hydrophilic, and therefore, IL-15 variants with such amino acid modifications may become more soluble. This alteration in the physiological and chemical properties of amino acids in the surface region adjacent to the receptor interaction surface of the IL-15 variant (e.g., the interleukin-15 receptor alpha (IL-15Rα) interaction surface) may also help to enhance the interaction between the IL-15 variant and the receptor. Therefore, IL-15 variants with such amino acid modifications may be more soluble in the bacterial chassis than wild-type IL-15, while also exhibiting biological activity.

[0058] In another preferred embodiment, the amino acid modification at amino acid position 49 is a hydrophobic-to-hydrophobic substitution. This amino acid modification may be present in conjunction with any other modifications described herein. Such a substitution involves the exchange of a hydrophobic amino acid for another hydrophobic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for another hydrophobic amino acid. For example, one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan may be substituted for one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan. However, the amino acid modification at amino acid position 49 may alternatively be a hydrophobic-to-hydrophilic substitution, preferably a valine-to-serine substitution.

[0059] The wild-type amino acid at amino acid position 49 in SEQ ID NO: 1 is valine. Preferred in this invention is that the hydrophobic-to-hydrophobic substitution at amino acid position 49 is a substitution from valine to alanine. The inventors of this invention have surprisingly found that the hydrophobic-to-hydrophobic mutation at V49 enhances the properties of the IL-15 variant. While we do not wish to be bound by theory, this relatively conserved amino acid modification may be able to act to optimize the region where the amino acid resides at position 49. In particular, 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 bulkier isopropyl side chain (-CH(CH3)2). Such substitutions to a smaller / less bulky amino acid may be able to help optimize the protein-protein interface for IL-15 / receptor interactions. Furthermore, alanine has lower hydrophobicity than valine, and therefore, this modification in the physiological and chemical properties of the amino acid in the surface region adjacent to the receptor interaction surface of the IL-15 variant may also help increase the solubility of the IL-15 receptor. By removing the hydrophobicity of valine in such substitution, the inventors of the present invention were able to assess the impact on the solubility / stability of the IL-15 variant. Thus, IL-15 variants having such amino acid modifications may have optimized solubility and functionality compared to wild-type IL-15.

[0060] In another preferred embodiment, the amino acid modification at amino acid position 52 is a hydrophobic to hydrophilic substitution. This amino acid modification may be present in conjunction with any other modifications described herein. Such substitutions involve the exchange of a hydrophobic amino acid for a hydrophilic amino acid. Any naturally occurring or non-naturally occurring hydrophobic amino acid may be exchanged or substituted for any present or non-naturally occurring hydrophilic amino acid. For example, any one of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan may be substituted for serine, threonine, cysteine, asparagine, glutamine, tyrosine, arginine, histidine, lysine, aspartic acid, or glutamic acid.

[0061] The wild-type amino acid at amino acid position 52 in sequence number 1 is leucine. In clarity, it is preferable that the hydrophobic-to-hydrophilic substitution at amino acid position 52 is a leucine-to-lysine substitution or a leucine-to-arginine substitution. While we do not wish to be bound by theory, this non-conservative amino acid modification may act to alter the physiochemical properties of the region where the amino acid at position 52 resides. In particular, the region may become more hydrophilic, and therefore, IL-15 variants having such amino acid modifications may become more water-soluble. This alteration in the physiochemical properties of amino acids in surface regions adjacent to the receptor interaction surface of the IL-15 variant (e.g., the interleukin-15 receptor alpha (IL-15Rα) interaction surface) may also help to enhance the interaction between the IL-15 variant and the receptor. Thus, IL-15 variants having such amino acid modifications may be more soluble than wild-type IL-15 while also preserving biological activity.

[0062] In some embodiments, the amino acids at positions 45, 49, and 52 do not need to be substituted with bulkier amino acids. The amino acids at positions 45, 49, and 52 are adjacent to the receptor binding surface, and therefore, substitution of these residues with bulkier residues may cause steric hindrance that could interfere with receptor binding. Aspartic acid, glutamic acid, asparagine, and glutamine are examples of bulky amino acids. Therefore, in some embodiments, the amino acid modification at amino acid positions 45, 49, and 52 does not need to be a substitution with any of aspartic acid, glutamic acid, asparagine, or glutamine.

[0063] As detailed herein, the IL-15 variant according to the present invention may include an amino acid modification at amino acid position 75. The amino acid modification may be present in conjunction with any other modifications described herein. Preferably in the present invention, the amino acid modification at amino acid position 75 is a serine-to-proline substitution (S75P). The serine-to-proline substitution at amino acid position 75 has been described in the Art (Behar, 2011). The inventors of the present invention selected the S75P IL-15 mutant (parent / M40 IL-15) as the starting sequence for generating the IL-15 variant because the S75P IL-15 mutant had been observed to be more soluble than unmodified IL-15 (Behar, 2011). The IL-15 variant including the S75P amino acid modification may have the sequence described in SEQ ID NO: 16, or a sequence having at least 70% identity to SEQ ID NO: 16. In another embodiment, the IL-15 variant may include an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 16; at least 80% sequence identity to SEQ ID NO: 16; at least 85% sequence identity to SEQ ID NO: 16; at least 90% sequence identity to SEQ ID NO: 16; at least 91% sequence identity to SEQ ID NO: 16; at least 92% sequence identity to SEQ ID NO: 16; at least 93% sequence identity to SEQ ID NO: 16; at least 94% sequence identity to SEQ ID NO: 16; at least 95% sequence identity to SEQ ID NO: 16; at least 96% sequence identity to SEQ ID NO: 16; at least 97% sequence identity to SEQ ID NO: 16; at least 98% sequence identity to SEQ ID NO: 16; at least 99% sequence identity to SEQ ID NO: 16; or at least 100% sequence identity to SEQ ID NO: 16, wherein the percentage of sequence identity to SEQ ID NO: 16 preserves the amino acid modification at amino acid position 75. It is understood that any of the amino acid modifications disclosed herein may exist in any combination with any of the other amino acid modifications disclosed herein.

[0064] Therefore, in a preferred embodiment, an IL-15 variant according to any aspect of the present 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 a more preferred embodiment, an IL-15 variant according to any aspect of the present invention may include (i) a leucine-to-serine substitution at amino acid position 45; and (ii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may include the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 2, wherein the % sequence identity with respect to SEQ ID NO: 2 preserves the amino acid modifications at amino acid positions 45 and 75.

[0065] In another embodiment, the IL-15 variant may include an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 2; at least 80% sequence identity to SEQ ID NO: 2; at least 85% sequence identity to SEQ ID NO: 2; at least 90% sequence identity to SEQ ID NO: 2; at least 91% sequence identity to SEQ ID NO: 2; at least 92% sequence identity to SEQ ID NO: 2; at least 93% sequence identity to SEQ ID NO: 2; at least 94% sequence identity to SEQ ID NO: 2; at least 95% sequence identity to SEQ ID NO: 2; at least 96% sequence identity to SEQ ID NO: 2; at least 97% sequence identity to SEQ ID NO: 2; at least 98% sequence identity to SEQ ID NO: 2; at least 99% sequence identity to SEQ ID NO: 2; or at least 100% sequence identity to SEQ ID NO: 2, wherein the percentage of sequence identity to SEQ ID NO: 2 preserves amino acid modifications at amino acid positions 45 and 75.

[0066] In another preferred embodiment, an IL-15 variant according to any aspect of the present 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 a more preferred embodiment, an IL-15 variant according to any aspect of the present invention may include (i) a leucine-to-serine substitution at amino acid position 45, (ii) a valine-to-alanine substitution at amino acid position 49, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may include the amino acid sequence described in SEQ ID NO: 3, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 3, wherein the % sequence identity with respect to SEQ ID NO: 3 preserves the amino acid modifications at amino acid positions 45, 49, and 75.

[0067] In another embodiment, the IL-15 variant may include an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 3; at least 80% sequence identity to SEQ ID NO: 3; at least 85% sequence identity to SEQ ID NO: 3; at least 90% sequence identity to SEQ ID NO: 3; at least 91% sequence identity to SEQ ID NO: 3; at least 92% sequence identity to SEQ ID NO: 3; at least 93% sequence identity to SEQ ID NO: 3; at least 94% sequence identity to SEQ ID NO: 3; at least 95% sequence identity to SEQ ID NO: 3; at least 96% sequence identity to SEQ ID NO: 3; at least 97% sequence identity to SEQ ID NO: 3; at least 98% sequence identity to SEQ ID NO: 3; at least 99% sequence identity to SEQ ID NO: 3; or at least 100% sequence identity to SEQ ID NO: 3, wherein the percentage of sequence identity to SEQ ID NO: 3 preserves amino acid modifications at amino acid positions 45, 49 and 75.

[0068] In another preferred embodiment, an IL-15 variant according to any aspect of the present 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, an IL-15 variant according to any aspect of the present invention may include (i) a leucine-to-serine substitution at amino acid position 45, (ii) a leucine-to-lysine substitution at amino acid position 52, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant is the amino acid sequence described in SEQ ID NO: 4, or relative to SEQ ID NO: 4. The amino acid sequence may contain at least 70% sequence identity, and the said % of sequence identity with respect to SEQ ID NO: 4 preserves the amino acid modifications at amino acid positions 45, 52, and 75.

[0069] In another embodiment, the IL-15 variant may include an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 4; at least 80% sequence identity to SEQ ID NO: 4; at least 85% sequence identity to SEQ ID NO: 4; at least 90% sequence identity to SEQ ID NO: 4; at least 91% sequence identity to SEQ ID NO: 4; at least 92% sequence identity to SEQ ID NO: 4; at least 93% sequence identity to SEQ ID NO: 4; at least 94% sequence identity to SEQ ID NO: 4; at least 95% sequence identity to SEQ ID NO: 4; at least 96% sequence identity to SEQ ID NO: 4; at least 97% sequence identity to SEQ ID NO: 4; at least 98% sequence identity to SEQ ID NO: 4; at least 99% sequence identity to SEQ ID NO: 4; or at least 100% sequence identity to SEQ ID NO: 4, wherein the percentage of sequence identity to SEQ ID NO: 4 preserves amino acid modifications at amino acid positions 45, 52 and 75.

[0070] In another preferred embodiment, an IL-15 variant according to any aspect of the present 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 a more preferred embodiment, an IL-15 variant according to any aspect of the present invention may include (i) a leucine-to-serine substitution at amino acid position 45, (ii) a leucine-to-arginine substitution at amino acid position 52, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may include the amino acid sequence described in SEQ ID NO: 5, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 5, wherein the % sequence identity with respect to SEQ ID NO: 5 preserves the amino acid modifications at amino acid positions 45, 52, and 75.

[0071] In another embodiment, the IL-15 variant may include an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 5; at least 80% sequence identity to SEQ ID NO: 5; at least 85% sequence identity to SEQ ID NO: 5; at least 90% sequence identity to SEQ ID NO: 5; at least 91% sequence identity to SEQ ID NO: 5; at least 92% sequence identity to SEQ ID NO: 5; at least 93% sequence identity to SEQ ID NO: 5; at least 94% sequence identity to SEQ ID NO: 5; at least 95% sequence identity to SEQ ID NO: 5; at least 96% sequence identity to SEQ ID NO: 5; at least 97% sequence identity to SEQ ID NO: 5; at least 98% sequence identity to SEQ ID NO: 5; at least 99% sequence identity to SEQ ID NO: 5; or at least 100% sequence identity to SEQ ID NO: 5, wherein the percentage of sequence identity to SEQ ID NO: 5 preserves amino acid modifications at amino acid positions 45, 52 and 75.

[0072] In another preferred embodiment, an IL-15 variant according to any aspect of the present 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 a more preferred embodiment, an IL-15 variant according to any aspect of the present invention may include (i) a leucine-to-threonine substitution at amino acid position 45, (ii) a valine-to-alanine substitution at amino acid position 49, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may include the amino acid sequence described in SEQ ID NO: 6, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6, wherein the % sequence identity to SEQ ID NO: 6 includes amino acid modifications at amino acid positions 45, 49, and 75. To hold.

[0073] In another embodiment, the IL-15 variant may include an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 6; at least 80% sequence identity to SEQ ID NO: 6; at least 85% sequence identity to SEQ ID NO: 6; at least 90% sequence identity to SEQ ID NO: 6; at least 91% sequence identity to SEQ ID NO: 6; at least 92% sequence identity to SEQ ID NO: 6; at least 93% sequence identity to SEQ ID NO: 6; at least 94% sequence identity to SEQ ID NO: 6; at least 95% sequence identity to SEQ ID NO: 6; at least 96% sequence identity to SEQ ID NO: 6; at least 97% sequence identity to SEQ ID NO: 6; at least 98% sequence identity to SEQ ID NO: 6; at least 99% sequence identity to SEQ ID NO: 6; or at least 100% sequence identity to SEQ ID NO: 6, wherein the percentage of sequence identity to SEQ ID NO: 6 preserves amino acid modifications at amino acid positions 45, 49 and 75.

[0074] In another preferred embodiment, an IL-15 variant according to any aspect of the present 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 a more preferred embodiment, an IL-15 variant according to any aspect of the present invention may include (i) a valine-to-alanine substitution at amino acid position 49, (ii) a leucine-to-arginine substitution at amino acid position 52, and (iii) a serine-to-proline substitution at amino acid position 75. In this embodiment, the IL-15 variant may include the amino acid sequence described in SEQ ID NO: 7, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 7, wherein the % sequence identity with respect to SEQ ID NO: 7 preserves the amino acid modifications at amino acid positions 49, 52, and 75.

[0075] In another embodiment, the IL-15 variant may include an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 7; at least 80% sequence identity to SEQ ID NO: 7; at least 85% sequence identity to SEQ ID NO: 7; at least 90% sequence identity to SEQ ID NO: 7; at least 91% sequence identity to SEQ ID NO: 7; at least 92% sequence identity to SEQ ID NO: 7; at least 93% sequence identity to SEQ ID NO: 7; at least 94% sequence identity to SEQ ID NO: 7; at least 95% sequence identity to SEQ ID NO: 7; at least 96% sequence identity to SEQ ID NO: 7; at least 97% sequence identity to SEQ ID NO: 7; at least 98% sequence identity to SEQ ID NO: 7; at least 99% sequence identity to SEQ ID NO: 7; or at least 100% sequence identity to SEQ ID NO: 7, wherein the percentage of sequence identity to SEQ ID NO: 7 preserves amino acid modifications at amino acid positions 49, 52 and 75.

[0076] In addition to any amino acid modification at any of amino acid positions 45, 49, and / or 52 and 75, IL-15 variants having one or more amino acid modifications at amino acid positions 48, 60, and / or 71 are also provided. In one embodiment, an IL-15 variant is provided that includes a hydrophilic-to-hydrophobic substitution at amino acid position 48, preferably a glutamine-to-isoleucine substitution or a glutamine-to-valine substitution at amino acid position 48. In another embodiment, the IL-15 variant may include a hydrophilic-to-hydrophobic substitution at amino acid position 60, preferably a histidine-to-valine substitution at amino acid position 60. In yet another embodiment, the IL-15 variant may include a hydrophilic-to-hydrophobic substitution at amino acid position 70, preferably an asparagine-to-isoleucine substitution at amino acid position 70. Any of these modifications may exist in combination with any of the other amino acid modifications described herein, including amino acid modifications at amino acid positions 45, 49, and / or 52. It is understood that, in particular, IL-15 variants may include amino acid modifications at amino acid positions 75 and 45; amino acid positions 75, 45 and 48; amino acid positions 75, 45 and 60; amino acid positions 75, 45 and 71; amino acid positions 75, 48 and 49; amino acid positions 75, 48 and 52; or any combination thereof.

[0077] As detailed herein, an IL-15 variant is provided comprising one or more amino acid modifications, wherein the amino acid modifications are located in a surface region adjacent to the receptor (e.g., IL-15Rα) interaction surface of the IL-15 variant (i.e., at one or more amino acid positions 45 and / or 49 and / or 52, compared to SEQ ID NO: 1). The receptor interaction surface of the IL-15 variant may interact with or bind to the IL-15 receptor. Three IL-15 receptors exist: IL-15Rβ, GammaC(γc), and IL-15Rα. IL-15Rβ may also be known as IL-2Rβ. These receptors form a complex with IL-15 via electrostatic interactions (Figure 7). Amino acids in the parent IL-15 sequence (SEQ ID NO: 16) that can be modified to enhance the stability of the complex are shown in bold and underlined. Sequence ID 16 [ka]

[0078] The inventors of the present invention have surprisingly found that the residues shown in the above sequence are located within the exposed region of IL-15. Sequence analysis revealed that lysine-41, leucine-45, glutamine-48, and leucine-52 give a tendency to aggregate and are therefore good candidates for amino acid modifications that enhance solubility and preserve function.

[0079] Accordingly, according to a second aspect of the present invention, an interleukin-15 (IL-15) variant is provided, wherein wild-type IL-15 comprises the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 1, and, compared to SEQ ID NO: 1, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, and the one or more amino acid modifications comprise any of i) a hydrophobic to hydrophilic substitution at amino acid position 45; ii) a hydrophobic to hydrophobic substitution at amino acid position 49; iii) a hydrophobic to hydrophilic substitution at amino acid position 52; and / or iv) a serine to proline substitution at amino acid position 75; preferably, the receptor interaction surface is the interleukin-15 receptor alpha (IL-15Rα) interaction surface.

[0080] In a particularly preferred embodiment, the substitution from hydrophobic to hydrophilic at amino acid position 45 is a substitution from leucine to serine or from leucine to threonine.

[0081] In another particularly preferred embodiment, the hydrophobic-to-hydrophilic substitution at amino acid position 52 is a leucine-to-lysine substitution or a leucine-to-arginine substitution.

[0082] In another particularly preferred embodiment, the hydrophobic-to-hydrophobic substitution at amino acid position 49 is a substitution from valine to alanine or from valine to serine.

[0083] The IL-15Rα receptor contains a protein-binding motif known in the art as the "sushi domain." According to the present invention, any of the present inventions detailed herein IL-15 variants in this embodiment may be fused to an IL-15R molecule, in particular an IL-15Rα molecule and / or an IL-15Rα sushi domain. The IL-15Rα molecule is a transmembrane protein having a hydrophobic transmembrane segment and can often be flexible and unstable. This makes the expression of the entire IL-15Rα molecule difficult. Therefore, in the present invention, it is preferred to fused the IL-15 variant to the IL-15Rα sushi domain, as opposed to the entire IL-15Rα molecule. The IL-15Rα sushi domain is soluble and is also a functional part of the receptor, which means that only the sushi domain is required for functionality and is also easier to express alone. As used herein, the term “fusion” refers to any linkage of two or more proteins. Proteins may be fused via covalent attachment, for example via a linker, and may be produced via the 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 of the IL-15 variants described herein (IL-15Rα sushi:linker:IL-15 variant), i.e., in some embodiments, the fusion is made to the amino(N) terminus of the IL-15 variant.

[0084] The IL-15 variant may (or may not) be fused to the sushi domain of the IL-15Rα molecule. Fusion of the sushi domain to the IL-15 variant generates a superagonist (SAg) molecule. Any other suitable protein or polypeptide may also be fused to the IL-15 variant and / or the IL-15 / IL-15Rα sushi domain complex.

[0085] Any IL-15 variant described herein in any aspect of the present invention may be fused to an IL-15Rα molecule or its sushi domain. Such fusions are referred herein as “superagonists,” “SAg,” “SAg variants,” or “IL-15 superagonists.” As used herein, the term “superagonist” refers to a type of cytokine that has the ability to induce a greater maximal immune response than the agonist (i.e., IL-15 variant) itself. Superagonists may be less soluble than their IL-15 variant counterparts.

[0086] Any of the IL-15 variants detailed herein may fuse to an IL-15Rα molecule or the sushi domain of an IL-15Rα molecule to form their superagonist counterparts. For example, in one embodiment, an IL-15 variant is provided which comprises the amino acid sequence described in any one of SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7, or an amino acid sequence having at least 70% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7, and which fuses to an IL-15Rα molecule or the sushi domain of an IL-15Rα molecule via a GS linker, for example, the GS linker described in SEQ ID NO: 17, to form a superagonist.In another embodiment, the amino acid sequence described in any one of SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7, or at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 80% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 91% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; at least 94% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7; 100% sequence identity for Preferably, the IL-15 variant is fused to the IL-15Rα sushi domain.

[0087] The term “relevant amino acid position” is intended to encompass the amino acid modifications described for each SEQ ID NO: 2 in this specification. For example, the amino acid modifications at the relevant amino acid position for SEQ ID NO: 2 are the amino acid modifications at amino acid positions 45 and 75.

[0088] In a preferred embodiment, the IL-15 superagonist may include the amino acid sequence described in any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, or an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13.

[0089] Sequence ID 8 corresponds to an amino acid sequence containing an IL-15 variant including the IL-15Rα sushi domain and the amino acid sequence described in Sequence ID 2 (i.e., a SAg01 superagonist containing GBL01). Sequence ID 9 corresponds to an amino acid sequence containing an IL-15 variant including the IL-15Rα sushi domain and the amino acid sequence described in Sequence ID 3 (i.e., a SAg15 superagonist containing GBL15). Sequence ID 10 corresponds to an amino acid sequence containing an IL-15 variant including the IL-15Rα sushi domain and the amino acid sequence described in Sequence ID 4 (i.e., a SAg17 superagonist containing GBL17). Sequence ID 11 corresponds to an amino acid sequence containing an IL-15 variant including the IL-15Rα sushi domain and the amino acid sequence described in Sequence ID 5 (i.e., a SAg18 superagonist containing GBL18). Sequence ID 12 corresponds to an amino acid sequence containing an IL-15 variant including the IL-15Rα sushi domain and the amino acid sequence described in Sequence ID 6 (i.e., a SAg25 superagonist containing GBL25). Sequence ID 13 corresponds to an amino acid sequence containing an IL-15 variant including the IL-15Rα sushi domain and the amino acid sequence described in Sequence ID 7 (i.e., a SAg50 superagonist containing GBL50). These superagonist sequences utilize the GS linker described in Sequence ID 17, but it will be understood by those skilled in the art that any suitable linker may be used to link the IL-15 variant to the IL-15Rα molecule or the sushi domain of the IL-15Rα molecule.

[0090] Therefore, in another embodiment, at least 75% sequence identity to any one of the amino acid sequences described in SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, or any combination thereof, or to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 80% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 85% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 90% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; Sequence identity of at least 91% for sequence numbers 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 92% for sequence numbers 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 93% for sequence numbers 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 94% for sequence numbers 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 95% for sequence numbers 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 96% for sequence numbers 8, 9, 10, 11, 12, and / or 13; sequence numbers 8, 9, 10, 11, 12, and / or 13 An IL-15 variant is provided, comprising an amino acid sequence having or containing at least 97% sequence identity to and / or 13; at least 98% sequence identity to sequence numbers 8, 9, 10, 11, 12, and / or 13; at least 99% sequence identity to sequence numbers 8, 9, 10, 11, 12, and / or 13; and / or at least 100% sequence identity to sequence numbers 8, 9, 10, 11, 12, and / or 13 (the aforementioned percentage of sequence identity to sequence numbers 8, 9, 10, 11, 12, and / or 13 retains the amino acids at the relevant amino acid positions).

[0091] However, it is preferable that the IL-15 superagonist may include the amino acid sequence described in any one of SEQ ID NOs: 9, 11, 12, and / or 13, or an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 9, 11, 12, and / or 13 (the aforementioned % sequence identity with respect to SEQ ID NOs: 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 superagonists are more soluble than superagonists of wild-type IL-15. In another embodiment, the IL-15 superagonist has at least 75% sequence identity to SEQ ID NOs. 9, 11, 12 and / or 13; at least 80% sequence identity to SEQ ID NOs. 9, 11, 12 and / or 13; at least 85% sequence identity to SEQ ID NOs. 9, 11, 12 and / or 13; at least 90% sequence identity to SEQ ID NOs. 9, 11, 12 and / or 13; at least 91% sequence identity to SEQ ID NOs. 9, 11, 12 and / or 13; at least 92% sequence identity to SEQ ID NOs. 9, 11, 12 and / or 13; at least 93% sequence identity to SEQ ID NOs. 9, 11, 12 and / or 13; The amino acid sequences may include at least 94% sequence identity to 13; at least 95% sequence identity to SEQ ID NOs: 9, 11, 12 and / or 13; at least 96% sequence identity to SEQ ID NOs: 9, 11, 12 and / or 13; at least 97% sequence identity to SEQ ID NOs: 9, 11, 12 and / or 13; at least 98% sequence identity to SEQ ID NOs: 9, 11, 12 and / or 13; at least 99% sequence identity to SEQ ID NOs: 9, 11, 12 and / or 13; and / or at least 100% sequence identity to SEQ ID NOs: 9, 11, 12 and / or 13, wherein the percentage of sequence identity preserves the amino acid modification at the relevant amino acid position.

[0092] Such fusions may be achieved by directly linking two or more proteins (i.e., direct fusion proteins) and / or by indirectly linking two or more proteins via any preferred form of linkage. The linkage may be covalent. However, direct linkage without a linker may lead to misfolding, low solubility, or impaired bioactivity 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 polypeptide linker. Flexible linkers, such as those using serine and glycine residues, allow the linked proteins to move relative to each other. The length of the linker may be increased to prevent steric hindrance between proteins. Short linkers may be about 4–6 amino acids long, intermediate-length linkers may be about 6–14 amino acids long, and long linkers may be about 14–21 or more amino acids long. The linker may be a naturally occurring linker or a synthetic linker. The linker may employ a secondary protein conformation, e.g., an alpha-helix and / or a beta-strand / coil / bend. In a more preferred embodiment, the linker is a glycine-serine linker (GS linker). The GS linker is a stretch of glycine and serine residues, e.g., (Ser-Gly-Gly-Gly-Gly) n , or (Ser-Gly-Gly-Gly) n It mainly contains the following. Any proportion of glycine and serine residues may be used, as well as other amino acids, such as leucine or glutamine. The copy number "n" may be adjusted to change the length of the GS linker to achieve proper separation of the linked proteins, and proper separation of the linked proteins may affect the functionality and interactions of the linked proteins. The copy number "n" typically ranges from 1 to 6. However, a preferred GS linker of the present invention may include the following sequence: Sequence ID 17 SGGGSGGGGSGGGGSGGGGSGGGSLQA

[0093] In a preferred embodiment, the linker used to fuse the IL-15Rα molecule or the sushi domain of the IL-15Rα molecule with an IL-15 variant includes the amino acid sequence described in SEQ ID NO: 17, or 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 to SEQ ID NO: 17.

[0094] In the present invention, the use of a flexible GS linker is preferred, but other flexible linkers, such as those containing additional amino acids, e.g., threonine, alanine, lysine, and / or glutamic acid, may also be used. Furthermore, a flexible linker consisting purely of glycine or serine, such as a (Gly)8 linker, may also be suitable.

[0095] In addition to the ligation of the IL-15Rα sushi domain, any other suitable protein or polypeptide may be ligated to the IL-15 variant and / or IL-15 superagonist. Such examples may include, but are not limited to, signal peptides, antibody fragments, antibodies, bispecific or polyspecific antibodies, tissue-targeting sequences (e.g., 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-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21)) or chemokines (e.g., CXCL9, CXCL10, CXCL11, CCL5, CCL2, CX3CL1).

[0096] In particular, therapeutic bacteria must be able to produce and secrete immunostimulatory cytokines in situ in order to elicit a strong immune response, for example, in the tumor microenvironment. Therefore, in preferred embodiments, IL-15 variants and / or IL-15 superagonists are fused to a signal peptide. As used herein, the term “signal peptide” refers to any signal sequence, localization sequence, or other polypeptide sequence that directs an IL-15 variant and / or IL-15 superagonist to a specific intracellular or extracellular location. The addition of a small (15-25 amino acid) sequence known as a signal peptide to a protein of interest may be used to direct it to a desired location.

[0097] Human IL-15 is produced as two isoforms through alternative splicing of mRNA transcripts (which occurs only in mammalian cells, not bacterial cells). One isoform contains a 21-amino acid short-chain signal peptide (SSP) (SEQ ID NO: 15) and remains in the cytoplasm. The other isoform contains a 48-amino acid long-chain signal peptide (LSP) (SEQ ID NO: 14) that enables secretion into the extracellular environment.

[0098] For expression within bacteria, a bacterial signal peptide is required to secrete the IL-15 variant out of the bacterial cytosol and, for example, into the periplasm. In a preferred embodiment, the signal peptide directs the IL-15 variant into the bacterial periplasm in a rapid and robust manner. Therefore, preferred in the present invention is that the signal peptide is a (bacterial) periplasmic signal peptide. A periplasmic signal peptide is a polypeptide sequence that enables the protein to which the signal peptide is attached to be transported, directed, localized, or targeted into the periplasm of a bacterial cell. There is no universal signal peptide that can efficiently translocate any protein, and therefore, the inventors of the present invention also screened for the optimal signal peptide for each IL-15 variant.

[0099] As those skilled in the art will understand, the periplasm is a concentrated matrix in the space defined between the inner plasma membrane and the outer bacterial membrane of Gram-negative bacteria. The periplasm is an oxidative compartment and ideal for the formation of disulfide bonds, which are ubiquitous in human cytokines. Chaperones that assist and promote disulfide bond formation are also present in the periplasm. In contrast, the cytosol is a reducing environment that does not work favorably for disulfide bond formation. Therefore, directing cytokines (IL-15 variants and their superagonists) to the periplasm via signal peptides proves beneficial for the production and correct folding of these proteins, as well as for their eventual secretion in situ.

[0100] In bacteria, there are major pathways for exporting proteins across the bacterial plasma membrane, namely the secretory (SEC) pathway or the twin arginine translocation (TAT) pathway. The SEC pathway allows unfolded proteins to pass through the inner membrane (IM), while the TAT pathway allows folded proteins to pass through the cytoplasm. Therefore, the signal peptide used to transport either the IL-15 variant or its superagonist according to the present invention may be an SEC or TAT signal peptide. However, the SEC pathway allows the protein to fold in a suitable environment, and therefore, in a preferred embodiment, the signal peptide is an SEC signal peptide. The SEC pathway is shared by both Gram-negative and Gram-positive bacteria. In Gram-negative bacteria, as a result of the SEC pathway, the protein is secreted into the periplasm. In Gram-positive bacteria, as a result of the SEC pathway, the protein is secreted directly into the external environment. In some embodiments, the signal peptide used to direct proteins to the periplasm is N-terminally fused to an IL-15 variant or IL-15 superagonist. However, the IL-15 variant or IL-15 superagonist may be transported extracellularly via other signal peptides, such as type I signal peptides. Therefore, in other embodiments, the signal peptide is C-terminally fused to an IL-15 variant or IL-15 superagonist.

[0101] From the periplasm, IL-15 variants or superagonists may then be promoted into the supernatant or other extracellular environment. In the fusion of a periplasmic signal peptide to either of the IL-15 variants and / or IL-15 superagonists described herein, when expressed in bacterial cells, IL-15 variants and / or IL-1 5. Superagonists can be efficiently transported into the periplasm of bacterial cells to achieve proper folding and subsequent secretion of IL-15 variants and / or IL-15 superagonists from bacterial cells. Such secretion allows for the delivery of these molecules to sites of interest where they are needed, including disease sites in the subject that may benefit from such enhancement of the immune response induced by IL-15 variants and / or IL-15 superagonists. For example, upon infection and invasion of host (e.g., eukaryotic) cells by Gram-negative bacteria expressing the IL-15 variant or superagonist of the present invention, the IL-15 variant or superagonist may be translocated into the cytoplasm of the host cell, where it may, for example, achieve therapeutic immunostimulatory effects.

[0102] In one embodiment, the signal peptide is fused to an IL-15 variant and / or superagonist via a spacer sequence. As used herein, the terms “spacer” and “spacer sequence” may be used interchangeably and refer to any amino acid sequence located between the signal peptide and the secreted cargo (IL-15 variant or its superagonist) (i.e., signal peptide:spacer:linker:IL-15 variant, and / or signal peptide:spacer:IL-15 variant). Preferably, the spacer is a very short amino acid sequence, typically 1 to 5 amino acids long (e.g., 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 4 amino acids, or 5 amino acids long). Any suitable amino acid sequence may be used as the spacer. More preferably, the spacer is located immediately after 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 improve the transport rate of unfolded proteins into the periplasm and to improve the cleavage of signal peptides so that mature proteins are released.

[0103] According to a third aspect of the present invention, a nucleic acid molecule encoding an interleukin-15 (IL-15) variant according to a second aspect of the present invention is provided. The present invention also provides a nucleic acid molecule encoding an IL-15 variant contained in a bacterium according to a first aspect of the present invention. As used herein, the term “nucleic acid molecule” means a nucleic acid molecule comprising DNA and / or RNA, which is recombinantly or synthetically produced, comprising a series of identified nucleic acid elements that enable the expression of a specific nucleic acid sequence in a host cell. In particular, the nucleic acid molecule may be expressed in bacterial cells, preferably Gram-negative bacterial cells. A nucleic acid molecule may include a sequence encoding an amino acid sequence that has at least 70% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, or any combination thereof (preferably any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13), or a sequence encoding an amino acid sequence that has at least 70% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 preserves amino acid modifications at the relevant amino acid positions. In some embodiments, IL-15 variant expression can be controlled via spatiotemporal activation and intensity modulation of promoters, i.e., by using promoters that are activated at specific locations (e.g., intracellular or extracellular), at specific times, or in response to specific chemicals.

[0104] In one embodiment, the nucleic acid molecule is a DNA molecule. Where used herein, the terms “DNA” and “deoxyribonucleic acid” are given their common meanings 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 general The 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 meanings 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 and refer to single-stranded RNA molecules involved in protein synthesis, which are transcribed from DNA and translated into amino acid sequences. Bacterial mRNA molecules are transcribed from uncompartmented DNA and translated in the cytosol in conjunction with transcription. 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 transported from the nucleus into the cytoplasm of the eukaryotic cell, where translation of the mRNA molecules into proteins takes place.

[0105] The nucleic acid may be expressed in a host cell, such as a bacterial cell, that has been manipulated or modified to express the nucleic acid. Alternatively, the nucleic acid may be expressed in a eukaryotic cell. In this embodiment, the nucleic acid (e.g., RNA) may be contained within a bacterial cell, which can then be delivered to a eukaryotic cell. The RNA molecule has a nucleotide-coding structure that, in the case of mRNA, allows transcription within the bacterium itself, and then, upon entry and replication of the bacterium in the target eukaryotic cell, allows for the translation and expression of the protein within the eukaryotic cell.

[0106] The nucleic acid molecule may contain a sequence encoding any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, or any combination thereof. In a preferred embodiment, the nucleic acid molecule encodes any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 9, 11, 12, 13, and / or 16, or any combination thereof. In a more preferred embodiment, the nucleic acid molecule encodes any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 9, 11, 12, and / or 13, or any combination thereof. In another embodiment, the nucleic acid molecule encodes an IL-15 variant comprising an amino acid sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, or a sequence having at least 70% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 preserves an amino acid modification at the relevant amino acid position.

[0107] In another embodiment, at least 75% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; at least 80% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; at least 85% sequence identity to SEQ ID NOs: 1, 2 Sequence identity of at least 90% for sequence numbers 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; sequence identity of at least 91% for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; sequence identity of at least 92% for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 At least 93% sequence identity for sequence numbers 1, 12, 13, and / or 16; at least 94% sequence identity for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 95% sequence identity for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 At least 96% sequence identity for; at least 97% sequence identity for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 98% sequence identity for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 99% sequence identity for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16;A nucleic acid molecule may be provided comprising a sequence encoding an amino acid sequence having at least 100% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 preserves an amino acid modification at the relevant amino acid position.

[0108] In the most preferred embodiment, the nucleic acid molecule has at least 70% identity to the amino acid sequence described in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; at least 75% sequence identity to 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, 1 Sequence identity of at least 80% for sequence numbers 1, 12, and / or 13; sequence identity of at least 85% for sequence numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 90% for sequence numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 91% for sequence numbers 2, 3, 4, 5, 6, 7, Sequence identity of at least 92% for sequences 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 93% for sequences 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 94% for sequences 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; sequence identity of at least 95% for sequences 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; sequence 2, 3, At least 96% sequence identity for sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; at least 97% sequence identity for sequence numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; at least 98% sequence identity for sequence numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13; at least 99% sequence identity for sequence numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13;Furthermore, it may include a sequence encoding an amino acid sequence having at least 100% sequence identity to sequence numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13, wherein the % sequence identity preserves the amino acid modification at the relevant amino acid position.

[0109] In a fourth aspect of the present invention, a bacterium is provided comprising an IL-15 variant according to a second aspect of the present invention, or a nucleic acid molecule according to a third aspect of the present invention. As will be understood by those skilled in the art, interleukins play diverse 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 found 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, superagonist counterparts of these IL-15 variants have the potential to induce maximum and persistent immune responses against a number of diseases in a subject. This is possible. Accordingly, according to a fifth aspect of the present invention, a bacterium according to a first or fourth aspect of the present invention, or an IL-15 variant according to a second aspect of the present invention, or a nucleic acid molecule according to a third aspect of the present invention is provided for therapeutic use. Any combination of the IL-15 variants and / or IL-15 superagonists disclosed herein has therapeutic applications.

[0110] Due to the broad involvement of interleukins in several different signaling pathways (including both pro-inflammatory and anti-inflammatory signaling), the present invention is understood to relate to the treatment, prevention, reduction, suppression, prevention of recurrence, or control of several diseases. In preferred embodiments, the disease is a human disease. The disease may include, but is 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 preferred embodiments, the disease may be a neoplastic disease. Accordingly, the present invention provides bacteria according to the first or fourth aspect of the present invention, or IL-15 variants according to the second aspect of the present invention, or nucleic acid molecules according to the third aspect of the present invention, for use in the treatment, prevention, reduction, suppression, prevention of recurrence, or control of neoplastic diseases in a subject.

[0111] The terms “tumor,” “cancer,” “malignant tumor,” and “neoplasm” are used interchangeably and refer to cells or cell populations whose growth, proliferation, or survival is greater than that of normal corresponding cells, e.g., impaired cell proliferation or differentiation. Typically, growth is uncontrolled. The term “malignant tumor” refers to invasion into nearby tissue. The term “metastasis” refers to the spread or dissemination of a tumor, cancer, or neoplasm to other sites, locations, or regions within a subject, where such sites, locations, or regions are distinct from the primary tumor or cancer.

[0112] In one embodiment, the neoplasm may be a solid tumor and / or hematological malignancy. Neoplasms, tumors, and cancers include benign, malignant, metastatic, and non-metastatic types, and include any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.) of a neoplasm, tumor, or cancer, or neoplasm, tumor, cancer, or metastasis that is progressing, worsening, stable, or in remission.

[0113] Cancers that can be treated according to the present invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, testes, tongue, or uterus. Additionally, cancer may be, but is not limited to, the following histological types: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant cell and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; piloerection carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; mixed hepatocellular carcinomas and cholangiocarcinomas; trabecular adenocarcinomas; adenoid cystic carcinomas; adenocarcinomas in adenomatous polyps; familial adenomatous polyposis; solid tumors; malignant carcinoid tumors; bronchioloalveolar adenocarcinomas; papillary adenocarcinomas; chromophobic carcinomas ;eosinophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; Serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; 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; Cellular tumors; malignant androblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; angioglobulosarcoma; malignant melanoma; achromatic melanoma; superficial melanoma; malignant melanoma within a giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonic rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor; Müller mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant Mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated germ cell tumor; embryonic carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant periangiocarcinoma; lymphangiosarcoma; osteosarcoma; paraosteal 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 glandoma; chordoma; malignant glioma; ependymoma; astrocytoma;Plasma astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; undifferentiated neuroectoderm; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurotumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's disease; paragranuloma; small lymphocytic lymphoma; diffuse large cell lymphoma; follicle Malignant lymphoma; mycosis fungoides; other certain non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; and hairy cell leukemia.

[0114] Preferably, solid tumors and / or hematological malignancies may be cancers 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, carcinoma, head and neck cancer, endometrial cancer, skin cancer, or sarcoma. More preferably, neoplasms may be associated with cancers 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.

[0115] In some embodiments, the neoplasm is a malignant neoplasm.

[0116] In some embodiments, the neoplasm is not a benign neoplasm.

[0117] In one preferred embodiment, a malignant tumor may be associated with a cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular carcinoma, lymphoma, leukemia, stomach cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, or sarcoma.

[0118] In another preferred embodiment of the present invention, the IL-15 variant or IL-15 superagonist is administered intratumorally, intravesically, intraperitoneally, or orally. In the most preferred embodiment, the IL-15 variant or IL-15 superagonist is administered intratumorally. However, it is also conceivable that other methods of administration may be used in some cases. Accordingly, in certain cases, the IL-15 variant or IL-15 superagonist of the present invention may be administered by injection, drop, infusion, continuous infusion, intradermally, intraarterially, intrafocally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intrapericardially, intraumbilically, intraocularly, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, by inhalation (e.g., aerosol inhalation), via catheter, via lavage, or by any combination of the foregoing, as is known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990). However, in the present invention, administration is preferred to be via topical drop, intraperitoneal, intrapleural, intravesical, peritumoral injection, intratumoral injection, or oral administration.

[0119] As used herein, the term “dropping” means that the composition is dropped into the relevant anatomical area. This refers to being introduced into and remaining there for a specific time until discharge, excretion, or removal. As used herein, the term “intraperitoneal” refers to the injection of a composition into the peritoneum of the subject. As used herein, the term “intrapleural” refers to the injection of a composition into the pleura or pleural cavity of the subject. As used herein, the term “intravesical” refers to the injection or dripping of a composition into the bladder of the subject via a catheter. As used herein, the term “peritumoral injection” refers to the injection of a composition around the site of a neoplasm, when the disease in question is a neoplasm. As used herein, the term “intratumoral injection” refers to the direct injection of an IL-15 variant or IL-15 superagonist into a neoplasm, when the disease in question is a neoplasm.

[0120] It is understood that the specific method of administration of the composition may depend on the disease being treated, for example, both the location and type of the disease. For example, if it is desired that a body cavity with a large surface area of ​​the target, such as the pleural cavity, be treated, administration by drip may be most appropriate. Alternatively, if the disease is located within the peritoneal cavity, administration by intraperitoneal injection may be most appropriate. Furthermore, it should be noted that if the disease being prevented and / or treated is, for example, a hematological malignancy, intratumoral injection may not be the chosen method of administration.

[0121] Apart from the direct administration of any of the IL-15 variants and / or their superagonists disclosed herein, bacteria containing any of the IL-15 variants and / or IL-15 variant superagonists disclosed herein, or any combination thereof, or nucleic acid molecules encoding the variants, are provided herein to achieve optimal and efficient expression, folding, and secretion of the IL-15 variants and / or their superagonists. The IL-15 variants and / or their superagonists disclosed herein may be expressed and secreted in a suitable expression system, and may also be isolated for direct administration to subjects requiring them. However, in preferred embodiments, the IL-15 variants and / or their superagonists may be administered together with bacteria, in which case the bacteria containing the IL-15 variants and / or their superagonists or nucleic acid molecules encoding the IL-15 variants and / or their superagonists are administered and express and secrete the IL-15 variants and / or their superagonists. In some embodiments, the bacteria may be Gram-negative bacteria. In another embodiment, the bacteria may be Gram-positive bacteria.

[0122] Any suitable bacterium capable of containing an IL-15 variant and / or its superagonist, or a nucleic acid molecule encoding an IL-15 variant and / or its superagonist, may be used. However, in a particularly preferred embodiment, a Gram-negative bacterium containing the nucleic acid construct according to the present invention is provided.

[0123] For example, the amino acid sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, or at least 70% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; at least 75% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; or at least 80% sequence identity with respect to 16; at least 85% sequence identity with respect to sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; at least 90% sequence identity with respect to sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; at least 91% sequence identity with respect to sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 Sequence identity of at least 92% for sequences 1, 12, 13, and / or 16; sequence identity of at least 93% for sequences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; sequence identity of at least 94% for sequences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; sequence identity of at least 95% for sequences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16; sequence identity of sequences 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; sequence identity of at least 96% for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; sequence identity of at least 98% for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16; sequence identity of at least 99% for sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16;Furthermore / or a bacterium, such as a Gram-negative bacterium, may be provided, comprising a nucleic acid molecule containing a sequence encoding a sequence having at least 100% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the % sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 preserves an amino acid modification at the relevant amino acid position. Preferably, the bacterium may comprise a nucleic acid molecule containing a sequence encoding the amino acid sequence described in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13.

[0124] Any suitable bacterium capable of containing the IL-15 variant may be used in accordance with the present invention. However, in a particularly preferred embodiment, the bacterium is a Gram-negative bacterium.

[0125] Any Gram-negative bacteria having the ability to express and secrete heterologous interleukins disclosed herein. Examples of Gram-negative bacteria for use in the present invention include, but are not limited to, Salmonella, Escherichia coli, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia, and Yersinia. Preferably, the Gram-negative bacteria are live attenuated Gram-negative bacteria. As used herein, the term “attenuated” in the context of the present invention refers to the modification of a microorganism to reduce pathogenicity while maintaining viability, thereby making it harmless to the host. This method is commonly used in vaccine development because it can induce a highly specific immune response while maintaining an acceptable safety profile. The development of such vaccines may involve numerous methods, including, but not limited to, the passage of pathogens under in vitro conditions until pathogenicity is lost, chemical mutagenesis, and genetic engineering techniques. Such attenuated microorganisms are preferably living attenuated microorganisms, but non-living attenuated microorganisms are also disclosed. As used herein, the term “inactivating mutation” refers to an alteration of the native genetic code of a particular gene or a gene promoter associated with that gene, for example, by alteration of the nucleotide code or deletion of a nucleotide portion or addition of a non-coding nucleotide or non-native nucleotide, such that the particular gene is not properly transcribed or translated, or is expressed as an inactive protein, and as a result the native function of the gene is lost or reduced to an immeasurable degree. Thus, a gene mutation inactivates the function of the gene or the protein encoded by that gene.

[0126] In other embodiments, the bacteria may be Gram-positive bacteria. Any Gram-positive bacteria capable of expressing and secreting heterologous cytokines disclosed herein may be used. Examples of Gram-positive bacteria for use in the present invention include, but are not limited to, Bacillus, Clostridium, Corynebacterium, Listeria, and Gardnerella. Again, it is preferable that the bacteria are live and attenuated.

[0127] As used herein, the term “non-natural bacteria” refers to bacterial (prokaryotic) cells that have been genetically modified or “engineered” to be altered compared to naturally occurring cells. Such genetic modification may, for example, involve the incorporation of additional genetic information into the cell, the modification of existing genetic information, or, in fact, the deletion of existing genetic information. This may be achieved, for example, through the transfection of recombinant plasmids into the cell or through modifications made directly to the bacterial genome. Additionally, bacterial cells may be genetically modified, for example, through chemical mutagenesis to achieve attenuation, methods well known to those skilled in the art. Thus, the term “non-natural bacteria” may refer to both recombinantly modified and non-recombinantly modified bacterial strains. As used herein, the terms “recombinant,” “recombinant strain,” or “recombinant bacteria” are interchangeable and, in the context of this invention, refer to bacterial strains that have been genetically engineered so that their bacterial DNA is altered by the introduction of new DNA. Recombinant DNA methods commonly involve the introduction of new DNA via a vector, such as a plasmid. Such methods are well known to those skilled in the art. The use of recombinant bacterial strains may confer advantageous properties to the bacterial strain, such as prolonged activity, induction of a stronger immune response in the target, or introduction of desired molecules.

[0128] Preferably, the Gram-negative bacteria of the present invention may be enterobacteriaceae (e.g., Escherichia coli, Klebsiella, Proteus, and Enterobacter), Salmonella, Sigella, Yersinia, and Campylobacter jejuni, but are not limited to these.

[0129] Preferably, the Gram-negative bacteria are Salmonella species. Examples of Salmonella species for use in this invention are Salmonella enterica and Salmonella bongori. Salmonella enterica may be further divided into different serotypes or serovariates. Examples of the serotypes or serovariants for use in the present invention include Salmonella enterica Typhi, Salmonella enterica Paratyphi A, Salmonella enterica Paratyphi B, Salmonella enterica Paratyphi C, Salmonella enterica Typhimurium, and Salmonella enterica enteritidis. In a preferred embodiment, the live attenuated Gram-negative bacterium is any attenuated, non-pathogenic Salmonella enterica seromutant cif or cifimurium strain. In the most preferred embodiment, the live attenuated Gram-negative bacterium is Salmonella enterica cif. In a more preferred embodiment, the live attenuated bacterium is Salmonella enterica cif ZH9 (as M01ZH09). (See also) Derivatives or variants of Salmonella enterica cifi ZH9 strain, including genetically modified variants, are also intended to be included.

[0130] Such bacteria are engineered to express one of the IL-15 variants and / or its superagonists disclosed herein. Numerous methods and techniques for genetically engineering bacterial strains are well known to those skilled in the art. These techniques include those required to introduce heterologous genes into bacteria either through chromosomal integration or the introduction of stable autosomal self-replicating gene elements. Exemplary methods for genetically modifying (also referred to as “transformation” or “engineering”) bacterial cells include bacteriophage infection, transduction, coupling, lipofection, or electroporation. Other techniques, including CRISPR and CRISPR-related proteins (e.g., Cas9), CasCLOVER, TALEN, retron, homing endonucleases or meganucleases, zinc finger nucleases, and transposon-based methods, are also intended to be included. General discussions of methods for gene manipulation in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); and Protein Methods (Bollag et al., John Wiley & Sons 1996) (these are incorporated herein by reference).

[0131] For example, a bacterium, such as a Gram-negative bacterium, is provided, comprising an IL-15 variant comprising an amino acid sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and / or 16, more preferably any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16, or even more preferably any one of SEQ ID NOs: 2, 3, 4, 5, 6, and / or 7, or an amino acid sequence having at least 70% sequence identity to any of these sequences, wherein the % sequence identity preserves an amino acid modification at the relevant amino acid position.In another embodiment, the amino acid sequence described in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16, or at least 75% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 80% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 85% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 91% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 92% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 93% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; A bacterium, such as a Gram-negative bacterium, is provided, comprising an IL-15 variant containing an amino acid sequence having at least 94% sequence identity to and / or 16; at least 95% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 96% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 97% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 98% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; at least 99% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16; and / or at least 100% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 6, 7, and / or 16, wherein the percentage of sequence identity preserves the amino acid modification at the relevant amino acid position.

[0132] In another preferred embodiment, a bacterium, such as a Gram-negative bacterium, is provided, comprising an IL-15 superagonist comprising an amino acid sequence described in any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, or an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, wherein the % sequence identity preserves an amino acid modification at the relevant amino acid position. In a more preferred embodiment, the bacterium may comprise an IL-15 superagonist comprising an amino acid sequence described in any one of SEQ ID NOs: 9, 11, 12, and / or 13.

[0133] In another embodiment, the amino acid sequence described in any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, or at least 75% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 80% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 85% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; SEQ ID NOs: 8 , 9, 10, 11, 12, and / or 13, with at least 90% sequence identity; at least 91% sequence identity for sequence numbers 8, 9, 10, 11, 12, and / or 13; at least 92% sequence identity for sequence numbers 8, 9, 10, 11, 12, and / or 13; at least 93% sequence identity for sequence numbers 8, 9, 10, 11, 12, and / or 13; sequence numbers 8, 9, 10, 11, 12, and / or A bacterium, such as a Gram-negative bacterium, is provided, comprising an IL-15 superagonist containing an amino acid sequence having or containing at least 94% sequence identity to 13; at least 95% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 96% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 97% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 98% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; at least 99% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13; and / or at least 100% sequence identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, wherein the percentage of sequence identity preserves amino acid modifications at the relevant amino acid positions. As will be understood by those skilled in the art, bacteria containing nucleic acid molecules encoding any of the amino acid sequences described herein, such as Gram-negative bacteria, are also intended to be included.

[0134] Bacteria containing IL-15 variants and / or their IL-15 superagonists (SAg variants) may be referred to herein as compositions unless otherwise specified. IL-15 variants and / or their SAg variants promote enhancement of the immune response, and the bacteria themselves additionally induce 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, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by such cells or the liver, resulting in selective damage to cancer cells, destruction of such cells, or elimination from the human body. As used herein, the term “cellular components of the immune system” refers to immune cells, such as lymphocytes, such as T and B lymphocytes, gamma-delta T cells, and NK cells, that are capable of recognizing specific antigens, such as prions, viruses, bacteria, yeasts, fungi, parasites, tumor-associated or tumor-specific antigens, or other antigens associated with specific diseases, disorders, or conditions. Other immune cells we refer to include leukocytes, 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.

[0135] The immune response may include, in particular, 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 nonspecific immune response, activation, proliferation, and / or differentiation of immune cells, or any combination thereof. As used herein, the terms “systemic immune response” and “systemic immunity” are used interchangeably and refer to broad, nonspecific immune activation, as well as a broad immune response throughout the body of a subject directed to an inducer, in contrast to a local, spatially limited response. Such a response involves complex interactions between different cells of the immune response, e.g., lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver, and, in the context of the present invention, is considered to prime the subject’s immune system so that the subject becomes more responsive to the second composition when a second composition containing live attenuated bacteria is administered topically to a site of neoplasm. The "systemic immune response" may therefore be measured and quantified through the analysis of various different immune cell types, including but not limited to neutrophils, monocytes, dendritic cells, T cells (e.g., CD4+ and / or CD8+ T cells) and natural killer cells. Methods by which these effects can be measured are well known to those skilled in the art, for example, flow cytometry. The "systemic immune response" may also be measured and quantified by the presence of antibodies, including but not limited to IgG and IgA isotype antibodies. Methods by which these antibodies can be measured are well known to those skilled in the art, for example, ELISA. Thus, the first composition may act to prime (as interchangeable with "conditioning," "boost," "amplify," "enhance," "improve," "increase," or "promote") the immune response of the target after administration of the second composition.

[0136] Therefore, the combination of an IL-15 variant and / or IL-15 superagonist expressed and secreted by bacteria and bacteria is synergistic and provides an enhanced immune response in the subject. The IL-15 variant or IL-15 superagonist of the present invention, delivered by bacteria as defined herein, e.g., Gram-negative bacteria (i.e., the composition), can enable the subject's immune system to elicit a potent and persistent, effective immune response against the disease, and consequently produce enhanced therapeutic benefits. The immune response initiated by administration of the composition may be therapeutic in itself or less therapeutic, requiring subsequent administration of further doses of the composition to exert a therapeutic effect.

[0137] Bacteria, e.g., Gram-negative, engineered to express any of the IL-15 variants and / or superagonists according to any aspect of the present invention act as therapeutic strains. Therefore, the bacteria disclosed herein may be used in therapeutic applications. These therapeutic strains may be administered to subjects requiring such therapeutic application. The terms “treatment” or “therapy” refer to the administration of an activator to a subject with the aim of curing, healing, reducing, alleviating, modifying, correcting, relieving, improving, or influencing a condition (e.g., a disease), its symptoms, or preventing or delaying the onset of symptoms, complications, or biochemical signs of a disease, or otherwise stopping or inhibiting further progression of the disease, condition, or disorder in a statistically significant manner. 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 enhancement of the immune response). The methods are particularly suitable for treating patients (preferably human patients) with disorders that can be treated by increasing the immune response. In specific embodiments, IL-15 variants and / or their superagonists are particularly suitable for in vivo cancer treatment.

[0138] The amount of bacteria administered to the target is a therapeutically effective dose of IL-15 variant and / or IL This is sufficient to deliver the IL-15 superagonist to the target. Those skilled in the art will readily understand that the precise amount administered depends on a number of factors, such as the disease being treated, the IL-15 variant and / or its IL-15 superagonist expressed and secreted, and the medical history of the subject being treated.

[0139] The terms “therapeutic effective dose” and “effective dose” refer to an amount of agent sufficient to deliver a desired biological or therapeutic outcome. This outcome may be a reduction, remission, temporary relief, shrinkage, delay, and / or mitigation of one or more signs, symptoms, or causes of a disease, or any other desired alteration of the biological system. With respect to cancer, an effective dose may contain an amount sufficient to cause tumor shrinkage and / or a decrease in the rate of tumor growth (e.g., suppression of tumor growth), or to prevent or delay other undesirable cell proliferation. In some embodiments, an effective dose is sufficient to delay progression, prolong survival, or induce stabilization of cancer or tumor. In some embodiments, a therapeutic effective dose is sufficient to prevent or delay recurrence. A therapeutic effective dose may be administered in one or more doses. A therapeutically effective dose of a drug or combination may result in: (i) a reduction in the number of cancer cells; (ii) a reduction in tumor size; (iii) any degree of inhibition, delay, slowing, and preferably inhibition of cancer cell infiltration into peripheral organs; (iv) inhibition of tumor metastasis (i.e., any degree of slowing and preferably inhibition); (v) inhibition of tumor growth; (vi) prevention or delay of tumor development and / or recurrence; and / or (vii) any degree of relief of one or more symptoms associated with cancer.

[0140] For example, for tumor treatment, a "therapeutably effective dose" may induce tumor shrinkage by at least approximately 5%, e.g., at least approximately 10%, approximately 20%, or more than approximately 60%, compared to the baseline measurement. The baseline measurement may be derived from an untreated subject.

[0141] A therapeutically effective dose of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Those skilled in the art can determine such a dose based on factors such as the size of the subject, the severity of the subject's symptoms, and the selected specific composition or route of administration.

[0142] Bacteria, 10 5 ~10 12 It may be administered in doses of CFU (where CFU is a colony-forming unit). For example, a preferred dose is 10 5 ~10 6 CFU, 10 5 ~10 7 CFU, 10 5 ~10 8 CFU, 10 5 ~10 9 CFU, 10 5 ~10 10 CFU, 10 5 ~10 11 CFU, 10 6 ~10 7 CFU, 10 6 ~10 8 CFU, 10 6 ~10 9 CFU, 10 6 ~10 10 CFU, 10 6 ~10 11 CFU, 10 6 ~10 12 CFU, 10 7 ~10 8 CFU, 10 7 ~10 9 CFU, 10 7 ~10 10 CFU, 10 7 ~10 11 CFU, 10 7 ~10 12CFU, 10 8 ~10 9 CFU, 10 8 ~10 10 CFU, 10 8 ~10 11 CFU, 10 8 ~10 12 CFU, 10 9 ~10 10 CFU, 10 9 ~10 11 CFU, 10 9 ~10 12 CFU, 10 10 ~10 11 CFU, 10 10 ~10 12 CFU, or 10 11 ~10 12 It may also be CFU. The composition may be administered in single or multiple doses. It is understood that the specific number of doses administered depends on the IL-15 variant delivered, as well as the specific symptom being treated.

[0143] Bacteria may be administered in any form considered to be in the vicinity of the disease. As used herein, the term “nearby” is intended to refer to a section or area surrounding the disease, up to a certain distance. For example, in one embodiment, the term “nearby” may refer to a section / area extending, for example, up to 10 mm, up to 5 mm, or up to 2.5 mm from the boundary of the disease. The distance from the site of the disease to which the composition is administered determines the biological effect (e.g., The mobilization and activation of various immune cell types allows for efficacy in the local environment of the disease in question, while minimizing or eliminating efficacy in tissues located in unrelated compartments of the body. Additionally, the composition may be administered "in" or "on" the tissue or surrounding tissue. Thus, the term "local administration" refers to any situation in which bacteria can come into contact with the disease or immediately surrounding tissue in order to have the desired effect. As used herein, the terms "locally" and "administered locally" are interchangeable and, in the context of the present invention, refer to the manner in which an IL-15 variant or its superagonist and / or bacteria are administered to a subject. Thus, the resulting immune response produced in the subject is also said to be local to the site of the disease, i.e., not a broad systemic immune response.

[0144] In preferred embodiments of the present invention, the bacteria described herein are administered intratumorally, intrabladderally, intravenously, intraperitoneally, or orally. In most preferred embodiments, the bacteria are administered intratumorally or orally. However, it is also conceivable that other methods of administration may be used in some cases. Thus, in certain specific cases, the bacteria of the present invention may be administered by injection, dripping, infusion, continuous infusion, intradermally, intraarterially, intrafocally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intrapericardially, intraumbilically, intraocularly, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, by inhalation (e.g., aerosol inhalation), via catheter, via lavage, or by any combination thereof, as is known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).

[0145] It is understood that the specific methods of administering the bacteria described herein may depend on the disease being treated, for example, both the location and type of the disease. For example, if it is desirable to treat a body cavity with a large surface area, such as the pleural cavity, administration by drip may be most appropriate. Alternatively, if the disease is located within the peritoneal cavity, administration by intraperitoneal injection may be most appropriate. Furthermore, it should be noted that if the disease being prevented and / or treated is, for example, a hematological malignancy, intratumoral injection may not be the preferred method of administration.

[0146] Due to the broad involvement of interleukins in several different signaling pathways (including both pro-inflammatory and anti-inflammatory signaling), the present invention is understood to relate to the treatment, prevention, reduction, suppression, prevention of recurrence, or control of several diseases. In a preferred embodiment, the disease is a human disease. The disease may include, but is 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. Accordingly, the present invention provides a bacterium according to a first or fourth aspect of the present invention, or an IL-15 variant according to a second aspect of the present invention, or a nucleic acid molecule according to a third aspect of the present invention, for use in the treatment, prevention, reduction, suppression, prevention of recurrence, or control of neoplastic diseases in a subject. Furthermore, in a sixth aspect of the present invention, the use of a bacterium according to a first or fourth aspect of the present invention, or an IL-15 variant according to a second aspect of the present invention, or a nucleic acid molecule according to a third aspect of the present invention, in the production of a therapeutic pharmaceutical.

[0147] When the disease being treated is a neoplasm, the bacteria, as well as the IL-15 variant and / or its SAg variant, may be administered in combination with other treatments. The causes of neoplasms are multifaceted and diverse, and it is widely known that in many cases this leads to preventive and treatment strategies involving multiple treatments to achieve optimal outcomes. Therefore, the present invention relates to the use of bacteria expressing IL-15 variants and / or IL-15 superagonists in combination with other treatments. This may be accompanied by combination with known cancer treatments. Preferably, bacteria expressing IL-15 variants and / or IL-15 superagonists may be administered in combination with immunotherapy, radiotherapy, chemotherapy, or anticancer agents. The term “anticancer agent,” as used herein, refers to any agent that is effective in killing cancer cells, stopping the division of cancer cells, or helping to prevent the recurrence of cancer cells, but is not considered an immunotherapy, radiotherapy, or chemotherapy. In a preferred embodiment, bacteria expressing IL-15 variants and / or their SAg variants may 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 manipulated cell immunotherapy. Bacteria expressing IL-15 variants and / or IL-15 superagonists may be administered separately from other known cancer treatments, concurrently, or sequentially (before and / or after).

[0148] In one embodiment, the neoplasm may be a solid tumor and / or hematological malignancy. Neoplasms, tumors, and cancers include benign, malignant, metastatic, and non-metastatic types, and include any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.) of a neoplasm, tumor, or cancer, or neoplasm, tumor, cancer, or metastasis that is progressing, worsening, stable, or in remission.

[0149] Cancers that can be treated according to the present invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, testes, tongue, or uterus. Additionally, cancer may be, but is not limited to, the following histological types: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant cell and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; piloerection carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; mixed hepatocellular carcinomas and cholangiocarcinomas; trabecular adenocarcinomas; adenoid cystic carcinomas; adenocarcinomas in adenomatous polyps; familial adenomatous polyposis; solid tumors; malignant carcinoid tumors; bronchioloalveolar adenocarcinomas; papillary adenocarcinomas; chromophobic carcinomas ;eosinophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; mucinous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; 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 carcinoma; malignant granular carcinoma Membrane cell tumor; malignant androblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; angioglobulosarcoma; malignant melanoma; achromatic melanoma; superficial melanoma; malignant melanoma within a giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonic rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor; Müller's mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant Mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated germ cell tumor; embryonic carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant periangiocarcinoma; lymphangiosarcoma; osteosarcoma; paraosteal 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 glandoma; chordoma; malignant glioma; ependymoma; astrocytoma;Plasma astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; undifferentiated neuroectoderm; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurotumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's disease; paragranuloma; small lymphocytic lymphoma; diffuse large cell lymphoma; follicle Malignant lymphoma; mycosis fungoides; other certain non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; and hairy cell leukemia.

[0150] Preferably, solid tumors and / or hematological malignancies may be cancers selected from prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, brain cancer, hepatocellular (liver) cancer, lymphoma, leukemia, stomach cancer, cervical cancer, endometrial cancer, ovarian cancer, mesothelioma, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, or sarcoma. More preferably, neoplasms may be associated with cancers selected from bladder cancer, lung cancer, mesothelioma, hepatocellular (liver) cancer, melanoma, esophageal cancer, stomach cancer, ovarian cancer, colorectal cancer, head and neck cancer, or breast cancer.

[0151] In some embodiments, the neoplasm is a malignant neoplasm.

[0152] In some embodiments, the neoplasm is not a benign neoplasm.

[0153] In one preferred embodiment, a malignant tumor may be associated with a cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, hepatocellular carcinoma, lymphoma, leukemia, stomach cancer, cervical cancer, ovarian cancer, vaginal cancer, endometrial cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer, or sarcoma.

[0154] A seventh aspect of the present invention provides a method for treating, suppressing, preventing or controlling a neoplastic disease in a subject, comprising administering to the subject a bacterium according to the first or fourth aspect of the present invention, an IL-15 variant according to the second aspect of the present invention, or a nucleic acid molecule according to the third aspect of the present invention.

[0155] The present invention therefore also provides methods for treating, suppressing, preventing or controlling neoplastic diseases in a subject. Thus, the methods of the present invention may be used to reduce or suppress the metastasis of a primary tumor or cancer to other sites, or the formation or establishment of metastatic tumors or cancers at other sites distal to the primary tumor or cancer, thereby suppressing or reducing the recurrence or progression of the tumor or cancer. Therefore, the present invention provides a detectable or measurable improvement in a given subject condition, e.g., reduction or remission of one or more adverse (physical) symptoms or consequences associated with cell proliferation or hyperproliferative disorders, the presence of neoplasms, tumors or cancers, 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 a condition or pathology, or reduction in the onset, severity, duration, or frequency of adverse symptoms associated with or caused by cell proliferation or hyperproliferative disorders, e.g., neoplasms, tumors or cancers, or metastasis. It may also lead to improved survival. A satisfactory clinical endpoint of the treatment method according to the present invention is achieved, for example, by incremental or partial reduction in the severity, duration, or frequency of one or more associated pathologies, adverse symptoms, or complications of a cell proliferation or hyperproliferative disorder, e.g., neoplasm, tumor, or cancer, or metastasis, or by suppression or reversal of one or more physiological, biochemical, or cellular signs or characteristics. Therapeutic benefits or improvements may therefore include, but are not limited to, the destruction of target proliferative cells (e.g., neoplasm, tumor, or cancer, or metastasis), or the elimination of one or more, almost or all pathologies, adverse symptoms, or complications associated with or caused by a cell proliferation or hyperproliferative disorder, e.g., neoplasm, tumor, or cancer, or metastasis.However, therapeutic benefits or improvements do not necessarily have to be the cure or complete destruction of all target proliferative cells (e.g., neoplasms, tumors or cancers, or metastases), or the elimination of all pathologies, adverse symptoms, or complications associated with or caused by a disorder of cell proliferation or hyperproliferation, such as neoplasms, tumors or cancers, or metastases. For example, partial destruction of a tumor or cancerous cell mass, or stabilization of a tumor or cancerous mass, size, or cell number by inhibiting the progression or worsening of the tumor or cancer, can reduce mortality and prolong life, even if only by a few days, weeks, or months, even if some or most of the tumor or cancerous mass, size, or cells remain.

[0156] Certain non-limiting examples of therapeutic benefits include reduction in volume (size or cell mass) or cell number of a neoplasm, tumor, or cancer or metastasis; inhibition or prevention (e.g., stabilization) of an increase in volume of a neoplasm, tumor, or cancer; slowing or inhibiting the progression, worsening, or metastasis of a neoplasm, tumor, or cancer; or inhibiting the proliferation, growth, or metastasis of a neoplasm, tumor, or cancer.

[0157] The method of the present invention does not necessarily have to produce an immediate effect. For example, there may be an increase in the number or mass of neoplasms, tumors, or cancer cells following the treatment, or a subsequent stabilization or reduction in the tumor cell mass, size, or number of cells in a given object over time may occur.

[0158] Additional adverse symptoms and complications associated with neoplasms, tumors, cancers, and metastases that can be suppressed, reduced, decreased, delayed, or prevented include, for example, nausea, loss of appetite, lethargy, pain, and discomfort. Thus, partial or complete reduction or mitigation in the severity, duration, or frequency of adverse symptoms or complications associated with or caused by cytohyperproliferative disorders, and improvements in the subject's quality of life and / or well-being, such as increased energy, appetite, and psychological well-being, are all specific and non-limiting examples of therapeutic benefits.

[0159] The therapeutic benefit or improvement may therefore also include a subjective improvement in the quality of life of the treated subject. In additional embodiments, the method prolongs or extends the lifespan (survival) of the subject. In further embodiments, the method improves the quality of life of the subject.

[0160] The therapeutic benefits may also include the prevention of recurrence of neoplasms, tumors, cancers, and metastases, for example, said neoplasms, tumors, cancers, and metastases, which may be surgically or chemically removed.

[0161] Bacteria expressing the IL-15 variant and / or IL-15 superagonist of the present invention are typically administered to a subject in a composition comprising an effective amount of bacteria, e.g., Gram-negative bacteria, e.g., Salmonella seromutant cifida ZH9 strain, and further comprising a pharmaceutically acceptable carrier / adjuvant / diluent or excipient. The terms “pharmaceutically” and “pharmacologically acceptable” mean that the molecular entities and compositions do not produce harmful, allergic, or other undesirable reactions when administered to animals, e.g., humans, as appropriate. Such preparations 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 is understood that the preparations should, where applicable, meet criteria for sterility, pyrogenicity, general safety, and purity.

[0162] The composition is intended to enhance the target immune response. Therefore, bacteria expressing IL-15 variants and / or IL-15 superagonists are used in vaccines or vaccines. It may be provided as a tin composition. Such terms are interchangeable and refer to a biological preparation to which a subject generates an immune response to the biological preparation, and thus provides active acquired immunity against a specific infectious disease, such as a disease caused by Salmonella. In the context of the present invention, the vaccine may contain a factor similar to the bacteria causing the infection, or an “exotic” factor, which is an attenuated or dead form of the bacteria, or any part or fragment of bacterial protein, capsule, DNA or RNA. Such an exotic factor is recognized by the immune system of the vaccinated person, and the immune system then destroys the factor and develops a “memory” against the bacteria, thereby inducing a certain level of sustained protection against future bacterial infections by the same or similar viruses. It is assumed that, through a route of vaccination including the vaccine composition of the present invention, when a vaccinated subject encounters the same bacteria or bacterial isolate against which the subject was previously vaccinated, the individual’s immune system may thereby recognize the bacteria or bacterial isolate and induce a more effective defense against infection. The active acquired immunity induced in a subject as a result of the vaccine may be humoral and / or cellular. The vaccine composition may further comprise an adjuvant, a pharmaceutically acceptable carrier, or an excipient.

[0163] As used herein, “pharmaceutically acceptable carriers / adjuvants / diluents / excipients” includes, but is not limited to, any and all solvents, dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobials, antifungals), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, similar materials and combinations thereof, as are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289–1329). Examples include, but are not limited to, disodium hydrogen phosphate, soy peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, calcium chloride, sucrose, borate buffers, sterile saline solution (0.9% NaCl), and sterile water.

[0164] Suitable aqueous and non-aqueous carriers that may be used in the vaccine composition of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Appropriate fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0165] The vaccine compositions disclosed herein may further contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of the presence of undesirable microorganisms may be ensured by both the aforementioned sterilization procedures and the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Additionally, sustained absorption of the injectable pharmaceutically acceptable form may be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin. The vaccine compositions may also optionally contain additional therapeutic agents, for example, those known to be effective in infectious diseases or neoplasms. Thus, the vaccine compositions disclosed herein may also contain antiretroviral drugs, antibiotics, antifungal agents, antiparasitic agents, and anticancer agents.

[0166] The vaccine composition may also contain additional components intended to enhance the immune response. Examples of such additional components include aluminum salts, e.g., aluminum hydroxide, aluminum oxide, and aluminum phosphate; oily adjuvants, e.g., Freund's complete adjuvant and Freund's incomplete adjuvant; mycolic acid-based adjuvants (e.g., trehalose dimycolate); bacterial lipopolysaccharides (LPS); peptidoglycans (e.g., murein, mucopeptides, or glycoproteins, e.g., N-Opaca, muramyl dipeptide [MDP], or MDP analogs); proteoglycans (e.g., extracted from Klebsiella pneumoniae); streptococcal preparations (e.g., OK432); muramyl dipeptides; and immunostimulatory complexes (EP 109 942, EP 180 564, and EP 231). "Iscoms" disclosed in 039), saponins, DEAE-dextran, neutral oils (e.g., migliol), vegetable oils (e.g., peanut oil), liposomes, polyols, Ribi adjuvant systems (e.g., GB-A-2 189) (See 141), vitamin E, carbopol, interferons (e.g., IFN-alpha, IFN-gamma, or IFN-beta), or other (unmodified) interleukins, particularly those that stimulate cell-mediated immunity (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21), or chemokines (e.g., CXCL9, CXCL10, CXCL11, CCL5, CCL2, CX3CL1).

[0167] The bacteria in the vaccine compositions disclosed herein may include any one or any combination of the characteristic features of the bacteria disclosed herein.

[0168] The use of options (e.g., "or") should be understood to mean one of the options, both, or any combination thereof. Where used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any listed or enumerated components.

[0169] Where used herein, “about” means within an acceptable margin of error for a particular value as determined by those skilled in the art, which in part depends on how that value is measured or determined, i.e., the limits of the measuring system. For example, “about” may mean within one standard deviation or within one standard deviation according to practice in the art. Alternatively, “about” may mean a range of up to 20%. Where a particular value is provided in this application and claims, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable margin of error for that particular value.

[0170] The present invention is further described with reference to the following non-limiting embodiments. [Examples]

[0171] Example 1: Identification and biological activity evaluation of soluble IL-15 sequence variants Variant Design We designed cytokine variants according to three approaches: • Targeted in silico screening: Computational analysis of cargo modeled against the reported crystal structure (PDB ID: 4GS7) in complex with essential receptors. The analysis examined improvements in solubility and stability without loss of activity. This resulted in five defined variants containing multiple mutations synthesized by IDT. • Semi-rational candidates: Using computational tools (e.g., Aggrescan3D), amino acids that give an aggregation tendency were identified, and then amino acid mutations were performed only at these positions. These resulted in two libraries, depending on the method used to generate the mutants: each variant was either 1 or 2 for IL-15M40. A library of 72 members containing one defined amino acid change, and a pool of 729 variants, each containing 0 to 6 random mutations. Over 85% of the variants in the pool had 3 to 6 mutations. The first library was synthesized by IDT, and the second library by TWIST. • Random Candidates: Using the Genemorph II kit (Agilent) and following the manufacturer's specifications, random mutants were generated via error-prone PCR to create two pools. One pool contained fragments expected to have 0–2 mutations, and the other pool contained fragments expected to have 2–4 mutations.

[0172] Soluble screening Each of the fragments generated during the design phase was cloned into a soluble reporter plasmid, pBRED, which was derived from a system reported by Foit et al. (2009 Molecular Cell 36 861-871) based on the survival of bacterial cells in penicillin antibiotics (e.g., carbenicillin, ampicillin, penicillin V) as a function of the stability of the protein of interest. pBRED contains a beta-lactamase gene divided into two independently folding domains (alpha and omega). Two flexible linkers between these domains allow for the fusion of arbitrary protein cargoes, resulting in the final protein being Bla アルファ -Linker-Cargo-Linker-Bla オメガIt is composed as follows: If the cargo is stable / soluble, the alpha and omega subdomains can work together to degrade penicillin-based antibiotics. On the other hand, if the cargo is unstable / insoluble and degraded, the two elements split, and the reporter bacterium cannot survive against the antibiotic. The viability of each experimental strain was tested in either liquid or solid medium. Both methods produced consistent and reproducible results, with the liquid assay providing a faster, more easily interpretable readout.

[0173] In the first replicate, screening of the parent sequence, IL-15M40, revealed that it induced carbenicillin-sensitive cells at a concentration of 1 mg / mL. The protein mScarlet, known to be stable, was used as a positive control or benchmark.

[0174] Four of the five sequences in the targeted library (1) were found to be more stable than the parent control. In the semi-rational library, of the 72 variants designed to contain 1-2 amino acid mutations, most single (75%) and double amino acid substitutions (72%) were found to improve stability. The pool generated so that 85% of the variants contained 3-6 mutations yielded 94 positive sequences (53%) out of 176 after screening, which were then sequenced. The number of mutations in the samples was normally distributed with an average of 4, and mutations were found to be most frequent at positions 45 and 49. Finally, of the 176 variants generated by error-prone PCR, a total of 42 (24%) were positive in the assay. The final pool of positive candidates contained 169 sequences.

[0175] Activity screening Cytokine variants found to be more stable than the parent sequence (IL-15M40) were evaluated for activity using three assays: • Promega IL-15 Kit: A rapid assay using the IL-15 Bioassay Cell (Promega) kit. Suitable for high-throughput assays, enabling sigmoid evaluation of activity. A first screening assay was performed on soluble candidates. In this assay, reporter cells were added to wells containing cytokine proteins immobilized at the bottom, and incubated for 6 hours as recommended by the producer. The samples were incubated. After incubation, the substrate was added to the plate according to the producer's recommendations, and the luminescence was measured using a Tecan plate reader. • NK Expansion Proliferation Assay: A customized assay based on 4-day incubation of isolated NK cells in Prokarium at 37°C and 5% CO2 after induction using candidate cells (see below). Positive activity results in NK cell proliferation, measured as higher optical density (OD). It provides a more accurate output of candidate activity, but the output range is narrow. A second screening assay was performed on soluble candidates found to be positive using the Bioassay Cell (Promega) kit. • IFN-gamma (IFN-y) Production Assay: A customized assay based on a 1-day incubation of isolated NK cells in Prokarium after induction using candidate cells (see below). Positive activity results in interferon-gamma production by NK cells, which is then quantified by ELISA. As an NK expansion proliferation assay, it provides a more accurate output of candidate activity. A third and final screening assay was performed on soluble candidates found to be positive using the Bioassay Cell (Promega) kit and the NK expansion proliferation assay.

[0176] 169 variants obtained by screening for solubility were selected from C-terminal His 10 Each tagged cargo of interest was constitutively expressed in a protein expression plasmid. Cells in 4 mL volumes containing each plasmid were then divided into approximately 1.0 OD units. 600The cells were grown to a certain size and then centrifuged. The culture supernatant was discarded, and the cell pellet was resuspended in 200 μL of NPI buffer (50 mM NaH2PO4, 300 mM NaCl) supplemented with 20 mM imidazole. The cells were then lysed using a PIXUL® Multi-Sample Sonicator. The insoluble fraction was separated from the soluble protein contents by centrifugation at 12000xg for 20 minutes and loaded into Ni-NTA HisSorb (QIAGEN) or HisPur (ThermoFisher) plates. The proteins were allowed to bind at room temperature for 1 hour. The plates were then washed 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. The plate was then used to perform the activity assay as described above.

[0177] Only variants obtained from a library of sequences containing one to two amino acid mutations were found to be active after screening against IL-15 Bioassay Cells.

[0178] A total of 438 IL-15 variants were screened. Of these variants, 169 were found to be 2 to 40 times more soluble than the reference sequence (Figures 8A and 8B). Of these soluble variants, 13 were also found to be biologically active using the high-throughput screening Bioassay Cells (Promega) bioassay (Figures 8C and 8D; Table 1). Seven variants listed in Table 1 were selected for further experimentation (GBL01, GBL15, GBL17, GBL18, GBL25, GBL50).

[0179] [Table 1]

[0180] Example 2: Generation of IL-15 superagonist (SAg) using the IL-15Rα sushi domain Fusion to IL-15Rα (sushi domain) via the Gly-Ser flexible linker (ILR) generated 13 superagonist counterparts for IL-15 soluble and functional variants (Table 2).

[0181] [Table 2]

[0182] As previously described, the superagonist was challenged in an antibiotic challenge assay after cloning in the reporter plasmid pBRED, with the parental IL-15M40 superagonist as the reference and the mScarlet fusion to the IL-15Rα-ILR construct as the positive control.

[0183] The superagonists were found to be less soluble than their IL-15 counterparts, requiring intermediate and low assay conditions to identify soluble partners. Five of the thirteen superagonists (38%) were found to be more soluble than the superagonists with parent IL-15. These soluble superagonists were SAg15, SAg17, SAg18, SAg25, and SAg50.

[0184] Example 3: Verification of the biological activity of the IL-15 / SAg variant Thirteen candidate IL-15 variants and superagonists SAg15, SAg18, SAg25, and SAg50 were further validated using IL-15 Bioassay Cells (Figure 9A), primary NK cell proliferation assay (Figure 9B), and interferon-gamma release assay (Figure 9C).

[0185] All IL-15 variants except GBL34 and GBL43 yielded reproducible measurements using the Bioassay Cell (Promega) kit. This assay suggested that SAg15, SAg18, and SAg25 may be biologically active (Figure 9A).

[0186] The same sample extracts used in the bioassay were used for the growth assay. To demonstrate the differences... Therefore, the samples were normalized against the values ​​of the mScarlet-negative control. Induction of NK cells using different IL-15 / SAg cargoes faithfully reflected the results obtained in the Bioassay Cells (Promega) assay (Figure 9B).

[0187] The results of the IFN-y production assay were consistent with other experiments and allowed for better quantification of activity than proliferation (Figure 9C).

[0188] Example 4: Protein purification of IL-15 variant and IL-15 / Sag variant We selected variants GBL01, GBL18, GBL25, SAg18, and SAg25 and performed more thorough characterization. The genes encoding these were identified by His 10 The variants were placed on expression plasmids that allowed fusion with a C-terminal tag and expressed in E. coli. A total of 1 L of culture per variant was centrifuged, and the resulting pellets were lysed by ultrasonic lysis. The soluble fractions were purified by Ni-NTA chromatography to obtain purified His-tagged IL-15, IL-15 variants, and superagonists (Figure 10B). The proteins were then quantified by HPLC / MS using commercial IL-15 as a sample for calculating standard curves. The concentrations of the purified proteins (Table 3) were calculated using the standard curve of IL-15 concentration against MS peak area (Figure 10A).

[0189] [Table 3]

[0190] Example 5: In vitro and ex vivo characterization of recombinant IL-15 variants Using a Bioassay Cells (Promega) assay, a concentration gradient (10 -2 The activity of recombinant purified IL-15 variants was determined by testing at concentrations of ~2.5 nM. IL-15 variants were incubated for 6 hours with reporter cell lines expressing luc2 in response to IL-15 signaling. Results in Figure 11A are shown as the mean (+ / -SD) of induction multipliers (sample / blank) of n=3 independent replicas. Figure 11B shows INF-γ release. Wild-type IL-15 protein showed the highest activity, as is evident from the lowest EC50, with GBL18 and GBL01 having the highest activity among the candidates, while SAg18 and Sag25 did not induce activation of the Bioassay reporter cell line (Figure 11A; Table 4). This indicated that wild-type IL-15 is the most active protein, although the activity of GBL1 and GBL18 was comparable. Variant GBL25 was found to require approximately twice as high a concentration to activate luminescence.

[0191] [Table 4]

[0192] The results in Figure 11 were validated by incubating purified variants at known concentrations with NK cells for 24 hours and measuring the concentration of IFN-γ in the resulting supernatant (Figure 12). In Figure 12, primary human NK cells were isolated from PBMCs using negative selection with magnetic microbeads. Purified IL-15 variants were incubated with primary NK cells for 24 hours at 71 nM (Experiment 1) and 58 nM (Experiment 2). The results in Figure 12 are shown as mean + / - SEM from stimulated replicas of a representative single experiment out of three experiments performed with two different cell donors. The supernatant was then evaluated for the presence of IFN-γ using ELISA. Peprotech is a commercially available recombinant IL-15. All candidates induced IFN-γ production from primary NK cells. Cells stimulated with WT IL-15 were the samples that produced the highest levels of IFN-γ, with similar concentrations obtained from SAg25-stimulated NK cells, as well as from GBL01 and GBL18-stimulated NK cells.

[0193] Finally, NK cells in mouse (naive balb / c) spleen cells were stimulated with the variants for 72 hours. The spleen cells were then stained and evaluated for NK cells and NK activation markers via flow cytometry (Table 5; Figure 13). GBL01 and GBL18 exhibited comparable behavior and were the most effective variants. In some measurements, they also showed improvement over wild-type measurements.

[0194] [Table 5]

[0195] Example 6: Long-term expression of IL-15 in ZH9 Salmonella strain in tumors Due to the difficulty in detecting the IL-15 cargo protein, pharmacokinetics were evaluated using IL-15 gene expression from Salmonella.

[0196] Mice harboring subcutaneous MC38 tumors were injected with either a PBS control, a ZH9 Salmonella strain, or a candidate IL-15 variant-expressing ZH9 variant strain. Tissue was collected and RNA was extracted at the indicated time points after treatment, and the Salmonella-derived IL-15 variant was also extracted. Gene copies were counted by TaqMan quantitative PCR. Gene copies were normalized with respect to tumor volume.

[0197] The results of this experiment show that il-15 mRNA can be detected from Salmonella in tumor tissue for a long period (more than 7 days) after intratumoral administration.

[0198] Additionally, wild-type human IL-15 was expressed in ZH9 Salmonella. Western blot analysis indicated that wild-type human IL-15 was mainly detected in the bacterial insoluble fraction.

[0199] Example 7: In vivo bladder tumor model study showed improved efficacy of IL-15 variants against chassis strains. Female C57BL / 6 mice were inoculated intravesically (IVES) with MB49-luc tumor cells and treated with various Salmonella strains (PBS control, ZH9 Salmonella strain, or candidate IL-15 variant-expressing ZH9 variant strain). The survival of these mice was monitored for 60 days. In all cases, mice injected with the candidate IL-15 variant-expressing ZH9 variant strain showed the greatest median survival compared to mice injected with the ZH9 Salmonella strain (i.e., the strain lacking the IL-15 variant).

[0200] The present invention may be described in any of the following embodiments.

[0201] Embodiment 1. An interleukin-15 (IL-15) variant, wherein, compared to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to the receptor interaction surface, preferably the receptor interaction surface being an interleukin-15 receptor alpha (IL-15Rα) interaction surface.

[0202] Embodiment 2. An IL-15 variant according to Embodiment 1, wherein wild-type IL-15 comprises the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 1.

[0203] Embodiment 3. The IL-15 variant according to Embodiment 2, wherein, compared to SEQ ID NO: 1, the amino acid modification is present at one or more amino acid positions 45 and / or 49 and / or 52, or any combination thereof.

[0204] Embodiment 4. An interleukin-15 (IL-15) variant, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 1, and, compared to SEQ ID NO: 1, the IL-15 variant comprises an amino acid modification at one or more amino acid positions 45 and / or 49 and / or 52, or any combination thereof.

[0205] Embodiment 5. An IL-15 variant according to any one of Embodiments 2 to 4, comprising the amino acid sequence described in 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 respect to SEQ ID NO: 1.

[0206] Embodiment 6. An IL-15 variant according to any one of Embodiments 2 to 5, comprising an amino acid modification at amino acid position 75.

[0207] Embodiment 7. An IL-15 variant according to Embodiment 3 or 4 or any one of its dependent embodiments, wherein the amino acid modification at amino acid position 45 is a substitution from hydrophobic to hydrophilic.

[0208] Embodiment 8. The IL-15 variant according to Embodiment 7, wherein the hydrophobic-to-hydrophilic substitution is a substitution from leucine to serine or a substitution from leucine to threonine.

[0209] Embodiment 9. An IL-15 variant according to Embodiment 3 or 4 or any one of its dependent embodiments, wherein the amino acid modification at amino acid position 49 is a hydrophobic to hydrophobic substitution.

[0210] Embodiment 10. The IL-15 variant according to Embodiment 9, wherein the hydrophobic-to-hydrophobic substitution is a substitution from valine to alanine.

[0211] Embodiment 11. An IL-15 variant according to Embodiment 3 or 4 or any one of its dependent embodiments, wherein the amino acid modification at amino acid position 52 is a substitution from hydrophobic to hydrophilic.

[0212] Embodiment 12. The IL-15 variant according to Embodiment 11, wherein the hydrophobic-to-hydrophilic substitution is a substitution from leucine to lysine or a substitution from leucine to arginine.

[0213] Embodiment 13. An IL-15 variant according to Embodiment 3 or 4 or any one of its dependent embodiments, wherein the amino acid modification at amino acid position 75 is a substitution of serine to proline.

[0214] Embodiment 14. The IL-15 variant according to Embodiment 13, wherein the IL-15 variant, which includes the serine-to-proline substitution at amino acid position 75, comprises the sequence described in SEQ ID NO: 16, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 16, and the % sequence identity with respect to SEQ ID NO: 16 preserves the amino acid modification at amino acid position 75.

[0215] Embodiment 15. i) Substitution of leucine to serine at amino acid position 45; and ii) Substitution of serine to proline at amino acid position 75 An IL-15 variant according to any one of Embodiments 3 or 4 or a dependent embodiment thereof, including the above.

[0216] Embodiment 16. The IL-15 variant according to Embodiment 15, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 2, and the aforementioned % of sequence identity with respect to SEQ ID NO: 2 preserves the amino acid modification at amino acid positions 45 and 75.

[0217] Embodiment 17. The IL-15 variant according to Embodiment 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 respect to SEQ ID NO: 2, and the aforementioned percentage of sequence identity with respect to SEQ ID NO: 2 preserves the amino acid modification at amino acid positions 45 and 75.

[0218] Embodiment 18. i) Substitution of leucine to serine at amino acid position 45; and ii) Substitution of valine to alanine at amino acid position 49; and iii) Substitution of serine to proline at amino acid position 75 An IL-15 variant according to any one of Embodiments 3 or 4 or a dependent embodiment thereof, including the above.

[0219] Embodiment 19. The IL-15 variant according to Embodiment 18, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 3, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 3, and the aforementioned % of sequence identity with respect to SEQ ID NO: 3 preserves the amino acid modification at amino acid positions 45, 49, and 75.

[0220] Embodiment 20. The IL-15 variant according to Embodiment 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 respect to SEQ ID NO: 3, and the aforementioned percentage of sequence identity with respect to SEQ ID NO: 3 preserves the amino acid modification at amino acid positions 45, 49, and 75.

[0221] Embodiment 21. i) Substitution of leucine to serine at amino acid position 45; and ii) Substitution of leucine to lysine at amino acid position 52; and iii) Substitution of serine to proline at amino acid position 75 An IL-15 variant according to any one of Embodiments 3 or 4 or a dependent embodiment thereof, including the above.

[0222] Embodiment 22. The IL-15 variant according to Embodiment 21, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 4, and the aforementioned % of sequence identity with respect to SEQ ID NO: 4 preserves the amino acid modification at amino acid positions 45, 52, and 75.

[0223] Embodiment 23. The IL-15 variant according to Embodiment 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 respect to SEQ ID NO: 4, and the aforementioned percentage of sequence identity with respect to SEQ ID NO: 4 preserves the amino acid modification at amino acid positions 45, 52, and 75.

[0224] Embodiment 24. i) Substitution of leucine to serine at amino acid position 45; and ii) Substitution of leucine to arginine at amino acid position 52; and iii) Substitution of serine to proline at amino acid position 75 An IL-15 variant according to any one of Embodiments 3 or 4 or a dependent embodiment thereof, including the above.

[0225] Embodiment 25. The IL-15 variant according to Embodiment 24, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 5, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 5, and the aforementioned % of sequence identity with respect to SEQ ID NO: 5 preserves the amino acid modification at amino acid positions 45, 52, and 75.

[0226] Embodiment 26. 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 to SEQ ID NO: 5, wherein said % sequence identity to SEQ ID NO: 5 retains said amino acid modifications at amino acid positions 45, 52 and 75 The IL-15 variant according to Embodiment 25.

[0227] Embodiment 27. i) a substitution of leucine to threonine at amino acid position 45; and ii) a substitution of valine to alanine at amino acid position 49; and iii) a substitution of serine to proline at amino acid position 75 The IL-15 variant according to any one of Embodiment 3 or 4, or an embodiment dependent thereon, comprising the same.

[0228] Embodiment 28. The IL-15 variant according to Embodiment 27, wherein the IL-15 variant comprises the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6, and said % sequence identity to SEQ ID NO: 6 retains said amino acid modifications at amino acid positions 45, 49 and 75.

[0229] Embodiment 29. The IL-15 variant according to Embodiment 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 to SEQ ID NO: 6, and said % sequence identity to SEQ ID NO: 6 retains said amino acid modifications at amino acid positions 45, 49 and 75.

[0230] Embodiment 30. i) a substitution of valine to alanine at amino acid position 49; and ii) a substitution of leucine to arginine at amino acid position 52; and iii) Substitution of serine to proline at amino acid position 75 An IL-15 variant according to any one of Embodiments 3 or 4 or a dependent embodiment thereof, including the above.

[0231] Embodiment 31. The IL-15 variant according to Embodiment 30, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 7, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 7, and the aforementioned % of sequence identity with respect to SEQ ID NO: 7 preserves the amino acid modification at amino acid positions 49, 52, and 75.

[0232] Embodiment 32. The IL-15 variant according to Embodiment 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 respect to SEQ ID NO: 7, and the aforementioned percentage of sequence identity with respect to SEQ ID NO: 7 preserves the amino acid modification at amino acid positions 49, 52, and 75.

[0233] Embodiment 33. An IL-15 variant according to any of the preceding embodiments, fused to an interleukin-15 receptor alpha (IL-15Rα) molecule.

[0234] Embodiment 34. An IL-15 variant according to any of the preceding embodiments, fused to the sushi domain of an interleukin-15 receptor alpha (IL-15Rα) molecule.

[0235] Embodiment 35. The IL-15 variant according to Embodiment 33 or 34, wherein the IL-15 variant is fused via a linker, preferably the linker being a glycine-serine linker.

[0236] Embodiment 36. The linker is in the sequence described in Sequence ID No. 17, or in relation to Sequence ID No. 17 An IL-15 variant according to Embodiment 35, comprising an array having at least 70% identity.

[0237] Embodiment 37. The IL-15 variant according to any one of Embodiments 33 to 36, wherein the IL-15 variant comprises the sequence described in any of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, or a sequence having at least 70% identity to SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, wherein the % sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13 preserves the amino acid modification at the relevant amino acid position.

[0238] Embodiment 38. The IL-15 variant according to Embodiment 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 respect to any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13, and the aforementioned percentage of sequence identity with respect to any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13 preserves the amino acid modification at the relevant amino acid position.

[0239] Embodiment 39. An IL-15 variant according to any of the prior embodiments, fused to a signal peptide.

[0240] Embodiment 40. The IL-15 variant according to Embodiment 39, wherein the signal peptide is a periplasmic signal peptide.

[0241] Embodiment 41. The IL-15 variant according to Embodiment 39 or 40, wherein the signal peptide is fused to the IL-15 variant via a spacer sequence, and preferably the spacer sequence is 1 to 5 amino acids long.

[0242] Embodiment 42. An IL-15 variant according to any of the prior embodiments for therapeutic use.

[0243] Embodiment 43. The IL-15 variant according to any one of the preceding embodiments, for use in the treatment, prevention, reduction, suppression, prevention of recurrence, or control of a neoplastic disease in a subject.

[0244] Embodiment 44. The IL-15 variant for use according to Embodiment 43, wherein the neoplastic disease is a solid cancer and / or a hematological malignancy.

[0245] Embodiment 45. The IL-15 variant for use according to Embodiment 44, wherein the solid cancer and / or the hematological malignancy is 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, carcinoma, head and neck cancer, endometrial cancer, skin cancer, or sarcoma.

[0246] Embodiment 46. The IL-15 variant for use according to any one of Embodiments 42 to 45, which is administered to the subject via local instillation, intraperitoneally, intrapleurally, intravesically, intravaginally, via peritumoral injection, via intratumoral injection, or orally.

[0247] Embodiment 47. A nucleic acid molecule encoding the IL-15 variant as defined in any one of Embodiments 1 to 41.

[0248] Embodiment 48. The nucleic acid molecule according to Embodiment 47, wherein the nucleic acid molecule encodes an IL-15 variant comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, or a sequence having at least 70% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein said % sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 retains said amino acid modification at the relevant amino acid position.

[0249] Embodiment 49. The nucleic acid molecule according to Embodiment 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 NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the aforementioned percentage of sequence identity with any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 preserves the amino acid modification at the relevant amino acid position.

[0250] Embodiment 50. A nucleic acid molecule according to any one of Embodiments 47 to 49, comprising a DNA molecule.

[0251] Embodiment 51. A nucleic acid molecule according to any one of Embodiments 47 to 50, comprising an RNA molecule.

[0252] Embodiment 52. The nucleic acid molecule according to Embodiment 51, wherein the RNA molecule includes an mRNA molecule.

[0253] Embodiment 53. A Gram-negative bacterium comprising an IL-15 variant described in any one of Embodiments 1 to 41, or a nucleic acid molecule described in any one of Embodiments 47 to 52.

[0254] Embodiment 54. The Gram-negative bacteria described in Embodiment 53, which are live, attenuated Gram-negative bacteria.

[0255] Embodiment 55. The Gram-negative bacterium according to Embodiment 54, wherein the live, attenuated Gram-negative bacteria are Salmonella.

[0256] Embodiment 56. The Gram-negative bacterium according to Embodiment 55, wherein the live attenuated Gram-negative bacteria are Salmonella enterica, and preferably, the live attenuated Gram-negative bacteria are Salmonella enterica seromutant cifii.

[0257] Embodiment 57. The Gram-negative bacterium according to Embodiment 56, wherein the live attenuated Gram-negative bacterium is Salmonella enterica seromutant cif ZH9.

[0258] Embodiment 58. Gram-negative bacteria according to any one of Embodiments 53 to 57 for therapeutic use.

[0259] Embodiment 59. A Gram-negative bacterium according to any one of Embodiments 53 to 58 for use in the treatment, prevention, reduction, suppression, prevention of recurrence, or control of neoplastic diseases in a subject.

[0260] Embodiment 60. The neoplasm is a solid tumor and / or hematological malignancy. Gram-negative bacteria for use as described in Embodiment 59.

[0261] Embodiment 61. Gram-negative bacteria for use according to Embodiment 60, wherein the solid tumor and / or hematological malignancy is 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, carcinoma, head and neck cancer, skin cancer, or sarcoma.

[0262] Embodiment 62. Gram-negative bacteria for use according to any one of Embodiments 53 to 61, administered to the subject via topical drop, intraperitoneal, intrapleural, intravenous, intrabladder, peritumoral injection, intratumoral injection, or orally.

[0263] Embodiment 63. Use of an IL-15 variant as defined in any one of Embodiments 1 to 41, or a nucleic acid molecule as defined in any one of Embodiments 47 to 52, or a Gram-negative bacterium as defined in any one of Embodiments 53 to 62, in the production of a therapeutic pharmaceutical.

[0264] Embodiment 64. The use according to Embodiment 63, wherein the treatment is the treatment, prevention, reduction, suppression, prevention of recurrence, or control of a neoplasm in the subject.

[0265] Embodiment 65. A method for treating, suppressing, preventing or controlling a neoplastic disease in a subject, comprising administering to the subject an IL-15 variant as defined in any one of Embodiments 1 to 41, a nucleic acid molecule as defined in any one of Embodiments 47 to 52, or a Gram-negative bacterium as defined in any one of Embodiments 53 to 62.

[0266] [ka] [ka] [ka]

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Claims

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

2. The bacterium according to claim 1, which is a living, attenuated bacterium.

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

4. The bacterium according to claim 3, wherein the live attenuated gram-negative bacteria are of the genus Salmonella (Salmonella spp), preferably Salmonella enterica, more preferably Salmonella enterica seromutant Typhi, and even more preferably Salmonella enterica seromutant Typhi ZH9.

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

16.

6. The bacterium according to claim 5, wherein the IL-15 variant comprises the amino acid sequence described in 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 respect to SEQ ID NO:

1.

7. The bacterium according to claim 5 or 6, wherein, compared to Sequence ID No. 1, the amino acid modification is present at one or more amino acid positions 45 and / or 49 and / or 52, or any combination thereof.

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

9. The bacterium according to claim 8, wherein the IL-15 variant, which includes the serine-to-proline substitution at amino acid position 75, comprises an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 16, and the aforementioned % of sequence identity with respect to SEQ ID NO: 16 preserves the amino acid modification at amino acid position 75.

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

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

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

13. The aforementioned IL-15 variant is i) Substitution of leucine to serine at amino acid position 45; and ii) Substitution of serine to proline at amino acid position 75 A bacterium according to any one of claims 7 to 12, including the bacterium described in any one of claims 7 to 12.

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

15. The bacterium according to 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 respect to SEQ ID NO: 2, and the aforementioned percentage of sequence identity with respect to SEQ ID NO: 2 preserves the amino acid modification at amino acid positions 45 and 75.

16. The aforementioned IL-15 variant is i) Substitution of leucine to serine at amino acid position 45; and ii) Substitution of valine to alanine at amino acid position 49; and iii) Substitution of serine to proline at amino acid position 75 A bacterium according to any one of claims 7 to 15, including the bacterium described in any one of claims 7 to 15.

17. The bacterium according to claim 16, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 3, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 3, and the aforementioned % of sequence identity with respect to SEQ ID NO: 3 preserves the amino acid modification at amino acid positions 45, 49, and 75.

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

19. The aforementioned IL-15 variant is i) Substitution of leucine to serine at amino acid position 45; and ii) Substitution of leucine to lysine at amino acid position 52; and iii) Substitution of serine to proline at amino acid position 75 A bacterium according to any one of claims 7 to 18, including the bacterium described in any one of claims 7 to 18.

20. The bacterium according to claim 19, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 4, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 4, and the aforementioned % of sequence identity with respect to SEQ ID NO: 4 preserves the amino acid modification at amino acid positions 45, 52, and 75.

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

22. The aforementioned IL-15 variant is i) Substitution of leucine to serine at amino acid position 45; and ii) Substitution of leucine to arginine at amino acid position 52; and iii) Substitution of serine to proline at amino acid position 75 A bacterium according to any one of claims 7 to 21, including the bacterium described in any one of claims 7 to 21.

23. The bacterium according to claim 22, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 5, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 5, and the aforementioned % of sequence identity with respect to SEQ ID NO: 5 preserves the amino acid modification at amino acid positions 45, 52, and 75.

24. The bacterium according to claim 23, wherein the IL-15 variant comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with respect to SEQ ID NO: 5, and the aforementioned percentages of sequence identity with respect to SEQ ID NO: 5 preserve the amino acid modification at amino acid positions 45, 52, and 75.

25. The aforementioned IL-15 variant is i) Substitution of leucine to threonine at amino acid position 45; and ii) Substitution of valine to alanine at amino acid position 49; and iii) Substitution of serine to proline at amino acid position 75 A bacterium according to any one of claims 7 to 24, including the bacterium described in any one of claims 7 to 24.

26. The bacterium according to claim 25, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 6, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 6, and the aforementioned % of sequence identity with respect to SEQ ID NO: 6 preserves the amino acid modification at amino acid positions 45, 49, and 75.

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

28. The aforementioned IL-15 variant is i) Substitution of valine to alanine at amino acid position 49; and ii) Substitution of leucine to arginine at amino acid position 52; and iii) Substitution of serine to proline at amino acid position 75 A bacterium according to any one of claims 7 to 27, including the bacterium described in any one of claims 7 to 27.

29. The bacterium according to claim 28, wherein the IL-15 variant comprises the amino acid sequence described in SEQ ID NO: 7, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 7, and the aforementioned % of sequence identity with respect to SEQ ID NO: 7 preserves the amino acid modification at amino acid positions 49, 52, and 75.

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

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

32. The bacterium according to any prior claim, wherein the IL-15 variant is fused to the sushi domain of the interleukin-15 receptor alpha (IL-15Rα) molecule.

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

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

17.

35. The bacterium according to any one of claims 31 to 34, wherein the IL-15 variant comprises a sequence described in any of sequence numbers 8, 9, 10, 11, 12, and / or 13, or a sequence having at least 70% identity to sequence numbers 8, 9, 10, 11, 12, and / or 13, wherein the % sequence identity to any one of sequence numbers 8, 9, 10, 11, 12, and / or 13 preserves the amino acid modification at the relevant amino acid position.

36. The bacterium according to 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 NOs: 8, 9, 10, 11, 12, and / or 13, and the aforementioned percentage of sequence identity with any one of SEQ ID NOs: 8, 9, 10, 11, 12, and / or 13 preserves the amino acid modification at the relevant amino acid position.

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

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

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

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

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

42. The bacterium according to 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 NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the aforementioned percentage of sequence identity with any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 preserves the amino acid modification at the relevant amino acid position.

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

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

45. An interleukin-15 (IL-15) variant wherein wild-type IL-15 comprises the amino acid sequence described in SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 1, and, compared to SEQ ID NO: 1, the IL-15 variant comprises one or more amino acid modifications in the surface region adjacent to the receptor interaction surface, and the one or more amino acid modifications are i) Substitution from hydrophobic to hydrophilic at amino acid position 45; ii) Substitution from hydrophobic to hydrophobic at amino acid position 49; iii) Substitution of the amino acid at position 52 from hydrophobic to hydrophilic; and / or iv) Substitution of serine to proline at amino acid position 75 Includes any of the following; Preferably, the receptor interaction surface is an interleukin-15 receptor alpha (IL-15Rα) interaction surface. IL-15 variant.

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

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

48. The IL-15 variant according to any one of claims 45 to 47, wherein the hydrophobic-to-hydrophobic substitution at amino acid position 49 is a substitution from valine to alanine or from 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 according to claim 49, wherein the nucleic acid molecule encodes an IL-15 variant comprising an amino acid sequence described in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, or a sequence having at least 70% identity with any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16, wherein the % sequence identity with any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and / or 16 preserves the amino acid modification at the relevant amino acid position.

51. The nucleic acid molecule according to 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 NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16, wherein the aforementioned percentage of sequence identity with any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 16 preserves the amino acid modification at the relevant amino acid position.

52. A nucleic acid molecule according to any one of claims 49 to 51, comprising 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 the RNA molecule comprises an mRNA molecule.

54. A bacterium comprising 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.

55. The bacterium according to claim 54, which is a living, attenuated bacterium.

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

57. The bacterium according to claim 56, wherein the live attenuated gram-negative bacteria are of the genus Salmonella, preferably Salmonella enterica, more preferably Salmonella enterica seromutant cifie, and even more preferably Salmonella enterica seromutant cifie ZH9.

58. A bacterium according to any one of claims 1 to 44 or 54 to 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, for therapeutic use.

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

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

61. The solid tumor and / or hematological malignancy is 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, carcinoma, head and neck cancer, skin cancer, or sarcoma, a bacterium, IL-15 variant, or nucleic acid molecule for use according to claim 60.

62. Bacteria, IL-15 variants, or nucleic acid molecules for use according to claim 61, administered to the subject via local drop, intraperitoneal, intrapleural, intravenous, intrabladder, peritumoral injection, intratumoral injection, or orally.

63. Use of a bacterium according to any one of claims 1 to 44 or 54 to 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, in the production of a therapeutic pharmaceutical.

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

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

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