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WO2025068401A1 - Variant d'interleukine 15 - Google Patents

Variant d'interleukine 15 Download PDF

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WO2025068401A1
WO2025068401A1 PCT/EP2024/077120 EP2024077120W WO2025068401A1 WO 2025068401 A1 WO2025068401 A1 WO 2025068401A1 EP 2024077120 W EP2024077120 W EP 2024077120W WO 2025068401 A1 WO2025068401 A1 WO 2025068401A1
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amino acid
seq
sequence identity
variant
bacterium
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Marc Biarnes CARRERA
Andrew Crosbie SCOTT
Georgia PETSIOU
Oluwatobiloba OKE
Nicholas Glanville
Livija DEBAN
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Prokarium Ltd
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Prokarium Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to interleukin-15 (IL-15) molecules and variants thereof.
  • IL-15 interleukin-15
  • Microbial immunotherapy has been shown to be effective as post-surgery treatment to reduce the incidence of tumour recurrence, with BCG treatment being the current standard of care for certain cancer types, in particular for NMIBC patients (Alhunaidi, 2019).
  • BCG therapy has limitations including the efficacy range (40-60% of patients will experience tumour recurrence within 5 years of surgery), adverse effects and safety concerns, complex manufacturing requirements, and a long treatment regimen (patients undergo 27 intra-vesical, transurethral instillations over 36 months).
  • microbial immunotherapy together with co-immunostimulatory molecules has been proposed.
  • Interleukin 15 IL-15
  • IL-15 agonist molecules are considered as promising immunotherapeutics because of their role in CD8 T cells and NK activation and proliferation (Guo, 2017; Robinson, 2017).
  • Clinical trials are currently ongoing to test a combination therapy of intravesical BCG plus ANKTIVA® (former ALT-803, N803), an IL-15 super-agonist molecule (developed by ImmunityBio, former Aitor BioScience).
  • IL-15 immuno-stimulatory anti-cancer treatment
  • cytokines expressed and purified from E. coli
  • a common problem with the use of bacterial systems to express eukaryotic cytokines is the formation of inclusion bodies.
  • Bacterial expression of cytokines generally results in the formation of non- functional inclusion bodies.
  • the depositing of cytokines in bacteria therefore imposes additional and laborious requirements of solubilising and refolding the proteins from inclusion bodies, which often leads to low yields of protein which is unsuitable for living therapeutics (Ferrer-Miralles, 2009; de Marco, 2009).
  • a high rate of protein expression or unfavourable reducing conditions in the cytoplasm may impair the folding of nascent peptide resulting in formation of insoluble protein aggregates.
  • the highly reducing environment of the bacterial cytoplasm hinders disulphide linkages forming between cysteine residues during protein folding - this can be problematic for the expression of cytokines (including IL-15) where there are disulphide bonds in the protein structure.
  • a further challenge presented by protein expression in Gram-negative strains of bacteria such as E. coli and Salmonella is that, even following the successful expression of cytokines, the proteins must be correctly folded and then transported across two cellular membranes, in between which is the bacterial periplasm, in order to be secreted from the cell.
  • the present invention provides IL-15 variants with both optimised solubility and preserved bioactivity.
  • the inventors of the present invention have surprisingly identified certain combinations of mutations in IL-15 that result in IL-15 variants which are more soluble than wild-type IL-15, and retain functionality.
  • the variants provided herein are therefore ideal candidates for expression in, and secretion from, efficient expression systems such as bacteria, for example Gram-negative bacteria and/or Gram-positive bacteria.
  • a bacterium comprising an interleukin-15 (IL-15) variant, wherein in comparison to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface.
  • IL-15 interleukin 15 receptor alpha
  • an interleukin-15 (IL-15) variant wherein wild-type IL-15 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, and wherein the one or more amino acid modifications comprise any of: i) a hydrophobic-to-hydrophilic substitution at amino acid position 45; ii) a hydrophobic-to-hydrophobic substitution at amino acid position 49; iii) a hydrophobic-to-hydrophilic substitution at amino acid position 52; and/or iv) a serine-to-proline substitution at amino acid position 75; preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL- 15Ra) interaction surface.
  • IL- 15Ra interleukin 15 receptor alpha
  • a nucleic acid molecule encoding the IL-15 variant as defined in the second aspect of the invention.
  • a bacterium comprising the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third aspect of the invention.
  • bacterium according to the first or fourth aspects of the invention or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third aspect of the invention, for use as a therapy.
  • a seventh aspect of the invention there is provided a method of treating, inhibiting, preventing recurrence, or controlling a neoplastic disease in a subject, wherein the method comprises administering to the subject the bacterium according to the first or fourth aspects of the invention, or the IL-15 variant according to the second aspect of the invention, or the nucleic acid molecule according to the third aspect of the invention.
  • Figure 1 shows the structure of an interleukin-15 (IL-15, PDB ID: 4GS7) variant, indicating amino acid modifications in a surface region adjacent to the IL-15Ra interaction surface. Also shown are IL-15Ra, IL-2Rp (IL-15Rp), and GammaC (YC).
  • IL-15Ra interleukin-15
  • IL-15Rp IL-2Rp
  • YC GammaC
  • Figure 2 shows a schematic representation of IL-15 trans-presentation and biological function.
  • Figure 3 shows the sequence-structure relationship of wild-type IL-15 and its interaction with cognate ligands. Short and long (mammalian) signal peptide sequences of pre-IL-15 (A), mature IL-15 (B), and IL-15 tertiary structure (C) are shown.
  • Figure 4 shows aggregation propensity and hydrophobicity score of IL-15 calculated using AggreScan and AggreScan 3D.
  • Figure 5 shows a summary of IL-15 and IL-15 agonists used in immunotherapy.
  • Figure 6 shows the structure of an IL-15 variant, indicating amino acid positions 45 and/or 49 and/or 52. Also shown are IL-15Ra, I L-2Rp, and GammaC (yc).
  • Figures 7A-7C show the structure of IL-15 in quaternary complex with IL-15Ra, IL-15R , and GammaC (yc) (PDB: 4GS7).
  • Figures 8A-8D demonstrate the solubility and functionality of different IL-15 variants.
  • A shows the total number of soluble variants obtained through different mutagenesis strategies.
  • B shows the solubility score distribution for each of the mutagenesis strategies, with in silico designs showing an overall higher success rate and epPCR variants showing the higher spread.
  • C shows the total number of active variants obtained through different mutagenesis strategies.
  • D shows the activity measurement as Relative Luminescence Units (RLU) from IL-15 Bioassay Cells (Promega) reporter cell line distributions for each mutagenesis strategy.
  • RLU Relative Luminescence Units
  • Figures 9A-9C demonstrate the biological activity of IL-15/SAg variants using three independent assays (IL-15 Bioassay Cells, NK proliferation assay, and IFNy production assay).
  • Figures 10A-10B show protein purification data confirming the isolation of different IL-15 variants and IL-15 SAg variants.
  • Figures 11A and 11B show pre-clinical candidate selection of IL-15 variants. The results of incubating a gradient of purified IL-15 variants for 6 hours with a reporter cell line are shown.
  • Figure 12 shows in vitro human cell assays. The results of incubating IL-15 variants with primary NK cells for 24 hours at 71 nM (experiment 1) and 58 nM (experiment 2) are shown.
  • Figure 13 shows the results of IL-15 variant mediated NK activation in mouse splenocytes after incubation with IL-15 variants for 72 hours.
  • the present invention provides optimised IL-15 variants (and/or super agonists thereof, i.e., SAg variants) which are both soluble and functional, and are therefore optimal candidates for expression in efficient secretion systems which allow the production and secretion of said IL-15 variants and SAg variants.
  • the invention provides a bacterium comprising an interleukin-15 (IL-15) variant, wherein in comparison to wild-type IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface ( Figure 1).
  • IL-15 interleukin 15 receptor alpha
  • interleukin refers to any member of the glycoprotein family which is expressed by leukocytes (white blood cells) and is involved in regulating immune responses. Interleukins may also be referred to as cytokines. Interleukins have diverse roles in the generation of immune cells, including immune cell activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. Interleukins can have pro- and antiinflammatory properties. Interleukins make up a large group of proteins which have the capacity to elicit several reactions in cells and tissues by binding to receptors known as interleukin receptors in order to trigger downstream signalling from the receptor.
  • Interleukin-15 is a four alpha-helix bundle cytokine belonging to the same family as IL-2, IL-4, IL-7, IL-9, and IL-21.
  • IL-15 is constitutively expressed by a wide variety of cells including dendritic cells, monocytes, macrophages, bone marrow stromal cells and intestinal epithelial cells.
  • Cells expressing IL-15 also express its high affinity receptor-a, IL-15Ra.
  • IL-15Ra Unlike most cytokines, which are secreted in soluble form, IL-15 has a unique form of expression whereby it is expressed in association with its high affinity receptor, IL-15Ra, and is shuttled as a heterodimeric complex to the surface of IL-15-producing cells.
  • IL-15 can also be secreted as a soluble IL-15 independently from IL-15Ra.
  • the IL-15/IL-15Ra complex is a cell surface complex which efficiently stimulates neighbouring cells through the I L-2/I L-15Rp and GammaC (yc) complex (I L-15R(3y) via a mechanism of trans-presentation ( Figure 2).
  • IL-15 preferentially stimulates NK (natural killer) and CD8+ T cells activation, proliferation and cytolytic activity (Guo, 2007; Santana-Carrero, 2019).
  • I L-15Ra/l L-15 complexes mediates cells responses during homeostasis.
  • I L-15Ra/l L-15 complexes are cleaved from the surface of presenting cells in response to numerous types of immune stimulation such as total body irradiation, TLR stimulation, virus infections, CD40 stimulation, type I IFNs (IFN-I) and activation of the stimulator of interferon genes (STING) pathway, thus releasing a soluble heterodimeric I L-15/I L-15Ra complex (Bergamaschi, 2012; Anthony, 2016).
  • Human IL-15 is a protein of 162 amino-acid (18,086 kDa) containing 4 helices, 2 disulphide bonds and 1 glycosylated site ( Figure 3). It has two isoforms produced by alternatively spliced transcripts: one form with a short signal peptide (SSP), which contains a 21 amino acid leader peptide and remains in the cell cytosol, and one other with a long signal peptide (LSP), which contains a 48 amino acid signal peptide that allows for secretion of the protein into the extracellular environment (Saeed and Revell, 2001 ; Duitman, 2008).
  • SSP short signal peptide
  • LSP long signal peptide
  • This secretion occurs through independent export into the Golgi Apparatus of the signal and the receptor alpha, followed by high affinity interaction between signal and receptor, and export into the environment of the complex via the receptor alpha.
  • the signal peptide is cleaved always on the same position, resulting in the same mature form of IL-15, possibly through the highly conserved sequence N-GLPKTEA/NW-C, although SSP IL-15 is already shown to be bioactive, indicating certain flexibility to the presence of N-terminus peptides (Bergamaschi, 2009).
  • the mature IL-15 is then folded in a four-helix bundle motif (the four a-helices are structured together lengthwise in antiparallel orientation), with 16-21 amino acids per a-chain, where alpha chain a1 interacts with a3 and a2 with a4 (interactions between helices represented as dashed lines in Figure 3, with key residues involved in proteinprotein interactions being highlighted).
  • interactions with yc show a more general interaction, without a highly determined chemical signature, highlighting the promiscuity of yc to interact with multiple cytokines.
  • Interaction with yc occurs via chains a1 and a4, with Q108, M109, and N112 being the key amino acids involved in this interaction.
  • An additional site of contact is made with the extended alpha chain to compensate for the smaller size of I L-15.
  • the amino acid sequence of IL-15 has been analysed by the inventors of the present invention with AggreScan, ProtScale, and AggreScan3D to identify potential aggregation sites, which would be responsible for protein aggregation and formation of inclusion bodies upon recombinant overexpression in E. coli or Salmonella Typhi.
  • the regions with higher propensity to aggregate are those forming the alpha helices ( Figure 4), as these are the regions involved in proteinprotein interactions.
  • IL-15 Given its role in the stimulation of proliferation and cytotoxic function of CD8 T cells and NK cells, IL-15 has been widely tested as a cancer immunotherapeutic agent. The efficacy of IL-15 is limited by the short half-life of the protein in vivo. A number of IL-15 variants have been generated to improve protein stability and efficacy.
  • Figure 5 depicts five (1-5) different IL-15-based agents that are used to stimulate cytotoxic T cell and NK cell responses.
  • rlL-15 was the first form of IL-15 to be examined in vivo. When administered, rlL-15 is believed to predominantly bind to cell surface IL- 15Ra where it is trans-presented to IL-15 responsive cells.
  • Heterodimeric IL- 15 is the natural form of IL-15 that is cleaved from cells and can stimulate responses independent of cell interactions. Heterodimeric hlL-15 is being produced as a therapeutic by Novartis.
  • RLI Cosmetic Pharmaceuticals
  • IL-15 is a fusion protein consisting of IL-15 linked to the sushi (cytokine-binding) domain of the IL-15Ra that can act as a soluble IL-15 agonist.
  • IL-15/IL-15Ra-Fc complexes are generated by mixing commercially available IL-15Ra-Fc chimeric fusion protein with rlL-15 and have been used extensively in preclinical studies.
  • ANKTIVA® (former ALT-803, N-803) (ImmunityBio, former Aitor Pharmaceutical) is an IL-15 superagonist consisting of mutated IL-15 (asparagine at position 72 within the mature IL-15 amino acid sequence replaced with an aspartic residue), which has an increased affinity for CD122-expressing immune cells, linked to the sushi domain of the IL-15Ra that is fused to an Fc fragment.
  • IL-15-based agents increases with increased abundance of agonist, increased in vivo half-life (imparted by IL-15 binding to IL-15Ra and presence of an Fc fragment), dimerization of the agonists, and increased affinity for the IL-2Rp/yC complex (imparted by IL-15 binding to the sushi domain and the mutation present in ALT-803).
  • the term “interleukin variant” refers to an IL-15 molecule which, in comparison to wild-type IL-15, comprises one or more amino acid modifications.
  • amino acid modifications refers to any change or mutation to a wild-type amino acid sequence.
  • An amino acid modification may or may not alter the structure, function, and/or physiochemical properties of the protein.
  • Amino acid modifications may include, but are not limited to deletions, substitutions, and insertions.
  • Deletion mutations involve the loss of an amino acid, resulting in a frameshift or decrease in the length of the amino acid sequence.
  • Insertion mutations involve the addition of an amino acid, resulting in a frameshift or increase in the length of the amino acid sequence.
  • substitution mutations involve the replacement of one amino acid for another. Substitution mutations can be conservative or non-conservative.
  • region adjacent may refer to surface amino acids which are in proximity to the receptor (e.g., IL-15Ra) interaction surface and may interact with IL-15Ra but do not interact with IL-15Rp or yc.
  • certain modifications may therefore act to enhance the interaction between IL-15 and a receptor (e.g., I L-15Ra), for example by increasing the affinity of binding.
  • modifications which alter the composition properties of amino acids on the surface of IL-15 may impact the solubility of IL-15.
  • the IL-15 variant may comprise an amino acid sequence comprising at least 75% sequence identity to SEQ ID NO: 1 ; at least 80% sequence identity to SEQ ID NO: 1 ; at least 85% sequence identity to SEQ ID NO: 1 ; at least 90% sequence identity to SEQ ID NO: 1 ; at least 91 % sequence identity to SEQ ID NO: 1 ; at least 92% sequence identity to SEQ ID NO: 1 ; at least 93% sequence identity to SEQ ID NO: 1 ; at least 94% sequence identity to SEQ ID NO: 1 ; at least 95% sequence identity to SEQ ID NO: 1 ; at least 96% sequence identity to SEQ ID NO: 1 ; at least 97% sequence identity to SEQ ID NO: 1 ; at least 98% sequence identity to SEQ ID NO: 1 ; or at least 99% sequence identity to SEQ ID NO: 1.
  • the composition is intended to enhance an immune response of a subject.
  • the bacterium expressing an IL-15 variant and/or IL-15 super agonist may be provided as a vaccine or vaccine composition.
  • Such terms are used interchangeably and refer to a biological preparation in which the subject produces an immune response to said biological preparation, therefore providing active acquired immunity to a particular infectious disease, for example, a disease caused by a Salmonella spp.
  • the vaccine may contain an agent, or “foreign” agent, that resembles the infection-causing bacteria, which is a weakened or killed form of said bacteria, or any portion of, or fragment of, a bacteria protein, capsule, DNA or RNA.
  • Such a foreign agent would be recognised by a vaccine-receiver’s immune system, which in turn would destroy said agent and develop “memory” against the bacteria, inducing a level of lasting protection against future bacterial infections from the same or similar viruses.
  • the individual’s immune system may thereby recognise said bacteria or bacterial isolate and elicit a more effective defence against infection.
  • the active acquired immunity that is induced in the subject as a result of the vaccine may be humoral and/or cellular in nature.
  • the vaccine composition may further comprise an adjuvant, a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable carrier/adjuvant/diluent/excipient includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavouring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329).
  • preservatives e.g., antibacterial agents, antifungal agents
  • isotonic agents e.g., absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavour
  • Examples include, but are not limited to disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulphate, calcium chloride, sucrose, borate buffer, sterile saline solution (0.9 % NaCI) and sterile water.
  • the vaccine compositions herein disclosed may further contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of presence of unwanted microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents.
  • the bacterium of the vaccine composition herein disclosed may include any one of, or any combination of, the features of the bacterium herein disclosed.
  • Random candidates Generation of random mutants via error-prone PCR using the Genemorph II kit (Agilent) and according to manufacturer specifications to create two pools, one with fragments expected to have 0 - 2 mutations and one with fragments expected to have 2 - 4 mutations.
  • the alpha and omega sub-domains can work together to degrade the penicillin-based antibiotic.
  • the cargo is unstable/insoluble and degraded, the two elements are split, and reporter bacteria cannot survive to the antibiotic. Survival of each experimental strain was tested either in liquid media or in solid. Both methods produced consistent and reproducible results, with the liquid assay providing a faster, easier to interpret, readout.
  • Cytokine variants found to be more stable than the parental sequence were evaluated for activity using three assays: • Promega IL-15 kit: Rapid assay using an IL-15 Bioassay Cell (Promega) kit. Allows a sigmoidal assessment of activity, suitable for high throughput assays. First screening assay performed on the soluble candidates. In this assay, reporter cells were added to wells containing immobilised cytokine protein on their bottom and incubated as recommended by the manufacturer for 6 h. After incubation, substrate was added into the plate following manufacturer recommendations and luminescence measured on a Tecan plate reader.
  • NK expansion assay Bespoke assay based on a 4-day incubation at 37 °C and 5 % CO2 of NK cells isolated at Prokarium after induction with candidates (see below). Positive activity results in proliferation of the NK cells, measured as higher Optical Density (OD). Offers a more accurate output of the activity of the candidates, albeit the output range being narrow. Second screening assay performed on soluble candidates found positive with the Bioassay Cell (Promega) Kit.
  • IFN-gamma (IFN-y) production assay Bespoke assay based on a 1-day incubation of NK cells isolated at Prokarium after induction with candidates (see below). Positive activity results in production of Interferon gamma by the NK cells, which is then quantified by ELISA. As the NK Expansion Assay, offers a more accurate output of the activity of the candidates. Third and last screening assay performed on soluble candidates found positive with the Bioassay Cell (Promega) Kit and the NK Expansion Assay.
  • the 169 variants obtained by screening for solubility were transferred into a protein expression plasmid that constitutively expressed each cargo of interest with a C-terminal His tag.
  • a volume of 4 mL of cells containing each plasmid were grown to OD 6 oo ⁇ 1.0 and spun down. Culture supernatant was discarded, and cell pellet re-suspended in 200 pL of NPI buffer (50mM NaH2PO4, 300mM NaCI) supplemented with 20 mM imidazole. The cells were then lysed on a PIXUL® Multi-Sample Sonicator.
  • the insoluble fraction was separated from the soluble protein content by centrifugation for 20 min at 12000xg and loaded into Ni- NTA HisSorb (QIAGEN) or HisPur (ThermoFisher) plates. Proteins were allowed to bind for 1 h at room temperature. Plates were then washed with NPI supplemented with 50 mM imidazole three times and with PBS (37 mM NaCI, 2.7 mM KCI, 8 mM Na 2 HPC>4, and 2 mM KH2PO4) two times to remove excess imidazole. Plates were then used to perform activity assays as described above.
  • the super agonists were challenged with the antibiotic challenge assay after being cloned into the reporter plasmid pBRED, using the super agonist of parental IL-15M40 as reference and the fusion of mScarlet to the IL-15Ra-ILR construct as positive control.
  • Super agonists were found to be less soluble than their IL-15 counterpart, requiring mid and low assay conditions to identify soluble partners. 5 out of 13 (38%) of super agonists were found to be more soluble than super agonists with parental IL-15. These soluble super agonists were SAg15, SAg17, SAg18, SAg25, and SAg50.
  • the variants GBL01 , GBL18, GBL25, SAg18, and SAg25 were selected to undergo more thorough characterisation.
  • the genes encoding for these were placed on the expression plasmid that allowed fusion with a His C-terminal tag and expressed in E coli.
  • a total of 1 L of culture per variant was centrifuged and the resulting pellet was lysed by sonication.
  • the soluble fraction was purified by Ni-NTA chromatography, yielding purified His-tagged IL-15, IL-15 variants, and super agonists (Figure 10B).
  • Protein was then quantified by HPLC/MS, using commercial IL-15 as sample to calculate a standard curve. Purified protein concentrations (Table 3) were calculated using a standard curve of IL-15 concentration against MS peak area ( Figure 10A).
  • Wild-type IL-15 protein showed the greatest activity, evident from the lowest EC50, GBL18 and GBL01 have the greatest activity of the candidates, and SAg18 and Sag25 do not induce activation of the Bioassay reporter cell line ( Figure 11 A; Table 4). While this showed that IL- 15 wild-type was the protein with highest activity, that of GBL1 and GBL18 was comparable. Variant GBL25 was found to require approximately 2x higher concentrations to activate luminescence.
  • the variants were used to stimulate NK cells in mouse (naive balb/c) splenocytes for 72 hours.
  • the splenocytes were then stained and assessed for NK cell and NK activation markers via flow cytometry (Table 5; Figure 13).
  • GBL01 and GBL18 had comparable behaviour and were the most effective variants. In some measurements, these were also improved upon wild-type measurements.
  • Example 6 Expression of IL-15 in ZH9 Salmonella strains in tumours for prolonged periods.
  • IL-15 gene expression from Salmonella was used to assess pharmacokinetics.
  • mice bearing subcutaneous MC38 tumours were injected intratumorally with a PBS control, a ZH9 Salmonella strain, or a candidate IL-15 variant-expressing ZH9 variant strain.
  • Tissues were harvested at the indicated timepoints after treatment, and RNA was extracted, and copies of the Sa/mone//a-derived IL-15 genes were enumerated by TaqMan quantitative PCR. Gene copies are normalised for tumour volume.
  • wild-type human IL-15 was expressed in ZH9 Salmonella.
  • Western blot analysis indicated that wild-type human IL-15 was detected mainly in the bacterial insoluble fraction.
  • Example 7 In vivo bladder tumour model studies showed improved efficacy for IL-15 variants versus chassis strain
  • mice Female C57BL/6 mice were inoculated intravesically (IVES) with MB49-luc tumour cells and treated with various Salmonella strains (PBS control, ZH9 Salmonella strain, or a candidate IL-15 variant-expressing ZH9 variant strain). The survival of these mice was monitored for 60 days. In all cases, mice injected with candidate IL-15 variant-expressing ZH9 variant strains showed greater median survival as compared to mice injected with ZH9 Salmonella strain (i.e., strains lacking the IL-15 variant).
  • Embodiment 1 An interleukin-15 (IL-15) variant, wherein in comparison to wildtype IL-15, the IL-15 variant comprises one or more amino acid modifications in a surface region adjacent to a receptor interaction surface, preferably wherein the receptor interaction surface is an interleukin 15 receptor alpha (IL-15Ra) interaction surface.
  • IL-15Ra interleukin 15 receptor alpha
  • Embodiment 2 The IL-15 variant according to Embodiment 1 , wherein wild-type IL-15 comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1.
  • Embodiment s The IL-15 variant according to Embodiment 2, wherein in comparison to SEQ ID NO: 1 , the amino acid modifications occur at one or more of amino acid positions 45 and/or 49 and/or 52, or any combination thereof.
  • Embodiment 4 An interleukin-15 (IL-15) variant, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 , and wherein in comparison to SEQ ID NO: 1 , the IL-15 variant comprises an amino acid modification at one or more of amino acid positions 45 and/or 49 and/or 52, or any combination thereof.
  • IL-15 interleukin-15
  • Embodiment 5 The IL-15 variant according to any one of Embodiments 2 to 4, wherein the IL-15 variant comprises an amino acid sequence according to SEQ ID NO: 1 , or an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1.
  • Embodiment 6 The IL-15 variant according to any one of Embodiments 2 to 5, wherein the IL-15 variant comprises an amino acid modification at amino acid position 75.
  • Embodiment 7 The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 45 is a hydrophobic-to-hydrophilic substitution.
  • Embodiment s The IL-15 variant according to Embodiment 7, wherein the hydrophobic-to-hydrophilic substitution is a leucine-to-serine substitution or a leucine-to-threonine substitution.
  • Embodiment 9 The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 49 is a hydrophobic-to-hydrophobic substitution.
  • Embodiment 10 The IL-15 variant according to Embodiment 9, wherein the hydrophobic-to- hydrophobic substitution is a valine-to-alanine substitution.
  • Embodiment 11 The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 52 is a hydrophobic-to-hydrophilic substitution.
  • Embodiment 12 The IL-15 variant according to Embodiment 11 , wherein the hydrophobic-to-hydrophilic substitution is a leucine-to-lysine substitution or a leucine-to-arginine substitution.
  • Embodiment 13 The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the amino acid modification at amino acid position 75 is a serine-to-proline substitution.
  • Embodiment 14 The IL-15 variant according to Embodiment 13, wherein the IL- 15 variant comprising the serine-to-proline substitution at amino acid position 75 comprises a sequence according to SEQ ID NO: 16, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 16, wherein the % sequence identity to SEQ ID NO: 16 retains the amino acid modifications at amino acid position 75.
  • Embodiment 15 The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the serine-to-proline substitution at amino acid position 75.
  • Embodiment 16 The IL-15 variant according to Embodiment 15, wherein the IL- 15 variant comprises an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO:
  • Embodiment 17 The IL-15 variant according to Embodiment 16, wherein the IL- 15 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2, wherein the % sequence identity to SEQ ID NO: 2 retains the amino acid modifications at amino acid positions 45 and 75.
  • Embodiment 18 The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the valine-to-alanine substitution at amino acid position 49; and iii) the serine-to-proline substitution at amino acid position 75.
  • Embodiment 19 The IL-15 variant according to Embodiment 18, wherein the IL- 15 variant comprises an amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO:
  • Embodiment 20 The IL-15 variant according to Embodiment 19, wherein the IL- 15 variant comprises an amino acid sequence comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3, wherein the % sequence identity to SEQ ID NO: 3 retains the amino acid modifications at amino acid positions 45, 49 and 75.
  • Embodiment 21 The IL-15 variant according to Embodiment 3 or 4 or any one of those pendent thereon, wherein the IL-15 variant comprises: i) the leucine-to-serine substitution at amino acid position 45; and ii) the leucine-to-lysine substitution at amino acid position 52; and iii) the serine-to-proline substitution at amino acid position 75.
  • Embodiment 22 The IL-15 variant according to Embodiment 21 , wherein the IL- 15 variant comprises an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence comprising at least 70% sequence identity to SEQ ID NO:
  • Embodiment 35 The IL-15 variant according to Embodiment 33 or 34, wherein the IL-15 variant is fused via a linker, preferably wherein the linker is a glycine-serine linker.

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Abstract

L'invention concerne une bactérie comprenant un variant d'interleukine-15 (IL-15), par rapport à l'IL-15 de type sauvage, le variant d'IL-15 comprenant une ou plusieurs modifications d'acide aminé dans une région de surface adjacente à une surface d'interaction de récepteur, de préférence la surface d'interaction de récepteur étant une surface d'interaction de récepteur alpha de l'interleukine 15 (IL-15Rα).
PCT/EP2024/077120 2023-09-28 2024-09-26 Variant d'interleukine 15 Pending WO2025068401A1 (fr)

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EP0109942A2 (fr) 1982-10-18 1984-05-30 Bror Morein Complexe protéinique ou peptidique immunogène, procédé pour préparer ce complexe et son usage comme immunostimulant et comme vaccin
EP0180564A2 (fr) 1984-11-01 1986-05-07 Bror Morein Complexe immunogénique, procédé de préparation et son utilisation comme immunostimulant, vaccins et réactifs
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