Mesoderm Killing (MK) cells

Abstract

The present invention relates to mesoderm killing (MK) cells and their use in therapeutic methods, in particular for the treatment of cancer.

Description

Technical Field

[0001] The present invention relates to mesoderm killing (MK) cells and their use in therapeutic methods, particularly for the treatment of cancer. Background Art

[0002] Mesodermal cells are derived from many tissues and serve as a supporting structure for other cell types. For example, bone marrow consists of hematopoietic cells and mesenchymal cells. Two major types of mesenchymal cells have been previously described and characterized, namely (i) mesenchymal stem cells (MSCs) and their precursors found in bone marrow and (ii) mesenchymal progenitor cells (MPCs). Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. MSCs differentiate into different specialized cells found in skeletal tissues. For example, they can differentiate into chondrocytes (chondrocyte bodies), osteocytes (osteoblasts), and adipocytes (fat cells).

[0003] MSCs have been used in a variety of therapeutic methods, such as the treatment of age-related macular degeneration (AMD) and myocardial infarction. Once administered to a subject, MSCs typically migrate (or home) to damaged tissues and exert their therapeutic effects by paracrine signaling and promoting the survival, repair, and regeneration of neighboring cells in the damaged tissues.

[0004] There is evidence that MSCs may possess certain immunosuppressive and immunostimulatory properties. Thus, MSCs can be used to manipulate the immune response and thereby treat diseases. However, current therapeutic methods typically involve the infusion of a mixture of MSC subtypes, most of which do not possess the desired immunomodulatory properties. This requires the use of high cell doses, which may lead to off-target side effects and volume-related side effects. In addition, MSCs are typically obtained from bone marrow, and thus it is difficult to obtain the large numbers of cells required for this method.

[0005] The applicant has also disclosed other mesodermal progenitor cells, namely progenitor cells of the mesodermal lineage (PML), immunomodulatory progenitor (iMP) cells, and immuno-oncology mesodermal progenitor (ioMP) cells. PML is disclosed in PCT / GB2012 / 051600 (published as WO 2013 / 005053). iMP cells are disclosed in PCT / GB2015 / 051673 (published as WO 2015 / 189587). ioMP cells are disclosed in PCT / GB2016 / 052447 (published as WO 2017 025729). Summary of the Invention

[0006] The present invention relates to a new cell type, mesodermal killer (MK) cells, which have not been previously identified or isolated. These MK cells are distinct from MSC, MPC, PML, iMP cells, and ioMP cells in their composition, function, and characteristics, and confer enhanced cytotoxicity and the ability to regulate natural killer (NK) cells. MK cells are capable of directly killing cancer cells, i.e., the cytotoxicity against cancer cells. MK cells are capable of triggering / activating NK cells (i.e., increasing the proliferation and / or cytotoxic activity of NK cells). MK preferably can attract immune cells to the site of inflammation. MK cells are named after NK cells because they exhibit similar natural killer characteristics, but they are engineered tissues derived from mesodermal cells, such as bone marrow. These MK cells are distinct from NK cells in their composition, function, and characteristics.

[0007] Surprisingly, the inventors have identified new mesodermal killer (MK) cells with a specific marker expression pattern. In particular, the MK cells express CD112, CD137L, CD178, CD253, and CD277. The MK cells may express CD16 and CD96. The MK cells do not express CD34 and CD45. The MK cells may not express CD56.

[0008] The MK cells of the present invention can be isolated from mononuclear cells (MNCs), such as bone marrow MNCs or peripheral blood MNCs. The MK cells are capable of increasing the cytotoxic activity of NK cells in vitro and in vivo. MK cells themselves are capable of killing cancer cells in vitro and in vivo. This is shown in the examples.

[0009] Accordingly, the present invention provides a mesodermal killer (MK) cell, wherein the cell expresses detectable levels of CD112, CD137L, CD178, CD253, and CD277, and wherein the cell does not express detectable levels of CD34 and CD45.

[0010] The present invention also provides a mesodermal killer (MK) cell, wherein the cell expresses detectable levels of CD16, CD96, CD112, CD137L, CD178, CD253, and CD277, and wherein the cell does not express detectable levels of CD34, CD45, and CD56.

[0011] The present invention also provides:

[0012] - a population of two or more MK cells of the present invention;

[0013] - A population of MK cells, wherein more than about 15% of the cells in the population express detectable levels of CD112, CD137L, CD178, CD253, and CD277, and wherein about 5% or fewer of the cells in the population express detectable levels of CD34 and CD45;

[0014] - A population of MK cells, wherein more than about 15% of the cells in the population express detectable levels of CD16, CD96, CD112, CD137L, CD178, CD253, and CD277, and wherein about 5% or fewer of the cells in the population express detectable levels of CD34, CD45, and CD56;

[0015] - A population of MK cells, wherein

[0016] (i) At least about 20% of the cells in the population express detectable levels of CD112,

[0017] (ii) At least about 80% of the cells in the population express detectable levels of CD137L,

[0018] (iii) At least about 20% of the cells in the population express detectable levels of CD178,

[0019] (iv) At least about 50% of the cells in the population express detectable levels of CD253, and

[0020] (v) At least about 50% of the cells in the population express detectable levels of CD277,

[0021] And wherein

[0022] (a) About 5% or fewer of the cells in the population express detectable levels of CD34, and

[0023] (b) About 5% or fewer of the cells in the population express detectable levels of CD45.

[0024] - A population of MK cells, wherein

[0025] (i) At least about 15% of the cells in the population express detectable levels of CD16,

[0026] (ii) At least about 50% of the cells in the population express detectable levels of CD96,

[0027] (iii) At least about 20% of the cells in the population express detectable levels of CD112,

[0028] (iv) At least about 80% of the cells in the population express detectable levels of CD137L,

[0029] (v) At least about 20% of the cells in the population express detectable levels of CD178,

[0030] (vi) At least about 50% of the cells in the population express detectable levels of CD253, and

[0031] (vii) At least about 50% of the cells in the population express detectable levels of CD277,

[0032] and wherein

[0033] (a) About 5% or fewer of the cells in the population express detectable levels of CD34,

[0034] (b) About 5% or fewer of the cells in the population express detectable levels of CD45, and

[0035] (c) About 5% or fewer of the cells in the population express detectable levels of CD56.

[0036] - A pharmaceutical composition comprising: (a) a population of MK cells according to the present invention and (b) a pharmaceutically acceptable carrier or diluent;

[0037] - A method for generating a population of MK cells according to the present invention, comprising: (a) culturing monocytes (MNC) under conditions that induce differentiation into immunomodulatory progenitor (iMP) cells, and (b) culturing the iMP cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides, and platelet lysate under hypoxic conditions and under conditions that permit the iMP cells to adhere and differentiate into MK cells;

[0038] - A method for generating a population of MK cells according to the present invention, comprising: culturing iMP cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides, and platelet lysate under hypoxic conditions and under conditions that permit the iMP cells to adhere and differentiate into MK cells;

[0039] - An in vitro method for priming a population of NK cells, comprising: incubating the population of NK cells with a population of MK cells according to the present invention under conditions that increase the activity of the NK cells;

[0040] - A population of primed NK cells generated by the method according to the present invention;

[0041] - A pharmaceutical composition comprising: (a) a population of primed NK cells according to the present invention and (c) a pharmaceutically acceptable carrier or diluent;

[0042] - An in vivo method for activating a population of NK cells, comprising: administering to a subject a population of MK cells or a pharmaceutical composition according to the present invention under conditions that increase the activity of the subject's NK cells; and

[0043] - A method for treating cancer in a subject, the method comprising: administering to the subject (a) a population of MK cells according to the present invention, (b) a population of activated NK cells according to the present invention, (c) a population of MK cells and NK cells according to the present invention, or (d) a pharmaceutical composition according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is shown that compared with IMP002 and IMP004, MK002 and MK004 show significantly increased cytotoxicity against K562 (chronic myeloid leukemia) and RPMI-8226 (plasma cell myeloma) (n = 3; t-test).

[0045] Figure 2 It is shown that incubation with MK002 and MK004 significantly increases the cytotoxicity of NK cells against K562 (chronic myeloid leukemia) and RPMI-8226 (plasma cell myeloma) (n = 3; t-test). MK002 = NK cell line activated with MK002. MK004 = NK cell line activated with MK004.

[0046] Figure 3 It is shown that incubation with MK004 significantly increases the cytotoxicity of primary NK cells against RPMI-8226 (plasma cell myeloma) and U266 (plasma cell myeloma) (n = 3; t-test). MK004 = primary NK cells activated with MK004.

[0047] Figure 4 The MK cells (MK002) of the present invention in culture are shown.

[0048] Figure 5 The amount of GROα secreted by the MK cells of the present invention when untreated and treated with IFN-γ (not significant in unpaired t-test) or TNF-α (unpaired t-test) is shown. Each bar represents data from 5 batches (mean ± SEM; n = 1).

[0049] Figure 6 The amount of IL-12 secreted by the MK cells of the present invention when untreated and treated with IFN-γ or TNF-α (not significant in unpaired t-test) is shown. Each bar represents data from 5 batches (mean ± SEM; n = 1).

[0050] Figure 7Shows the amount of IL-2Ra secreted by the MK cells of the present invention when untreated and treated with IFN-γ (unpaired t-test) or TNF-α (not significant in unpaired t-test). Each bar represents data from 5 batches (mean ± SEM; n = 1).

[0051] Figure 8 Shows the amount of IL-8 secreted by the MK cells of the present invention when untreated and treated with IFN-γ or TNF-α (unpaired t-test). Each bar represents data from 4 batches (data of MKPC not included as it is not relevant to the standard curve; mean ± SEM; n = 1).

[0052] Figure 9 Shows the amount of soluble TRAIL secreted by the MK cells of the present invention when untreated and treated with IFN-γ or TNF-α (not significant in unpaired t-test). Each bar represents data from 5 batches (mean ± SEM; n = 1).

[0053] Figure 10 Shows the amount of IL-6 secreted by the MK cells of the present invention when untreated and treated with IFN-γ or TNF-α (not significant in unpaired t-test). Each bar represents data from all 5 batches (mean ± SEM; n = 1).

[0054] Figure 11 Shows the percentage of NK cells present in the air sacs of (A) control mice, (B) mice treated with untreated MK cells of the present invention, (C) mice treated with MK cells of the present invention treated with IFN-γ, or (D) mice treated with MK cells of the present invention treated with TNF-α (MK006; mean ± SEM; n = 5; unpaired t-test).

[0055] Figure 12 Shows the percentage of monocytes present in the air sacs of (A) control mice, (B) mice treated with untreated MK cells of the present invention, (C) mice treated with MK cells of the present invention treated with IFN-γ, or (D) mice treated with MK cells of the present invention treated with TNF-α (MK006; mean ± SEM; n = 5; unpaired t-test).

[0056] Figure 13 Shows that incubation with MK002 and MK004 significantly increases the cytotoxicity of primary NK cells against K562 (chronic myeloid leukemia) (mean ± SEM; n = 3; unpaired t-test).

[0057] Figure 14 Shows that all batches of MK cells tested showed cytotoxicity against MCF7 (mean ± SEM; n = 2).

[0058] Figure 15 Shows the fold change in the amount of GZMB mRNA expressed by MK004 after co - culturing (CC) with RPMI - 8226 for 6 hours, 12 hours, or 24 hours compared to monoculture (MC) (mean ± SD; n = 2 for MC and CC at each time point; unpaired t - test).

[0059] Figure 16 Shows the fold change in the amount of GZMH mRNA expressed by MK004 after co - culturing (CC) with RPMI - 8226 for 6 hours, 12 hours, or 24 hours compared to monoculture (MC) (mean ± SD; n = 2 for MC and CC at each time point; unpaired t - test).

[0060] Figure 17 Shows the fold change in the amount of GZMM mRNA expressed by MK004 after co - culturing (CC) with RPMI - 8226 for 6 hours, 12 hours, or 24 hours compared to monoculture (MC) (mean ± SD; n = 2 for MC and CC at each time point; unpaired t - test).

[0061] Figure 18 Shows the fold change in the amount of GZMA mRNA expressed by MK004 after co - culturing (CC) with RPMI - 8226 for 6 hours, 12 hours, or 24 hours compared to monoculture (MC) (mean ± SD; n = 2 for MC and CC at each time point; unpaired t - test).

[0062] Figure 19 Shows the fold change in the amount of GZMK mRNA expressed by MK004 after co - culturing (CC) with RPMI - 8226 for 6 hours, 12 hours, or 24 hours compared to monoculture (MC) (mean ± SD; n = 2 for MC and CC at each time point; unpaired t - test).

[0063] Figure 20 Shows the fold change in the amount of perforin mRNA expressed by MK004 after co - culturing (CC) with RPMI - 8226 for 6 hours, 12 hours, or 24 hours compared to monoculture (MC) (mean ± SD; n = 2 for MC and CC at each time point; unpaired t - test).

[0064] Figure 21 Shows the cytotoxicity of MK002, MK004, and MK006 against MCF7 when untreated (A) or treated with 0.5 mM EGTA (B), 1.0 mM EGTA (C), or 2.0 mM (D) for 24 hours (E:T = 5.1; 24 hours) (mean ± SEM; n = 3; unpaired t - test).

[0065] Figure 22 Shows the cytotoxicity of MK004 and MK006 against MCF7 (E:T = 5.1; 24 hours) (mean ± SEM; n = 3; unpaired t-test) in the case of untreated (A) or treated with scrambled / non-targeting (NT) siRNA (A), specific siRNA against CD178 / FasL (B), or specific siRNA against CD253 / TRAIL (D) for 48 hours. Detailed Description

[0066] It should be understood that different applications of the disclosed products and methods can be adjusted according to the specific needs in the art. It should also be understood that the terms used herein are for the purpose of describing particular embodiments of the invention only and are not intended to be limiting.

[0067] Furthermore, the singular forms "a", "an", and "the" used in this specification and the appended claims include plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes "a plurality of cells", reference to "a tissue" includes two or more such tissues, reference to "a subject" includes two or more such subjects, and so on.

[0068] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0069] MK cells of the present invention

[0070] The present invention provides a mesoderm killing (MK) cell. MK cells express detectable levels of CD112, CD137L, CD178, CD253, and CD277. MK cells do not express detectable levels of CD34 and CD45. MK cells preferably do not express detectable levels of one or more of (a) CD45RA, (b) CD45RB, and (c) CD45RO, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b), and (c). In the context of the present invention, not expressing detectable levels means that approximately 5% or less of the MK cells express the relevant marker.

[0071] MK cells preferably express detectable levels of CD16 and / or CD96. As discussed in more detail below, when treated with interferon γ (IFN-γ), MK cells preferably express detectable levels of CD16 and / or CD96.

[0072] MK cells preferably do not express detectable levels of CD56. MK cells preferably express detectable levels of CD16 and / or CD96 and do not express detectable levels of CD56.

[0073] In a preferred embodiment, MK cells express detectable levels of CD16, CD96, CD112, CD137L, CD178, CD253, and CD277. Preferred MK cells do not express detectable levels of CD34, CD45, and CD56.

[0074] The term MK cell can be interchanged herein with mesodermal progenitor killer (MPK) cell, bone marrow-derived cell, or bone marrow-derived killer cell.

[0075] MK cells preferably express detectable levels of CD112, CD137L, CD178, CD253, and CD277 on their surface. MK cells preferably express detectable levels of CD16 and / or CD96 on their surface. MK cells preferably express detectable levels of CD16, CD96, CD112, CD137L, CD178, CD253, and CD277 on their surface. MK cells preferably do not express detectable levels of CD34 and CD45 on their surface. MK cells preferably do not express detectable levels of one or more of (a) CD45RA, (b) CD45RB, and (c) CD45RO as defined above on their surface. MK cells preferably do not express detectable levels of CD56 on their surface. MK cells preferably do not express detectable levels of CD34, CD45, and CD56 on their surface. MK cells preferably express or do not express detectable levels of any of the markers listed below. Similarly, a population of MK cells can express / not express any of the listed markers on their surface.

[0076] CD16 (also known as FcγRIIIA) plays a role in antibody-dependent cell-mediated cytotoxicity (ADCC; WeiHseun Yeap et al. Scientific Reports Volume 6, Article number: 34310 (2016)). Expression of this marker also differentiates MK cells from ioMP cells and MSCs. Expression of this marker can be increased by treatment / stimulation with IFN-γ.

[0077] CD96 (also known as TACTILE) plays a role in NK cell adhesion and stimulates cytotoxicity of activated NK cells (Fuchs et al. J Immunol April 1, 2004, 172(7)3994-3998). Expression of this marker also differentiates MK cells from MSCs. Expression of this marker can be increased by treatment / stimulation with IFN-γ.

[0078] CD112 (also known as PRR2 and nectin-2) is involved in NK cell priming / activation (Deuss et al. J Biol Chem. July 7, 2017; 292(27):11413-11422). Expression of this marker also differentiates MK cells from MSCs. Expression of this marker can be increased by treatment / stimulation with IFN-γ.

[0079] CD137L (also known as 4-1BB L) is involved in NK cell priming / activation (Zhang et al. J Immunother. March 2011; 34(2):187–195.).

[0080] CD178 (also known as FasL and CD95L) is involved in NK cell cytotoxicity (Zamai et al. J Exp Med. December 21, 1998; 188(12):2375–2380). Expression of this marker also differentiates MK cells from MSCs. Expression of this marker can be increased by treatment / stimulation with IFN-γ and / or tumor necrosis factor-α (TNF-α).

[0081] CD253 (also known as TRAIL and TNFSF10) is involved in NK cell cytotoxicity (Zamai et al. J Exp Med. December 21, 1998; 188(12):2375–2380). Expression of this marker also differentiates MK cells from ioMP cells.

[0082] The results in Example 15 indicate that CD253 forms part of the mechanism by which the MK cells of the present invention are cytotoxic.

[0083] CD277 (also known as BT3.1 and butyrophilin SF3 A1) regulates immune cell function (Messal et al. Eur J Immunol. December 2011; 41(12):3443-54). Expression of this marker also differentiates MK cells from ioMP cells, iMPs, and MSCs.

[0084] CD34 (also known as HPCA1) is a key MNC marker. Absence of expression of this marker differentiates MK cells from MNCs.

[0085] CD45 (also known as LCA) is a key MNC marker. Absence of expression of this marker also differentiates MK cells from MNCs and NK cells.

[0086] CD56 (also known as NCAM) is a key NK cell marker (Zamai et al. J Exp Med. December 21, 1998; 188(12):2375–2380). The absence of expression of this marker distinguishes MK cells from NK cells.

[0087] The MK cells of the present invention have many advantages. The key advantages will be summarized here. However, further advantages will become apparent from the following discussion.

[0088] The MK cells of the present invention can be advantageously used to treat diseases in a subject. For example, MK cells can be used to treat cancer in a subject.

[0089] The MK cells of the present invention can treat diseases through their direct action. For example, MK cells can kill cancer cells by contact-dependent cell lysis. Preferably, MK cells kill tumor cells by contact-dependent cell lysis. MK cells can also induce cancer cell death through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0090] The MK cells of the present invention can regulate the immune response. In other words, MK cells can have an immunomodulatory effect. For example, MK can increase the activity (especially cytotoxicity) of NK cells in vitro or in vivo. For example, MK cells can be used to generate a population of primed or activated NK cells in vitro. The primed or activated NK cells can be used to treat diseases in a subject, such as cancer. The primed or activated NK cells can be administered to the subject alone or in combination with MK cells. MK cells can also prime or activate endogenous NK cells in the subject.

[0091] A key advantage of the MK cells of the present invention is that they are mesodermal cells and they are generally safe in vivo. There is ample evidence that iMP cells (allogeneic mesodermal cells) generated in a manner similar (but different) to the MK cells of the present invention are safe in human subjects (Anastasiadis et al. J Cardiovasc Transl Res. June 2016; 9(3):202-13). MK cells are cytotoxic, but are not expected to induce any side effects of other cytotoxic cell therapies, such as chimeric antigen receptor-T (CAR-T) cells. In particular, MK cells are not expected to induce cytokine release syndrome (CRS; also known as cytokine storm), macrophage activation syndrome (MAS), and off-target effects.

[0092] As discussed in more detail below, MK cells are generated from mononuclear cells (MNCs) taken from an individual, such as a human individual, such as bone marrow MNCs. Since MK cells are generated from MNCs, they can be readily generated (e.g., from bone marrow) and can be autologous to the subject to be treated, thus avoiding the risk of immune rejection caused by the subject.

[0093] In principle, an unlimited number of MK cells can be generated from a single individual because various MNC samples (i.e., various bone marrow samples) can be obtained. It is certainly possible to generate a large number of MK cells from a single individual. Thus, the MK cells of the present invention can be prepared in large quantities.

[0094] The MK cells of the present invention are generated under clinically relevant conditions, e.g., in the absence of trace endotoxins and other environmental contaminants and animal products such as fetal bovine serum. This makes the MK cells of the present invention particularly suitable for administration to a subject.

[0095] Prior to the commencement of any other therapy, such as chemotherapy or radiotherapy, a population of a large number of MK cells of the present invention can be generated from a single sample taken from the subject. Thus, the MK cells of the present invention are able to avoid any adverse effects of these treatments.

[0096] The MK cells of the present invention can be rapidly prepared. MK cells can be generated from MNCs in less than 34 days, such as in about 33 days, about 32 days, about 31 days, about 30 days, about 29 days, about 28 days, about 27 days, about 26 days or about 25 days. MK cells can also be frozen and cryopreserved and thawed for use.

[0097] Generating MK cells from MNCs avoids the ethical and moral implications associated with using mesenchymal stem cells (MSCs) derived from human embryonic stem cells (hESCs).

[0098] The MK cells of the present invention are generally generated from human MNCs. Thus, the MK cells of the present invention are generally human. The markers discussed above and below are generally human markers. Alternatively, MK cells can be generated from MNCs from other animals or mammals, such as from commercially farmed animals such as horses, cows, sheep or pigs, from laboratory animals such as mice or rats, or from pets such as cats, dogs, rabbits or guinea pigs.

[0099] The MK cells of the present invention can be identified as mesodermal killer cells using standard methods known in the art, including the expression of lineage-restricted markers, structural and functional characteristics. MK cells will express detectable levels of cell surface markers known to be characteristic of MK cells. These will be discussed below.

[0100] The MK cells of the present invention are not stem cells. In particular, they are not MSCs. They are progenitor cells as they replicate / self-renew in vitro. Although they can be forced to differentiate under appropriate in vitro conditions, such as into chondrocytes or osteocytes, they generally do not differentiate in vivo. The MK cells of the present invention preferably have their anti-cancer effects through: (i) direct action, such as contact-dependent cell lysis or ADCC, (ii) modulation of the immune response or immune cell activity (i.e., immunomodulatory effect), and in particular the priming / activation of NK cells, and (ii) attraction of immune cells to the cancer site, in particular NK cells and monocytes. The untreated MK cells of the present invention (i.e., the MK cells of the present invention not treated with IFN-γ and / or TNF-α) generally have effects (i) and (ii). The MK cells of the present invention treated with IFN-γ generally have effects (i), (ii), and (iii). In contrast, stem cells generally treat diseases by differentiating into replacement tissues.

[0101] The MK cells of the present invention are typically characterized by a spindle-shaped morphology. The MK cells are generally fibroblast-like, i.e., they have a small cell body with several elongated cell processes. The diameter of the cells is typically about 10 μm to about 20 μm. This is as Figure 4 shown.

[0102] The MK cells of the present invention are distinguished from known cells by their marker expression patterns. The MK cells express detectable levels of CD16, CD96, CD112, CD137L, CD178, CD253, and CD277. The MK cells preferably express detectable levels of CD16, CD96, CD112, CD137L, CD178, CD253, and CD277. Compared to known cells such as ioMP cells, iMP cells, and MSCs, the MK preferably expresses increased amounts of these markers. Compared to those cells, the MK cells preferably express increased amounts of all the markers. This can be determined by comparing the expression level / amount of the markers in the MKs of the present invention with the expression level / amount in known cells using the same technique under the same conditions. As discussed in more detail below, the percentage of cells expressing the MK markers is increased in the population of MK cells compared to the populations of ioMP cells, iMP cells, and MSCs. As discussed above, ioMP and iMP cells are known in the art (and data on these cells are presented in the Examples). Suitable MSCs are commercially available. The MSCs used for comparison are preferably human MSCs. Human MSCs can be obtained commercially from Ltd, Osiris Inc. or commercially. The human MSCs are preferably obtained from Obtained. Such cells were used for comparison in the examples. MSCs can be derived from any animal or mammal discussed above.

[0103] MK cells do not express detectable levels of CD34 and CD45. MK cells preferably do not express detectable levels of CD34, CD45, and CD56. MK cells preferably do not express detectable levels of one or more of (a) CD45RA, (b) CD45RB, and (c) CD45RO, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b), and (c).

[0104] Standard methods known in the art can be used to determine the detectable or increased expression of the various markers discussed above (and below). Suitable methods include, but are not limited to, immunocytochemistry, immunoassays, flow cytometry (such as fluorescence-activated cell sorting (FACS)), and polymerase chain reaction (PCR) (such as reverse transcription PCR (RT-PCR)). Suitable immunoassays include, but are not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT assay), enzyme-multiplied immunoassay technique, radioallergosorbent (RAST) test, radioimmunoassay, radioligand assay, and immunofluorescence. Western blotting, ELISA, and RT-PCR are all quantitative and can therefore be used to measure the expression levels of various markers, if present. The use of high-throughput FACS (HT-FACS) is disclosed in the examples. Flow cytometry, FACS, or HT-FACS is preferably used for the expression or increased expression of any marker disclosed herein. Antibodies and fluorescently labeled antibodies for all the various markers discussed herein are commercially available.

[0105] The MK cells of the present invention preferably do not express detectable levels of CD14. CD14 is a key MNC marker. The absence of the expression of this marker distinguishes MK cells from MNCs.

[0106] The MK cells of the present invention preferably express detectable levels of CD25 (also known as IL-2R α , Tac, and p55). The expression of this marker also distinguishes MK cells from ioMP cells, iMPs, and MSCs. The expression of this marker can be increased by treatment / stimulation with IFN-γ and / or TNF-α.

[0107] The MK cells of the present invention preferably express detectable levels of CD136 (also known as MSP-R and RON). The expression of this marker also distinguishes MK cells from ioMP cells, iMPs, and MSCs. The expression of this marker can be increased by treatment / stimulation with IFN-γ.

[0108] The MK cells of the present invention preferably express a detectable level of CD155 (also known as PVR). CD155 is involved in NK cell priming / activation (Chan et al. J Immunol, January 15, 2010, 184(2) 902-911).

[0109] The MK cells of the present invention preferably express a detectable level of CD183 (also known as CXCR3). CD183 is involved in NK accumulation in cancer (Wendel et al. Cancer Res. October 15, 2008; 68(20):8437-45). The expression of this marker also differentiates MK cells from ioMP cells, iMPs, and MSCs. The expression of this marker can be increased by treatment / stimulation with IFN-γ.

[0110] The MK cells of the present invention preferably express a detectable level of CD205 (also known as DEC-205). The expression of this marker also differentiates MK cells from ioMP cells, iMPs, and MSCs. The expression of this marker can be increased by treatment / stimulation with IFN-γ.

[0111] The MK cells of the present invention preferably express a detectable level of CD332 (also known as FGFR2, BEK, and KGFR). CD332 (also known as FGFR2, BEK, and KGFR) regulates immune cell function (Messal et al. Eur J Immunol. December 2011; 41(12):3443-54). The expression of this marker also differentiates MK cells from ioMP cells, iMPs, and MSCs. The expression of this marker can be increased by treatment / stimulation with IFN-γ.

[0112] The MK cells of the present invention (a) preferably do not express detectable levels of CD102 and / or CD127. The MK cells of the present invention (b) preferably do not express detectable levels of CD104. The MK cells of the present invention (c) preferably do not express detectable levels of one or more of CD50, CD62E, CD62L, and CD62P, and preferably all of CD50, CD62E, CD62L, and CD62P. The MK cells of the present invention can be (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b), and (c). This marker expression pattern also distinguishes MK cells from PML. All references herein (including Tables 1 or 2 below) to one or more of (i) CD50, (ii) CD62E, (iii) CD62L, and (iv) CD62P can refer to (i), (ii), (iii), (iv), (i) and (ii), (i) and (iii), (i) and (iv), (ii) and (iii), (ii) and (iv), (iii) and (iv), (i), (ii) and (iii), (i), (ii) and (iv), (i), (iii) and (iv), (ii), (iii) and (iv), or (i), (ii), (iii) and (iv). The lack of expression of CD50, CD102, and CD127 also distinguishes MK cells from MSCs.

[0113] The MK cells of the present invention preferably express detectable levels of CD328. CD328 (also known as Siglec-7 (sialic acid-binding immunoglobulin-like lectin-7)) is not expressed by CD56-negative NK cells, which represent an abnormal NK cell subset found in small amounts in healthy individuals and at elevated levels in individuals chronically infected with HIV-1 and HCV (Brunetta, E. et al. (2009) Blood 114, 3822–3830). Thus, the expression of CD328 also distinguishes MK cells from CD56-negative NK cells.

[0114] The MK cells of the present invention preferably express detectable levels of one or more NK activating receptors and / or one or more NK inhibitory receptors. The activating and inhibitory receptors can be any of the receptors discussed below with reference to NK cells.

[0115] In terms of activating receptors, the MK cells of the present invention preferably express one or more of detectable levels of CD158d (also known as KIR2DL4), CD158i (also known as KIR2DS4), CD160 (also known as BY55), CD314 (also known as NKG2D and KLR), and CD337 (also known as NKp30 and Ly117), preferably all of CD158d, CD158i, CD160, CD314, and CD337. The MK cells of the present invention preferably express a detectable level of CD159c (also known as NKG2C).

[0116] In terms of inhibitory receptors, the MK cells of the present invention preferably express one or more of detectable levels of (a) CD158b2 (also known as KIR2DL3), (b) CD158f (also known as KIR2DL5), and (c) CD159a (also known as NKG2A), preferably all of (a) CD158b2, (b) CD158f, and (c) CD159a. The cell can express detectable levels of (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b), and (c).

[0117] The MK cells of the present invention preferably do not express a detectable level of CD159c (also known as NKG2C). The MK cells of the present invention preferably do not express one or more of (a) CD244, (b) CD335, and (c) CD352, such as (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b), and (c). The MK cells of the present invention preferably do not express one or more of (a) CD244, (b) CD335, and (c) CD352 (in any defined manner) and CD159c. The absence of the expression of these markers differentiates MK cells from NK cells.

[0118] The MK cells of the present invention preferably do not express CD140a. The MK cells of the present invention preferably do not express one or more of (i) CDH6, (ii) CD129, (iii) CD200, and (iv) CD271. The MK cells preferably do not express any number and combination of (i) to (iv), such as (i), (ii), (iii), (iv), (i) and (ii), (i) and (iii), (i) and (iv), (ii) and (iii), (ii) and (iv), (iii) and (iv), (i), (ii) and (iii), (i), (ii) and (iv), (i), (iii) and (iv), (ii), (iii) and (iv), or (i), (ii), (iii) and (iv).

[0119] The MK cells of the present invention are not MSCs. The expression patterns of typical markers of MSCs are listed in Table 1 of Zhao et al., Stem Cells & Regenerative Medicine, Springer Science 2011 (10.1007 / 978-1-60761-860-7_12). The MK cells preferably express one or more of CD11b, CD11c, CD49d, CD51, CD86, CD106, CD117, CD202b, and CD309 at detectable levels, such as 2, 3, 4, 5, 6, 7, or 8 or more of them. The MK cells preferably express CD11b, CD25, CD49d, CD51, CD86, CD106, CD117, CD202b, and CD309 at detectable levels. The MK cells preferably express CD11b, CD11c, CD25, CD49d, CD51, CD86, CD106, CD117, CD202b, and CD309 at detectable levels. The MK cells preferably express one or more TRAIL receptors at detectable levels, and the MK cells preferably express one or more of CD261, CD262, CD263, and CD264 at detectable levels, such as 2 or 3 or more of them. The MK cells preferably express CD261, CD262, and CD264 at detectable levels. The MK cells preferably express CD261, CD262, CD263, and CD264 at detectable levels. The MK cells preferably do not express one or more of CD184, CD195, CD197, and CD282 at detectable levels, such as 2 or 3 or more of them. The MK cells preferably do not express CD184, CD195, CD197, and CD282 at detectable levels.

[0120] The MK cells preferably express one or more Toll-like receptors (TLRs) at detectable levels, particularly TLR3, TLR4, TLR6, TLR8, TLR9, and TLR10. The MK cells preferably express one or more of CD283, CD284, CD286, CD288, CD289, and CD290 at detectable levels, such as 2, 3, 4, 5, or more of them.

[0121] The MK cells of the present invention preferably express one or more granzymes at a detectable level. Granzymes are a family of serine proteases that are expressed by cytotoxic T lymphocytes and NK cells and are involved in their cytotoxicity. After the formation of a receptor-mediated conjugate between a cell containing granzymes and an infected or transformed target cell, the granzymes enter the target cell by endocytosis and induce apoptosis (Trapani, J. A. Granzymes: a family of lymphocyte granule serine proteases. Genome Biol 2, review 3014.1 (2001) doi:10.1186 / gb-2001-2-12-review 3014). The MK cells of the present invention preferably express one or more of (a) granzyme B (GZMB), (b) granzyme H (GZMH), (c) granzyme M (GZMM), (d) granzyme A (GZMA), and (e) granzyme K (GZMK), such as (a); (b); (c); (d); (e); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (b) and (c); (b) and (d); (b) and (e); (c) and (d); (c) and (e); (d) and (e); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (c) and (d); (a), (c) and (e); (a), (d) and (e); (b), (c) and (d); (b), (c) and (e); (b), (d) and (e); (c), (d) and (e); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (d) and (e); (a), (c), (d) and (e); (b), (c), (d) and (e); and (a), (b), (c), (d) and (e). The expression of one or more granzymes by MK cells can be increased by exposing the MK cells to cancer cells (any cancer cells as described herein). The MK cells of the present invention preferably express perforin (PRF1). Perforin is a pore-forming cytolytic protein that is found in the granules of cytotoxic T lymphocytes and NK cells (Osińska I, Popko K, Demkow U. Perforin: an important player in immune response. Cent Eur J Immunol. 2014;39(1):109–115. doi:10.5114 / ceji.2014.42135). The expression of perforin by MK cells can be increased by exposing the MK cells to cancer cells (any cancer cells as described herein). Any of the above methods can be used to detect the expression of one or more granzymes and / or perforin.The results in Examples 13 and 14 demonstrate at least a partial role of one or more granzymes and / or perforins in MK cytotoxicity.

[0122] The MK cells of the present invention are generally capable of having a cytotoxic effect on cancer cells (i.e., being able to kill cancer cells). The ability of the MK cells of the present invention to have a cytotoxic effect can be measured using standard assays known in the art. The present invention preferably uses a chromium-51 ( 51 51Cr) release assay, which measures the 51 51Cr released from cells (such as cancer cells) after lysis of the MK cells. The MK cells can be incubated with cancer cells as discussed below and in the Examples. The present invention also preferably uses an europium (Eu3+) release assay. Suitable cancers are discussed in more detail.

[0123] The MK cells of the present invention generally also secrete a variety of cytokines and other molecules that contribute to their cytotoxic and NK-priming functions. The cytokines and other molecules can be measured using methods known in the art. Suitable methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. One particular method is an assay commercially available from Life Technologies.

[0124] MK cells preferably secrete detectable levels of one or more of (a) chemokine (C-X-C motif) ligand 1 (CXCL1, also known as GROα), (b) interleukin-12 (IL-12), (c) soluble IL-2 receptor (IL-2Ra), (d) IL-8, (e) soluble TRAIL, and (f) IL-6. MK cells can secrete detectable levels of all of (a) to (f). The secretion of these detectable molecules can be measured as described above. In the definitions of (a) to (f) given above, any combination and permutation of one or more of (a) to (f) can be secreted.For example, for each of the definitions (a) through (f), the MK cell can secrete detectable levels of (a); (b); (c); (d); (e); (f); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (b) and (c); (b) and (d); (b) and (e); (b) and (f); (c) and (d); (c) and (e); (c) and (f); (d) and (e); (d) and (f); (e) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f); (a), (e) and (f); (b), (c) and (d); (b), (c) and (e); (b), (c) and (f); (b), (d) and (e); (b), (d) and (f); (b), (e) and (f); (c), (d) and (e); (c), (d) and (f); (c), (e) and (f); (d), (e) and (f); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (b), (d) and (e); (a), (b), (d) and (f); (a), (b), (e) and (f); (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (d), (e) and (f); (b), (c), (d) and (e); (b), (c), (d) and (f); (b), (c), (e) and (f); (b), (d), (e) and (f); (c), (d), (e) and (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (b), (c), (e) and (f); (a), (b), (d), (e) and (f); (a), (c), (d), (e) and (f); (b), (c), (d), (e) and (f); or (a), (b), (c), (d) and (e). Combinations of (i) through (vii) can be independently selected from this list. The MK cell preferably secretes detectable levels of (a) GROα, (b) interleukin-12 (IL-12), (c) IL-2 receptor α chain (IL-2Rα), (d) IL-8, (e) soluble TRAIL, and (f) IL-6.

[0125] CXCL1 (also known as GROa) is a chemokine capable of attracting neutrophils (Moser et al., J Exp Med. May 1, 1990; 171(5):1797–1802). Secretion of GROa by MK cells is increased by TNF-α.

[0126] IL-12 is a pro-inflammatory cytokine that promotes the differentiation of naive T cells into Th1 cells (Hsieh et al. (April 1993) Science. 260(5107):547–9) and increases the cytotoxic activity (“priming”) of NK cells (Lehmann et al., Br J Haematol. September 2001; 114(3):660-5) and CD8+ T cells.

[0127] IL-2Ra is the soluble form of the IL-2 receptor. It can produce a pro-inflammatory effect by binding to IL-2. For example, it can enhance the development of the Th17 response in mice (Russell SE, Moore AC, Fallon PG, Walsh PT (2012) Soluble IL-2Rα (sCD25) Exacerbates Autoimmunity and Enhances the Development of Th17 Responses in Mice. PLoS ONE 7(10):e47748. https: / / doi.org / 10.1371 / journal.pone.0047748). Secretion of L-2Ra by MK cells is increased by IFN-γ.

[0128] IL-8, also known as neutrophil chemotactic factor, attracts and activates neutrophils within the inflammatory region (Bickel, J Periodontol. May 1993; 64(5 Suppl):456-60). Secretion of IL-8 by MK cells is decreased by IFN-γ and increased by TNF-α.

[0129] Soluble TRAIL (the soluble extracellular domain of TRAIL) can induce apoptosis in many tumor cell lines without affecting most normal cells. For example, overexpression of soluble TRAIL induces apoptosis of human lung adenocarcinoma and inhibits the growth of tumor xenografts in nude mice (Shi et al Cancer Res 2005; 65:(5). March 1, 2005).

[0130] Although IL-6 is generally considered an anti-inflammatory cytokine, during an active immune response, it can also promote lymphocyte activation, proliferation, and survival (Fisher et al., Semin Immunol. February 2014; 26(1):38-47).

[0131] MK cells preferably secrete detectable levels of IL-15 and / or C-X-C motif chemokine 10 (CXCL10, also known as interferon-gamma-inducible protein 10 (IP-10)). MK cells preferably secrete detectable levels of IL-15 and / or CXCL10 (IP-10) in combination with one or more of the above (a) GROa, (b) interleukin-12 (IL-12), (c) IL-2Ra, (d) IL-8, (e) soluble TRAIL, and (f) IL-6. Detectable secretion of these molecules can be measured as described above.

[0132] IL-15 stimulates the proliferation of T, B, and NK cells and induces stem, central, and effector memory CD8 T cells, and clinical trials using IL-15 and related molecules are being initiated (Waldmann, 2014, Expert Review of Clinical Immunology, Volume 10, 2014 - Issue 12).

[0133] CXCL10 (IP-10) is involved in the chemotaxis of monocytes, macrophages, T cells, NK cells, and dendritic cells, promotes the adhesion of T cells to endothelial cells, anti-tumor activity, and the inhibition of bone marrow colony formation and angiogenesis (Dufour et al. 2002, Journal of Immunology. 168(7):3195–204; and Angiolillo et al., 1995, The Journal of Experimental Medicine. 182(1):155–62).

[0134] MK cells preferably secrete detectable levels of one or more of interleukin-6 (IL-6), IL-8, chemokine (C-C motif) ligand 2 (CCL2; monocyte chemoattractant protein-1; MCP-1), and chemokine (C-C motif) ligand 5 (CCL5; regulated on activation, normal T cell expressed and secreted; RANTES). MK cells can secrete any amount of these factors and any combination of these factors. MK cells preferably secrete all of these markers.

[0135] The MK cells of the present invention can secrete a detectable level of one or more of (i) vascular endothelial growth factor (VEGF), (ii) transforming growth factor β (TGF-β), (iii) insulin-like growth factor-1 (IGF-1), (iv) fibroblast growth factor (FGF), (v) tumor necrosis factor α (TNF-α), (vi) IFN-γ, and (vii) interleukin-1α (IL-1α). The detectable secretion of these markers can be measured as described above.

[0136] In the definitions of (i) to (vii) given above, any combination of one or more of (i) to (vii) can be secreted. For example, for each of the definitions of (i) to (vii), MK cells can secrete detectable levels of (i); (ii); (iii); (iv); (v); (vi); (vii); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (i) and (vii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (ii) and (vii); (iii) and (iv); (iii) and (v); (iii) and (vi); (iii) and (vii); (iv) and (v); (iv) and (vi); (iv) and (vii); (v) and (vi); (v) and (vii); (vi) and (vii); (i), (ii) and (iii); (i), (ii) and (iv); (i), (ii) and (v); (i), (ii) and (vi); (i), (ii) and (vii); (i), (iii) and (iv); (i), (iii) and (v); (i), (iii) and (vi); (i), (iii) and (vii); (i), (iv) and (v); (i), (iv) and (vi); (i), (iv) and (vii); (i), (v) and (vi); (i), (v) and (vii); (i), (vi) and (vii); (ii), (iii) and (iv); (ii), (iii) and (v); (ii), (iii) and (vi); (ii), (iii) and (vii); (ii), (iv) and (v); (ii), (iv) and (vi); (ii), (iv) and (vii); (ii), (v) and (vi); (ii), (v) and (vii); (ii), (vi) and (vii); (iii), (iv) and (v); (iii), (iv) and (vi); (iii), (iv) and (vii); (iii), (v) and (vi); (iii), (v) and (vii); (iii), (vi) and (vii); (iv), (v) and (vi); (iv), (v) and (vii); (iv), (vi) and (vii); (v), (vi) and (vii); (i), (ii), (iii) and (iv); (i), (ii), (iii) and (v); (i), (ii), (iii) and (vi); (i), (ii), (iii) and (vii); (i), (ii), (iv) and (v); (i), (ii), (iv) and (vi); (i), (ii), (iv) and (vii);(i), (ii), (v) and (vi); (i), (ii), (v) and (vii); (i), (ii), (vi) and (vii); (i), (iii), (iv) and (v); (i), (iii), (iv) and (vi); (i), (iii), (iv) and (vii); (i), (iii), (v) and (vi); (i), (iii), (v) and (vii); (i), (iii), (vi) and (vii); (i), (iv), (v) and (vi); (i), (iv), (v) and (vii); (i), (iv), (vi) and (vii); (i), (v), (vi) and (vii); (ii), (iii), (iv) and (v); (ii), (iii), (iv) and (vi); (ii), (iii), (iv) and (vii); (ii), (iii), (v) and (vi); (ii), (iii), (v) and (vii); (ii), (iii), (vi) and (vii); (ii), (iv), (v) and (vi); (ii), (iv), (v) and (vii); (ii), (iv), (vi) and (vii); (ii), (v), (vi) and (vii); (iii), (iv), (v) and (vi); (iii), (iv), (v) and (vii); (iii), (iv), (vi) and (vii); (iii), (v), (vi) and (vii); (iv), (v), (vi) and (vii); (i), (ii), (iii), (iv) and (v); (i), (ii), (iii), (iv) and (vi); (i), (ii), (iii), (iv) and (vii); (i), (ii), (iii), (v) and (vi); (i), (ii), (iii), (v) and (vii); (i), (ii), (iii), (vi) and (vii); (i), (ii), (iv), (v) and (vi); (i), (ii), (iv), (v) and (vii); (i), (ii), (iv), (vi) and (vii); (i), (ii), (v), (vi) and (vii); (i), (iii), (iv), (v) and (vi); (i), (iii), (iv), (v) and (vii); (i), (iii), (iv), (vi) and (vii); (i), (iii), (v), (vi) and (vii); (i), (iv), (v), (vi) and (vii); (ii), (iii), (iv), (v) and (vi); (ii), (iii), (iv), (v) and (vii);(ii), (iii), (iv), (vi) and (vii); (ii), (iii), (v), (vi) and (vii); (ii), (iv), (v), (vi) and (vii); (iii), (iv), (v), (vi) and (vii); (i), (ii), (iii), (iv), (v) and (vi); (i), (ii), (iii), (iv), (v) and (vii); (i), (ii), (iii), (iv), (vi) and (vii); (i), (ii), (iii), (v), (vi) and (vii); (i), (ii), (iv), (v), (vi) and (vii); (i), (iii), (iv), (v), (vi) and (vii); (ii), (iii), (iv), (v), (vi) and (vii); or (i), (ii), (iii), (iv), (v), (vi) and (vii). The combinations of (i) to (vii) can be independently selected from this list.;

[0137] The MK cells of the present invention preferably secrete detectable levels of IFN-γ. IFN-γ expression or secretion can be determined using the methods described above.

[0138] As discussed in more detail below, MK cells are capable of priming / activating NK cells (i.e., increasing the proliferation and / or cytotoxic activity of NK cells).

[0139] The MK cells of the present invention preferably are capable of migrating to specific tissues of a subject. In other words, when the cells are administered to a subject suffering from a disease such as cancer, the cells are capable of migrating or homing to the desired one or more tissues. The tissue can be a tissue that is normally present in a healthy subject. Alternatively, the tissue can be a tumor. This migratory ability of MK cells is advantageous because it means that the cells can be infused via standard routes, such as intravenously, and then will target the diseased site. The cells do not have to be delivered to the diseased tissue.

[0140] The specific tissue can be any of the tissues discussed above. This applies not only to migration, but also to adhesion, reverse migration, proliferation, anti-tumor effects, immunomodulatory effects, pro-inflammatory effects, and anti-inflammatory effects as discussed in more detail above and below.

[0141] The ability of the MK cells of the present invention to migrate to diseased tissues can be measured using standard assays known in the art. Suitable methods include, but are not limited to, genomic reverse transcription polymerase chain reaction (RT-PCR with or without a reporter gene) and labeling techniques. Alternatively, the MK cells of the present invention can be stained with a dye of interest (such as a fluorescent dye) and can be monitored in a subject by the signal from the dye. Such methods are routine methods in the art.

[0142] Migration (or homing) is typically determined by measuring the number of cells that reach the damaged tissue. It can also be measured indirectly by observing the number of cells that accumulate in the lung (instead of the damaged tissue).

[0143] The MK cells of the present invention preferably have the ability to adhere to a specific diseased tissue of a subject. Adherence and adhesion assays are known in the art (Humphries, Methods Mol Biol. 2009; 522:203-10).

[0144] The MK cells of the present invention preferably have the ability to migrate through vascular endothelium to a specific diseased tissue of a subject. Transmigration assays are known in the art (Muller and Luscinskas, Methods Enzymol. 2008; 443:155–176).

[0145] The MK cells of the present invention preferably have the ability to attract or chemoattract immune cells to an inflammatory site. More preferably, the MK cells of the present invention have the ability to attract or chemoattract immune cells to cancer or a tumor. The MK cells of the present invention preferably have the ability to induce the migration of immune cells to an inflammatory, cancerous, or tumor site. The MK cells of the present invention are preferably pro-inflammatory. Preferably, the MK cells having any of these attracting / chemoattracting / pro-inflammatory effects are being or have been treated with IFN-γ. Any of the methods discussed above for measuring cell migration or movement can be used to measure the attraction / chemoattraction / migration of immune cells. The immune cells can be lymphocytes (such as T cells, B cells, or NK cells), neutrophils, or monocytes / macrophages. The immune cells are preferably NK cells and / or immune cells.

[0146] The MK cells of the present invention are preferably autologous. In other words, the cells are preferably derived from the subject to whom the cells will be administered. Alternatively, the MK cells are preferably allogeneic. In other words, the cells are preferably derived from a different subject / donor or from a subject / donor who is immunologically compatible with the subject to whom the cells will be administered.

[0147] The MK cells of the present invention can be isolated, substantially isolated, purified or substantially purified. An MK cell is isolated or purified if it is completely free of any other components, such as culture medium, other cells of the present invention or other cell types. An MK cell is substantially isolated if it is mixed with a carrier or diluent (such as culture medium) that does not interfere with its intended use. Alternatively, as described below, the MK cells of the present invention can be present in a growth matrix or immobilized on a surface.

[0148] A variety of techniques can be used to isolate the MK cells of the present invention, including antibody-based techniques. Based on the binding of monoclonal antibodies to those surface markers present on MK cells (see above), negative and positive selection techniques can be used to isolate the cells. Thus, MK cells can be isolated using any antibody-based technique, including fluorescence-activated cell sorting (FACS) and magnetic bead separation.

[0149] As discussed in more detail below, MK cells can be processed ex vivo. Thus, the cells can be loaded or transfected with a therapeutic or diagnostic agent and then used therapeutically in the methods of the present invention.

[0150] Population of the present invention

[0151] The present invention also provides a population of MK cells of the present invention. The present invention also provides a population of two or more MK cells of the present invention. The MK cells can be any of those cells defined above. Any number of cells can be present in the population. The populations of the present invention can include at least about 5,000 cells, such as at least about 6,000 cells, at least about 7,000 cells, at least about 8,000 cells, at least about 9,000 cells, at least about 10,000 cells, at least about 20,000 cells, at least about 30,000 cells, at least about 40,000 cells, at least about 50,000 cells, at least about 100,000 cells, at least about 200,000 cells or at least about 250,000 cells. The populations of the present invention preferably include at least about 5×10 5 MK cells of the present invention. The population more preferably includes at least about 1×10 6 at least about 2×10 6 at least about 2.5×10 6 at least about 5×10 6 at least about 1×10 7 at least about 2×10 7 at least about 5×10 7 at least about 1×10 8 at least about 2×10 8MK cells of the present invention. In certain cases, the population can include at least about 1.0×10 7 cells, at least about 1.0×10 8 cells, at least about 1.0×10 9 cells, at least about 1.0×10 10 cells, at least about 1.0×10 11 cells or at least about 1.0×10 12 cells of the present invention or even more.

[0152] A population comprising MK cells of the present invention can include other cells in addition to the MK cells of the present invention. However, at least about 70% of the cells in the population are preferably MK cells of the present invention. More preferably, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 97%, at least about 98% or at least about 99% of the cells in the population are preferably MK cells of the present invention. In a preferred embodiment, at least about 70%, at least about 75%, at least about 80% or at least about 85% of the cells in the population are MK cells, expressing the positive MK markers defined above and not expressing the negative MK markers defined above. In another preferred embodiment, at least about 90% of the cells in the population are MK cells, expressing the positive MK markers defined above and not expressing the negative MK markers defined above. In another preferred embodiment, at least about 95% of the cells in the population are MK cells, expressing the positive MK markers defined above and not expressing the negative MK markers defined above.

[0153] The present invention also provides a population of MK cells of the present invention, wherein more than about 15% of the cells in the population express detectable levels of CD112, CD137L, CD178, CD253 and CD277, and wherein about 5% or less of the cells in the population express detectable levels of CD34 and CD45. In these populations, more than about 15% of the cells in the population (or any of the percentages discussed above) can express detectable levels of the above specific markers detectably expressed by the MK cells of the present invention. Similarly, about 5% or less (or any of the lower percentages discussed above) can express detectable levels of the above specific markers not detectably expressed by the MK cells of the present invention. These populations can contain any of the above numbers of cells. Table 1 lists specific populations of the present invention.

[0154] Table 1 - Preferred Populations of the Present Invention (wherein "one or more" is as defined above with reference to specific markers, which definition applies to the use of terms related to those markers in the table; * = combination of markers in the left column of the relevant population)

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] In the populations discussed above and listed in Table 1, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% of the cells in the population preferably express a detectable level of a relevant marker (in particular, the markers in column 2 of Table 1). In the populations discussed above and listed in Table 1, about 60% or more of the cells in the population more preferably express a detectable level of a relevant marker (in particular, the markers in column 2 of Table 1). In the populations discussed above and listed in Table 1, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or more of the cells in the population more preferably express a detectable level of a relevant marker (in particular, the markers in column 2 of Table 1). In the populations discussed above and listed in Table 1, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less of the cells can express a detectable level of a relevant marker (in particular, the markers in column 3 of Table 1).

[0161] The present invention also provides a population of MK cells of the present invention, wherein more than about 15% of the cells in the population express a detectable level of CD16, CD96, CD112, CD137L, CD178, CD253, and CD277, and wherein about 5% or less of the cells in the population express a detectable level of CD34, CD45, and CD56. In these populations, more than about 15% of the cells (or any percentage % discussed above) in the population can express a detectable level of the above-mentioned specific markers detectably expressed by the MK cells of the present invention. Similarly, about 5% or less (or any lower percentage % discussed above) can express a detectable level of the above-mentioned specific markers not detectably expressed by the MK cells of the present invention. These populations can contain any of the above numbers of cells. Table 2 lists specific populations of the present invention.

[0162] Table 2 - Preferred Populations of the Invention (wherein, "one or more" is as defined above with reference to the specific markers, and this definition applies to the use of terms related to those markers in the table; *= combination of markers in the left column of the relevant population)

[0163]

[0164]

[0165]

[0166]

[0167] In the populations discussed above and listed in Table 2, in the population, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% of the cells preferably express a detectable level of the relevant marker (especially the markers in the second column of Table 2). In the populations discussed above and listed in Table 2, in the population, about 60% or more of the cells more preferably express a detectable level of the relevant marker (especially the markers in the second column of Table 2). In the populations discussed above and listed in Table 2, in the population, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or more of the cells more preferably express a detectable level of the relevant marker (especially the markers in the second column of Table 2). In the populations discussed above and listed in Table 2, in the population, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less of the cells may express a detectable level of the relevant marker (especially the markers in the third column of Table 2).

[0168] The invention also provides specific populations of MK cells. The invention provides a population of MK cells, wherein

[0169] (i) at least about 20% (such as at least about 25%, at least about 27%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 61% or at least about 62%) of the cells in the population express a detectable level of CD112,

[0170] (ii) at least about 80% (such as at least about 90%, at least about 95%, at least about 96% or at least about 97%) of the cells in the population express a detectable level of CD137L,

[0171] (iii) At least about 20% (such as at least about 21%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%) of the cells in the population express a detectable level of CD178,

[0172] (iv) At least about 50% (such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 92%, or at least about 93%) of the cells in the population express a detectable level of CD253, and

[0173] (v) At least about 50% (such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, or at least about 97%) of the cells in the population express a detectable level of CD277,

[0174] And wherein

[0175] (a) About 5% or less (such as about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less) of the cells in the population express a detectable level of CD34, and

[0176] (b) About 5% or less (such as about 4% or less, about 3% or less, about 2% or less, or about 1% or less) of the cells in the population express a detectable level of CD45.

[0177] The present invention also provides a population of MK cells, wherein

[0178] (i) At least about 15% (such as at least about 18%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, or at least about 66%) of the cells in the population express a detectable level of CD16,

[0179] (ii) At least about 50% (such as at least about 58%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, or at least about 86%) of the cells in the population express a detectable level of CD96,

[0180] (iii) At least about 20% (such as at least about 25%, at least about 27%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 61%, or at least about 62%) of the cells in the population express a detectable level of CD112,

[0181] (iv) At least about 80% (such as at least about 90%, at least about 95%, at least about 96%, or at least about 97%) of the cells in the population express a detectable level of CD137L,

[0182] (v) At least about 20% (such as at least about 21%, at least about 30%, at least about 40%, at least about 50% or at least about 60%) of the cells in the population express a detectable level of CD178,

[0183] (vi) At least about 50% (such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 92% or at least about 93%) of the cells in the population express a detectable level of CD253, and

[0184] (vii) At least about 50% (such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96% or at least about 97%) of the cells in the population express a detectable level of CD277,

[0185] and wherein

[0186] (a) About 5% or less (such as about 4% or less, about 3% or less, about 2% or less, about 1% or less or about 0.5% or less) of the cells in the population express a detectable level of CD34,

[0187] (b) About 5% or less (such as about 4% or less, about 3% or less, about 2% or less or about 1% or less) of the cells in the population express a detectable level of CD45, and

[0188] (c) About 5% or less (such as about 4% or less or about 3% or less) of the cells in the population express a detectable level of CD56.

[0189] Preferably, wherein one or more of the following, or more preferably all of the following:

[0190] —— About 10% or less (such as about 9% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less or about 1% or less) of the cells in the population express a detectable level of CD14;

[0191] —— At least about 5% (such as at least about 7%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% or at least about 54%) of the cells in the population express a detectable level of CD25;

[0192] —— At least about 10% (such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 69%, at least about 70% or at least about 80%) of the cells in the population express a detectable level of CD136;

[0193] ——At least about 90% (such as at least about 95%, at least about 97%, at least about 98%, or at least about 99%) of the cells in the population express a detectable level of CD155;

[0194] ——At least about 20% (such as at least about 30%, at least about 40%, at least about 50%, or at least about 51%) of the cells in the population express a detectable level of CD183;

[0195] ——At least about 10% (such as at least about 15%, at least about 20%, at least about 30%, or at least about 32%) of the cells in the population express a detectable level of CD205;

[0196] ——At least about 9% (such as at least about 10%, at least about 20%, at least about 25%, or at least about 29%) of the cells in the population express a detectable level of CD332;

[0197] ——About 2% or less (such as about 1% or less or about 0.5% or less) of the cells in the population express a detectable level of CD102;

[0198] ——About 2% or less (such as about 1% or less or about 0.5% or less) of the cells in the population express a detectable level of CD127;

[0199] ——About 10% or less (such as about 9% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less) of the cells in the population express a detectable level of CD104;

[0200] ——About 60% or less (such as about 50% or less, about 46% or less, about 30% or less, or about 20%) of the cells in the population express a detectable level of CD126;

[0201] ——At least about 15% (such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%) of the cells in the population express a detectable level of CD126;

[0202] ——About 3% or less (such as about 2% or less or about 1% or less) of the cells in the population express a detectable level of CD62E;

[0203] ——About 5% or less (such as about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less) of the cells in the population express a detectable level of CD62L;

[0204] ——About 1% or less (such as about 0.5% or less) of the cells in the population express a detectable level of CD62P;

[0205] ——At least about 30% (such as at least about 33%, at least about 40%, at least about 50%, at least about 55%, or at least about 59%) of the cells in the population express a detectable level of CD158d;

[0206] ——At least about 22% (such as at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 61%) of the cells in the population express a detectable level of CD158i;

[0207] ——At least about 30% (such as at least about 40%, at least about 45%, at least about 50%, or at least about 51%) of the cells in the population express a detectable level of CD160;

[0208] ——At least about 40% (such as at least about 45%, at least about 48%, at least about 50%, or at least about 54%) of the cells in the population express a detectable level of CD314;

[0209] ——At least about 30% (such as at least about 35%, at least about 40%, or at least about 50%, at least about 60%, at least about 70%, or at least about 72%) of the cells in the population express a detectable level of CD337;

[0210] ——At least about 6% or at least about 10% of the cells in the population express a detectable level of CD159c;

[0211] ——At least about 7% (such as at least about 10%, at least about 15%, at least about 20%, or at least about 23%) of the cells in the population express a detectable level of CD158b2;

[0212] ——At least about 30% (such as at least about 40%, at least about 41%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 87%) of the cells in the population express a detectable level of CD158f; and

[0213] ——At least about 8% (such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 51%) of the cells in the population express a detectable level of CD159a.

[0214] Preferably, about 3% or less (such as about 2.5% or less) of the cells in the population express a detectable level of CD159c.

[0215] A specific population of the present invention can be defined as above with reference to any combination of the markers shown in Table 1 or 2.

[0216] The present invention also provides specific populations of the present invention based on MK002 and MK004 in Example 3. The present invention preferably provides a population of MK cells, wherein

[0217] (i) at least about 62% of the cells in the population express a detectable level of CD112,

[0218] (ii) at least about 97% of the cells in the population express a detectable level of CD137L,

[0219] (iii) at least about 60% of the cells in the population express a detectable level of CD178,

[0220] (iv) at least about 93% of the cells in the population express a detectable level of CD253, and

[0221] (v) at least about 97% of the cells in the population express a detectable level of CD277,

[0222] and wherein

[0223] (a) about 0.5% or less of the cells in the population express a detectable level of CD34, and

[0224] (b) about 4% or less of the cells in the population express a detectable level of CD45.

[0225] The present invention preferably provides a population of MK cells, wherein

[0226] (i) at least about 66% of the cells in the population express a detectable level of CD16,

[0227] (ii) at least about 86% of the cells in the population express a detectable level of CD96,

[0228] (iii) at least about 62% of the cells in the population express a detectable level of CD112,

[0229] (iv) at least about 97% of the cells in the population express a detectable level of CD137L,

[0230] (v) at least about 60% of the cells in the population express a detectable level of CD178,

[0231] (vi) at least about 93% of the cells in the population express a detectable level of CD253, and

[0232] (vii) at least about 97% of the cells in the population express a detectable level of CD277,

[0233] and wherein

[0234] (a) about 0.5% or less of the cells in the population express a detectable level of CD34,

[0235] (b) Approximately 4% or fewer cells in the population express detectable levels of CD45, and

[0236] (c) Approximately 3% or fewer cells in the population express detectable levels of CD56.

[0237] Preferably, one or more of the following, or more preferably all of the following:

[0238] —— Approximately 9% or fewer cells in the population express detectable levels of CD14;

[0239] —— At least approximately 54% of the cells in the population express detectable levels of CD25,

[0240] —— At least approximately 80% of the cells in the population express detectable levels of CD136;

[0241] —— At least approximately 99% of the cells in the population express detectable levels of CD155;

[0242] —— At least approximately 51% of the cells in the population express detectable levels of CD183;

[0243] —— At least approximately 32% of the cells in the population express detectable levels of CD205;

[0244] —— At least approximately 29% of the cells in the population express detectable levels of CD332;

[0245] —— Approximately 2% or fewer cells in the population express detectable levels of CD102;

[0246] —— Approximately 2% or fewer cells in the population express detectable levels of CD127;

[0247] —— Approximately 9% or fewer cells in the population express detectable levels of CD104;

[0248] —— Approximately 46% or fewer cells in the population express detectable levels of CD126, or at least approximately 45% of the cells in the population express detectable levels of CD126;

[0249] —— Approximately 3% or fewer cells in the population express detectable levels of CD62E;

[0250] —— Approximately 5% or fewer cells in the population express detectable levels of CD62L;

[0251] —— Approximately 1% or fewer cells in the population express detectable levels of CD62P;

[0252] —— At least approximately 59% of the cells in the population express detectable levels of CD158d;

[0253] — At least about 61% of the cells in the population express detectable levels of CD158i;

[0254] — At least about 40% of the cells in the population express detectable levels of CD160;

[0255] — At least about 54% of the cells in the population express detectable levels of CD314;

[0256] — At least about 72% of the cells in the population express detectable levels of CD337;

[0257] — At least about 10% of the cells in the population express detectable levels of CD159c;

[0258] — At least about 23% of the cells in the population express detectable levels of CD158b2;

[0259] — At least about 87% of the cells in the population express detectable levels of CD158f; and

[0260] — At least about 51% of the cells in the population express detectable levels of CD159a.

[0261] A specific population of the present invention can be defined with reference to any combination of the markers shown in Table 1 or 2. This population most preferably has the marker expression pattern of MK002 shown in Table 7.

[0262] The present invention preferably provides a population of MK cells, wherein

[0263] (i) At least about 27% of the cells in the population express detectable levels of CD112,

[0264] (ii) At least about 97% of the cells in the population express detectable levels of CD137L,

[0265] (iii) At least about 21% of the cells in the population express detectable levels of CD178,

[0266] (iv) At least about 93% of the cells in the population express detectable levels of CD253, and

[0267] (v) At least about 96% of the cells in the population express detectable levels of CD277,

[0268] And wherein

[0269] (a) About 0.5% or less of the cells in the population express detectable levels of CD34, and

[0270] (b) About 1% or less of the cells in the population express detectable levels of CD45.

[0271] The present invention preferably provides a population of MK cells, wherein

[0272] (i) at least about 18% of the cells in the population express detectable levels of CD16,

[0273] (ii) at least about 58% of the cells in the population express detectable levels of CD96,

[0274] (iii) at least about 27% of the cells in the population express detectable levels of CD112,

[0275] (iv) at least about 97% of the cells in the population express detectable levels of CD137L,

[0276] (v) at least about 21% of the cells in the population express detectable levels of CD178,

[0277] (vi) at least about 93% of the cells in the population express detectable levels of CD253, and

[0278] (vii) at least about 96% of the cells in the population express detectable levels of CD277,

[0279] and wherein

[0280] (a) about 0.5% or fewer of the cells in the population express detectable levels of CD34,

[0281] (b) about 1% or fewer of the cells in the population express detectable levels of CD45, and

[0282] (c) about 5% or fewer of the cells in the population express detectable levels of CD56.

[0283] Preferably, one or more of the following, or more preferably all of the following:

[0284] —about 1% or fewer of the cells in the population express detectable levels of CD14;

[0285] —at least about 7% of the cells in the population express detectable levels of CD25,

[0286] —at least about 69% of the cells in the population express detectable levels of CD136;

[0287] —at least about 99% of the cells in the population express detectable levels of CD155;

[0288] —at least about 20% of the cells in the population express detectable levels of CD183;

[0289] ——At least about 15% of the cells in the population express a detectable level of CD205;

[0290] ——At least about 9% of the cells in the population express a detectable level of CD332;

[0291] ——About 0.5% or less of the cells in the population express a detectable level of CD102;

[0292] ——About 2% or less of the cells in the population express a detectable level of CD127;

[0293] ——About 2% or less of the cells in the population express a detectable level of CD104;

[0294] ——About 20% or less of the cells in the population express a detectable level of CD126, or at least about 19% of the cells in the population express a detectable level of CD126;

[0295] ——About 1% or less of the cells in the population express a detectable level of CD62E;

[0296] ——About 0.5% or less of the cells in the population express a detectable level of CD62L;

[0297] ——About 0.5% or less of the cells in the population express a detectable level of CD62P;

[0298] ——At least about 33% of the cells in the population express a detectable level of CD158d;

[0299] ——At least about 22% of the cells in the population express a detectable level of CD158i;

[0300] ——At least about 51% of the cells in the population express a detectable level of CD160;

[0301] ——At least about 48% of the cells in the population express a detectable level of CD314;

[0302] ——At least about 35% of the cells in the population express a detectable level of CD337;

[0303] ——About 2.5% or less of the cells in the population express a detectable level of CD159c;

[0304] ——At least about 7% of the cells in the population express a detectable level of CD158b2;

[0305] ——At least about 41% of the cells in the population express a detectable level of CD158f; and

[0306] ——At least about 8% of the cells in the population express a detectable level of CD159a.

[0307] The specific population of the present invention can be defined by any combination of the markers shown in Table 1 or 2. Most preferably, the population has the marker expression pattern of MK004 shown in Table 7.

[0308] The present invention preferably provides a population of MK cells, wherein

[0309] (i) at least about 46% of the cells in the population express a detectable level of CD112,

[0310] (ii) at least about 91% of the cells in the population express a detectable level of CD137L,

[0311] (iii) at least about 65% of the cells in the population express a detectable level of CD178,

[0312] (iv) at least about 88% of the cells in the population express a detectable level of CD253, and

[0313] (v) at least about 96% of the cells in the population express a detectable level of CD277,

[0314] and wherein

[0315] (a) about 1.5% or less of the cells in the population express a detectable level of CD34, and

[0316] (b) about 4.5% or less of the cells in the population express a detectable level of CD45.

[0317] The present invention preferably provides a population of MK cells, wherein

[0318] (i) at least about 34% of the cells in the population express a detectable level of CD16,

[0319] (ii) at least about 83% of the cells in the population express a detectable level of CD96,

[0320] (iii) at least about 46% of the cells in the population express a detectable level of CD112,

[0321] (iv) at least about 91% of the cells in the population express a detectable level of CD137L,

[0322] (v) at least about 65% of the cells in the population express a detectable level of CD178,

[0323] (vi) at least about 88% of the cells in the population express a detectable level of CD253, and

[0324] (vii) at least about 96% of the cells in the population express a detectable level of CD277,

[0325] and wherein

[0326] (a) Approximately 1.5% or fewer cells in the population express detectable levels of CD34,

[0327] (b) Approximately 4.5% or fewer cells in the population express detectable levels of CD45, and

[0328] (c) Approximately 1% or fewer cells in the population express detectable levels of CD56.

[0329] Preferably, one or more of the following, or more preferably all of the following:

[0330] —— Approximately 4% or fewer cells in the population express detectable levels of CD14;

[0331] —— At least approximately 43% of the cells in the population express detectable levels of CD25,

[0332] —— At least approximately 79% of the cells in the population express detectable levels of CD136;

[0333] —— At least approximately 99% of the cells in the population express detectable levels of CD155;

[0334] —— At least approximately 39% of the cells in the population express detectable levels of CD183;

[0335] —— At least approximately 46% of the cells in the population express detectable levels of CD205;

[0336] —— At least approximately 23% of the cells in the population express detectable levels of CD332;

[0337] —— Approximately 1.5% or fewer cells in the population express detectable levels of CD102;

[0338] —— At least approximately 6% of the cells in the population express detectable levels of CD127;

[0339] —— At least approximately 16% of the cells in the population express detectable levels of CD104;

[0340] —— At least approximately 54% of the cells in the population express detectable levels of CD126;

[0341] —— Approximately 4% or fewer cells in the population express detectable levels of CD62E;

[0342] —— At least approximately 11% of the cells in the population express detectable levels of CD62L;

[0343] —— Approximately 2.5% or fewer cells in the population express detectable levels of CD62P;

[0344] — At least about 37% of the cells in the population express a detectable level of CD158d;

[0345] — At least about 44% of the cells in the population express a detectable level of CD158i;

[0346] — At least about 78% of the cells in the population express a detectable level of CD160;

[0347] — At least about 76% of the cells in the population express a detectable level of CD314;

[0348] — At least about 49% of the cells in the population express a detectable level of CD337;

[0349] — At least about 14% of the cells in the population express a detectable level of CD159c;

[0350] — At least about 21% of the cells in the population express a detectable level of CD158b2;

[0351] — At least about 48% of the cells in the population express a detectable level of CD158f; and

[0352] — At least about 34% of the cells in the population express a detectable level of CD159a.

[0353] A specific population of the present invention can be defined with reference to any combination of the markers shown in Table 1 or 2. This population most preferably has the marker expression pattern of IFN-γ-treated MK004 shown in Table 11.

[0354] The present invention preferably provides a population of MK cells, wherein

[0355] (i) At least about 23% of the cells in the population express a detectable level of CD112,

[0356] (ii) At least about 79% of the cells in the population express a detectable level of CD137L,

[0357] (iii) At least about 30% of the cells in the population express a detectable level of CD178,

[0358] (iv) At least about 77% of the cells in the population express a detectable level of CD253, and

[0359] (v) At least about 82% of the cells in the population express a detectable level of CD277,

[0360] And wherein

[0361] (a) Approximately 0.5% or less of the cells in the population express detectable levels of CD34, and

[0362] (b) Approximately 2% or less of the cells in the population express detectable levels of CD45.

[0363] The present invention preferably provides a population of MK cells, wherein

[0364] (i) At least about 16% of the cells in the population express detectable levels of CD16,

[0365] (ii) At least about 45% of the cells in the population express detectable levels of CD96,

[0366] (iii) At least about 23% of the cells in the population express detectable levels of CD112,

[0367] (iv) At least about 79% of the cells in the population express detectable levels of CD137L,

[0368] (v) At least about 30% of the cells in the population express detectable levels of CD178,

[0369] (vi) At least about 77% of the cells in the population express detectable levels of CD253, and

[0370] (vii) At least about 82% of the cells in the population express detectable levels of CD277,

[0371] wherein

[0372] (a) Approximately 0.5% or less of the cells in the population express detectable levels of CD34, and

[0373] (b) Approximately 2% or less of the cells in the population express detectable levels of CD45,

[0374] and wherein

[0375] Approximately 10% or less of the cells in the population express detectable levels of CD56, or at least about 10% of the cells in the population express detectable levels of CDCD56.

[0376] Preferably, one or more of the following, or more preferably all of the following:

[0377] —— Approximately 2% or less of the cells in the population express detectable levels of CD14;

[0378] —— At least about 12% of the cells in the population express detectable levels of CD25,

[0379] —— At least about 52% of the cells in the population express detectable levels of CD136;

[0380] ——At least about 99% of the cells in the population express detectable levels of CD155;

[0381] ——At least about 19% of the cells in the population express detectable levels of CD183;

[0382] ——At least about 11% of the cells in the population express detectable levels of CD205;

[0383] ——At least about 9% of the cells in the population express detectable levels of CD332;

[0384] ——About 1.5% or less of the cells in the population express detectable levels of CD102;

[0385] ——About 5% or less of the cells in the population express detectable levels of CD127;

[0386] ——About 3.5% or less of the cells in the population express detectable levels of CD104;

[0387] ——At least about 18% of the cells in the population express detectable levels of CD126;

[0388] ——About 1.5% or less of the cells in the population express detectable levels of CD62E;

[0389] ——About 3.5% or less of the cells in the population express detectable levels of CD62L;

[0390] ——About 2% or less of the cells in the population express detectable levels of CD62P;

[0391] ——At least about 24% of the cells in the population express detectable levels of CD158d;

[0392] ——At least about 18% of the cells in the population express detectable levels of CD158i;

[0393] ——At least about 52% of the cells in the population express detectable levels of CD160;

[0394] ——At least about 39% of the cells in the population express detectable levels of CD314;

[0395] ——At least about 31% of the cells in the population express detectable levels of CD337;

[0396] ——About 3.5% or less of the cells in the population express detectable levels of CD159c;

[0397] ——At least about 9% of the cells in the population express detectable levels of CD158b2;

[0398] ——At least about 33% of the cells in the population express a detectable level of CD158f; and

[0399] ——At least about 9% of the cells in the population express a detectable level of CD159a.

[0400] A specific population of the present invention can be defined with reference to any combination of the markers shown in Table 1 or 2. This population most preferably has the marker expression pattern of TNF-α-treated MK004 shown in Table 11.

[0401] In any of the populations discussed above, about 5% or fewer (such as about 4% or fewer, about 3% or fewer, about 2% or fewer, or about 1% or fewer) of the cells in the population preferably express one or more of (a) CD45RA, (b) CD45RB, and (c) CD45RO, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b), and (c).

[0402] In any of the populations discussed above, about 5% or fewer (such as about 4% or fewer, about 3% or fewer, about 2% or fewer, or about 1% or fewer) of the cells in the population preferably express CD140a, such as on their surface. In any of the populations discussed above, about 5% or fewer (such as about 4% or fewer, about 3% or fewer, about 2% or fewer, or about 1% or fewer) of the cells in the population preferably express one or more of (i) CDH6, (ii) CD129, (iii) CD200, and (iv) CD271, such as (i), (ii), (iii), (iv), (i) and (ii), (i) and (iii), (i) and (iv), (ii) and (iii), (ii) and (iv), (iii) and (iv), (i), (ii) and (iii), (i), (ii) and (iv), (i), (iii) and (iv), (ii), (iii) and (iv), or (i), (ii), (iii) and (iv).

[0403] The cells in these preferred populations can further express a detectable level of any of the above markers of the MK of the present invention. The cells in these preferred populations can have any of the advantageous properties of the above MK cells.

[0404] In any of the above embodiments, where a population is defined with reference to the percentage % of cells expressing certain markers, the population preferably comprises at least about 5,000 cells, such as at least about 6,000 cells, at least about 7,000 cells, at least about 8,000 cells, at least about 9,000 cells, at least about 10,000 cells, at least about 20,000 cells, at least about 30,000 cells, at least about 40,000 cells, at least about 50,000 cells, at least about 100,000 cells, at least about 200,000 cells, at least about 250,000 cells or at least about 500,000 cells. The population is more preferably at least about 5000 cells, at least about 50,000 cells or at least about 250,000 cells. These populations can contain any number of the above cells.

[0405] Any population of the invention preferably secretes a detectable level of one or more of (a) chemokine (C-X-C motif) ligand 1 (CXCL1 also known as GROa), (b) interleukin-12 (IL-12), (c) soluble IL-2 receptor (IL-2Ra), (d) IL-8, (e) soluble TRAIL and (f) IL-6. MK cells can secrete any combination and permutation of the above (a) to (f). Any population of the invention preferably secretes a detectable level of IL-15 and / or CXCL10 (IP-10). The population preferably secretes a detectable level of IL-15 and / or CXCL10 (IP-10) in combination with one or more of the above (a) GROa, (b) interleukin-12 (IL-12), (c) IL-2Ra, (d) IL-8, (e) soluble TRAIL and (f) IL-6.

[0406] Any population of the cells disclosed herein can be diluted with other cells prior to use. For example, the population can be combined with the subject's blood, MNC, MSC, NK cells, PML, iMP cells, ioMP cells or a combination thereof.

[0407] The populations of the invention are advantageous for the treatment methods as described above. The ability to generate populations of safe MK cells of the invention in large numbers is one of the key advantages of the invention. The invention allows treating a subject with a population of cells that are capable of effectively migrating to the tissue of interest and having an anti-tumor effect once there. This allows the use of low cell doses and avoids side effects associated with CAR-T cells and volume-related side effects.

[0408] The populations of the invention are preferably homogeneous. In other words, all iMP cells in the population are preferably identical genotypically and phenotypically. The population is preferably autologous or allogeneic as defined above.

[0409] However, the population can also be semi-allogeneic. A semi-allogeneic population is typically generated from MNCs from two or more subjects. In other words, all the cells in the population are preferably genetically identical or genetically similar enough. Since the MK cells of the present invention can be from a subject, they can be autologous to the subject to be treated.

[0410] The population of the present invention can be isolated, substantially isolated, purified or substantially purified. A population is isolated or purified if it is completely free of any other components, such as media and other cells. A population is substantially isolated if it is mixed with a carrier or diluent (such as media) that does not interfere with its intended use. Other carriers and diluents are discussed in more detail below. A substantially isolated or substantially purified population does not contain cells other than the MK cells of the present invention. In some embodiments, the population of the present invention can be present in a growth matrix or immobilized on a surface as described below.

[0411] The population is typically cultured in vitro. Techniques for culturing cells are well known to those skilled in the art. The cells can be cultured in serum-free media under standard conditions of 37 °C and 5% CO 2 2. The cells are preferably cultured with platelet lysate under hypoxic conditions as discussed in more detail below. The cells can be cultured in any suitable flask or container, including multi-well plates, such as standard 6-well plates. Such plates are commercially available from Fisher Scientific, VWR suppliers, Nunc, Starstedt or Falcon. The wells typically have a capacity of about 1 mL to about 4 mL.

[0412] The flask, container or well containing or culturing the population can be modified to facilitate the handling of the MK cells. For example, the flask, container or well can be modified to facilitate cell culture, such as by including a growth matrix. The flask, container or well can be modified to allow the MK cells to attach or to allow the MK cells to be immobilized on a surface. One or more surfaces can be coated with extracellular matrix proteins, such as laminin or collagen or any other capture molecule that binds to the cells and immobilizes or captures them on the surface.

[0413] The population can be modified ex vivo using any of the techniques described herein. For example, the population can be transfected or loaded with a therapeutic or diagnostic agent. The population can then be used in the treatment methods discussed in more detail below.

[0414] Method for generating MK cells of the present invention

[0415] The present invention also provides a method for generating the population of cells of the present invention. The method comprises culturing monocytes (MNCs) under conditions that induce the differentiation of MNCs into iMP cells (step (a)). This step is disclosed in PCT / GB2015 / 051673 (published as WO 2015 / 189587). The method then involves culturing the iMP cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides, and platelet lysate under hypoxic conditions and under conditions that permit the iMP cells to adhere and differentiate into MK cells (step (b)). MK cells have the marker expression profile as discussed above for the cells of the present invention. The cells can be harvested and frozen or used immediately using conventional techniques (such as those disclosed in the Examples).

[0416] Monocytes (MNCs) and methods for isolating them are known in the art. The MNCs are preferably primary MNCs isolated from bone marrow. The MNCs can preferably be peripheral blood MNCs (PBMCs), such as lymphocytes, monocytes, and / or macrophages. Hydrophilic polysaccharides (such as ) can be used to isolate MNCs from bone marrow or blood. For example, (a commercially available density medium) can be used to isolate MNCs, as disclosed in the Examples.

[0417] In all steps of the method, the cells are cultured in a serum-free medium under standard conditions of 37 °C and 5% CO 2 .

[0418] As described in PCT / GB2015 / 051673 (published as WO 2015 / 189587), in step (a), the MNCs are typically cultured in nucleoside-free Minimum Essential Medium (MEM) Alpha (ThermoFisher; product code: 32561-102) having the components listed in Table 5 to form iMP cells. MEM can be commercially obtained from various sources including Thermofisher and Sigma-Aldrich. Step (a) preferably comprises culturing monocytes (MNCs) in a medium lacking ribonucleosides and deoxyribonucleosides under conditions that induce the differentiation of MNCs into iMP cells. The ribonucleosides and deoxyribonucleosides can be any of those discussed below.

[0419] The culture medium in step (a) preferably further contains heparin and / or penicillin / streptomycin (P / S). The culture medium in step (a) is supplemented with platelet lysate. Step (a) preferably includes: culturing mononuclear cells (MNCs) in a culture medium lacking ribonucleosides and deoxyribonucleosides and containing platelet lysate under conditions for inducing the differentiation of MNCs into iMP cells. The ribonucleosides and deoxyribonucleosides can be any of those discussed below. Platelet lysate refers to a combination of natural growth factors contained in platelets released by lysing these platelets. The lysis can be accomplished chemically (i.e., CaCl 2 ), osmotically (using distilled water), or by a freeze / thaw process. The platelet lysate can be from whole blood, as described in U.S. Patent No. 5,198,357. The platelet lysate is preferably prepared as described in PCT / GB12 / 052911 (published as WO 2013 / 076507). The platelet lysate is preferably prepared by 4 freeze / thaw cycles using liquid nitrogen at each freezing stage. The plasma lysate is preferably human plasma lysate. The culture medium preferably contains about 20% or less platelet lysate by volume, such as about 15% or less or about 10% or less by volume. The culture medium preferably contains about 5% to about 20% platelet lysate by volume, such as about 10% to about 15% by volume. The culture medium preferably contains about 10% platelet lysate by volume.

[0420] Step (a) of the method of the present invention generally includes culturing MNCs for a sufficient time to induce the differentiation of MNCs into iMP cells. This sufficient time is generally about 15 days to about 25 days, preferably about 18, 19, 20, 21, 22, 23 or 24 days. The cells can be passaged and the culture medium can be changed after about 8 days. When the cells are nearly confluent, the cells can be passaged again and the culture medium can be changed after about another 4, 5 or 6 days (a total of about 12, 13 or 14 days). Then the iMP cells can be harvested after about 6, 7 or 8 days of near confluence (a total of about 18 to 22 days).

[0421] Step (a) generally includes culturing MNCs under conditions that allow MK cells to adhere. Culture flasks and 6-well, 12-well, 24-well and 96-well plates of different sizes that allow cell adhesion are commercially available from various sources, such as and

[0422] In step (a), the MNCs are preferably cultured under hypoxic conditions. Hypoxic conditions refer to less than 20.95% oxygen present in the atmosphere. The MNCs are preferably cultured under less than about 20.5% oxygen (O 2) Cultured under, such as below about 20%, below about 19%, below about 18%, below about 17%, below about 16%, below about 15%, below about 14%, below about 13%, below about 12%, below about 11%, below about 10%, below about 9%, below about 8%, below about 7%, below about 6%, below about 5%, below about 4%, below about 3%, below about 2% or below about 1% oxygen (O 2 ). MNCs can be cultured under about 0% to about 19% O 2 , such as about 1% to about 15% O 2 , about 2% to about 10% O 2 or about 5% to about 8% O 2 . MNCs are most preferably cultured under about 16% to about 19% O 2 . The numbers of % oxygen (or % O 2 ) cited above refer to the volume percentage of oxygen in the gas in the incubator during the culturing process. This method is generally implemented in an incubator that does not actively supply oxygen to the cells. Even if the incubator does not actively supply oxygen, there will still be oxygen in the atmosphere. This is generally about 16% to about 19%. This method can include culturing cells under about 16% to about 19% oxygen (O 2 ). Specialized hypoxic incubators can be used to further reduce the oxygen level.

[0423] In step (a), MNCs are most preferably cultured in the presence of platelet lysate and under hypoxic conditions.

[0424] In step (a), MNCs differentiate into iMP cells. This is described in PCT / GB2015 / 051673 (WO 2015 / 189587). iMP cells express detectable levels of MIC A / B, CD304 (neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363, (sphingosine-1-phosphate receptor 1), CD99, CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal growth factor receptor (EGF-R), CXCR2 and CD126. iMP cells generally also express detectable levels of CD29, CD44, CD73, CD90, CD105 and CD271, and do not express detectable levels of CD14, CD34 and CD45. Any of the culturing conditions of step (a) discussed above can be used to differentiate MNCs into iMP cells, and the culturing conditions include any one of platelet lysate, adhesion and hypoxia, preferably including all of them.

[0425] In step (b), the method preferably further comprises culturing the iMP cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides, and platelet lysate under hypoxic conditions and under conditions that permit the iMP cells to adhere and differentiate into MK cells. The one or more ribonucleosides are preferably one or more of (i) adenosine, (ii) cytidine, (iii) guanosine, and (iv) uridine. The one or more deoxyribonucleosides are preferably one or more of (i) 2'-deoxyadenosine, (ii) 2'-deoxycytidine hydrochloride, (iii) 2'-deoxyguanosine, and (iv) thymidine. In both cases, the medium can comprise any number and combination of (i) to (iv), such as (i), (ii), (iii), (iv), (i) and (ii), (i) and (iii), (i) and (iv), (ii) and (iii), (ii) and (iv), (iii) and (iv), (i), (ii) and (iii), (i), (ii) and (iv), (i), (iii) and (iv), (ii), (iii) and (iv), or (i), (ii), (iii) and (iv). The medium preferably comprises adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine hydrochloride, 2'-deoxyguanosine, and thymidine. The medium in step (b) preferably further comprises L-glutamine rather than L-alanyl-L-glutamine.

[0426] In step (b), the method more preferably further comprises culturing the iMP cells in a medium comprising the components listed in Table 6 and comprising platelet lysate under hypoxic conditions and under conditions that permit the iMP cells to adhere and differentiate into MK cells. The medium comprising the components listed in Table 6 is preferably MEM Alpha (ThermoFisher; product code: 12571-063) with nucleosides used in the examples.

[0427] Step (b) typically takes about 6, 7, or 8 days. Once the MK cells are nearly confluent, the MK cells can be harvested. Steps (a) and (b) typically take a total of about 24 days to about 30 days, such as about 25, 26, 27, 28, or 29 days. Step (b) can include culturing the MK cells for about 6, 7, or 8 days, passaging (re-seeding) the MK cells and culturing them for an additional about 2, 3, 4, 5, 6, 7, or 8 days. In this case, steps (a) and (b) typically take a total of about 24 days to about 34 days, such as about 25, 26, 27, 28, 29, 30, 31, 32, or 33 days.

[0428] Any embodiments regarding platelet lysate and hypoxic conditions discussed above for step (a) are equally applicable to step (b). The platelet lysate used in step (b) is preferably prepared as described in PCT / GB12 / 052911 (published as WO 2013 / 076507). The platelet lysate is preferably prepared by 4 freeze / thaw cycles using liquid nitrogen at each freezing stage. The culture medium in step (b) preferably further comprises heparin and / or penicillin / streptomycin (P / S).

[0429] Step (b) may further comprise supplementing the culture medium with IFN-γ and / or TNF-α. Any amount of IFN-γ may be used, such as from about 100 μg / mL to about 1000 μg / mL. The culture medium is preferably supplemented with 500 μg / mL. Any amount of TNF-α may be used, such as from about 1 ng / mL to about 100 ng / mL. The culture medium is preferably supplemented with 10 ng / mL. Step (b) preferably further comprises supplementing the culture medium with IFN-γ and / or TNF-α for 24 hours / day. Step (b) more preferably further comprises supplementing the culture medium with IFN-γ and / or TNF-α for 24 hours / day and then removing IFN-γ and / or TNF-α from the culture medium for 2 days before harvesting the MK cells. For example, if step (b) takes about 6 days, it preferably comprises supplementing the culture medium with IFN-γ and / or TNF-α on day 4 and removing IFN-γ and / or TNF-α on days 5 and 6. If step (b) comprises culturing the MK cells for about 6 days, passaging (re-seeding) the MK cells and culturing them for another about 6 days, it preferably comprises supplementing the culture medium with IFN-γ and / or TNF-α on day 10 and removing IFN-γ and / or TNF-α on days 11 and 12. Those skilled in the art can apply this concept to other timings of step (b) described above.

[0430] The present invention also provides a method for generating a population of the MK cells of the present invention, which only comprises step (b). The method comprises culturing iMP cells in a culture medium comprising one or more ribonucleosides, one or more deoxyribonucleosides and platelet lysate under hypoxic conditions and under conditions allowing the iMP cells to adhere and differentiate into MK cells. All of the above embodiments are equally applicable to this method.

[0431] It is clear from the above discussion that the method of the present invention is carried out under clinically relevant conditions, i.e., in the absence of trace endotoxins and other environmental contaminants, such as lipopolysaccharides, lipopeptides and peptidoglycans, etc. This makes the MK cells of the present invention particularly suitable for administration to a subject.

[0432] MNCs are preferably obtained from the subject or an allogeneic donor. The present invention also provides a method for generating a population of the present invention suitable for administration to a subject, which method comprises (a) culturing MNCs obtained from the subject under conditions that induce the differentiation of MNCs into iMP cells, and (b) culturing the iMP cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides and platelet lysate under hypoxic conditions and under conditions that permit the adhesion and differentiation of the iMP cells into MK cells suitable for administration to the subject. The present invention also provides a method for generating a population of the present invention suitable for administration to a subject, which method comprises culturing iMP cells derived from the subject in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides and platelet lysate under hypoxic conditions and under conditions that permit the adhesion and differentiation of the iMP cells into MK cells suitable for administration to the subject. The population will be autologous to the subject and thus will not be rejected upon implantation. The present invention also provides a population of the present invention suitable for administration to a subject and generated in this manner.

[0433] Alternatively, the present invention also provides a method for generating a population of the present invention suitable for administration to a subject, which method comprises (a) culturing MNCs obtained from a different subject under conditions that induce the differentiation of MNCs into iMP cells, and (b) culturing the iMP cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides and platelet lysate under hypoxic conditions and under conditions that permit the adhesion and differentiation of the iMP cells into MK cells suitable for administration to the subject. The present invention also provides a method for generating a population of the present invention suitable for administration to a subject, which method comprises culturing iMP cells derived from a different subject in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides and platelet lysate under hypoxic conditions and under conditions that permit the adhesion and differentiation of the iMP cells into MK cells suitable for administration to the subject. The population will be allogeneic to the subject. There is substantial evidence that allogeneic mesenchymal cells are safe in human subjects (Anastasiadis et al J Cardiovasc Transl Res. June 2016;9(3):202-13). The present invention also provides a population of the present invention suitable for administration to a subject and generated in this manner.

[0434] In vitro method

[0435] The MK cells or populations of the present invention can be used in an in vitro method for modulating the activity of immune cells. Specifically, the present invention provides an in vitro method for priming a population of NK cells, comprising: incubating a population of NK cells with a population of MK cells of the present invention under conditions that increase the activity of NK cells. The method preferably increases the cytotoxic activity of NK cells. Methods for measuring cytotoxicity are disclosed above. The method can further increase the proliferation of NK cells. In other words, priming preferably includes increasing the cytotoxicity and / or proliferation of NK cells. The activity of NK cells can be evaluated during or after incubation.

[0436] The method can also include incubating the MK cells and NK cells with an agent that primes / activates NK cells (such as interleukin-18 (IL-8)). Other such agents are known in the art.

[0437] A population of NK cells can comprise any number of NK cells, including any number discussed above with reference to the MK cells of the present invention.

[0438] NK cells are generally granular lymphocytes. This can be determined using standard microscopy techniques. NK cells typically have a diameter of about 10 μm to about 30 μm, such as a diameter of about 14 μm to about 20 μm.

[0439] NK cells preferably express low but detectable levels of CD56 (also known as CD56 暗 (CD56 dim )) on their surface. NK cells can express detectable levels of CD56 (also known as CD56 亮 (CD56 bright )) on their surface.

[0440] NK cells preferably express detectable levels of CD16 (also known as CD16 亮 ) on their surface. NK cells can express low but detectable levels of CD16 (also known as CD16 暗 ) on their surface.

[0441] NK cells preferably do not express detectable levels of CD3 (also known as CD3 - ) on their surface.

[0442] NK cells preferably do not express detectable levels of TCR (also known as TCR - ) on their surface. NK cells preferably do not express detectable levels of TCRαβ. NK cells preferably do not express detectable levels of TCRγδ on their surface.

[0443] NK cells are preferably CD56 暗 / CD16 亮, and more preferably CD56 暗 / CD16 亮 / CD3 - / TCR - 。

[0444] The NK cells can be CD56 亮 / CD16 暗 , and more preferably CD56 亮 / CD16 暗 / CD3 - / TCR - 。

[0445] NK cells typically also express detectable levels of one or more activating receptors on their surface. The activating receptors bind to target ligands present on infected or transformed cells and activate the NK cells. In the context of the present invention, activation of the NK cells via these receptors can correspond to an increase in cell proliferation and / or cytotoxic activity. Both can be measured using standard methods in the art. The activating receptors can stimulate or increase the proliferation and / or cytotoxic activity of the NK cells, such as when it binds to its target ligand. Table 3 below summarizes one or more activating receptors that can be expressed by NK cells and their target ligands.

[0446] Table 3 - NK activating receptor genes, their receptor products, and the target ligands recognized by these receptor products (HLA = human leukocyte antigen)

[0447]

[0448]

[0449] NK cells can express any number and any combination of these activating receptors at detectable levels on their surface. In this case, detectable level means that more than 5% of the population of NK cells express the relevant receptor.

[0450] NK cells typically also express detectable levels of one or more inhibitory receptors on their surface. The inhibitory receptors inhibit the activation of NK cells when they bind to their target ligands. Table 4 below summarizes one or more inhibitory receptors that can be expressed by NK cells and their target ligands.

[0451] Table 4 - NK inhibitory receptor genes, their receptor products, and the target ligands recognized by these receptor products (HLA = human leukocyte antigen)

[0452]

[0453] NK cells can express any number and any combination of these inhibitory receptors at detectable levels on their surface. In this context, detectable levels mean that more than 5% of the population of NK cells express the relevant receptor.

[0454] The NK cells are preferably human. The NK cells are derived from any of the above animals. Human NK cells are typically derived from a human subject. Human NK cells can be derived in any manner. Human NK cells can be isolated from the peripheral blood of a human subject. Methods of so doing are known in the art. For example, leukocytes can be isolated from the peripheral blood, and NK cells can be isolated or selected based on markers on their surface. Any of the markers discussed above can be used, such as CD56 亮 / CD3 - . Leukocytes isolated from the peripheral blood can be subjected to immunomagnetic bead selection.

[0455] NK cells can be generated from CD34 + hematopoietic progenitor cells. CD34 + hematopoietic progenitor cells can be isolated from the peripheral blood or bone marrow. Alternatively, CD34 + hematopoietic progenitor cells are commercially available, for example, from PromoCell. CD34 + hematopoietic progenitor cells are capable of differentiating into NK cells using interleukin-15 (IL-15).

[0456] NK cells can be derived from human induced pluripotent stem (iPS) cells. Such cells can be identified based on the presence of one or more transcription factors used to induce pluripotency. Such transcription factors include, but are not limited to, Oct-3 / 4, Sox1, Sox2, Sox3, Sox15, Sox18, Klf2, Klf4, c-Myc, n-Myc, l-Myc, Nanog, LIN28, and Glis1. Such cells can also include evidence of a mechanism for delivering such transcription factors.

[0457] NK cells can be autologous. In other words, the cells can be derived from the subject to whom the cells will be administered. The NK cells are preferably allogeneic. In other words, the cells are preferably derived from a different subject. Administration of autologous or allogeneic NK cells to a human subject has been well documented.

[0458] Then, the isolated NK cells can be cultured in vitro using methods known in the art. Interleukin-2 (IL-2) can be used to induce the differentiation and proliferation of NK cells. Anti-CD3 antibodies can also be used to increase the in vitro expansion of NK cells induced by IL-2. Thus, NK cells can be cultured in a medium containing IL-2 and optionally anti-CD3 antibodies. Such antibodies are available to those skilled in the art. The medium can also contain IL-15. The medium can also contain one or more of IL-1, IL-4, IL-7, IL-12, and tumor necrosis factor (TNF).

[0459] NK cells can be co-cultured with accessory cells to provide additional signals to promote proliferation. Suitable accessory cells include, but are not limited to, irradiated EBV-transformed lymphoblastoid cells, HFWT (Wilm's tumor-derived cell line), and the BCR-ABL1 chronic myeloid leukemia cell line K652.

[0460] The NK cells can be an NK cell line, such as NK-92 (Gong; JH, Maki; G, Klingemann; HG, Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells, Leukaemia, Vol. 8, No. 4, 1994, pp. 658-658) or KHYG-1 (Yagita; M, Huang; CL, Umehara; H, Matsuo; Y, Tabata; R, Miyake; M, Konata; Y, Takatsuki; K, A novel natural killer cell line (KHYG-1) from a subject with aggressive natural killer cell leukemia carrying a p53 point mutation, Leukaemia, Vol. 14, No. 5, 2000, pp. 922-930).

[0461] The MK cells and NK cells can be incubated for any period of time. The period can be any time from about 30 seconds to about 3 days. For example, the period can be about 30 seconds, about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 1 day, about 2 days, or about 3 days. The MK cells and NK cells are preferably incubated for one day.

[0462] MK cells and NK cells can be incubated in the insert. MK cells and NK cells can be incubated in MK cell medium (see above) or NK cell medium (see above).

[0463] Activated NK cells

[0464] The present invention also provides a population of NK cells primed / activated using the present invention. The activity of the primed NK cells is increased. Preferably, the cytotoxic activity of the NK cells is increased. Methods for measuring it are disclosed above. The proliferation of the NK cells can be increased. The NK cells can be any of the cells discussed above. The population of primed NK cells can comprise any number of NK cells, including any number discussed above with reference to the MK cells of the present invention.

[0465] In vivo method

[0466] The MK cells or population of the present invention can be used in in vivo methods of modulating the activity of immune cells. Specifically, the present invention provides an in vivo method of priming a population of NK cells, comprising: administering to a subject, under conditions that increase the activity of the NK cells of the subject, a population of MK cells of the present invention or a pharmaceutical composition of the present invention comprising a population of cells comprising MK cells. The dosage of the cells, the dosage of the pharmaceutical composition, and the route of administration are discussed in more detail below.

[0467] NK cells can be extracted and isolated from a subject as described above. The method preferably increases the cytotoxic activity of the NK cells. Methods for measuring it are disclosed above. The method can increase the proliferation of the NK cells.

[0468] Pharmaceutical compositions and administration

[0469] The present invention also provides a pharmaceutical composition comprising: (a) a population of MK cells of the present invention and (b) a pharmaceutically acceptable carrier or diluent. The population of MK cells can be any of those discussed above. The pharmaceutical composition can also comprise a population of NK cells. The NK cells can be any of those discussed above, including a population of unprimed NK cells or a population of primed NK cells of the present invention discussed above. The MK cells and NK cells can be present in any ratio. The MK cells and NK cells are preferably present in approximately equal ratios, such as about 1:about 1. Other ratios, including but not limited to about 1:about 2, about 1:about 3, about 1:about 5, about 1:about 10, about 1:about 20, about 1:about 50, about 1:about 100 or more, are also contemplated by the present invention. Suitable cell numbers are discussed above and below.

[0470] The present invention also provides a pharmaceutical composition comprising: (a) a population of primed NK cells of the present invention and (c) a pharmaceutically acceptable carrier or diluent. The population of primed NK cells can be any of those discussed above. The pharmaceutical composition can also comprise a population of MK cells of the present invention. The cells can be in any ratio discussed above.

[0471] The various compositions of the present invention can be formulated using any suitable method. The formulation of cells with standard pharmaceutically acceptable carriers and / or excipients can be carried out using conventional methods in the pharmaceutical art. The exact nature of the formulation will depend on several factors, including the cells to be administered and the desired route of administration. The types of suitable formulations are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pennsylvania, USA.

[0472] The cells can be formulated so that they can be administered by any route. Suitable routes include but are not limited to intravenous, intramuscular, subcutaneous, intraperitoneal, endocardial myocardial, epicardial myocardial, intraventricular, intracoronary, retrograde coronary sinus, intraarterial, intrapericardial, intraosseous or intralung routes. The cells can also be directly administered to the tissue of interest, such as liver, kidney or lung tissue. The cells can be directly administered into the tumor.

[0473] The composition can be prepared with a physiologically acceptable carrier or diluent. Generally, such compositions are prepared as a liquid suspension of the cells. The cells can be mixed with pharmaceutically acceptable excipients that are compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, analogs and combinations thereof.

[0474] In addition, if desired, the pharmaceutical compositions of the present invention can contain small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents and / or adjuvants that enhance potency. The composition preferably contains human serum albumin.

[0475] A suitable carrier or diluent is Plasma-Lyte This is a sterile, pyrogen-free, isotonic solution for intravenous administration. Each 100 mL contains: 526 mg sodium chloride, USP (NaCl); 502 mg sodium gluconate (C6H11NaO7); 368 mg sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg potassium chloride, USP (KCl); and 30 mg magnesium chloride, USP (MgCl2·6H2O). It does not contain antibacterial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

[0476] MK cells can be included within one or more liposomes and / or one or more microbubbles. Suitable liposomes are known in the art. Suitable liposomes are disclosed, for example, in Akbarzadeh et al., Nanoscale Research Letters 2013, 8:102 and Meghana et al., International Journal Of Pharmaceutical And Chemical Sciences, 2012, 1(1):1-10. Suitable lipids for forming liposomes are discussed below with reference to microbubbles.

[0477] Microbubbles, their formation and biomedical uses are known in the art (e.g., Sirsi and Borden, Bubble Sci Eng Technol. November 2009; 1(1-2):3–17). Microbubbles are gas bubbles having a diameter of less than 1 millimeter and greater than 1 micron. The microbubbles used in the present invention preferably have a diameter of 8 μm or less, such as a diameter of 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less or 2 μm or less. Microbubbles can be formed from any substance. The general composition of microbubbles is a gas core stabilized by a shell. The gas core can contain air or a heavy gas, such as perfluorocarbon, nitrogen or perfluoropropane. Heavy gases are less water-soluble and are thus less likely to leak from the microbubbles and cause the microbubbles to dissolve. Microbubbles having a heavy gas core generally persist in the circulation for a longer time. The shell can be formed from any material. The shell material preferably comprises a protein, surfactant, lipid, polymer or a mixture thereof.

[0478] The cells can be administered in a manner compatible with the dosage formulation and in an amount that will be therapeutically effective. The dosage depends on the subject to be treated, the capacity of the subject's immune system and the degree of repair required. The precise amount of cells to be administered can depend on the judgment of the practicing physician and can be specific to each subject.

[0479] Any suitable number of cells can be administered to the subject. For example, it can be administered at least about 0.2×10 6 、about 0.25×10 6 、about 0.5×10 6 、about 1.5×10 6 、about 4.0×10 6 or about 5.0×10 6 cells per kg of subject. For example, it can be administered at least about 10 5 、about 10 6 、about 10 7 、about 10 8 、about 10 9cells. As a guide, the number of cells of the invention to be administered can be about 10 5 to about 10 9 , preferably about 10 6 to about 10 8 . Generally, up to about 2 × 10 8 cells are administered to each subject. Any of the specific numbers discussed above with reference to the populations of the invention can be administered.

[0480] In the case of the administration or presence of cells, a culture medium can be present to promote cell survival. In certain cases, the cells of the invention can be provided as frozen aliquots, and a substance such as DMSO can be present to promote survival during freezing. Such frozen cells are typically thawed and then placed in a buffer or culture medium for maintenance or administration. Specific cryopreservation media are also commercially available, such as those commercially available from BioLife Solutions and there is evidence that the cells contained in these media are capable of being administered directly to a subject after thawing.

[0481] Drugs, methods and therapeutic uses

[0482] The MK cells of the invention can be used in a method of treatment of a human or animal body. Accordingly, the invention provides the MK cells of the invention, a population of MK cells of the invention or a pharmaceutical composition of the invention for use in a method of treating a human or animal body by therapy.

[0483] The primed NK cells of the invention can be used in a method of treatment of a human or animal body. The invention provides a population of primed NK cells of the invention or a pharmaceutical composition of the invention for use in a method of treating a human or animal body by therapy.

[0484] The invention provides a method of treating cancer in a subject, the method comprising: administering to the subject (a) a population of MK cells of the invention, (b) a population of primed NK cells of the invention or (c) a pharmaceutical composition of the invention. The invention provides (a) a population of MK cells of the invention, (b) a population of primed NK cells of the invention or (c) a pharmaceutical composition of the invention for use in treating cancer in a subject. The invention provides the use of (a) a population of MK cells of the invention, (b) a population of primed NK cells of the invention or (c) a pharmaceutical composition of the invention in the manufacture of a medicament for treating cancer in a subject.

[0485] The population of MK cells can be any of those discussed above. The population of NK cells can be any of those discussed above. The pharmaceutical composition can be any of those discussed above and can comprise (i) a population of MK cells of the present invention, (ii) a population of primed NK cells of the present invention, (iii) a population of MK cells of the present invention and a (any) population of NK cells or (iv) a population of MK cells of the present invention and a population of primed NK cells of the present invention.

[0486] The cancer can be any cancer. The cancer can be a carcinoma, sarcoma, melanoma, lymphoma or leukemia. Preferably, the cancer is anal cancer, biliary tract cancer (cholangiocarcinoma), bladder cancer, blood cancer, bone cancer, bowel cancer, brain tumor, breast cancer, colorectal cancer, cervical cancer, endocrine tumor, eye cancer (such as uveal melanoma), fallopian tube cancer, gallbladder cancer, head and / or neck cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymph node cancer, lymphoma, melanoma, mesothelioma, myeloma, neuroendocrine tumor, ovarian cancer, esophageal cancer, pancreatic cancer, penile cancer, primary peritoneal cancer, prostate cancer, pseudomyxoma peritonei, skin cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, tracheal cancer, cancer of unknown primary, vaginal cancer, vulvar cancer or endometrial cancer. The leukemia is preferably acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia or chronic myeloid / myelogenous leukemia. The lymphoma is preferably Hodgkin's lymphoma or non-Hodgkin's lymphoma. The cancer is preferably primary cancer or secondary cancer. The cancer is preferably chronic myeloid leukemia or plasma cell myeloma. The cancer is preferably breast cancer.

[0487] The method may also involve administering a population of MK cells of the invention and a population of NK cells, such as a population of primed NK cells of the invention. In these cases, the MK cells and NK cells can be administered simultaneously (e.g., in the same pharmaceutical composition), sequentially, or separately. The MK cells can be administered before or after the NK cells. For example, the MK cells can be administered to the subject about 1 day to about 28 days, such as about 3 days to about 25 days, about 6 days to about 22 days, about 9 days to about 18 days, or about 12 days to about 15 days before or after administering the NK cells. The MK cells can be administered to the subject up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, up to about 25 days, up to about 26 days, up to about 27 days, or up to about 28 days before or after administering the NK cells. Suitable cell numbers and ratios of MK cells to NK cells were discussed above.

[0488] A population of MK cells of the invention, a population of NK cells, and / or a pharmaceutical composition of the invention can be administered to the subject once. Alternatively, a population of MK cells of the invention, a population of NK cells, and / or a pharmaceutical composition of the invention can be administered to the subject at least about two times, such as at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times. The interval between two administrations can be about 1 day to about 28 days, such as about 3 days to about 25 days, about 6 days to about 22 days, about 9 days to about 18 days, or about 12 days to about 15 days. Preferably, the interval between two administrations is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0489] In all cases, the MK cells and / or NK cells are preferably derived from the subject or an allogeneic donor. Obtaining MK cells and NK cells from the subject should ensure that the cells themselves are not rejected by the subject's immune system. Any differences between the donor and the recipient will ultimately result in the clearance of the MK cells and NK cells, but not before they have at least partially treated the disease.

[0490] The invention relates to administering a therapeutically effective amount of MK cells and / or NK cells to a subject. A therapeutically effective amount is an amount that alleviates one or more symptoms of a disease. A therapeutically effective amount is preferably an amount that treats a disease. Suitable cell numbers were discussed in more detail above.

[0491] MK cells and / or NK cells can be administered to any suitable subject. The subject is typically a human subject. The subject can be any of the above animals or mammals.

[0492] The subject can be an infant, adolescent or adult. The subject can be known to have a disease or suspected of having a disease. The subject can be susceptible or at risk of the relevant disease. For example, the subject can be genetically predisposed to cancer.

[0493] The present invention can be used in combination with other measures and substances for treating diseases. In some cases, MK cells and / or NK cells can be administered simultaneously, sequentially or separately with other substances intended to treat a disease or alleviate the symptoms of a disease or provide pain relief. MK cells and / or NK cells can be used in combination with existing disease treatments, for example, can simply be mixed with such treatments. Thus, the present invention can be used to enhance the efficacy of existing disease treatments.

[0494] Mixture composition

[0495] One or more MK cells of the present invention can form part of a hybrid composition comprising one or more biocompatible fibers and one or more MK cells of the present invention. The one or more biocompatible fibers can be any of those disclosed in PCT / GB2015 / 051672 (published as WO 2015 / 189586). The hybrid composition can also comprise one or more NK cells, such as one or more primed NK cells of the present invention.

[0496] One or more MK cells of the present invention can form part of a hybrid composition disclosed in PCT / GB2015 / 051672 (published as WO 2015 / 189586) and are preferably administered to a subject as part of such a composition. Specifically, the present invention provides a hybrid composition comprising:

[0497] (a) one or more biocompatible fibers;

[0498] (b) one or more MK cells of the present invention; and

[0499] (c) one or more biocompatible components that (i) attach one or more MK cells to one or more fibers and / or embed one or more MK cells and one or more fibers and / or (ii) are capable of attaching the composition to tissue. The hybrid composition can also comprise one or more NK cells, such as one or more primed NK cells of the present invention.

[0500] The following examples illustrate the present invention.

[0501] Examples

[0502] Example 1 - Expansion of bone marrow and MK cells (batch CLXR-H-17-002RG)

[0503] The human bone marrow sample was diluted with Hank's balanced salt solution and layered on Ficoll-Paque to isolate mononuclear cells (MNCs) by centrifugation. The MNCs were then resuspended in Hank's balanced salt solution and counted using a 0.4% trypan blue exclusion assay to assess cell viability. The cells were seeded (day 0) in culture flasks with nucleoside-free MEM Alpha GlutaMAX (ThermoFisher; product code: 32561-102) containing penicillin-streptomycin, platelet lysate, and heparin, and incubated at 37 °C, 5% CO 2 2. The supplemented medium was the same as that used in the examples of PCT / GB2012 / 051600 (published as WO 2013 / 005053) and PCT / GB2015 / 051673 (published as WO 2015 / 189587).

[0504] In these examples, in all cases, platelet lysate was generated as described in PCT / GB2012 / 051600 (published as WO 2013 / 005053) and PCT / GB2012 / 052911 (published as WO 2013 / 076507): 4 freeze / thaw cycles using liquid nitrogen at each freezing stage.

[0505] Table 5 - Formulation of nucleoside-free MEM Alpha GlutaMAX (ThermoFisher; product code: 32561-102, 32561-029, 32561-037, or 32561-094, depending on country)

[0506]

[0507]

[0508] On day 8, the cells were passaged (re-seeded) and the medium was changed. On day 12, the cells were passaged (re-seeded) and the medium was changed.

[0509] On day 19, the cells were iMP cells (the subject of PCT / GB2015 / 051673; WO 2015 / 189587), and were passaged (re-seeded) with fresh medium MEM Alpha with nucleosides (ThermoFisher; product code: 12571-063; see components below) containing penicillin-streptomycin, platelet lysate, and heparin, and incubated at 37 °C, 5% CO 2 2. Six days later (day 25), the cells were MK cells (seeFigure 4 ) and harvested using a cell dissociation solution according to the manufacturer's instructions. The cells were cryopreserved at -80 °C in medium supplemented with 10% dimethyl sulfoxide and stored in liquid nitrogen for later use. The MK cells generated from this batch (CLXR-H-17-002RG) were designated MK002.

[0510] Table 6 - MEM Alpha with nucleosides (ThermoFisher; product codes: 12571-063, 12571-048, 12571-071, or 12571-089, depending on country). The shaded rows show where the components differ from those in nucleoside-free MEM Alpha GlutaMAX (ThermoFisher; product codes: 32561-102, 32561-029, 32561-037, or 32561-094, depending on country) in Table 5. The amounts of some components also differ between the two media.

[0511]

[0512]

[0513] Example 2 - Expansion of bone marrow and MK cells (batch CLXR-H-17-004)

[0514] Example 1 above was repeated using different bone marrow samples (and thus different batches), with only some differences in the timing of cell passage (re-seeding). Human MNCs (prepared as in Example 1) were seeded (day 0) in culture flasks with αMEM GlutaMAX containing penicillin-streptomycin, platelet lysate, and heparin and incubated at 37 °C, 5% CO 2 2. Platelet lysate was generated as described in PCT / GB2012 / 051600 (published as WO 2013 / 005053): 4 freeze / thaw cycles using liquid nitrogen at each freezing stage.

[0515] On day 8, the cells were passaged (re-seeded) and the medium was changed. On day 14, the cells were passaged (re-seeded) and the medium was changed.

[0516] On day 21, the cells were iMP cells (the subject of PCT / GB2015 / 051673; WO 2015 / 189587), passaged (re-seeded) with fresh medium (MEM Alpha with nucleosides containing penicillin-streptomycin, platelet lysate, and heparin (ThermoFisher; product code: 12571-063; see components above)) and incubated at 37 °C, 5% CO 2Incubate below. After 6 days (on day 27), the cells were MK cells and harvested using a cell dissociation solution according to the manufacturer's instructions. The cells were cryopreserved at -80 °C in medium supplemented with 10% dimethyl sulfoxide and stored in liquid nitrogen for later use. The MK cells generated from this batch (CLXR-H-17-004) were designated MK004.

[0517] Example 3 - HT-FACS analysis

[0518] High-throughput fluorescence-activated cell sorting (HT-FACS) analysis is a high-throughput screening platform that can rapidly characterize the cell surface phenotypes of suspended cells. Currently, there are approximately 380 cell surface markers in the panel. This platform has been extensively validated and has been implemented on various types of human tissues and cells. The panel consists of approximately 380 human cell surface-specific antibodies arranged in 96-well plates.

[0519] The aim was to determine the surface antigen expression profile of the human MK cells of the present invention. Two batches of MK cells (MK002 and MK004) were thawed and seeded into culture flasks containing supplemented MEM Alpha (ThermoFisher; product code: 12571-063) with nucleosides as defined in Examples 1 and 2. The cells were grown for 5 days and the medium was changed on day 2. To harvest the cells, the medium was removed and the cells were washed twice with PBS. The cells were treated with 5 mL of 0.25% trypsin until detached. Medium (8 mL) was added to inactivate the trypsin and the cells were collected. The cells were centrifuged at 400 g for 5 minutes. The cell pellets were resuspended (single cell suspension) in a total of 5 mL of HBSS (Hank's balanced salt solution minus calcium / magnesium, supplemented with 2 mM EDTA and 1% BSA). An aliquot (10 μl) of the sample was used to determine the total number of viable cells by using an exclusion dye (0.2% trypan blue).

[0520] 100 μl of the sample was loaded into each well (approximately 40,000 cells per well, ensuring 10,000 to 20,000 events were collected in FACS). The samples were run on a BD FACSDiva upgraded with a BD high-throughput sampler (autosampler). FlowJo software was used for the analysis of flow cytometry data. The results were provided as graphs and an Excel spreadsheet containing the percentage of positive cells for each antibody.

[0521] Table 7 - HT-FACS analysis results showing the percentage of cells expressing each cell surface marker (* presents the corresponding data for ioMP cells, iMP cells, and BM-MSC (Lonza) from PCT / GB2016 / 052447 (published as WO2017025729))

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531] Example 4 - MK cell cytotoxicity

[0532] For the MK cytotoxicity, two batches of MK cells (MK002 and MK004) were thawed and seeded in triplicate in 6-well plates containing supplemented MEM Alpha (ThermoFisher; product code: 12571-063) with nucleosides as defined in Examples 1 and 2. The cells were grown for 3 days before harvest (as described in Examples 1 and 3). The MK cells were washed with Cr release assay medium (AIM) and resuspended in Cr release assay medium, then seeded on 96-well plates and exposed to the target cells K562 (chronic myeloid leukemia) and U266 (plasma cell myeloma) at the desired E:T ratio of 10:1. The plates were applied to a standard 4-hour Cr release assay to evaluate the killing activity. iMP cells of the same batches as MK002 and MK004 were prepared as described in PCT / GB2015 / 051673; WO 2015 / 189587 (using the same method as in Examples 1 and 2, except that in the final culture step, supplemented αMEM GlutaMAX was not replaced by supplemented MEM Alpha with nucleosides) and used as controls. The cytotoxicity results are as Figure 1 shown. The MK cells showed a significantly increased cytotoxicity compared to the iMP cells.

[0533] Example 5 - MK activation of NK cells

[0534] For NK priming, two batches of MK cells (MK002 and MK004) were thawed and seeded in triplicate in 6-well plates containing supplemented MEM Alpha with nucleosides (ThermoFisher; product code: 12571-063) as defined in Examples 1 and 2. The cells were grown for 2 days, then the medium was removed and the cell monolayer was rinsed once with warm HBSS. NK-specific medium (GM.1 or X-Vivo 10) was added to the monolayer. NK cells were added to Transwell inserts placed within the wells, and separately cultured NK cells were used as controls. The plates were incubated for 1 day. The NK cells were harvested, washed with Cr release assay medium (AIM) and resuspended in Cr release assay medium, then seeded onto 96-well plates and exposed to target cells K562 (chronic myeloid leukemia), RPMI-8226 (plasma cell myeloma), and U266 (plasma cell myeloma) at the desired E:T ratio of 10:1. The plates were applied to a standard 4-hour Cr release assay to evaluate killing activity. The NK priming results are shown in Figure 2 and 3 . The MK cells primed the NK cells and increased their cytotoxicity.

[0535] Example 6 - MNC and C14, CD34 and CD45

[0536] Flow cytometry was used to evaluate the expression of CD14, CD34, and CD45 on MNCs from batches CLXR-H-17-002RG and CLXR-H-17-004. The cells were counted using a NucleoCounter and prepared in 1% BSA / PBS solution to have 2×10 5 cells / 25 μL. The following reagents were used.

[0537] Table 8 - Flow cytometry reagents

[0538] Reagents Manufacturer Product number Water for molecular biology Sigma-Aldrich W4502-1L Antibody: CD45 PE R&D Systems From kit: FMC002 Isotype control: PE IgG1 mouse R&D System From kit: FMC002 Antibody: CD34 PerCP BD Ref:345803 Isotype control: PerCP mouse IgG1-k BD 550672 Antibody: CD14 FITC BD Pharmingen 555397 Isotype control: FITC IgG2A BD Pharmingen 555573 Nucleocassettes Chemometec 941-0001 5mL FAC tubes FALCON 352054 50mL centrifuge tubes VWR 21008-178

[0539] Table 9 - Summary results for batch 002

[0540] Antibody % MNC CD14+ 13.2％ CD34+ 6.17％ CD45+ 77.3％

[0541] Table 10 - Summary results for batch 004

[0542] Antibody % MNC CD14+ 7.59％ CD34+ 5.1％ CD45+ 81.5％

[0543] Example 7 - HT-FACS analysis of MK cells treated with IFN-γ and TNF-α

[0544] Repeat Example 3 using MK004, and add 500 ug / mL IFN-γ or 10 ng / ml TNF-α to the culture medium when changing the culture medium on the 2nd day during the 5-day growth. After treating with IFN-γ or TNF-α for 1 day, change the culture medium again to remove IFN-γ or TNF-α. On the 5th day, collect and test the cells as described in Example 3. The results of the specific markers of the present invention are shown in Table 11 below.

[0545] Table 11 - Results of HT-FACS analysis showing the percentage of MK004 cells expressing each cell surface marker after stimulation with IFN-γ or TNF-α. The untreated values were taken from Example 3.

[0546]

[0547]

[0548]

[0549] Example 8 - Generation of MK cells from additional batches

[0550] Repeat Examples 1 and 2 using the following 3 additional batches (batches are numbered or named according to the bone marrow samples from which they were derived):

[0551] -CLXR-H-17-001RG generates MK001

[0552] -CLXR-H-17-006RG generates MK006

[0553] -PC generates MKPC.

[0554] Example 9 - Secretome analysis of MK cells

[0555] The purpose was to determine the secretome profile of the human MK cells of the present invention. Five different batches of MK cells (MK001, MK002, MK004, MK006, and MKPC) were thawed and seeded into culture flasks containing supplemented MEMAlpha with nucleosides (ThermoFisher; product code: 12571-063) as defined in Examples 1 and 2. The cells were grown for 5 days, and the culture medium was changed on the 2nd day (untreated). In an alternative embodiment, when changing the culture medium on the 2nd day, 500 ug / mL IFN-γ or 10 ng / mL TNF-α (IFN-γ-treated or TNF-α-treated) was added to the culture medium. After treating with IFN-γ or TNF-α for 1 day, the culture medium was changed again to remove IFN-γ or TNF-α.

[0556] In all cases, the conditioned medium was collected on the 5th day. Using (bead-based) The cytokine profile (human 48-plex format) measures the levels of GROα, IL-12, IL-2Rα, IL-8, soluble TRAIL, and IL-6 in each sample. Samples are added to a 96-well plate containing magnetic beads conjugated with antibodies against each cytokine / chemokine or growth factor. Fluorescent signals are captured from each well and the concentrations are determined.

[0557] The experiment was performed once. The results (mean ± SEM) for all 5 batches are shown in Figures 5 - 10 (except as described in the legend above Figure 8 ). The MK cells of the present invention secrete all the tested cytokines at detectable levels. IFN-γ stimulation significantly increased the secretion of IL-2Rα and significantly decreased the secretion of IL-8. TNF-α stimulation significantly increased the secretion of GROα and IL-8.

[0558] Example 10 - Carrageenan for air sac model

[0559] The purpose was to determine the ability of the MK cells of the present invention to attract immune cells to the site of inflammation using the carrageenan air pouch model.

[0560] Under anesthesia, sterile air was injected into the back of mice (immunocompetent BALB / c). The mice were left for 4 - 5 days to allow air pouch formation and, if needed, the air pouches were reinflated in the middle. 0.5 mL of 1% carrageenan (an inflammation inducer) was injected into each air pouch and left for several hours to allow the inflammatory response to develop. Then the air pouches were treated with untreated MK cells of the present invention, IFN-γ-treated MK cells of the present invention, or TNF-α-treated MK cells of the present invention (all from batch MK006) and prepared as described in Examples 8 and 9. Each mouse received 1 million MK cells in 0.2 mL of physiological saline per air pouch. Untreated control (carrageenan only control).

[0561] The air pouches were punctured by injecting phosphate-buffered saline (PBS), gently kneaded to mix the cells, and washed with additional PBS to extract all the cells. The extracted cells were analyzed by FACS and the percentages of NK cells and monocytes present were determined. The experiment was repeated five times (5 air pouches per treatment).

[0562] The results are shown in Figure 11 and 12 The IFN-γ-treated MK cells of the present invention significantly increased the percentages of NK cells and monocytes in the air pouches.

[0563] Example 11 - MK activation of NK cells

[0564] The method of Example 5 was repeated 3 times (n = 3) using untreated MK cells, primary NK cells, and K562 (chronic myeloid leukemia) from batches MK002 and MK004. The results are shown in Figure 13 . MK cells primed primary NK cells and significantly increased their cytotoxicity.

[0565] Example 12 - MK cell cytotoxicity

[0566] The method of Example 4 was repeated twice (n = 2) with 4 different batches of the MK cells of the present invention (MK002, MK004, MK006, MKPC) and the breast cancer cell line MCF7 at the required E:T ratio of 10:1. The cytotoxicity results are shown in Figure 14 . MK cells from all batches showed cytotoxicity against MCF7.

[0567] Example 13 - Expression of granzyme and perforin in MK cells

[0568] RNA was isolated from MK cells of batch MK004 using the RNeasy kit (Qiagen). cDNA was synthesized using the cDNA synthesis kit (Roche). Specific primers for granzyme B (GZMB), H (GZMH), M (GZMM), A (GZMA), and K (GZMK) and perforin in a specific thermal profile were used to amplify the amplicons. The amplicon products were electrophoresed on a 2% gel, and the bands were detected using ETBr. It was confirmed that MK cells expressed GZMB, GZMH, GZMM, GZMA, GZMK, and perforin (data not shown).

[0569] RNA was also isolated from MK cells of batch MK004 from separate culture (monoculture: MC) or MK cells co-cultured (co-culture: CC) with RPMI-8226 cells at the required E:T ratio of 10:1 for 6 hours, 12 hours, and 24 hours. cDNA was synthesized using the cDNA synthesis kit (Roche). Quantitative PCR signals for GZMB, GZMH, GZMM, GZMA, GZMK, perforin, and GADPH were detected using the Sybr green master mix. The fold change in the expression of each gene was determined under the CC condition relative to MC, and the results are shown in Figures 15 - 20 (n = 2 for each RNA at each time point for MC and CC). The GZM and perforin values were normalized to the GADPH value. The value of CC relative to MC (CC - MC) obtained each time was studied. To obtain the fold change (FC) value, the following formula was applied: 2^(-(CC - MC)). A value greater than 1 indicates an increase in the expression of the gene in CC relative to MC. As Figures 15 - 20As shown, co-culture with RPMI-8226 cells increased the expression of GZMB, GZMH, GZMM, GZMA, GZMK, and perforin.

[0570] Example 14 - Inhibition of MK cell cytotoxicity using EGTA

[0571] EGTA is a non-specific inhibitor of granule exocytosis. MK cells from three batches (MK002, MK004, and MK006) were pretreated with different concentrations of EGTA (0.5 mM, 1.0 mM, 2.0 mM) for 24 hours, rinsed with sterile HBSS, and exposed to MCF7 cells (at the desired 5:1 E:T ratio) for an additional 24 hours, and their killing activity was evaluated using a chromium release assay (as described in Examples 4 and 12). As Figure 21 shown, EGTA significantly reduced the cytotoxicity of all three batches of MK cells. This indicates that granule exocytosis plays at least a partial role in the cytotoxicity of MK cells.

[0572] Example 15 - Inhibition of MK cell cytotoxicity using siRNA

[0573] Using Lipofectamine RNAiMAX, MK cells from batches MK004 and MK006 were treated with 25 pM of specific siRNA against CD178 / FasL (siFasL) or CD253 / TRAIL (siTRAIL) or scrambled / non-targeting (NT) siRNA. After 48 hours, the cells were washed with sterile HBSS and exposed to MCF7 cells (at the desired 5:1 E:T ratio) for an additional 24 hours, and their killing activity was evaluated using a chromium release assay (as described in Examples 4 and 12). Analysis was performed in triplicate (3 wells per condition). The results are shown in Figure 22 which shows that inhibition of TRAIL significantly reduced the cytotoxicity of MK006 (compared to scrambled / NT siRNA), indicating the role of this surface marker in the cytotoxicity of MK cells.

Claims

1. A mesoderm killing cell, wherein, the mesoderm killing cell is obtained by the following method, the method comprising: culturing the immunomodulatory progenitor cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides and platelet lysate under hypoxic conditions and under conditions that permit the immunomodulatory progenitor cells to adhere and differentiate into the mesoderm killing cell, wherein the mesoderm killing cell expresses detectable levels of CD112, CD137L, CD178, CD253 and CD277, and wherein the mesoderm killing cell does not express detectable levels of CD34 and CD45.

2. The mesoderm killing cell according to claim 1, wherein, the mesoderm killing cell expresses detectable levels of CD16 and CD96, and wherein the mesoderm killing cell does not express detectable levels of CD56.

3. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell does not express detectable levels of CD14.

4. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of CD25.

5. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of CD136.

6. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of CD155.

7. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of CD183.

8. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of CD205.

9. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of CD332.

10. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell (a) does not express detectable levels of CD102 and / or CD127, (b) does not express detectable levels of CD104, (c) does not express detectable levels of one or more of CD50, CD62E, CD62L and CD62P, or (d) any combination of (a) to (c).

11. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of CD328.

12. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of one or more of CD158d, CD158i, CD160, CD314 and CD337.

13. The mesoderm killing cell according to claim 1 or 2, wherein, the mesoderm killing cell expresses detectable levels of CD159c.

14. The mesoderm killing cell according to claim 1 or 2, wherein, The mesoderm killer cells express one or more of detectable levels of CD158b2, CD158f, and CD159a.

15. The mesoderm killer cells according to claim 1 or 2, wherein, the mesoderm killer cells secrete one or more of detectable levels of (a) chemokine ligand 1, (b) interleukin-12, (c) soluble IL-2 receptor, (d) IL-8, (e) soluble TRAIL, and (f) IL-6.

16. A population of two or more mesoderm killer cells according to any one of claims 1 to 15.

17. The population of mesoderm killer cells according to claim 16, wherein, more than 15% of the mesoderm killer cells in the population express detectable levels of CD16, CD96, CD112, CD137L, CD178, CD253, and CD277, and wherein 5% or less of the mesoderm killer cells in the population express detectable levels of CD34, CD45, and CD56.

18. A population of mesoderm killer cells, wherein, the mesoderm killer cells are obtained by a method comprising culturing the immunomodulatory progenitor cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides, and platelet lysate under hypoxic conditions and under conditions that permit the immunomodulatory progenitor cells to adhere and differentiate into the mesoderm killer cells, wherein more than 15% of the mesoderm killer cells in the population express detectable levels of CD112, CD137L, CD178, CD253, and CD277, and wherein 5% or less of the mesoderm killer cells in the population express detectable levels of CD34 and CD45.

19. A population of mesoderm killer cells, wherein, the mesoderm killer cells are obtained by a method comprising culturing the immunomodulatory progenitor cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides, and platelet lysate under hypoxic conditions and under conditions that permit the immunomodulatory progenitor cells to adhere and differentiate into the mesoderm killer cells, wherein (i) at least 20% of the mesoderm killer cells in the population express detectable levels of CD112, (ii) at least 80% of the mesoderm killer cells in the population express detectable levels of CD137L, (iii) at least 20% of the mesoderm killer cells in the population express detectable levels of CD178, (iv) at least 50% of the mesoderm killer cells in the population express detectable levels of CD253, and (vi) at least 50% of the mesoderm killer cells in the population express detectable levels of CD277, and wherein (a) 5% or less of the mesoderm killer cells in the population express detectable levels of CD34, and (b) 5% or less of the mesoderm killer cells in the population express detectable levels of CD45.

20. The population of mesoderm killer cells according to claim 19, wherein (i) At least 15% of the mesodermal killer cells in the population express a detectable level of CD16, (ii) At least 50% of the mesodermal killer cells in the population express a detectable level of CD96, (iii) At least 20% of the mesodermal killer cells in the population express a detectable level of CD112, (iv) At least 80% of the mesodermal killer cells in the population express a detectable level of CD137L, (v) At least 20% of the mesodermal killer cells in the population express a detectable level of CD178, (vi) At least 50% of the mesodermal killer cells in the population express a detectable level of CD253, and (vii) At least 50% of the mesodermal killer cells in the population express a detectable level of CD277, and wherein (a) 5% or less of the mesodermal killer cells in the population express a detectable level of CD34, (b) 5% or less of the mesodermal killer cells in the population express a detectable level of CD45, and (c) 5% or less of the mesodermal killer cells in the population express a detectable level of CD56.

21. A pharmaceutical composition comprising: (a) a population of mesodermal killer cells according to any one of claims 16 to 20 and (b) a pharmaceutically acceptable carrier or diluent.

22. The pharmaceutical composition according to claim 21, wherein, the composition further comprises a population of NK cells.

23. A method of generating a population of mesodermal killer cells according to any one of claims 16 to 20, comprising: (a) culturing monocytes under conditions that induce their differentiation into immunomodulatory progenitor cells, and (b) culturing the immunomodulatory progenitor cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides, and platelet lysate under hypoxic conditions and under conditions that permit the immunomodulatory progenitor cells to adhere and differentiate into the mesodermal killer cells.

24. A method of generating a population of mesodermal killer cells according to any one of claims 16 to 20, comprising: culturing immunomodulatory progenitor cells in a medium comprising one or more ribonucleosides, one or more deoxyribonucleosides, and platelet lysate under hypoxic conditions and under conditions that permit the immunomodulatory progenitor cells to adhere and differentiate into the mesodermal killer cells.

25. An in vitro method of priming a population of NK cells, comprising: incubating the population of NK cells with a population of mesodermal killer cells according to any one of claims 16 to 20 under conditions that increase the activity of the NK cells.

26. Use of a population of mesodermal killer cells according to any one of claims 16 to 20 in the manufacture of a medicament for treating cancer in a subject, the treatment comprising: administering the population of mesodermal killer cells to the subject, wherein the cancer is selected from chronic myeloid leukemia and plasma cell myeloma.

27. Use of a population of mesodermal killer cells according to any one of claims 16 to 20 in the manufacture of a medicament for treating cancer in a subject, wherein, (a) The preparation includes incubating a population of NK cells with the population of mesoderm killing cells to generate a population of primed NK cells, and (b) The treatment includes: administering the population of primed NK cells to the subject, wherein the cancer is selected from chronic myelogenous leukemia and plasma cell myeloma.

28. Use of a population of mesoderm killing cells and a population of NK cells according to any one of claims 16 to 20 in the preparation of a medicament for treating cancer in a subject, the treatment comprising: administering to the subject a population of mesoderm killing cells and the population of NK cells according to any one of claims 16 to 20, wherein the cancer is selected from chronic myelogenous leukemia and plasma cell myeloma.

29. Use of a pharmaceutical composition according to claim 21 or 22 in the preparation of a medicament for treating cancer in a subject, the treatment comprising: administering the pharmaceutical composition to the subject, wherein the cancer is selected from chronic myelogenous leukemia and plasma cell myeloma.

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