HK1233307A1 - Modified natural killer cells and uses thereof - Google Patents

Abstract

The present invention provides, in certain aspects, a natural killer (NK) cell that expresses all or a functional portion of interleukin-15 (IL-15), and methods for producing such cells. The invention further provides methods of using a natural killer (NK) cell that expresses all or a functional portion of interleukin-15 (IL-15) to treat cancer in a subject or to enhance expansion and/or survival of NK cells.

Description

Modified natural killer cells and uses thereof

RELATED APPLICATIONS

This application claims benefit of U.S. provisional application No.61/993,494 filed on 5, 15, 2014. The entire teachings of the above application are incorporated herein by reference.

Background

NK cells require stimulation by cytokines (e.g., IL-2 and IL-15) for survival and proliferation in vivo. For example, after injection into immunodeficient mice, activated NK cells become undetectable after 1 week, but remain detectable for up to one month if human IL-2 is also administered. Therefore, clinical protocols using NK cell infusion typically rely on IL-2 administration to prolong NK cell survival in patients. However, IL-2 can have considerable side effects. In addition to fever and chills, IL-2 administration can lead to more serious and potentially fatal consequences, such as capillary leak syndrome (capillary leak syndrome). Reducing the dose of IL-2 should reduce the risk of side effects, but may result in stimulation of regulatory T cells that can inhibit NK cell function and possibly negate their anti-cancer effects.

Therefore, it would be important to develop alternative means of promoting NK cell expansion and activity in vitro and/or in vivo.

Brief Description of Drawings

This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

The foregoing will be apparent from the following more particular description of some example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating some embodiments of the invention.

FIGS. 1A-1C: design and expression of IL-15 constructs. Schematic of wild-type and membrane-bound IL-15 constructs ("wtIL 15" and "mbIL 15") used in this study. Expression of IL-15 on the surface of NK cells transduced with mbiL 15. Expanded NK cells were transduced with wtIL15, mbIL15 or with vectors containing GFP only ("Mock"). Flow cytometry dot plots show the expression of GFP and IL-15 as detected by anti-IL 15 antibodies (R & D Systems) and goat anti-mouse secondary antibodies conjugated to phycoerythrin (phytoerythrin) (Southern Biotechnology Associates). The percentage of cells in each quadrant (> 98% CD56+ CD3-NK cells) is shown. IL-15 secretion from NK cells transduced with wtIL 15. NK cells from 3 different donors were tested in triplicate. Bars represent the mean ± SD of ELISA measurements performed in supernatants collected after 24 and 48 hours of incubation without IL-2. No IL-15 was detected in the supernatant of mock-transduced cells.

FIGS. 2A-2C: survival and expansion of NK cells expressing IL-15 in vitro. Percentage NK cell recovery compared to IL-2 free transfused cells from mock transduced and mbIL15 transduced cells from 15 donors (left panel) and IL-2 free 7-day row cultured transfused cells from mbIL15 or wtIL15 from 9 donors. The horizontal bars represent median values. The results of the paired t-test are shown. The results of the incubation with IL-2(10 and 100IU/mL) are shown in supplementary FIG. S1. Mock transduction from 6 donors with low dose IL-2(10IU/mL) and survival and expansion of mbIL15 transduced NK cells. Expansion and long-term survival of NK cells transduced or mock transduced with mbIL15, wtIL15 cultured without IL-2 or with low dose IL-2 (results for 100IU/mL IL2 are shown in figure 6) from one donor. Percentage NK cell recovery at the indicated days of culture is shown.

FIGS. 3A-3C: survival and expansion of NK cells expressing mb-IL15 in vivo. Absolute number of human CD45+ cells in peripheral blood of mice injected with or without mock-transduced or mbIL 15-transduced NK cells (16 mice total) 7 and 11 days post infusion (P ═ 0.004 at day 7 without IL-2, P ═ 0.021 with IL-2, P ═ 0.044 and 0.026 at day 11). Flow cytogram shows the presence of human CD45+, GFP + NK cells in peripheral blood of mice treated without IL-2 (upper) and with IL-2 (lower). The percentage of human CD45+ cells with or without GFP expression is shown. Percentage of human CD45+ cells in various tissues of mice injected with mock-transduced or mbIL 15-transduced NK cells with or without IL-2 collected on day 11 post-injection. Overall, the percentage of mbIL15 human CD45+ cells was significantly higher (P < 0.001 without IL-2 and P ═ 0.002 with IL-2).

FIGS. 4A-4C: nature of NK cells expressing mbIL 15. Relative proportion of GFP + cells before and after 7 days of culture in a population of NK cells transduced or mock transduced with mbIL 15. Results for NK cells from 13 donors are shown; for mbIL15P < 0.001, not significant for the simulation. Immunophenotypic characterization of mbil 15-transduced NK cells. NK cells cultured for 48 hours in the absence of IL-2 were subjected to cell marker analysis by flow cytometry. All results are summarized in the table. Mock-transduced and mbIL 15-transduced NK cells were cultured in the absence of IL-2 for 48 hours and cell lysates were analyzed by Kinex antibody microarray (Kinexus). Of the 809 anti-phosphoprotein antibodies tested, those with a Z ratio of signal > 0.5 and a margin of error% < 100 were shown. Bars represent the percent change in signal in NK cells expressing mbIL15 compared to the normalized intensity in mock-transduced NK cells.

5A-5D antitumor Capacity of NK cells expressing mbIL 15. 5A. the results of 24 hour cytotoxicity assays with 1: 4 and 1: 1E: T ratios (15 experiments at each ratio; two ratios of P < 0.001) of mbIL15 from 9 donors and mock-transduced NK cells against Nalm-6, U937, K562, Daudi, SK-BR-3 and ES8 cell lines the results obtained with a single cell line in the 4 hour and 24 hour cytotoxicity assays are shown in FIG. 7. 5B. NK cells expressing mbIL15 release lytic particles in the presence of target cells increase. the percentage of CD107a + NK cells after 4 hour cytotoxicity assay at 1: 1E: T. the results of NK cells from 3 donors against 2 cell lines (P0.007). 5℃ NK cells expressing mbIL15 show in vivo antitumor activity in SCLII-2 RGD-5910. RGD-10. RG.5910⁴U937 cells labeled with luciferase in 3 mice, no treatment ("No NK"), while 4 mice received mock-transduced NK cells on days 3 and 7 (1 × 10)⁷i.p.) and 4 additional mice received mbIL15 transduced NK cells at the same dose and schedule in vivo imaging results showing tumor growth (abdominal images) 5d overall survival comparison of mice in different treatment groups when bioluminescence reached 1 × 10¹¹Mice were euthanized at one photon/second. P values for the log rank test for 3 curves and comparisons between each of the 2 curves are shown.

FIGS. 6A-6C: survival and expansion of NK cells expressing IL-15 in vitro. The expansion of NK cells expressing mbIL15 in the absence of IL-2 was inhibited by anti-IL-15 neutralizing antibodies. The symbols show the mean NK cell recovery during culture compared to the input cells in experiments with NK cells transduced with mbIL15(± SD; n ═ 3). Percentage of NK cell recovery compared to input cells after 7-day row culture of mock transduced, mbIL15 transduced, and wtIL15 transduced cells from 6 donors with low dose (10IU/mL) and high dose (100IU/mL) IL-2. The horizontal bars represent median values. The results of the paired t-test are shown. Expansion and long-term survival of NK cells from one donor transduced with mbIL15 or wtIL15 and cultured with 100IU/mL IL 2. Percentage NK cell recovery at the indicated days of culture is shown.

FIGS. 7A-7B: antitumor capacity of NK cells expressing mbIL 15. Results of 4 hour (7A) and 24 hour (7B) cytotoxicity assays for mbIL15 and mock-transduced NK cell anti-Naim-6, U937, K562, Daudi, SK-BR-3, and ES8 cell lines at 1: 4, 1: 2, and 1: 1E: T ratios are shown. Each symbol represents the mean. + -. SD cytotoxicity in experiments with NK cells from 3 different donors for U937, K562, ES8 and NK cells from 2 donors for Nalm-6, Daudi and SK-BR-3, all experiments performed in triplicate (P < 0.001 for all experiments).

FIGS. 8A-8C antitumor Capacity of NK cells expressing mbiL15 injection of luciferase-labeled 1 × 10 into NOD-SCID-IL2RGnull mice i.p⁵In 7 mice, no treatment ("no NK"), while 11 mice received mock-transduced NK cells on day 3 (1 × 10)⁷i.p.) and another 12 mice received mbIL15 transduced NK cells at the same dose and schedule 8a. in vivo imaging results of tumor growth. abdominal images of 4 mice with the highest tumor signal in each group are shown 8b. in vivo imaging results of tumor growth each symbol corresponds to one bioluminescence measurement (3 measured photons/second on relevant days in each mouse) 8c. overall survival comparison of mice in different treatment groups when bioluminescence reaches 1 × 10¹⁰Mice were euthanized at one photon/second. P values for the log rank test for 3 curves and comparisons between each of the 2 curves are shown.

FIG. 9 shows the nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of membrane-bound IL-15.

FIG. 10 shows the nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of human IL-15(NCBI reference sequence: NM-000585.4).

FIGS. 11A-11C: mbIL15 stimulated NK cells by cis-presentation. NK92 cells were transduced with mbIL15 (left) or wtIL15 (right) in GFP-containing vectors, sorted to obtain 100% GFP + cells, and co-cultured with untransduced NK92 cells at a 1: 1 ratio. The percentage recovery of cells after culture for GFP + and GFP-cells relative to the number of cells at the beginning of the culture (. + -. SD; n. sub.3) is shown. In the combinations shown, mbIL 15-expressing or untransduced NK92 cells were co-cultured with K562 cells ("K") transduced or untransduced with mbIL15 in a ratio of 1: 2. K562 cells were labeled with PKH26(Sigma) and treated with Streck cell preservative (Streck, Omaha, NE) to prevent cell division prior to culture. The percentage recovery (. + -. SD; n. sub.3) of NK92 cells after culture relative to the number of cells at the start of culture is shown. Proliferation of NK92 cells expressing mbIL15 compared to the proliferation of untransduced NK92 cells in the presence of increasing concentrations of exogenous IL-15. Cultures were either incubated in the absence of IL-2 (left) or with 10IU/mL (middle) or 100IU/mL (right) of IL-2. The percentage of cells recovered after culture (. + -. SD; n. sub.3) relative to the number of cells at the start of culture is shown.

FIG. 12A-12℃ mb15-NK cell KIR expression and function. NK cell subpopulations defined by KIR expression before and after transduction thereof and mock transduction or mb15 transduction. Flow cytograms show the results of staining with anti-KIR antibodies in CD56+ CD 3-cells from 2 donors. The percentage of KIR + cells is shown. Results of CD107a expression in CD158a positive and CD158a negative subpopulations after 4 hours of culture of 721.221 cells or the same cells expressing CD158a bound to Cw6 HLA. The mean (. + -. SD) of 4 independent experiments of NK cells from 3 donors (P < 0.0001;. P0.0002) is shown. 12C. results of IFN γ secretion in the same experiment shown in 12B (P < 0.0001).

Fig. 13A and 13B: antibody-dependent cellular cytotoxicity (ADCC) of NK cells expressing mbiL 15. Results of 4 hour ADCC assays of mbIL15 and mock-transduced NK cells against (13A) Daudi and (13B) SK-BR-3, respectively, in the presence of rituximab or trastuzumab; IgG of the same concentration of immunotherapeutic antibody (1. mu.g/mL) was used as a control. Each symbol indicates the mean ± SD cytotoxicity in triplicate experiments of NK cells from each donor. In the presence of immunotherapeutic antibodies, mbIL15-NK cells showed significantly higher ADCC than mock-transduced cells (P < 0.001 for either donor in the assay with Daudi or SK-BR-3). The cytotoxicity of antibody-free mbIL15-NK cells was also significantly higher (P < 0.001 for either donor in the test with Daudi or SK-BR-3).

Detailed description of the invention

Some example embodiments of the invention are described below.

The well-known anti-leukemic activity of Natural Killer (NK) cells suggests therapeutic potential for NK cell infusion. NK cell survival (and hence cytotoxicity) requires cytokine support. Described herein are experiments to investigate whether expression of a non-secreted membrane-bound form of interleukin-15 (IL-15) could sustain NK cell growth. The human IL15 gene was linked to a gene encoding the CD8 α transmembrane domain ("mbIL 15"). Following retroviral transduction, human NK cells expressed mbIL-15 on the cell surface, but IL-15 secretion was negligible. Survival and expansion of IL-2-free mbIL15-NK cells was greatly superior to that of mock-transduced cells (P < 0.0001, n-15 after 7 days of culture) and to NK cells secreting non-membrane bound IL-15 (P-0.025, n-9); viable mbIL15-NK cells were detectable for up to 2 months. In immunodeficient mice, mbIL15-NK cells were expanded in the absence of IL-2 and could be detected in much higher numbers (P < 0.001) in all examined tissues (except brain) than mock-transduced NK cells. In the presence of IL-2, in vitro and in vivo expansion was further improved. The primary mechanism of mbIL15 stimulation is autocrine; it activates IL-15 signaling and anti-apoptotic signaling. The cytotoxicity of cell lines against leukemia, lymphoma and solid tumors was consistently higher than that of mbIL15-NK cells. The 24 hour cytotoxicity was 71% at the median 1: 4E: T, while 22% for mock-transduced cells, 99% and 54% at 1: 1E: T (P < 0.0001). Enhanced antitumor capacity was also evident in immunodeficient mice transplanted with leukemia (U937) or sarcoma (ES8) cells. Therefore, mbIL15 enabled NK cells to grow independently and enhance their anti-tumor capacity. Infusion of mbIL15-NK cells allowed NK cell therapy without the adverse effects of IL-2.

Thus, provided herein is a cell(s) expressing all or a functional portion of interleukin-15 (IL-15), wherein the cell is an IL-15 responsive cell. Cells that respond to IL-15 include cells in which one or more activities are modulated by IL-15. Examples of such cells include Natural Killer (NK) cells, T cells, dendritic cells, and monocytes. One or more (e.g., isolated) cells can express all or a functional portion of IL-15 as a membrane-bound polypeptide, as a secreted protein, or as a combination thereof.

In one aspect, the invention relates to Natural Killer (NK) cells expressing all or a functional portion of interleukin-15 (IL-15). One or more (e.g., isolated) NK cells may express all or a functional portion of IL-15 as a membrane-bound polypeptide, as a secreted protein, or as a combination thereof.

As used herein, "natural killer cell" ("NK cell") refers to a cytotoxic lymphocyte of the immune system. NK cells provide a rapid response to virus-infected cells and respond to transformed cells. Generally, immune cells detect peptides from pathogens that are presented by Major Histocompatibility Complex (MHC) molecules on the surface of infected cells, triggering cytokine release, causing lysis (lysis), or apoptosis. However, NK cells are unique in that they have the ability to recognize stressed cells regardless of whether peptides from pathogens are present on MHC molecules. They are named "natural killer cells" because they do not require the initial insight of prior activation to kill the target. NK cells are Large Granular Lymphocytes (LGL) and are known to differentiate and mature in the bone marrow where they then enter the circulation.

In some aspects, the NK cell is a mammalian NK cell. Examples of "mammal" or "mammal" include primates (e.g., humans), canines (canines), felines (felines), rodents, swine, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cattle, sheep, goats, rabbits, guinea pigs, rats, and mice. In a specific aspect, the mammalian NK cell is a human NK cell.

As used herein, "interleukin-15" ("IL-15") refers to a cytokine that regulates the activation and proliferation of T and NK cells. The cytokine and interleukin 2 share many biological activities. They were found to bind to a common receptor subunit and could compete for the same receptor and thus down-regulate each other's activity. It was shown that the number of CD8+ memory cells was controlled by the balance between IL-15 and IL-2. This cytokine induces the activation of JAK kinases and the phosphorylation and activation of the activators of transcription STAT3, STAT5, and STAT6, and can increase the expression of the apoptosis inhibitor BCL2L1/BCL-x (L) by the transcriptional activation activity of STAT6, and thus prevent apoptosis.

A "functional portion" ("biologically active portion") of IL-15 refers to a portion of IL-15 that retains one or more functions of full-length or mature IL-15. Such functions include promoting NK cell survival, regulating NK cell and T cell activation and proliferation, and maintaining NK cell development from hematopoietic stem cells.

As will be appreciated by those skilled in the art, the sequence of a variety of IL-15 molecules is known in the art. In one aspect, the IL-15 is a wild-type IL-15. In some aspects, IL-15 is a mammalian IL-15 (e.g., human (Homo sapiens) interleukin 15(IL15), transcript variant 3, mRNA, NCBI reference sequence: NM-000585.4; canine (Canis lupusfamiliaris) interleukin 15, mRNA, NCBI reference sequence: NM-001197188.1; feline (Felis cat) interleukin 15(IL15), mRNA, NCBI reference sequence: NM-001009207.1). Examples of "mammal" or "mammal" include primates (e.g., humans), canines, felines, rodents, swine, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cattle, sheep, goats, rabbits, guinea pigs, rats, and mice. In a specific aspect, the mammalian IL-15 is human IL-15.

All or a functional portion of IL-15 can be expressed by one or more NK cells (as membrane-bound and/or secreted polypeptides) in a variety of ways. For example, all or a functional portion of IL-15 may be expressed within and secreted by NK cells and/or may be linked (conjugated; fused) directly or indirectly (e.g., ionically, non-ionically, covalently) to the surface of NK cells (e.g., on the surface or within the membrane of NK cells) using any of a variety of linkers known in the art (Hermanson, G., Bioconjugate technologies, Academic Press 1996). In some particular aspects, all or a functional portion of IL-15 is linked to all or a portion of a transmembrane protein. In one aspect, the NK cell expresses a fusion protein comprising all or a portion of IL-15 fused to all or a portion of the transmembrane protein. In a particular aspect, the portion of the transmembrane protein comprises all or a portion of the transmembrane domain of the transmembrane protein.

As used herein, a "transmembrane protein" or "membrane protein" is a protein located at and/or within a membrane, such as a phospholipid bilayer of a biological membrane (e.g., a biological membrane such as a cell membrane). Membrane proteins enable membranes to perform their unique activities. The complement of proteins (complement) attached to the membrane varies according to cell type and subcellular localization. Some proteins bind only to the membrane surface, while others have one or more regions embedded in the membrane and/or domains on one or both sides of the membrane. Protein domains on the surface of the outer membrane of cells are often involved in cell-cell signaling or interactions. The domains located on the cytoplasmic surface of the membrane have a wide range of functions, from anchoring cytoskeletal proteins to the membrane to triggering intracellular signaling pathways. Regions within the membrane are referred to herein as "transmembrane domains," particularly those that form channels and pores for the movement of molecules through the membrane. A "transmembrane domain" is a three-dimensional protein structure that is thermodynamically stable in a membrane (e.g., the membrane of a vesicle (e.g., a cell)). Examples of transmembrane domains include a single alpha helix, a stable complex of several transmembrane alpha helices, a transmembrane beta barrel structure (betabarrel), the beta helix of gramicidin a, or any other structure. Transmembrane helices are typically about 20 amino acids long.

Generally, membrane proteins are divided into two broad classes, integral (intrinsic) and peripheral (extrinsic) based on the nature of membrane-protein interactions. Most biological membranes contain two types of membrane proteins.

Integral membrane proteins (also known as intrinsic proteins) have one or more fragments embedded in a phospholipid bilayer. Integral membrane proteins include transmembrane proteins and lipid-anchored proteins. Most integrins contain residues with hydrophobic side chains that interact with fatty acyl groups of membrane phospholipids, anchoring the protein to the membrane. Most integrins span the entire phospholipid bilayer. These transmembrane proteins comprise one or more transmembrane domains and a domain that extends into the aqueous medium on each side of the bilayer that is 4 to several hundred residues long. Typically, the transmembrane domain is one or more (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more) alpha helices and/or beta strands (β strand). Transmembrane alpha helical domains are typically embedded in the membrane by hydrophobic interactions with lipids inside the bilayer and possibly also by ionic interactions with polar head groups of phospholipids (e.g., glycophorin). The structure of the beta chain is usually in the form of a transmembrane barrel-shaped structure (e.g., porin). Some integrins are anchored to one of the membrane leaflets (leaflets) by covalently bound fatty acids. In these proteins, the bound fatty acids are embedded in the membrane, but the polypeptide chains do not enter the phospholipid bilayer. Some cell surface proteins are anchored to the outer lipid membrane (exolaser face) of the plasma membrane by complex glycosylated phospholipids (e.g., glycosylphosphatidylinositol, alkaline phosphatase) attached to the C-terminus. Some cytoplasmic proteins are anchored to the cytoplasmic face of the membrane by a hydrocarbon moiety (e.g., prenyl, farnesyl and geranylgeranyl groups) covalently linked to a cysteine near the C-terminus. In another group of lipid-anchored cytosolic proteins, a fatty acyl group (e.g., myristate or palmitate) is linked to the N-terminal glycine residue through an amide bond.

The peripheral membrane proteins or exogenous proteins do not interact with the hydrophobic core of the phospholipid bilayer. Instead, they are usually bound to the membrane indirectly through interaction with integral membrane proteins or directly through interaction with lipid polar head groups. The peripheral proteins localized to the cytoplasmic surface of the plasma membrane include the cytoskeletal proteins spectrin and actin and the enzyme protein kinase C in erythrocytes. This enzyme shuttles between the cytosol and the cytoplasmic surface of the plasma membrane and plays a role in signal transduction. Other peripherins, including certain proteins of the extracellular matrix, localize to the outer (extralipid) surface of the plasma membrane.

Examples of transmembrane proteins include receptors, ligands, immunoglobulins, glycophorins or combinations thereof. Specific examples of transmembrane proteins include CD8 α, CD4, CD3, CD3 γ, CD3, CD3 ζ, CD28, CD137, FcRI γ, T cell receptors (TCRs, e.g., TCR α and/or TCR β), nicotinic acetylcholine receptors, GABA receptors, or combinations thereof. Specific examples of immunoglobulins include IgG, IgA, IgM, IgE, IgD, or combinations thereof. Specific examples of glycophorin include glycophorin A, glycophorin D, or combinations thereof.

In addition to being linked to all or a portion of the transmembrane protein, all or a functional portion of IL-15 may be linked to other components such as: signal peptides (e.g., CD 8a signal sequence), leader sequences, secretion signals, markers (e.g., reporter genes), and the like. In a particular aspect, all or a functional portion of IL-15 is fused to all or a portion of the signal peptide of CD8 α and the transmembrane domain of CD8 α.

In another aspect, the invention relates to a method of producing Natural Killer (NK) cells that express all or a functional portion of interleukin-15 (IL-15). All or a portion of IL-15 may be expressed as a membrane-bound polypeptide, a secreted polypeptide, or a combination thereof. The method comprises introducing a nucleic acid encoding all or a functional portion of IL-15 into one or more NK cells. In one aspect, a nucleic acid encoding all or a functional portion of IL-15 is linked (e.g., fused) to all or a portion of a transmembrane protein. Alternatively or in addition, a nucleic acid encoding all or a functional portion of IL-15 is introduced into NK cells (e.g., wild-type IL-15). As will be apparent to those skilled in the art, aspects in which nucleic acids encoding all or a functional portion of IL-15 and all or a functional portion of IL-15 fused to all or a portion of a transmembrane protein are introduced into NK cells may be so conducted using a single nucleic acid or multiple (e.g., separate; two) nucleic acids. Maintaining the NK cells under conditions wherein all or a functional portion of IL-15 is expressed as a membrane-bound polypeptide and/or as a secreted polypeptide, thereby producing NK cells expressing all or a functional portion of IL-15 as a membrane-bound polypeptide and/or as a secreted polypeptide. In a specific aspect, a nucleic acid encoding all or a functional portion of IL-15 is fused to a signal peptide of CD8 α and all or a portion of the transmembrane domain of CD8 α is introduced into NK cells.

In yet another aspect, the invention relates to methods of enhancing the expansion and/or survival (e.g., in vitro, ex vivo, and/or in vivo) of NK cells. The method comprises introducing a nucleic acid encoding all or a functional portion of IL-15. Nucleic acids encoding all or a portion of IL-15 (e.g., wild-type IL-15) and/or encoding all or a functional portion of IL-15 fused to all or a portion of a transmembrane protein may be introduced into NK cells. Thus, NK cells may express all or a functional portion of IL-15 as a membrane-bound polypeptide, a secreted polypeptide, or as a combination thereof. Maintaining the NK cells under conditions wherein all or a portion of IL-15 is expressed as a membrane-bound polypeptide, a secreted polypeptide, or a combination thereof and wherein the NK cells proliferate. In a specific aspect, a nucleic acid encoding all or a functional portion of IL-15 is fused to a signal peptide of CD8 α and all or a portion of the transmembrane domain of CD8 α is introduced into NK cells. In some aspects, the methods can further comprise contacting an NK cell comprising membrane-bound IL-15 and/or secreted IL-15 with IL-2. In some aspects, the concentration of IL-2 is from about 10IU/ml to about 1000 IU/ml. In other aspects, the concentration of IL-2 is about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980 IU/ml.

It will be apparent to those skilled in the art that a variety of methods (e.g., transfection, transduction, and/or transposon systems) for introducing nucleic acids encoding all or a functional portion of IL-15 as a transmembrane polypeptide and/or as a secreted polypeptide into NK cells may be used. Examples of such methods include chemical-based methods (e.g., involving the use of calcium phosphate; highly branched organic compounds (e.g., dendrimers), liposomes (lipofection), and/or cationic polymers (e.g., DEAE dextran; polyethyleneimine)), non-chemical-based methods (e.g., electroporation; cell extrusion; sonoporation; optical transfection; puncture transfection; hydrodynamic delivery), particle-based methods (e.g., gene guns; magnetic transfection; particle bombardment), vector-based methods (e.g., vectors comprising viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, etc.), nuclear transfection, transposon-based methods (e.g., sleepingbauty, PiggyBAC, etc.), and/or RNA transfection.

Also apparent to those skilled in the art are various methods for maintaining NK cells under the following conditions: wherein (i) all or a functional portion of IL-15 is expressed as a membrane-bound polypeptide and/or as a secreted polypeptide and/or (ii) NK cells comprising membrane-bound IL-15 and/or a secreted IL-15 proliferator may be used. For example, the NK cells can be at a suitable temperature and gas mixture (e.g., about 25 ℃ to about 37 ℃, about 5% CO in a cell culture incubator)₂) Growth and/or maintenance. Culture conditions can vary widely, and changes in conditions of particular cell types can result in different phenotypes. A factor that is often varied in culture systems, in addition to temperature and gas mixture, is the cell growth medium. The formulation of the growth medium may vary in terms of pH, glucose concentration, growth factors and the presence of other nutrients. The growth factors used to supplement the culture medium are typically derived from animal blood serum, such as Fetal Bovine Serum (FBS), calf serum, horse serum, pig serum, and/or human platelet lysate (hPL). Other factors considered for maintaining cells include graftingSeed density (number of cells per volume of medium) and growth of cells in suspension or adherent culture.

The methods can further comprise isolating or isolating one or more NK cells produced by the methods provided herein. In addition, the method may further comprise culturing the one or more NK cells. In some aspects, an NK cell line is generated.

The invention also encompasses Natural Killer (NK) cell(s) or cell lines produced by the methods described herein and compositions comprising NK cells provided herein. In a particular aspect, the composition is a pharmaceutical composition comprising one or more NK cells or cell lines provided herein. The pharmaceutical composition can also comprise all or a functional portion of IL-2 (e.g., all or a functional portion of the IL-2 protein(s); a nucleic acid encoding all or a functional portion of IL-2).

As used herein, "IL-2" refers to a member of the cytokine family that also includes IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2 signals through a receptor complex consisting of three chains (termed α, β and γ). The gamma chain is shared by all members of the cytokine receptor family. IL-2 (which is similar to IL-15) promotes the production of immunoglobulins produced by B cells and induces the differentiation and proliferation of NK cells. The major difference between IL-2 and IL-15 was found in the adaptive immune response. For example, IL-2 is essential for adaptive immunity to foreign pathogens because it is the basis for the development of immunological memory. On the other hand, IL-15 is essential for maintaining a highly specific T cell response by maintaining the survival of CD8 memory T cells.

In another aspect, the invention relates to a method of treating a disease and/or disorder involving NK cell therapy in an individual in need thereof, comprising administering to the individual Natural Killer (NK) cells expressing all or a functional portion of interleukin-15 (IL-15). In some particular aspects, the NK cells express all or a functional portion of IL-15 as a membrane-bound polypeptide and/or as a secreted polypeptide. As known in the art, diseases and/or disorders involving NK cell therapy include NK cell deficiency, cancer, autoimmune diseases, infectious diseases, and the like.

In a particular aspect, the invention relates to a method of treating cancer (e.g., a tumor) in an individual in need thereof, comprising administering to the individual Natural Killer (NK) cells that express all or a functional portion of interleukin-15 (IL-15). All or a functional portion of IL-15 may be expressed as a membrane-bound polypeptide and/or a secreted polypeptide.

The methods can further comprise administering one or more antibodies, antigen fragments, and/or fusions thereof specific for a cancer (e.g., a tumor). For example, the method may further comprise administering one or more antibodies against one or more tumor antigens. As will be understood by those of skill in the art, the one or more antibodies can be polyclonal antibodies, monoclonal antibodies, multivalent (e.g., bivalent, trivalent) antibodies, chimeric antibodies, humanized antibodies, and the like, and combinations thereof. A variety of antigenic fragments and/or fusions are also known in the art and include Fab ', F (ab')₂Single-chain variable fragments (scFv), multivalent scFv (e.g., di-scFv, tri-scFv), single domain antibodies (nanobodies), and the like.

In some aspects, the cancer is a leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), myelodysplastic syndrome, a lymphoma (e.g., B-cell non-Hodgkin lymphoma, T-cell lymphoblastic lymphoma, anaplastic large cell lymphoma), a solid tumor (e.g., breast cancer, prostate cancer, gastric cancer, colon cancer, hepatocellular carcinoma, nasopharyngeal cancer, neuroblastoma, high grade glioma), a sarcoma (e.g., Ewing sarcoma, rhabdomyosarcoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma).

The method of treating cancer may further comprise administering IL-2 (all of the IL-2 protein) to the subjectOr a functional moiety; nucleic acid encoding all or a functional portion of IL-2). In one aspect, IL-2 is a mammalian IL-2, such as human IL-2. In a particular aspect, a low dose of IL-2 is administered to an individual. As used herein, a "low dose" of IL-12 refers to about 1 million IU/m²Or smaller doses (e.g., about 800,000IU/m²；600,000IU/m²；400,000IU/m²；200,000IU/m²；100,000IU/m²；80,000IU/m²；60,000IU/m²；40,000IU/m²；20,000IU/m²；10,000IU/m²；8,000IU/m²；6,000IU/m²；4,000IU/m²；2,000IU/m²；1,000IU/m²；800IU/m²；600IU/m²；400IU/m²；200IU/m²；100IU/m²) IL-2 of (1). In contrast, the normal dose of IL-2 is about 1 million IU/m²To about 5 million IU/m²。

One or more Natural Killer (NK) cells (e.g., therapeutic compounds; pharmaceutical compositions) that express all or a functional portion of interleukin-15 (IL-15) are administered in a therapeutically effective amount (i.e., an amount sufficient to treat cancer, e.g., by alleviating a symptom associated with cancer, preventing or delaying the onset of cancer, also reducing the severity or frequency of a cancer symptom and/or preventing, delaying or overcoming cancer metastasis). The amount therapeutically effective in the treatment of a particular individual will depend on the symptoms and severity of the condition (e.g., cancer) and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be used to help determine the optimal dosage range. The precise dose to be used in the formulation will also depend on the route of administration and the severity of the cancer and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose response curves derived from in vitro or animal model test systems.

The therapeutic compound may be delivered in a composition (e.g., a pharmaceutical composition) as described above or by itself. They may be administered systemically or may be targeted to specific tissues. Therapeutic compounds can be produced in a variety of ways, including, for example, chemical synthesis; recombinant production; produced in vivo (e.g., transgenic animals such as U.S. Pat. No.4,873,316 to Meade et al) and can be isolated using standard means such as those described herein. Combinations of any of the above treatments may also be used.

The compounds for use in the methods described herein can be formulated with physiologically acceptable carriers or excipients to prepare pharmaceutical compositions. The carrier and composition may be sterile. The formulation should be suitable for the mode of administration.

Suitable pharmaceutically acceptable carriers include, but are not limited to: water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates (e.g., lactose), amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oils, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and the like, and combinations thereof. If desired, the pharmaceutical preparations can be mixed with auxiliaries, for example lubricants which do not deleteriously react with the active compounds, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffers, colorants, flavorants and/or aromatic substances and the like.

The composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

Methods of introducing these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, topical (topical), oral, and intranasal. Other suitable methods of introduction may also include gene therapy (as described below), reloadable or biodegradable devices, particle acceleration devices ("gene guns"), and sustained release polymer devices. The pharmaceutical compositions of the present invention may also be administered as part of a combination therapy with other compounds.

The compositions may be formulated according to conventional procedures as pharmaceutical compositions suitable for administration to humans. For example, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. If necessary, the composition may further comprise a solubilizing agent and a local anesthetic to relieve pain at the injection site. Generally, the ingredients are provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate, in a sealed container such as an ampoule or sachet (sachette) indicating the amount of active compound. In the case of administration of the composition by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. In the case of administration of the composition by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

For topical application, non-sprayable forms ranging from viscous to semi-solid or solid forms comprising a carrier compatible with topical application and preferably having a kinematic viscosity greater than water may be used. Suitable formulations include, but are not limited to: solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sol (sol), liniments, salves (salves), aerosols, etc., which are, if desired, sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.).

The compounds described herein may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, as well as those formed with free carboxyl groups such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, iron hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

In other aspects, the invention relates to pharmaceutical compositions comprising one or more NK cells expressing all or a functional portion of interleukin-15 (IL-15) as a membrane-binding polypeptide. The invention also relates to compositions (e.g., pharmaceutical compositions) for use as medicaments in therapy. For example, the agents identified herein may be used to treat cancer. In addition, the agents identified herein can be used in the preparation of a medicament for the treatment of cancer.

As used herein, "individual" refers to an animal, and in a particular aspect, to a mammal. Examples of mammals include primates, canines, felines, rodents, and the like. Specific examples include humans, dogs, cats, horses, cattle, sheep, goats, rabbits, guinea pigs, rats, and mice. The term "subject in need thereof" refers to a subject in need of treatment or prevention as determined by a researcher, veterinarian, medical doctor or other clinician. In one embodiment, the individual in need thereof is a mammal, e.g., a human.

As used herein, an "isolated", "substantially pure" or "substantially pure and isolated" NK cell(s) refers to cells of an NK: it is isolated (as opposed to substantially isolated) from the complex cellular environment in which it naturally occurs, or from the culture medium when produced by recombinant techniques, or from chemical precursors or other chemicals when chemically synthesized. In some cases, the isolated material will form part of a composition (e.g., a crude extract containing other materials), a buffer system, or a mixture of reagents. In other cases, the material may be purified to substantial homogeneity, e.g., as determined by agarose gel electrophoresis or column chromatography (e.g., HPLC). Preferably, the NK cells comprise at least about 50%, 80%, 90%, 95%, 98% or 99% (on a molar basis) of all macromolecular species present.

Unless indicated to the contrary or otherwise apparent from the context, the terms "a", "an", and "the" mean "one or more".

The phrase "and/or" as used in the specification and claims herein should be understood to mean "either or both" of the elements so combined. Multiple elements listed with "and/or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. Other elements may optionally be present in addition to the elements specifically identified by the "and/or" clause. As used herein in the specification and claims, "or" is understood to have the same meaning as "and/or" as defined above. For example, when used in a list of elements, "or" and/or "should be interpreted as being inclusive, i.e., including at least one, but optionally more than one, of the list of elements, and optionally additional unlisted elements. Only the opposite terms, such as "only one of" or "exactly one of," are explicitly indicated to include exactly one of the plurality or list of elements. Unless indicated to the contrary, claims including "or" between one or more members of a group are taken to satisfy that one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Some embodiments are provided in which exactly one member of a group is present in, employed with, or otherwise relevant to a given product or process. Some embodiments are provided in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Any one or more claims may be amended to expressly exclude any embodiment, aspect, feature, element, or characteristic, or any combination thereof. Any one or more claims may be modified to exclude any agent, composition, amount, dose, route of administration, cell type, target, cellular marker, antigen, targeting moiety, or combination thereof.

Examples of the invention

Materials and methods

Tumor cell lines

The human cell lines Nalm-6 (B-line acute lymphoblastic leukemia), Daudi (B-cell lymphoma), K562 and U937 (acute myeloid leukemia) and SK-BR-3 (breast cancer) were obtained from the American type culture Collection (American type culture Collection), Ewing sarcoma cell line ES8 from the St.Jude Children's Research Hospital tissue Bank. All cell lines were transduced with MSCV-Internal Ribosome Entry Site (IRES) -GFP retroviral Vector (from St.Jude Vector development and Production Shared Resource) containing the firefly luciferase gene. Transduced cells were selected for their GFP expression using either MoFlo (Beckman Coulter, Miami, FL) or FACSAria (BD Biosciences, San Jose, CA). All cell lines were maintained using RPMI-1640(Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, Mass.) and antibiotics. Cell lines are characterized by molecular and/or gene expression characteristics by the supplier; cell marker profiles of leukemia and lymphoma cell lines were tested regularly by flow cytometry to ensure no changes occurred and ES8 was verified by DNA fingerprinting (finger printing) at DSMZ (Braunschweig, Germany).

Human NK cell expansion

Peripheral blood samples were obtained as waste by-products from platelet collections from healthy adult donors. Mononuclear cells were purified by centrifugation on Accu-Prep density step (Accurate, Westbury, NY) and washed twice in RPMI-1640. To amplify CD56+ CD3-NK cells, as previously described in Fujisaki et al, Cancer Res, 69 (9): 4010 and 4017 (2009); imai et al, Blood, 106: 376-383(2005)), peripheral blood mononuclear cells were co-cultured with the genetically modified K562-mb15-41BBL cell line. Briefly, peripheral blood mononuclear cells were cultured with 100Gy irradiated K562-mb15-41BBL cells in a ratio of 1.5: 1 in 6-well tissue culture plates in SCGM (CellGenix, Freiburg, Germany) containing 10% FBS, antibiotics and 10IU/mL recombinant human interleukin-2 (IL-2; Roche, Mannheim, Germany). The tissue culture medium was replaced in portions every 2 days. After 7 days of co-culture, residual T cells were removed using Dynabeads CD3(Invitrogen) to give a cell population containing > 95% CD56+ CD3-NK cells.

Plasmid, viral production and gene transduction

pMSCV-IRES-GFP, pEQ-PAM3(-E) and pRDF were obtained from St.Jude Vector Development and production Shared Resource. Interleukin-15 (IL-15) with a long signal peptide was subcloned by Polymerase Chain Reaction (PCR) from the human spleen cDNA library used as template (from Dr neighbor, St Jud Children's Research Hospital). The cDNA encoding the signal peptide of CD8 α, the mature peptide of IL-15, and the transmembrane domain of CD8 α were assembled by splicing by overlapping extension PCR (SOE-PCR) to encode a membrane-bound form of IL-15 ("mbIL 15"); wild-type forms of IL-15 (not linked to the CD 8. alpha. transmembrane domain; "wtIL 15") were also tested. The resulting expression cassette (expression cassette) was subcloned into the EcoRI and XhoI sites of the murine Stem cell Virus internal ribosome entry site, Green fluorescent protein (MSCV-IRES-GFP).

To generate RD144 pseudotype retroviruses, X-tremagene 9DNA (Roche, Mannheim, Germany) was used to transfect 3.0 × 10⁶293T cells, together with 3.5. mu.g of cDNA encoding the mbiL15 construct, 3.5. mu.g of pEQ-PAM3(-E) and 3. mu.g of pRDF were kept in a 10cm tissue culture dish for 18 hours. After 24 hours medium exchange with RPMI-1640 with 10% FBS and antibiotics, conditioned medium containing retrovirus was harvested at 36-96 hours and added to a polypropylene tube coated with RetroNectin (Takara, Otsu, Japan), which was centrifuged at 1400g for 10 minutes and at 37 ℃ and 5% CO₂Incubated for 4 hours, after additional centrifugation and removal of supernatant, expanded NK cells (0.5-1 × 10)⁶) Added to the tube and left at 37 ℃ for 12 hours; these steps were repeated up to 6 times in 2-3 days. The cells were then maintained in RPMI-1640 with FBS, antibiotics and 100IU/ml IL-2. The transduced cells were assayed 3-29 days after transduction.

Surface expression of mbIL-15 was analyzed by flow cytometry using anti-human IL-15 antibodies (R & D, Minneapolis, MN) and phycoerythrin conjugated goat anti-mouse IgG1(Southern Biotech, Birmingham, AL). Antibody staining was detected with a Fortessa flow cytometer (Becton Dickinson). The level of IL-15 in the culture supernatant was measured by Quantikine immunoassay (R & D).

Functional analysis of NK cells in vitro

To assess NK cell survival and growth in vitro, transduced NK cells (1 × 10)⁶Individual cells/mL) were resuspended in RPMI-1640 with 10% FBS and antibiotics, plated in wells of 24-well or 96-well plates (Costar, Corning, NY), and cultured without or with IL-2(10-100 IU/mL). After staining with propidium iodide, the number of viable GFP + cells was determined with an Accuri C6 flow cytometer (Becton Dickinson). In some experiments, cells were incubated with neutralizing anti-IL-15 antibody (R) prior to culturing&D) Or isotype matched non-reactive antibody for 10 minutes.

NK cell immunophenotyping was performed using the antibodies listed in the table, displayed with a Fortessa flow cytometer, and analyzed by diva (Becton Dickinson) and FlowJo (TreeStar, Ashland, OR) software for phosphoprotein analysis, mock-transduced and mbiL 15-transduced NK cells (1 × 10) were analyzed in the absence of IL-2⁷) Culture for 48 hours cell lysates were prepared using lysis buffer containing 20mM 3- (N-morpholino) propanesulfonic acid, 2mM EGTA, 5mM EDTA, 30mM sodium fluoride, 60mM β -glycerophosphate, 20mM sodium pyrophosphate, 1mM sodium orthovanadate, 1% Triton X-100, Complete Mini protease inhibitor cocktail (Roche, Mannheim, Germany) and 1mM dithiothreitol after sonication the lysates were frozen at-80 ℃ and shipped to Kinexus (Vancouver, CA) on dry ice for Kinex antibody microarray analysis.

For cytotoxicity assays, luciferase-labeled target cells and NK cells (cultured for 48 hours in the absence of IL-2) were treated with multiple effectors: target (E: T) ratio was seeded in 96-well flat bottom black viewplates (Corning) and cultured for 4 or 24 hours. Adherent cell lines were plated at 37 ℃ and 5% CO prior to NK cell addition₂Incubate for 4 hours to allow the cells to attach. For antibody-dependent cytotoxicity assays, rituximab (Rituxan, Roche; Mannheim, Germany) was added before NK cells) Trastuzumab (Herceptin, Roche) or purified human IgG (R)&D Systems, Minneapolis, MN) (all 1. mu.g/mL). At the end of the incubation, an equal volume of Bright-Gl was then added to each test well_oLuciferase reagent (Promega, Madison, WI), 5 minutes later luminescence was measured using a plate reader and analyzed with gen52.00 software (all from BioTek, Tucson, AZ). In each plate, the luminescence signal of wells containing only target cells was used to calculate target cell viability. All experiments were performed in triplicate.

To measure the release of lytic granules, NK cells (cultured in the absence of IL-2 for 48 hours) were co-cultured with K562, U937 or 721.221 cells and their variants expressing Cw6 for 4 hours. PE-conjugated or PE-Cy 7-conjugated anti-CD 107a antibody (BD Biosciences) was added at the beginning of the culture, and GolgiStop (0.15. mu.L; BDbiosciences) was added after 1 hour. The percentage of CD107a + NK cells was determined by flow cytometry.

Expansion and cytotoxicity of NK cells in immunodeficient mice

To test NK cell expansion in vivo, transduction or mock transduction with mbiL15 (6-9 × 10 per mouse)⁶Individual cell) transduced human NK cells were injected into nod^scidIL2rg^tm1Wj1In the tail vein of a/SzJ (NOD/scid IL2RGnull) mouse (Jackson Laboratories, Bar Harbor, ME). In some mice 20000IU of IL-2 were injected intraperitoneally (i.p.) 3 times a week. On days 7 and 11, blood cells were counted using a cell counter (Beckman Coulter); after treatment of cells with erythrocyte blood lysis solution (Invitrogen) and staining with phycocyanin-conjugated mouse anti-human CD45 and phycoerythrin-conjugated rat anti-mouse CD45 antibodies (both from BD Biosciences), human and mouse CD45+ cells were counted by flow cytometry. After euthanasia, human NK cells in bone marrow, liver, spleen, kidney, lung and brain were counted as described above. All Animal experiments were performed according to protocols approved by the Institutional Animal Care and use committee of National University of Singapore institute (National University of Singapore institute) of Singapore.

To test tumor cell killing in mice, two xenograft models were prepared. In the first, luciferase-expressing U937 cells were i.p. injected into NOD.Cg-Prkdc^scidIL2rg^tm1Wj1/SzJ (NOD/scid IL2RGnull) mice (1 × 10 per mouse⁴Individual cells) after 3 days, NK cells transduced with MSCV vector containing GFP or mbIL15 only (1 × 10 per mouse) were injected i.p⁷Individual cells), repeat NK cell injection on day 7 as a control, one group of mice received tissue culture medium instead of NK cells, in a second model, the mice were subjected to ES8 cells (i.p.; 1 × 10 per mouse)⁵Individual cells) and then 1 NK cell injection was performed on day 3 as described above. Tumor transplantation and progression were evaluated using the Xenogen IVIS-200 system (calipers life Sciences, Hopkinton, MA) and imaging was started 5 minutes after i.p. injection of D-fluorescein potassium salt in water (3 mg per mouse). Photons emitted by luciferase expressing cells were quantified using the Living Image 4.3.1 software program.

Results

Design of IL-15 constructs and expression in NK cells

As described herein, two forms of IL15 gene are expressed in human NK cells: membrane-bound forms derived from a construct in which the human IL15 gene is linked to a gene encoding the transmembrane domain of CD8 α ("mbIL 15"), and wild-type unmodified forms ("wtIL 15"). Both constructs were inserted into a GFP-containing MCSV retroviral vector (FIG. 1A) used to transduce proliferating NK cells obtained after culturing peripheral blood mononuclear cells with the stimulatory cell line K562-mb15-41 BBL.28. At the end of the culture, the residual T cells were depleted with anti-CD 3 immunomagnetic beads prior to retroviral transduction, yielding CD56+ CD 3-cells of > 95% purity. Median GFP expression for constructs containing mbIL15 was 71% (23% -97%, n ═ 60), while median GFP expression for constructs containing wtIL15 was 69% (range, 20% -91%, n ═ 25). Median GFP expression among NK cells from the same donor also transduced with GFP-only vectors was 84% (53% -98%, n ═ 60) (fig. 1B).

After transduction with mbIL15, IL-15 was expressed on NK cell membranes: between 40% and 63% (median, 52; n ═ 7) of GFP + NK cells had IL-15 as detected by anti-IL 15 antibody (fig. 1B). In contrast, no IL-15 was detected in cells transduced with wtIL15 (n-4) or mock-transduced NK cells (n-7). The production of soluble IL-15 by transduced NK cells was determined by testing supernatants collected after 24 and 48 hours of culture. As shown in figure 1C, cells expressing wtIL15 secreted significant amounts of IL-15, which was minimal in mbIL15-NK cells and undetectable in mock-transduced NK cells.

NK cells expressing IL-15 with autonomous survival and expansion capabilities

To determine whether expression of IL-15 could replace exogenous IL-2 to maintain NK cell survival, NK cells from 15 donors were transduced with the mbIL15 construct and cultured in the absence of IL-2; the number of cells after culture was then compared to the number of cells cultured in parallel with mock-transduced NK cells. As shown in fig. 2A, expression of mbIL-15 significantly improved NK cell survival: after 7 days of culture, the median cell recovery was 85%, while virtually no viable mock-transduced NK cells were detected (< 1%; P < 0.0001 by paired t test). The effect of mbIL15 was significantly reduced if anti-IL-15 neutralizing antibodies were added to the cultures (fig. 6A-6B). Recovery of mbIL15NK cells was also compared to recovery of NK cells expressing wtIL15 in 9 of 15 donors: it was significantly higher than the former (median, 85% vs 56%, P ═ 0.026; fig. 2A).

In parallel experiments, the supporting effect of IL15 expression in the presence of exogenous IL-2 was determined. When the culture contained 10IU/mL of IL-2, the 7-day recovery of NK cells expressing mbIL15 or wtIL15 remained significantly higher than that of mock-transduced cells; under these conditions, no significant difference was observed between the 2 forms of IL15 (fig. 6A-6B). Only when exogenous IL-2 was present at high concentration (100IU/mL), the 7-day recovery of mock-transduced NK cells matched the 7-day recovery of NK cells transduced with IL15 (fig. 6A).

In experiments with expanded NK cells from 6 of 9 donors, the ability of mbIL15 to maintain NK cell survival for more than 7 days with low dose IL-2(10IU/mL) was determined. Maintaining or increasing mbIL15NK cell numbers in 4 of 6 cultures on day 14; in 2 of 6 cultures, these cells were further expanded before day 21. Of 6 cultures of mock-transduced NK cells from the same donor, only 2 maintained cell numbers at day 14 and day 21, and no cell growth was observed; at day 21, median cell recovery was 205% for mbIL15NK cells, while 80% for mock-transduced NK cells. Thus, expression of mbIL15 confers significant survival and growth advantages even in the presence of low doses of IL-2.

In cultures of NK cells from one donor, particularly high cell recovery was observed on day 7 when IL-15 was expressed (261% for mbiL15 and 161% for wtIL15 in the absence of IL-2; 266% and 188% in the presence of 10IU/mL IL-2). These cultures were monitored for 2 months and significant improvement in cell expansion and survival due to mbIL15 expression was observed (fig. 2C). Even in the absence of IL-2, mbIL-15NK cells continued to survive until day 21 and they were detectable 75 days after the start of culture, whereas mock-transduced cells became undetectable at day 7 and wtIL 15-transduced NK cells became undetectable at day 42. In the presence of low concentrations (10IU/mL) of IL-2, the number of NK cells expressing mbiL15 2 months after the start of culture was the same as that initially seeded, while live mock-transduced and wtIL15 transduced NK cells had already begun to decline early. As shown in FIG. 6B, NK cells transduced with mbIL155 or wtIL15 had similar persistence characteristics when high doses (100IU/mL) of IL-2 were added to the cultures, even though under these conditions, both cell types survived longer than mock-transduced NK cells.

Expansion and homing (homing) of mbIL15NK cells in vivo

Experiments performed in vitro showed that IL15 expression improved NK cell survival and expansion, and mbIL15 produced overall better stimulation. It was then determined whether mbIL15 expression would maintain expansion of human NK cells in NOD/scid IL2RGnull mice. Activated NK cells from 4 donors were transduced with mbIL15 (52% -74% GFP positive) and injected into 4 mice (one mouse per donor); 4 control mice were injected with mock transduced NK cells from the same donor. NK cells expressing mbIL15 expanded much more than mock-transduced NK cells: 7 days post-injection, median mbIL15NK cells/μ l blood was 44.5 (range, 42-60), while mock-transduced NK cells were 6.5(0-12) (P ═ 0.004) (fig. 3A). Parallel experiments were performed with the same cells, this time also with 20,000IU human IL-2 administered i.p. every 2 days (FIG. 3A). Under these conditions mbIL15NK cells expanded even more (median NK cells/. mu.l, 101; range, 60-167), while mock-transduced cells remained very low (median, 18; range, 6-20; P ═ 0.021).

On day 11 post-injection, mbIL15NK cells contained 168.5 cells/μ l (range, 94-355) of peripheral blood mononuclear cells in the absence of IL-2, and 382 cells/μ l (151-710) when IL-2 was also administered (fig. 3A, B). In contrast, human CD45 cells were barely detectable in the absence of IL-2 in mice injected with mock-transduced NK cells, whereas human CD45 cells were present at low levels (median 27; range 9-207; P ═ 0.026) when IL-2 was also injected. Human CD45+ cells also expressed CD56 and lacked CD3 (not shown). Notably, the proportion of GFP + increased from 66.5% + -9.9% prior to injection to 93.8% + -4.4% on day 7 and 94.8% + -3.4% on day 11 (P < 0.01 for both comparisons).

After euthanasia on day 11, 3 of 4 mice were examined for the presence of human CD45+ cells in multiple tissues. If mbIL15 was expressed, a significant number of human NK cells were detected in bone marrow, liver, spleen, kidney and lung; in all tissues, the number was significantly higher than that observed with mock-transduced cells (fig. 3C): the mean (± SD) percentage of mbIL15 expressing CD45+ cells without IL-2 was 1.2% ± 1.5% compared to 0.04% ± 0.09% and 0.4% ± 0.6% of mock transduced cells, while that with IL-2 was 3.0% ± 4.3% (P < 0.001 and P ═ 0.002, respectively). The only exception was the brain, where neither NK cells transduced with mbIL15 nor mock transduced NK cells could be detected.

mbIL15 stimulation mechanism

To determine whether mbIL15 stimulated cells primarily in trans (trans) (IL-15 present on one NK cell stimulating neighboring cells (a mechanism reported to occur physiologically)) or cis (cis) (by direct binding of mbIL15 to receptors expressed in the same cell), the proportion of GFP + and GFP-NK cells in the culture was evaluated after 7 days of culture. If the trans mechanism is dominant, the ratio between GFP + and GFP-NK cells should remain unchanged during culture; if cis is predominant, the proportion of GFP + cells should be increased. Fig. 4A shows the results of such an analysis: if mbIL15 is expressed, the percentage of GFP + cells in NK cells examined after 7 days of culture in the absence of IL-2 consistently increased, while it did not consistently increase in cultures with mock-transduced cells: GFP + cells at day 7 constituted 95.9% ± 3.3% and 57.5% ± 18.6% of the total cell population (P < 0.0001) compared to 71.2% ± 19.0% and 80.5% ± 17.1% at day 0. Therefore, the major stimulatory mechanism of mbIL-15 expressed in NK cells is autocrine.

Cells expressing IL15 substantially retained the immunophenotype of activated NK cells. However, when examined 2 days after IL-2 withdrawal compared to mock-transduced NK cells, mbiL15NK cells expressed moderately higher levels of activating receptors NKG2D, NKp44(CD336) and NKp30(CD337) and moderately higher levels of CD16 and CD56, whereas the expression of NKp46(CD335) was reduced and the expression of other molecules such as DNAM-1(CD226) remained unchanged (FIG. 4B; Table). The signaling pathway activated by expression of mbIL15 was also determined. As shown in fig. 4C, mbIL-15NK cells have several highly phosphorylated molecules compared to mock-transduced NK cells. These include molecules known to phosphorylate in response to IL-15 signaling, such as the transcription factors STAT1, STAT3 and STAT5, kinase src, Erk1/2 and Mek 1. Notably, significant phosphorylation of Bad and phosphorylation of caspases (caspases) 7 and 9 were observed, collectively accounting for the anti-apoptotic effect. Other highly phosphorylated molecules in mbIL15NK cells with unclear roles in IL-15 signaling include CDK6 and RafA.

Effect of mbIL-15 on NK cell antitumor cytotoxicity in vitro and in vivo

The improvement in NK cell survival and proliferation caused by expression of mbIL15 suggests that NK-mediated tumor cell killing may also be increased. This insight was first tested by comparing the tumor cell cytotoxicity exhibited by mbIL15-NK cells with the cytotoxicity of mock transduced NK cells from the same donor. Experiments were performed with NK cells from 9 donors targeting leukemia cell lines Nalm-6 (B-line acute lymphoblastic leukemia), U937 and K562 (acute myeloid leukemia) as well as Daudi (B-cell lymphoma), SKBR3 (breast cancer) and ES8 (ewing's sarcoma) at different E: T ratios and co-culture times for a total of 90 experiments. Fig. 5A shows the results of the 24 hour assay: at 1: 4E: T, the median cytotoxicity of mock-transduced NK cells was 22%, and at 1: 1E: T, 54%; with mbIL15NK cells, 71% and 99%, respectively (P < 0.0001). The results for the individual cell lines are shown in FIGS. 7A-7B. Although increased cytotoxicity may be associated with increased survival of NL cells in culture, increased lytic granules (P ═ 0.0067; fig. 5B) released by mbIL15-NK cells were also observed, as shown by staining with CD107a after culture with K562 or U937 cells.

The increase in vitro cytotoxicity associated with expression of mbIL15 was reflected in the experiments with NOD/scid IL2RGnull mice transplanted with human tumor cells. In one set of experiments, mice were injected with the human Acute Myeloid Leukemia (AML) cell line U937 and then treated with mbIL15 or mock-transduced NK cells. As shown in fig. 5C and 5D, mice receiving mbIL15 transduced NK cells had slower tumor growth and significantly longer survival than untreated mice and mice treated with mock transduced NK cells (P ═ 0.014, log rank test of trend). Cells were also tested in a second xenograft model in which NOD/scid IL2RGnull mice were injected with the ewing sarcoma cell line ES8, which had a slower growth rate, and the mice were treated with one NK cell injection. As shown in figures 8A-8C, the results of mice treated with mbIL15NK cells (n-12) were superior to those of mice treated with mock-transduced NK cells (n-11) and untreated mice (n-7): median survival was 162, 49 and 21 days (P ═ 0.005), respectively.

Discussion of the related Art

Of the factors that determine the success of NK cell-based therapies for cancer, perhaps the most basic one is that NK cells persist in sufficient numbers to achieve an E: T ratio that may cause a reduction in tumor cells. It is demonstrated herein that expression of membrane-bound forms of IL-15 in human NK cells maintains their autonomous expansion and prolonged survival in the absence of IL-2. NK cells expressing mbIL15 could be maintained in vitro in the absence of exogenous IL-2 for up to 2 months. NK cells expressing mbIL15 can expand and infiltrate multiple tissues in immunodeficient mice where they can be present in greater numbers than mock-transduced cells. In vitro and in vivo, the expansion of mbIL-15NK cells was further enhanced by low concentrations of IL-2. Expression of mbIL15 did not impair the cytotoxic capacity of NK cells. Indeed, mbIL15NK cells showed stronger anti-cancer activity than mock-transduced cells in the xenograft model, suggesting that this approach may improve the anti-tumor capacity of NK cell infusions while avoiding the side effects of IL-2 administration.

The findings herein indicate that ectopic expression of IL-15 in human NK cells results in a stronger survival promoting effect when IL-15 is present in a membrane-bound form rather than in a secreted form. However, it is noteworthy that mbIL15 expressed in NK cells is preferentially stimulated in cis rather than in trans when IL-15 is provided by other cells. That is, mbIL15 appears to preferentially bind to the IL-15 receptor on the same cell, resulting in autocrine stimulation. This mechanism illustrates the IL-15 expression pattern consistently observed when labeling mbIL 15-transduced NK cells with anti-IL-15 antibodies, showing a significant proportion of cells with strong GFP expression but lacking IL-15 on the surface (ostensibly) (fig. 1B). It is speculated that in these cells, IL-15 is expressed but not accessible to the antibody because it binds to its receptor and/or internalizes. The ability of mbIL15 to promote NK cell viability may account for the increased cytotoxicity exhibited by these cells, particularly in 24 hour in vitro assays and in vivo. However, the advantage of mbIL15-NK cells was also evident in the short-term (4 hours) assay, and these cells also released more lytic granules according to CD107a test. Thus, expression of mbIL15 might otherwise increase NK cell cytotoxicity (possibly by enhancing its activation state).

Clinical administration of NK cells often relies on IL-2 to support their survival and expansion in vivo. However, a number of side effects associated with IL-2 administration are potentially severe and often render the cytokine poorly tolerated for administration. Stopping IL-2 administration or reducing its dose can lead to reduced NK cell expansion and inadequate antitumor effects that can be further inhibited by stimulation of regulatory T cells. For this reason, the replacement of IL-2 by IL-15 is potentially attractive, but clinical formulations of IL-15 are still being tested. Although generally well tolerated when administered to rhesus macaques, adverse effects including diarrhea, vomiting, weight loss, transient neutropenia, elevated transaminases, and hyponatremia were observed in some animals. In addition to T and NK cell expansion, expansion of regulatory T cells has been observed. In contrast to NK cells transduced with wtIL15, those transduced with mbIL15 released very small amounts of IL-15 in the supernatant. Therefore, any potential side effects that may be caused by the interaction of IL-15 with cells other than NK cells should be minimized by this approach. Notably, chronic exposure of murine large granular lymphocytes to IL-15 results in leukemic growth. This poses potential safety issues for IL-15 administration in patients and the use of NK cells expressing IL-15, particularly if such cells are administered to patients with a low risk of relapse. However, in the experiments described herein, NK cells expressing mbIL15 generally survived for a much shorter time than the one year or longer reported for T cell clones expressing soluble IL-15. Furthermore, no sustained NK expansion was observed in immunodeficient mice upon follow-up over 9 months.

There is considerable clinical evidence to support the anticancer potential of NK cells. NK cells also play a key role in mediating antibody-dependent cellular cytotoxicity in patients treated with monoclonal antibodies. Therefore, infusion of NK cells may be beneficial in a variety of situations. Large ex vivo expansion of human NK cells is feasible; robust large-scale methods for this purpose have been established and are being used in clinical trials. Genetic modification of NK cells by retroviral transduction or electroporation is also possible. Thus, it is practical to convert the methods described herein to clinical grade conditions and it is ensured by excellent expansion and cytotoxicity of mbIL15-NK cells.

Expression of surface markers in mock-transduced and mbIL 15-transduced NK cells 1

¹Cell markers were analyzed after 48 hours of culture in the absence of IL-2. Antibodies were from BDbiosciences (CD56 PE, CD16 PE-Cy7, CD69 PE, CD25 PE-Cy7, CD122 BV421, CD158b PE), Beckman Coulter (CD335 PE, CD336 PE, CD337 PE, CD158ah PE, CD159a PE), Miltenyi Biotech (CD226PE, CD158e APC), R&D Systems(NKG2D PE)，Biolegend(CD132APC)。

²Percentages refer to GFP + cells expressing the marker.

³The over-expressed markers are highlighted in bold.

MFI means mean fluorescence intensity

The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

While the present invention has been particularly shown and described with reference to certain example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (20)

1. A Natural Killer (NK) cell that expresses all or a functional portion of interleukin-15 (IL-15).

2. The NK cell of claim 1, wherein the IL-15 is expressed as a membrane-bound polypeptide and/or a secreted polypeptide.

3. The NK cell of any one of claims 1 and 2, wherein all or a functional portion of the IL-15 is fused to all or a portion of a transmembrane protein.

4. A method of generating Natural Killer (NK) cells that express all or a functional portion of interleukin-15 (IL-15), comprising:

a) introducing into the NK cell a nucleic acid encoding all or a functional portion of IL-15; and

b) maintaining said NK cells under conditions wherein all or a functional portion of said IL-15 is expressed,

thereby producing NK cells expressing all or a functional portion of IL-15.

5. The method of claim 4, wherein the nucleic acid introduced into the NK cell comprises a signal peptide of CD 8a, all or a functional portion of IL-15, and all or a portion of the transmembrane domain of CD 8a.

6. The method of any one of claims 4 and 5, wherein the NK cell is transduced with a vector that expresses all or a functional portion of the IL-15 linked (e.g., fused) to all or a portion of the transmembrane domain.

7. The method of claim 6, wherein the vector is a viral vector.

8. A Natural Killer (NK) cell produced by the method of any one of claims 4 to 7.

9. A composition comprising the NK cell of any one of claims 1 to 3 or 8.

10. A pharmaceutical composition comprising the NK cell of any one of claims 1 to 3 or 8.

11. The pharmaceutical composition of claim 10, further comprising all or a functional portion of IL-2.

12. A method of treating cancer in an individual in need thereof, comprising administering to the individual Natural Killer (NK) cells that express all or a functional portion of interleukin-15 (IL-15).

13. The method of claim 12, wherein the cancer is leukemia (e.g., acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia), myelodysplastic syndrome, lymphoma (e.g., B-cell non-hodgkin's lymphoma, T-cell lymphoblastic lymphoma, anaplastic large cell lymphoma), solid tumors (e.g., breast cancer, prostate cancer, gastric cancer, colon cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, neuroblastoma, high grade glioma), sarcoma (e.g., ewing's sarcoma, rhabdomyosarcoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma).

14. The method of any one of claims 12 and 13, further comprising administering IL-2 to the individual.

15. The method of any one of claims 12 to 14, further comprising administering to the individual one or more antibodies against the cancer.

16. A method of enhancing NK cell expansion and/or survival comprising:

a) introducing a nucleic acid encoding all or a functional portion of IL-15; and

b) maintaining said NK cells under conditions wherein all or a functional portion of said IL-15 is expressed and said NK cells proliferate,

thereby enhancing the expansion and/or survival of said NK cells.

17. The method of claim 16, wherein the nucleic acid introduced into the NK cell comprises a signal peptide of CD 8a, all or a functional portion of IL-15, and all or a portion of the transmembrane domain of CD 8a.

18. The method of any one of claims 16 and 17, wherein the NK cell is transduced with a vector that expresses all or a functional portion of the IL-15 linked (e.g., fused) to all or a portion of the transmembrane domain.

19. The method of claim 18, wherein the vector is a retroviral vector.

20. The method of any one of claims 16 to 19, further comprising contacting the NK cells with IL-2.