How to generate NK cells

Abstract

The present invention relates to novel methods for obtaining NK cells. The present invention further relates to NK cell populations and their use for the treatment of diseases, including cancer.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to methods for increasing the number of NK cells, particularly in a subject in need thereof, and for generating CD3-CD56+ NK cells for the treatment of cancer and infectious diseases, NK cell populations, and their use as medicaments. [Background technology]

[0002] 2. Background of the Invention NK cells are innate immune cells that are cytotoxic and responsible for killing tumor or infected cells. NK cells are produced in the bone marrow from hematopoietic stem cells. NK cell precursors are CD3-CD161+CD56- and differentiate into immature NK cells expressing CD3-CD161+CD56+. Although maturation of immature NK cells occurs primarily in the bone marrow, immature NK cells can also leave the bone marrow and mature into secondary lymphoid organs. During maturation, NK cells acquire a specific profile of membrane receptor expression that is associated with functional NK cells.

[0003] NK cells recognize tumor cells and infected cells through receptors expressed on their membranes (i.e., activating and inhibitory receptors). Inhibitory receptors recognize major histocompatibility complex (MHC) I molecules expressed by all cells of the body (i.e., self cells). Activating receptors can recognize non-self molecules expressed by either self or non-self cells (e.g., tumor cells or infected cells). In response to signals received through inhibitory and / or activating receptors, NK cells then release components of intracellular granules (including, e.g., perforin and granzymes) and / or express the TNF receptor ligands Fas Ligand (FasL), TNF and TRAIL, which bind to their corresponding receptors on target cells, and / or produce inflammatory cytokines (e.g., TNF-α and IFNγ), which then lyse target cells (including, e.g., cancer cells and infected cells).

[0004] T lymphocytes expressing chimeric antigen receptors are today a promising therapeutic tool for targeting and killing tumor cells. However, extensive secondary effects can be associated with these T cell-based immunotherapies, including, for example, cytokine storm and graft-versus-host disease (GVHD). Due to these drawbacks, new therapeutic strategies need to be investigated. NK cells, which do not cause cytokine storm and GVHD, are an interesting alternative to T cells.

[0005] In vitro methods for obtaining NK cells have been described in the art. However, the yield and purity of the obtained NK cells are usually low, which requires a cell selection step prior to in vivo infusion. Moreover, these methods usually require about one month to generate NK cells. In addition, NK cells usually do not express activating receptors well, exhibit senescence, and are difficult to genetically modify.

[0006] Therefore, there is a need to develop a new method for generating high quality NK cells in a short period of time.In the present invention, the applicants provide an in vitro method for generating CD3-CD56+ NK cells, which allows obtaining functional cytotoxic NK cells that express activation-promoting receptors and lack the expression of some inhibitory receptors in a short period of about 2 weeks. Summary of the Invention

[0007] The present invention relates to a) culturing CD34+ cells in the presence of TNF-α or a fragment thereof and of a Notch ligand or a fragment thereof, thereby obtaining a first population of cells; and b) culturing the population of cells obtained in step a) in a cytokine-containing medium. The present invention relates to an in vitro method for generating NK cells, comprising:

[0008] In one embodiment, in step a), the cells are cultured in the presence of TNF-α or a fragment thereof and of a Notch ligand or a fragment thereof for more than 5 days and less than 9 days, preferably for about 7 days.

[0009] In one embodiment, in step a), the Notch ligand is Delta-like 4 ligand or a fragment thereof, preferably the soluble domain of Delta-like 4 ligand.

[0010] In one embodiment, in step a), the cells are also exposed to a fibronectin fragment comprising RGDS, linking segment 1 (CS-1) and / or a heparin binding domain, preferably the fibronectin fragment is CH-296.

[0011] In one embodiment, the CD34+ cells are isolated from an adult donor or from umbilical cord blood cells.

[0012] In one embodiment, the cytokine-containing medium of step (b) contains at least three, preferably five, cytokines selected from the group consisting of interleukin-7 (IL-7), stem cell factor (SCF), interleukin-15 (IL-15), interleukin-2 (IL-2) and Flt3 ligand (FLT3L).

[0013] In one embodiment, in step b), the cells are cultured in the cytokine-containing medium for more than 7 days and less than 21 days.

[0014] In one embodiment, the in vitro method comprises the additional step of transducing cells with the vector, preferably during or prior to step (a).

[0015] In one embodiment, the vector encodes a chimeric antigen receptor (CAR).

[0016] Another object of the present invention is a NK cell population likely to be obtained by the in vitro method of the present invention, wherein more than 60% of the cells are CD3-CD56+.

[0017] In one embodiment, the cells of the NK cell population are CD3-CD56+ cells and do not express at least one inhibitory receptor selected from the group including KIR3DL1 / DL2, KIR2DL2 / DL3 and KLRG1.

[0018] In one embodiment, the cells of the NK cell population are CD3-CD56+ cells and express at least one molecule selected from CD161 and an activating receptor selected from the group including NKp30, NKp44, NKp46, DNAM-1 and NKG2D. In one embodiment, the cells of the NK cell population are CD3-CD56+ cells and express CD161, NKp30, NKp44, NKp46, DNAM-1 and NKG2D.

[0019] In one embodiment, the cells of the NK cell population are CD3-CD56+ cells and express high levels of CD161 and at least one molecule selected from the group including or consisting of NKp30, NKp44, NKp46, DNAM-1 and NKG2D.

[0020] In one embodiment, the cells of the NK cell population are CD3-CD56+ cells, and at least 85% of said CD3-CD56+ cells express CD161 and at least one activating receptor selected from the group including NKp30, NKp44, NKp46, DNAM-1 and NKG2D.

[0021] The present invention further relates to a population of NK cells which expresses CD161, NKp30, NKp44, NKp46, DNAM-1 and NKG2D, but does not express KIR3DL1 / DL2, KIR3DL2 / DL3, KLRG1.

[0022] Another object of the present invention is a population of NK cells as described herein for use as a medicament.Another object of the present invention is a population of NK cells as described herein for increasing the number of NK cells in a subject in need thereof.

[0023] Another object of the present invention is the NK cell population described herein for treating cancer, persistent viral infections and parasitic diseases.

[0024] In one embodiment, the cancer is selected from the group including, but not limited to, leukemia (i.e., acute myeloid leukemia), lymphoma (e.g., B lymphoma), non-Hodgkin's lymphoma, multiple myeloma, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, thyroid cancer, melanoma, uterine cancer, kidney cancer, sarcoma, carcinoma, non-small cell lung cancer, oral and oropharyngeal cancer, methylcholanthrene-induced sarcoma, and colorectal cancer.

[0025] In one embodiment, the cancer is a leukemia (e.g., acute myeloid leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia), lymphoma (e.g., B lymphoma, peripheral T-cell lymphoma), non-Hodgkin's lymphoma, glioblastoma, neuroblastoma, multiple myeloma, cervical cancer, breast cancer (e.g., Thyroid cancer, ovarian ... The cancer may be selected from the group including, but not limited to, ripple-negative breast cancer), ovarian cancer, bladder cancer, prostate cancer, pancreatic cancer, gastric cancer, thyroid cancer, melanoma, uterine cancer, kidney cancer, liver cancer (e.g., hepatocellular carcinoma), sarcoma, carcinoma (e.g., renal cell carcinoma, breast cancer), small cell lung cancer, non-small cell lung cancer, pediatric solid tumors, CD133+ cancer stem cells, NKGDL+ cancer cells, PD-L1+ cancer cells, oral and oropharyngeal cancer (e.g., tongue cancer, esophageal cancer, laryngeal cancer, pharyngeal cancer), methylcholanthrene-induced sarcoma, and colorectal cancer.

[0026] In one embodiment, the viral infection is selected from the group including, but not limited to, human immunodeficiency virus (HIV), herpes virus (e.g., herpes simplex virus-1, cytomegalovirus (CMV)), influenza, retrovirus, human papilloma virus (HPV), enterovirus (e.g., Coxsackie B3 virus).

[0027] In one embodiment, the parasitic disease is selected from the group including, but not limited to, toxoplasmosis, trypanosomiasis, leishmaniasis and malaria. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] definition In the present invention, the following terms have the following meanings.

[0029] The term "about" when referring to a measurable value, such as an amount, a time period, or the like, is meant to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, as such variations are appropriate for performing the disclosed methods.

[0030] The term "NK cells" or "natural killer cells" refers to cytotoxic lymphocytes that play an important role in innate immunity. NK cells are constantly in contact with other cells. NK cells express activating and inhibitory receptors on their cell surface. This mechanism allows NK cells to recognize whether a cell should be eliminated (such as a tumor cell or an infected cell). In one embodiment, NK cells can be defined as "immature" (immature NK cells are generally defined by the absence of expression of CD16 and KIR receptors) or "mature" (mature NK cells are generally defined by the expression of CD16 and KIR receptors). Mature NK cells include memory-like NK cells.

[0031] The term "pharmaceutically acceptable excipient" (which may also be referred to as "pharmaceutically acceptable carrier") refers to an excipient that does not produce adverse, allergic, or other unpleasant reactions when administered to a mammal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory authorities such as the FDA or EMA.

[0032] The terms "expressing", "positive", or "+" and "not expressing", "negative", or "-" are well known in the art and refer to the expression level of a cellular marker of interest, where a "+" corresponds to a high, medium or low expression level of the cellular marker (i.e., the cellular marker is expressed or present on the cell surface) and a "-" corresponds to no expression level of the cellular marker (i.e., the cellular marker is not expressed or present on the cell surface).

[0033] The term "subject" is intended to include organisms in which an immune response can be elicited (e.g., mammals, particularly humans, primates, dogs, cats, horses, sheep, etc.). In one embodiment, the subject is a human. In one embodiment, the subject may be a "patient", i.e., a warm-blooded animal, preferably a human, waiting to undergo medical treatment, or undergoing medical treatment, or has been / is / will be the subject of medical treatment, or is monitored for the development of a target disease or condition, such as, for example, cancer or an infectious disease (e.g., a persistent viral infection or a parasitic disease). In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is suffering from, and preferably diagnosed with, a target disease or condition, such as, for example, cancer, an infectious disease (e.g., a persistent viral infection or a parasitic disease). In one embodiment, the subject is at risk of developing a target disease or condition, such as, for example, cancer, an infectious disease (e.g., a persistent viral infection or a parasitic disease), etc. Examples of risk factors include, but are not limited to, a genetic predisposition or a family history of the target disease or condition.

[0034] The term "therapeutically effective amount" refers to an amount of cells or compositions described herein that is effective to achieve a particular biological result. Thus, the term "therapeutically effective amount" refers to a level or amount of a composition or number of cells that aims to (1) delay or prevent the onset of a target disease or condition; (2) delay or halt the progression, severity, or worsening of one or more symptoms of a target disease or condition; (3) bring about an improvement in the symptoms of a target disease or condition; (4) reduce the severity or incidence of a target disease or condition; or (5) cure a target disease or condition, without causing significant negative or harmful side effects to the target. A therapeutically effective amount may be administered prior to the onset of a target disease or condition for a prophylactic or preventive effect. Alternatively, or in addition, a therapeutically effective amount may be administered after the onset of a target disease or condition for a therapeutic effect.

[0035] The terms "transfection" or "transduction" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid, and includes the primary subject cell and its progeny.

[0036] The term "treatment" or "treating" refers to both therapeutic and prophylactic or preventative measures, the purpose being to prevent or slow down (alleviate) the targeted disease or condition. Those in need of treatment include those already in the condition, as well as those prone to the condition, or those whose condition is to be prevented. A subject is successfully "treated" for a disease or condition if, after receiving a therapeutic dose of the cells or compositions described herein, the subject shows an observable and / or measurable improvement in one or more of the following: a reduction in the number of pathogenic cells; a reduction in the percentage of total cells that are pathogenic; some alleviation of one or more symptoms associated with the particular condition; a reduction in morbidity and mortality, and / or an improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in a condition are readily measurable by routine procedures familiar to physicians.

[0037] Detailed Description Applicants have previously observed that culturing CD34+ cells in the presence of TNF-α and Notch ligands allows generating CD34-CD7+ progenitor T cells after 7 days of culture (WO2018 / 146297). However, as shown in FIG. 8, these cells strongly express transcription factors known in the art to be specifically switched on after T cell commitment in the lymphohematopoietic process, namely GATA3 and BCL11B. Given this expression pattern, NK cell development of these cells may not be expected a priori. However, in the present invention, applicants have surprisingly provided a method for the in vitro production of NK cells, in which in a first step CD34+ cells are cultured in the presence of TNF-α and Notch ligands or fragments thereof.

[0038] The first object of the present invention is therefore to a) culturing CD34+ cells in the presence of TNF-α or a fragment thereof and of a Notch ligand or a fragment thereof, thereby obtaining a first population of cells; and b) culturing the first population of cells in a cytokine-containing medium The present invention is an in vitro method for generating NK cells, comprising:

[0039] In one embodiment, the CD34+ cells are isolated from cord blood cells.

[0040] In one embodiment, CD34+ cells are collected from adult donors. In one embodiment, CD34+ cells are obtained from bone marrow aspirates of peripheral blood of adult donors. Because the number of CD34+ cells is low in adult peripheral blood (i.e., 0.15% of cells), CD34+ cells may be mobilized from bone marrow to the periphery (e.g., blood) to increase the number of CD34+ cells in peripheral blood. In one embodiment, to mobilize CD34+ cells, adult donors may be treated with granulocyte colony-stimulating factor (G-CSF) and / or plerixafor, preferably G-CSF. Other examples of mobilizing agents include, but are not limited to, agonists of CXCR2 (e.g., MGTA 145) and analogs of plerixafor.

[0041] In one embodiment, CD34+ cells are isolated from mobilized peripheral blood.

[0042] Methods for isolating CD34+ cells are well known in the art and include, but are not limited to, methods using beads coated with an antibody that recognizes CD34. In one embodiment, CD34+ cells are isolated using an indirect CD34 microbead kit (Miltenyi).

[0043] In one embodiment, the CD34+ cell population used in the methods of the invention is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% pure. In one embodiment, the CD34+ cells do not express markers of mature cells, preferably the CD34+ cells do not express at least one marker selected from the group comprising or consisting of CD3, CD56, CD14 / 15, CD11b, more preferably the CD34+ cells do not express all of these markers.

[0044] In one embodiment, the CD34+ cells are derived from iPSCs (induced pluripotent stem cells). Examples of methods for deriving CD34+ cells from iPSC cells are known to the skilled artisan and have been described, for example, in 2020 by John F. Tisdale (Hematopoietic stem cells from pluripotent stem cells: Clinical potential, challenges, and future perspectives, Stem Cells Translational Medicine, Vol. 9, No. 12, December 2020, pp. 1549-1557) and by Rao et al. (Hematopoietic Cells from Pluripotent Stem Cells: Hope and Promise for the Treatment of Inherited Blood Disorders. Cells 2022, 11, 557).

[0045] In one embodiment, the CD34+ cells are from about 10 6 ~10 7 Cells are seeded at concentrations ranging from 100 to 200 mL of medium.

[0046] In one embodiment, the CD34+ cells are present at about 5,000 to about 30,000 cells / cm. 2 At a concentration in the range of medium, preferably about 10,000 to about 30,000 cells / cm 2 The seeds are seeded at concentrations ranging from 0.1 to 1.0 μg / ml.

[0047] In one embodiment, at the start of step (a), the CD34+ cells are about 10 6 ~10 7 Cells are seeded at concentrations ranging from 100 to 200 mL of medium.

[0048] In one embodiment, at the start of step (a), the CD34+ cells are present at about 5,000 to about 30,000 cells / cm. 2At a concentration in the range of medium, preferably about 10,000 to about 30,000 cells / cm 2 The seeds are seeded at concentrations ranging from 0.1 to 1.0 μg / ml.

[0049] In one embodiment, the culture vessel is selected from conventional culture vessels, including but not limited to 6-96 well culture plates, Petri dishes, flasks, stirrer bottles, microtiter plates, test tubes, hollow fiber devices, cell foams and bags. The amount of cells to be seeded can be adapted by those skilled in the art depending on the culture vessel used.

[0050] In one embodiment, the media used in step (a) and step (b) are different.

[0051] In one embodiment, the medium used in step a) of the method of the invention is adapted for the culture of CD34+ cells. Examples of media adapted for the culture of CD34+ cells include, but are not limited to, α-MEM, DMEM, RPMI 1640, IMDM, BME, McCoy's 5A, SFII (StemCell Technologies) medium, Fischer medium and X-VIVO™ medium (Lonza, Basel, Switzerland). In one embodiment, CD34+ cells are cultured in α-MEM medium (Thermo Fischer, MA, USA).

[0052] In one embodiment, the medium used in the present invention (particularly the medium in step (a) and / or the medium in step (b)) does not contain feeder cells. In particular, in one embodiment of the present invention, the medium used in the present invention (particularly the medium in step (a) and / or the medium in step (b)) does not contain OP-9 feeder cells.

[0053] In one embodiment, the medium used in the present invention (particularly the medium in step (a) and / or the medium in step (b)) is serum-free. In one embodiment, the medium used in the present invention (particularly the medium in step (a) and / or the medium in step (b)) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% v / v fetal bovine serum (FBS) or fetal calf serum (FCS).

[0054] In one embodiment, the TNF-α is human TNF-α, for example having the sequence of SEQ ID NO: 1 (Uniprot accession number: P01375).

[0055] SEQ ID NO:1 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANG VELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0056] TNF-α is produced primarily as a type II transmembrane protein that is arranged into stable homodimers, with each monomer containing 233 amino acids in humans. In humans, the soluble portion of human TNF-α consists of amino acids 77-233 of SEQ ID NO:1.

[0057] In one embodiment, the first medium (i.e., the medium of step (a)) comprises full-length TNF-α or a soluble fragment thereof, wherein said soluble fragment may comprise or consist of amino acids 77 to 233 of SEQ ID NO:1.

[0058] In one embodiment, the TNF-α or fragment thereof is added on day 0 of the culture. In one embodiment, the TNF-α or fragment thereof is present in the medium from day 0 and for at least about 1, 2, 3, 4, 5, 6, or 7 days. In one embodiment, the TNF-α or fragment thereof is present in the medium from day 0 of step (a) until the end of step (a).

[0059] In one embodiment, TNF-α or a fragment thereof is used at a concentration ranging from about 1 to about 300 ng / mL, for example at least about 1, 5, 10, 20, 30, 40, 50, 100, 200 or 300 ng / mL. In one embodiment, TNF-α or a fragment thereof is used at a concentration of about 10 ng / mL. However, other concentrations are suitable, such as about 5, 10, 20 or 50 ng / mL.

[0060] Notch proteins are transmembrane receptors that regulate cellular responses to numerous environmental signals. Four Notch receptors (Notch 1-4) and five ligands (Delta-like 1, Delta-like 3, Delta-like 4, Jagged-1, and Jagged-2) have been described in mammals (Weinmaster Curr Opin Genet Dev 2000:10:363-369).

[0061] In one embodiment the medium comprises Delta-like 4, preferably human Delta-like 4 (also known as DL-4, Uniprot Accession Number: Q9NR61, SEQ ID NO: 2), or a fragment thereof.

[0062] SEQ ID NO:2 MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSY RVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQ EDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFV GSRCEFPVGLPPSFPWVAVSLGVGLAVLLVLLGMVAVAVRQLRLRRPDDGSREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGLDKSNCGKQQNHTLDYNLAPGPLGRGTMPGKFPHSDKSLGEKAPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNECVIATEV

[0063] In one embodiment, the medium used in the present invention, preferably the medium used in step (a), comprises at least one soluble domain of a Notch ligand, which in one embodiment represents the extracellular portion of said ligand.

[0064] In one embodiment, the Notch ligand or a fragment thereof (preferably the soluble domain of the Notch ligand) is fused to a protein, allowing the Notch ligand to be immobilised on a support.

[0065] In one embodiment, the Notch ligand or a fragment thereof (preferably the soluble domain of the Notch ligand) is fused to biotin.

[0066] In one embodiment, the Notch ligand or a fragment thereof (preferably the soluble domain of the Notch ligand) is fused to the Fc region of an IgG protein, such as, for example, a human IgG protein. In one embodiment, the Notch ligand or a fragment thereof (preferably the soluble domain of the Notch ligand) is fused to the Fc region of an IgG2 protein, such as, for example, a human IgG2 protein (NCBI Accession Number: 4HAF_A, SEQ ID NO: 3).

[0067] SEQ ID NO:3 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0068] In one embodiment the medium used in the present invention, preferably the medium used in step (a), comprises DL-4 or a fragment thereof, preferably a fragment comprising or consisting of the soluble domain of DL-4.

[0069] In one embodiment, the soluble domain of DL-4 comprises or consists of amino acids 1 to 526 of SEQ ID NO: 2. In another embodiment, the soluble domain of DL-4 comprises or consists of amino acids 1 to 525 of SEQ ID NO: 2. In another embodiment, the soluble domain of DL-4 comprises or consists of amino acids 1 to 524 of SEQ ID NO: 2.

[0070] In one embodiment, DL-4 or a soluble domain thereof is fused to the Fc receptor region of an IgG protein, such as a human IgG protein, in particular an IgG2 protein, preferably human IgG2. An example of a protein comprising the soluble domain of DL-4 fused to the Fc receptor region of a human IgG2 protein is SEQ ID NO:4.

[0071] SEQ ID NO:4 MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLC KKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPSTMVRSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0072] An example of a protein comprising the soluble domain of DL-4 fused to the Fc receptor region of an IgG1 protein (such as a human IgG1 protein) is a commercially available product (Sino Biologicals) that contains the extracellular domain (Met 1-Pro 524) of human DLL4 (full-length DLL4 accession number NP_061947.1) fused at its C-terminus to the Fc region of human IgG1.

[0073] In one embodiment, the Notch ligand or a fragment thereof is immobilized (i.e., bound to a solid support) on the culture vessel used for the culture, although it is possible that certain elements may be found in solution. In one embodiment, the Notch ligand or a fragment thereof is immobilized on a surface, preferably the inner surface, of the culture vessel. Without being bound by any theory, the applicants suggest that the immobilization of the Notch ligand or a fragment thereof may stabilize it to facilitate interaction with CD34+ cells and thus enable activation of the Notch receptor of the CD34+ cells. In another embodiment, the Notch ligand or a fragment thereof is immobilized on a surface present in the culture medium, such as beads, preferably microbeads, or polymeric or magnetic beads (generally composed of 1-5 μm in diameter).

[0074] The binding of the Notch ligand or a fragment thereof (e.g., to a bead or to the surface of the culture vessel) may or may not be covalent. The binding of the Notch ligand may be performed non-covalently by allowing the Notch ligand or a fragment thereof to be adsorbed to the surface of the culture vessel or bead. Methods for attaching proteins or peptides to beads or culture vessels are known in the art and include, but are not limited to, fragment crystallizable (Fc) regions of immunoglobulin molecules (such as human IgG); and biotin-streptavidin / neutravidin / avidin conjugation methods; and click chemistry conjugation methods.

[0075] Methods for coating culture vessels or beads with Notch ligands are disclosed in WO2016 / 055396. In one embodiment, according to WO2016 / 055396, about 75% of the Notch ligand, particularly DL-4, is attached to the culture vessel surface or to the bead surface when 5 μg / ml is used. In one embodiment, the composition used to coat the culture vessel or beads with Notch ligand comprises a concentration of Notch ligand of 1.25 μg / ml or more, and preferably in the range of 2.5 and 5 μg / ml.

[0076] In one embodiment, the medium used in step (a) further comprises a cytokine.

[0077] In one embodiment, the medium used in step (a) comprises at least one, two or three cytokines selected from the group comprising or consisting of SCF (Stem Cell Factor), Flt3-L (Flt3 Ligand), and IL-7. In one embodiment, the medium used in step (a) comprises at least one, two or three cytokines selected from the group comprising or consisting of human SCF, human Flt3-L, and human IL-7.

[0078] In one embodiment, the medium used in step (a) comprises at least one, two, three or four cytokines selected from the group comprising or consisting of SCF (Stem Cell Factor), Flt3-L (Flt3 Ligand), TPO (Thrombopoietin) and IL-7 (Interleukin 7).

[0079] In one embodiment, the medium used in step (a) comprises at least one, two, three or four cytokines selected from the group consisting of or including hSCF (e.g., stem cell factor, corresponding to uniprot accession number: P21583), hFlt3-L (e.g., Flt3 ligand, corresponding to uniprot accession number: P49771), hTPO (e.g., thrombopoietin, corresponding to uniprot accession number: P40225) and hIL-7 (e.g., human interleukin 7, corresponding to uniprot accession number: P13232). hSCF, hFlt3-L, hTPO and hIL-7 are provided, for example, by Peprotech.

[0080] In one embodiment, the medium used in step (a) comprises SCF, (preferably hSCF). In one embodiment, the medium used in step (a) comprises Flt3-L, (preferably hFlt3-L). In one embodiment, the medium used in step (a) comprises TPO, (preferably hTPO). In one embodiment, the medium used in step (a) comprises IL-7, (preferably hIL-7).

[0081] In one embodiment, the medium used in step (a) comprises SCF (preferably hSCF) and Flt3-L (preferably hFlt3-L). In one embodiment, the medium used in step (a) comprises SCF (preferably hSCF) and TPO (preferably hTPO). In one embodiment, the medium used in step (a) comprises SCF (preferably hSCF) and IL-7 (preferably hIL-7). In one embodiment, the medium used in step (a) comprises Flt3-L (preferably hFlt3-L) and TPO (preferably hTPO). In one embodiment, the medium used in step (a) comprises Flt3-L (preferably hFlt3-L) and IL-7 (preferably hIL-7). In one embodiment, the medium used in step (a) comprises TPO (preferably hTPO) and IL-7 (preferably hIL-7).

[0082] In one embodiment, the medium comprises SCF (preferably hSCF), Flt3-L (preferably hFlt3-L) and TPO (preferably hTPO). In one embodiment, the medium comprises SCF (preferably hSCF), Flt3-L (preferably hFlt3-L) and IL-7 (preferably hIL-7). In one embodiment, the medium comprises SCF (preferably hSCF), TPO (preferably hTPO) and IL-7 (preferably hIL-7). In one embodiment, the medium comprises Flt3-L (preferably hFlt3-L), TPO (preferably hTPO) and IL-7 (preferably hIL-7).

[0083] In one embodiment, the medium comprises SCF (preferably hSCF), Flt3-L (preferably hFlt3-L), TPO (preferably hTPO) and IL-7 (preferably hIL-7).

[0084] In one embodiment, the concentration of hSCF ranges from about 2 to about 300 ng / mL, preferably from about 40 to about 200 ng / mL, and more preferably is about 100 ng / mL.

[0085] In one embodiment, the concentration of hFlt3-L ranges from about 2 to about 300 ng / mL, preferably from about 40 to about 200 ng / mL, and more preferably is about 100 ng / mL.

[0086] In one embodiment, the concentration of hTPO ranges from about 2 to about 300 ng / mL, preferably from about 40 to about 200 ng / mL, and more preferably is about 100 ng / mL.

[0087] In one embodiment, the concentration of hIL-7 ranges from about 2 to about 300 ng / mL, preferably from about 40 to about 200 ng / mL, and more preferably is about 100 ng / mL.

[0088] In one embodiment, the medium in step (a) does not contain IL-3.

[0089] In one embodiment, the medium comprises fibronectin or a fibronectin fragment (fibronectin may have a sequence corresponding to uniprot accession number: P02751, SEQ ID NO: 5). In one embodiment, the fibronectin fragment comprises or consists of an RGDS motif, a linking segment 1 (CS-1) motif and / or a heparin binding domain, preferably, the fibronectin fragment comprises or consists of an RGDS motif, a CS-1 motif and a heparin binding domain.

[0090] Fibronectin is a protein, which in its native form is a V-shaped large dimer with a length of 100 nm and a mass of 460 kDa. The two monomers are linked by two disulfide bridges at their C-termini. The term "fibronectin" or "fibronectin fragment" is understood to mean not only the native fibronectin protein (i.e., any isoform produced by alternative splicing), but also a monomer of this protein, or a fragment of this protein (containing the RGDS motif, the CS-1 motif and the heparin binding site, if specified).

[0091] An example of a fibronectin fragment that is particularly suitable for carrying out the processes disclosed herein is RetroNectin®. This protein corresponds to a fragment of human fibronectin (CH-296 fragment, Kimizuka et al., J Biochem., 1991 Aug. 110(2):284-91, Chono et al., J Biochem 2001 Sep 130(3):331-4) and contains a cell-binding C domain (containing the RGDS motif, the heparin-binding domain and the CS-1 motif). This protein is sold, among others, by the companies Takara Bio (Shiga, Japan), Clinisciences (Nanterre, France, also called NovoNectin®) and Fisher scientific (Hampton, United States).

[0092] The term "RGDS motif" is intended to refer to any peptide or protein that contains the RGDS (SEQ ID NO: 6) pattern such that it can bind to the integrin VLA-5. Such peptides or proteins can be tested for their ability to bind VLA-5 integrin by methods known and reported in the art. The RGDS motif binds to the integrin VLA-5 (Very Late Antigen-5), a dimer composed of CD49e (alpha 5) and CD29 (beta 1).

[0093] Heparin-binding domains are known in the art and are present in many proteins that bind heparin. Their sequences are generally XBBXBX or XBBBXXBX (B=basic amino acid; X=hydropathic amino acid; Cardin and Weintraub, Arterioscler Thromb Vasc Biol. 1989;9:21-32, SEQ ID NO:7 and SEQ ID NO:8). The presence of such a heparin-binding domain is particularly preferred when CD34+ cells are exposed to viral (especially retroviral) vectors to transduce them and obtain T cell precursors expressing a transgene.

[0094] The CS-1 motif is a 25 amino acid peptide (DELPQLVTLPHPNLHGPEILDVPST, SEQ ID NO: 9) as described by Wayner et al., 1989, J. Cell Biol. 109:1321. The CS-1 motif binds to the VLA-4 (Very Late Antigen-4) receptor. VLA-4 is a dimeric integrin composed of CD49d (alpha 4) and CD29 (beta 1).

[0095] In one embodiment, the fibronectin or fibronectin fragment is immobilized (i.e., bound to a solid support). The binding of the fibronectin or fibronectin fragment (e.g., to a bead or to the surface of the culture vessel) may or may not be covalent. In one embodiment, the fibronectin or fibronectin fragment is immobilized on the inner surface of the culture vessel (although it is possible that certain elements may be found in solution). In another embodiment, the fibronectin or fibronectin fragment is immobilized on a surface such as a bead, preferably a microbead, or a polymeric or magnetic bead (generally having a diameter comprised between 1 and 5 μm). In one embodiment, the Notch ligand or fragment thereof and the fibronectin or fragment thereof are immobilized on the same bead. In another embodiment, the Notch ligand or fragment thereof and the fibronectin or fragment thereof are immobilized on separate beads.

[0096] In one embodiment, the immobilization of fibronectin or a fibronectin fragment is performed non-covalently by allowing the fibronectin or a fragment thereof to be adsorbed onto the inner surface of the culture vessel or onto the surface of a bead. Methods for attaching proteins or peptides to beads or to the surface of a culture vessel are known in the art and are listed herein.

[0097] A method for coating a culture vessel or bead with fibronectin or a fragment thereof is disclosed in WO2016 / 055396. In one embodiment, the composition used to coat a culture vessel or bead with fibronectin or a fragment thereof comprises a concentration of fibronectin or a fragment thereof in the range of 10-100 μg / ml, preferably about 25 μg / ml.

[0098] In one embodiment, the CD34+ cells are cultured in the presence of TNF-α or a fragment thereof and a Notch ligand or a fragment thereof for at least about 4, 5, 6, 7, 8, 9 or 10 days, preferably at least about 5 or 6 days. In one embodiment, the CD34+ cells are cultured in the presence of TNF-α or a fragment thereof and a Notch ligand or a fragment thereof for about 4, 5, 6, 7, 8, 9 or 10 days, preferably about 7 days. In one embodiment, the CD34+ cells are cultured in the presence of TNF-α or a fragment thereof and a Notch ligand or a fragment thereof for up to about 10 days.

[0099] In one embodiment, the population of cells obtained in step (a) may be infused in vivo into a human subject to generate mature NK cells.

[0100] In one embodiment, the cytokine-containing medium used in step b) of the method of the invention is adapted for the culture of NK cell precursors. In one embodiment, the medium is selected from the group comprising RPMI Glutamax medium (Thermo Fischer, MA, USA), StemSpan serum-free medium (Stem Cell Technologies, Vancouver, Canada), serum-free CellGro SCGM medium (Bioz, CA, USA), CellGro DC medium (CellGenix, Freiburg, Germany), Glycostem Basal Growth Medium (Clear Cell Technologies, Beernem, Belgium) and alphaMEM (Thermo Fischer, MA, USA), preferably RPMI Glutamax medium (Thermo Fischer, MA, USA).

[0101] In one embodiment, the cytokine-containing medium used in step (b) does not contain feeder cells.

[0102] In one embodiment, the cytokine-containing medium used in step (b) is serum-free. In one embodiment, the cytokine-containing medium used in step (b) is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20%, preferably about 10%, fetal bovine serum (FBS) or fetal calf serum (FCS).

[0103] In one embodiment, in step (b), the cytokine-containing medium does not comprise TNF-α or a fragment thereof. In one embodiment, in step (b), the cytokine-containing medium does not comprise a Notch ligand or a fragment thereof. In one embodiment, in step (b), the cytokine-containing medium does not comprise TNF-α or a fragment thereof or a Notch ligand or a fragment thereof.

[0104] In one embodiment, the cytokine-containing medium comprises IL-15, preferably hIL-15. In one embodiment, the cytokine-containing medium comprises SCF, preferably hSCF. In one embodiment, the cytokine-containing medium comprises Flt3-L, preferably hFlt3-L. In one embodiment, the cytokine-containing medium comprises IL-7, preferably hIL-7. In one embodiment, the cytokine-containing medium comprises IL-2, preferably hIL-2.

[0105] In one embodiment, the cytokine-containing medium comprises one, two, three, four or five cytokines selected from the group including or consisting of IL-15, SCF, Flt3-L, IL-7, and IL-2.

[0106] In one embodiment, the cytokine-containing medium comprises one, two, three, four or five cytokines selected from the group including or consisting of hIL-15 (e.g., human interleukin-15, which may be provided by Peprotech, corresponding to accession number P40933), hSCF (Stem Cell Factor), hFlt3-L, hIL-7, and hIL-2 (e.g., human interleukin-2, which may be provided by Novartis, corresponding to accession number P60568).

[0107] In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15) and SCF (preferably hSCF). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15) and Flt3-L (preferably hFlt3-L). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15) and IL-7 (preferably hIL-7). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises SCF (preferably hSCF) and Flt3-L (preferably hFlt3-L). In one embodiment, the cytokine-containing medium comprises SCF (preferably hSCF) and IL-7 (preferably hIL-7). In one embodiment, the cytokine-containing medium comprises SCF (preferably hSCF) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises Flt3-L (preferably hFlt3-L) and IL-7 (preferably hIL-7). In one embodiment, the cytokine-containing medium comprises Flt3-L (preferably hFlt3-L) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises IL-7 (preferably hIL-7) and IL-2 (preferably hIL-2).

[0108] In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), SCF (preferably hSCF) and Flt3-L (preferably hFlt3-L). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), SCF (preferably hSCF) and IL-7 (preferably hIL-7). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), SCF (preferably hSCF) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), Flt3-L (preferably hFlt3-L) and IL-7 (preferably hIL-7). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), Flt3-L (preferably hFlt3-L) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), IL-7 (preferably hIL-7) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises SCF (preferably hSCF), Flt3-L (preferably hFlt3-L) and IL-7 (preferably hIL-7). In one embodiment, the cytokine-containing medium comprises SCF (preferably hSCF), Flt3-L (preferably hFlt3-L) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises SCF (preferably hSCF), IL-7 (preferably hIL-7) and hIL-2. In one embodiment, the cytokine-containing medium comprises Flt3-L (preferably hFlt3-L), IL-7 (preferably hIL-7) and IL-2 (preferably hIL-2).

[0109] In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), SCF (preferably hSCF), Flt3-L (preferably hFlt3-L) and IL-7 (preferably hIL-7). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), SCF (preferably hSCF), Flt3-L (preferably hFlt3-L) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), Flt3-L (preferably hFlt3-L), IL-7 (preferably hIL-7) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises IL-15 (preferably hIL-15), SCF (preferably hSCF), IL-7 (preferably hIL-7) and IL-2 (preferably hIL-2). In one embodiment, the cytokine-containing medium comprises SCF (preferably hSCF), Flt3-L (preferably hFlt3-L), IL-7 (preferably hIL-7) and IL-2 (preferably hIL-2).

[0110] In one embodiment, the cytokine-containing medium comprises IL-15, SCF (stem cell factor), Flt3-L, IL-7, and IL-2.

[0111] In one embodiment, the cytokine-containing medium comprises hIL-15, hSCF (stem cell factor), hFlt3-L, hIL-7, and hIL-2.

[0112] In one embodiment, the cytokine-containing medium does not include IL-12 (preferably human IL-12) and / or IL-18 (preferably human IL-18). In one embodiment, the cytokine-containing medium does not include IL-12 (preferably human IL-12) or IL-18 (preferably human IL-18).

[0113] In one embodiment, the cytokine-containing medium further comprises IL-12, preferably human IL-12. In one embodiment, the cytokine-containing medium further comprises IL-18, preferably human IL-18. In one embodiment, the cytokine-containing medium further comprises IL-12 (preferably human IL-12) and IL-18 (preferably human IL-18).

[0114] In one embodiment, the cytokine-containing medium contains one, two, three, four, five, six or seven cytokines selected from the group comprising or consisting of interleukin-7 (preferably hIL-7), stem cell factor (preferably hSCF), interleukin-15 (preferably hIL-15), interleukin-2 (preferably hIL-2), interleukin-18 (preferably hIL-18), interleukin-12 (preferably hIL-12) and Flt3 Ligand (preferably hFLT3L). In one embodiment, the cytokine-containing medium contains interleukin-7 (preferably hIL-7), stem cell factor (preferably hSCF), interleukin-15 (preferably hIL-15), interleukin-2 (preferably hIL-2), interleukin-18 (preferably hIL-18), interleukin-12 (preferably hIL-12) and Flt3 Ligand (preferably hFLT3L).

[0115] In one embodiment, the concentration of the cytokine hSCF ranges from about 10 to about 200 ng / mL, preferably from about 20 to about 50 ng / mL, and more preferably is about 50 ng / mL.

[0116] In one embodiment, the concentration of the cytokine hFlt3-L ranges from about 10 to about 200 ng / mL, preferably from about 20 to about 50 ng / mL, and more preferably is about 50 ng / mL.

[0117] In one embodiment, the concentration of the cytokine hIL-7 ranges from about 10 to about 200 ng / mL, preferably from about 20 to about 50 ng / mL, and more preferably is about 20 ng / mL.

[0118] In one embodiment, the concentration of the cytokine hIL-15 ranges from about 10 to about 200 ng / mL, preferably from about 20 to about 50 ng / mL, and more preferably is about 20 ng / mL.

[0119] In one embodiment, the concentration of the cytokine hIL-2 ranges from about 200 IU / mL to about 1000 IU / mL, preferably about 500 IU / mL.

[0120] In one embodiment, the concentration of the cytokine hIL-12 is in the range of about 0.01 ng / mL to about 100 ng / mL, preferably in the range of about 0.1 ng / mL to about 50 ng / mL or about 0.1 ng / mL to about 10 ng / mL, and more preferably about 10 ng / mL.

[0121] In one embodiment, the concentration of the cytokine hIL-18 is in the range of about 0.1 ng / mL to about 200 ng / mL, preferably in the range of about 0.5 ng / mL to about 100 ng / mL, and more preferably about 100 ng / mL.

[0122] In one embodiment, in step (b), the cells are cultured for at least about, or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, preferably for a period ranging from about 7 to about 14 days, more preferably for about 10, 11, 12, 13, or 14 days, thereby obtaining NK cells.

[0123] In one embodiment, at the start of step (b), the CD34+ cells are about 10 6 ~10 7 Cells are seeded at a range of concentrations / 1 mL cytokine-containing medium.

[0124] In one embodiment, the method of the invention further comprises a washing step of the cells obtained at the end of step (b).

[0125] In one embodiment, the NK cells obtained in step (b) are frozen according to methods known in the art, in one embodiment, the NK cells are centrifuged (e.g., at 1500 rpm for 5 minutes) and resuspended in freezing medium (e.g., containing 90% v / v FBS and 10% v / v DMSO).

[0126] In one embodiment, the NK cells obtained in step (b) are thawed prior to use, for example in a water bath at 37°C.

[0127] In one embodiment, the method of the invention may also include a step of conditioning the NK cells obtained in step (b) in a pouch for injection into a patient. In one embodiment, the NK cells are reconditioned in saline containing 5% HSA, such as albunorm™ 5% 50 g / L (Octopharma, Lingolsheim, France).

[0128] In one embodiment, during or after step (b) of the method of the invention, the cells may be further cultured in a medium adapted for maturation of NK cells. Thus, in one embodiment, the method of the invention further comprises a step (b') of culturing the cells during or after step (b) in a maturation medium.

[0129] In one embodiment, step (b') is performed after step (b).

[0130] In one embodiment, step (b') is performed during step (b), wherein step (b) comprises: - Step (b1) of culturing the cells in a cytokine-containing medium - a step (b') of culturing the cells in a maturation medium, and - Step (b2) of culturing the cells in a cytokine-containing medium means that it contains

[0131] In one embodiment, in step (b'), the cells are cultured in a maturation medium for at least about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours, preferably for a period ranging from about 6 to about 12 hours, more preferably for about 6 hours, thereby obtaining mature NK cells.

[0132] In one embodiment, step (b') is carried out in maturation medium for at least about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours, preferably about 12 hours, prior to step (b) as a pre-activation culture step.

[0133] In one embodiment, in step (b1), the cells are cultured in the cytokine-containing medium for at least about or about 4, 5, 6, 7, 8 days, preferably for a period ranging from about 4 to about 7 days, more preferably for about 7 days.

[0134] In one embodiment, in step (b2), the cells are cultured in the cytokine-containing medium for at least about or about 4, 5, 6, 7, 8 days, preferably for a period ranging from about 4 to about 7 days, more preferably for about 7 days.

[0135] In one embodiment, the maturation medium is selected from the group comprising RPMI (Thermo Fischer, MA, USA), StemSpan serum-free medium (Stem Cell Technologies, Vancouver, Canada), serum-free CellGro SCGM medium (Bioz, CA, USA), CellGro DC medium (CellGenix, Freiburg, Germany), Glycostem Basal Growth Medium (Clear Cell Technologies, Beernem, Belgium) and alphaMEM (Thermo Fischer, MA, USA), preferably RPMI (Thermo Fischer, MA, USA).

[0136] In one embodiment, the maturation medium does not contain feeder cells.

[0137] In one embodiment, the maturation medium is serum-free. In one embodiment, the maturation medium is supplemented with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%, preferably about 10%, fetal bovine serum (FBS) or fetal calf serum (FCS).

[0138] In one embodiment, in step (b'), the maturation medium does not comprise TNF-α or a fragment thereof. In one embodiment, in step (b'), the maturation medium does not comprise a Notch ligand or a fragment thereof. In one embodiment, in step (b'), the maturation medium does not comprise TNF-α or a fragment thereof or a Notch ligand or a fragment thereof.

[0139] In one embodiment, the maturation medium comprises IL-12, IL-15 and / or IL-18.

[0140] In one embodiment, the maturation medium comprises hIL-12 (e.g. human interleukin-12, which may comprise a dimer of p35 and p40 proteins corresponding to uniprot accession numbers: P29459 and P29460), hIL-15 and / or hIL-18 (e.g. human interleukin-18 corresponding to uniprot accession number: Q14116). hIL12 may be provided, for example, by Peprotech or Miltenyi Biotech, and hIL18 may be provided, for example, by MBL International Corporation or R&D Biosystems.

[0141] In one embodiment, the mature cytokine-containing medium comprises IL-12 (preferably hIL-12). In one embodiment, the mature cytokine-containing medium comprises IL-15 (preferably hIL-15). In one embodiment, the mature cytokine-containing medium comprises IL-18 (preferably hIL-18).

[0142] In one embodiment, the mature cytokine-containing medium comprises IL-12 (preferably hIL-12) and IL-15 (preferably hIL-15). In one embodiment, the mature cytokine-containing medium comprises IL-12 (preferably hIL-12) and IL-18 (preferably hIL-18). In one embodiment, the mature cytokine-containing medium comprises IL-15 (preferably hIL-15) and IL-18 (preferably hIL-18).

[0143] In one embodiment, the maturation cytokine-containing medium comprises IL-12 (preferably hIL-12), IL-15 (preferably hIL-15) and IL-18 (preferably hIL-18).

[0144] In one embodiment, the concentration of the cytokine hIL-12 ranges from about 5 to 200 ng / mL, preferably from about 5 to 50 ng / mL, and more preferably is about 10 ng / mL.

[0145] In one embodiment, the concentration of the cytokine hIL-12 is in the range of about 0.01 ng / mL to about 100 ng / mL, preferably in the range of about 0.1 ng / mL to about 50 ng / mL or about 0.1 ng / mL to about 10 ng / mL, more preferably about 10 ng / mL.

[0146] In one embodiment, the concentration of the cytokine hIL-15 ranges from about 10 to 200 ng / mL, preferably from about 20 to 100 ng / mL, and more preferably is about 50 ng / mL.

[0147] In one embodiment, the concentration of the cytokine hIL-18 ranges from about 10 to 200 ng / mL, preferably from about 20 to 100 ng / mL, and more preferably is about 50 ng / mL.

[0148] In one embodiment, the concentration of the cytokine hIL-18 ranges from about 0.1 ng / mL to 200 ng / mL, preferably from about 0.5 ng / mL to 100 ng / mL, and more preferably is about 100 ng / mL.

[0149] In one embodiment, the maturation medium comprises interleukin-7 (preferably hIL-7), stem cell factor (preferably hSCF), interleukin-15 (preferably hIL-15), interleukin-2 (preferably hIL-2), Flt3 ligand (preferably hFLT3L), interleukin-18 (preferably hIL-18) and interleukin-12 (preferably hIL-12). In one embodiment, the concentrations of cytokines in the maturation medium are equivalent to the concentrations of cytokines present in the cytokine-containing medium and are detailed herein above.

[0150] In one embodiment, the method of the invention further comprises a washing step of the cells obtained at the end of step (b') or (b2).

[0151] In one embodiment, the NK cells obtained in step (b') or (b2) are frozen according to methods known in the art. In one embodiment, the NK cells are centrifuged (e.g., at 1500 rpm for 5 minutes) and resuspended in freezing medium (e.g., 90% v / v FBS and 10% v / v DMSO).

[0152] In one embodiment, the NK cells obtained in step (b') or (b2) are thawed prior to use, for example in a water bath at 37°C.

[0153] In one embodiment, the method of the invention may also comprise a step of conditioning the NK cells (preferably mature NK cells) obtained in step (b') or (b2) in a pouch for injection into a patient. In one embodiment, the NK cells are reconditioned in saline containing 5% HSA, such as albunorm™ 5% 50 g / L (Octopharma, Lingolsheim, France).

[0154] In one embodiment, the method of the present invention further comprises a genetic modification step. In one embodiment, the method of the present invention further comprises one or more genetic modification step(s).

[0155] In one embodiment, the genetic modification step(s) corresponds to a gene disruption step, a gene correction step or a gene addition step, preferably a gene addition step. In one embodiment, the genetic modification step(s) is / is performed by a method selected from the group including, but not limited to, transfection, transduction or gene editing.

[0156] Examples of gene editing methods that can be used in the present invention include, but are not limited to, modified nuclease-based methods, recombinant adeno-associated virus (i.e., AAV)-based methods, transposon-based methods (e.g., Sleeping Beauty transposon system), homologous recombination-based methods, conditional targeting using site-specific recombinase (e.g., Cre-LoxP and Flp-FRT systems), and multiplex automated genome engineering (MAGE).Other examples of gene editing methods that can be used in the present invention include, but are not limited to, nickase-based methods.

[0157] Non-limiting examples of engineered nucleases include, but are not limited to, clustered regularly interspaced short palindromic repeats (CRISPR) transcription activator-like effector nucleases (TALENs), zinc finger endonucleases (ZFNs), meganucleases (mn, also known as homing endonucleases), or megaTALs (combining a TAL effector with an mn cleavage domain). Other non-limiting examples of engineered nucleases include base editors or prime editors.

[0158] In one embodiment, an exogenous nucleic acid sequence expressing a gene of interest is introduced into the cell, preferably before or during step (a) of the method.

[0159] In one embodiment, transduction or transfection of the CD34+ cells is carried out prior to step (a) of the method of the invention.

[0160] In one embodiment, prior to step (a) of the method, the CD34+ cells are preactivated for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours, preferably overnight, before being transduced. In one embodiment, prior to step (a) of the method, the CD34+ cells are preactivated for at least about 1 day before being transduced. In one embodiment, the preactivation comprises or consists of cell culture in a medium equivalent to the medium of step (a) but lacking TNF-α or a fragment thereof. In another embodiment, the preactivation comprises or consists of cell culture in a medium equivalent to the medium of step (a). According to this embodiment, the duration of the combination of the genetic modification step and step a) is at least about 4, 5, 6, 7, 8, 9 or 10 days, preferably at least about 5 or 6 days. In one embodiment, the duration of the combination of the genetic modification step and step a) is about 4, 5, 6, 7, 8, 9 or 10 days, preferably about 7 days.

[0161] In one embodiment, the transduction or transfection of CD34+ cells is carried out during step (a) of the method of the invention.

[0162] In one embodiment, transduction is performed in the absence of DL-4 and fibronectin. In one embodiment, transduction is performed in the presence of DL-4 and fibronectin. In one embodiment, transduction is performed in the presence of TNF-α or a fragment thereof. In one embodiment, transduction is performed in the absence of TNF-α or a fragment thereof.

[0163] In one embodiment, transduction is performed in the medium of step (a). In one embodiment, transduction is performed in the presence of at least one cytokine selected from the group comprising or consisting of SCF (preferably hSCF), Flt3-L (preferably hFlt3-L), and IL-7 (preferably hIL-7), hIL3, more preferably with three cytokines. In one embodiment, transduction is performed in the presence of at least one cytokine selected from the group comprising or consisting of SCF (preferably hSCF), TPO (preferably hTPO), Flt3-L (preferably hFlt3-L), and IL-7 (preferably hIL-7), hIL3, more preferably with four cytokines. In one embodiment, transduction is performed in the presence of at least one cytokine selected from the group comprising hSCF, hTPO, hFlt3-L, hIL-7, hIL3, more preferably with five cytokines. In one embodiment, the cytokine is used at a concentration of at least 20 ng / mL to 300 ng / mL, more preferably the cytokine is used at a concentration of 20 ng / mL, 100 ng / mL or 300 ng / mL.

[0164] In one embodiment, the transduction is carried out for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours, preferably for at least 6 hours.

[0165] In one embodiment, for the transduction step, CD34+ cells are cultured at about 10 6 ~10 7 Cells are seeded at concentrations ranging from 100 to 200 cells / mL.

[0166] In one embodiment, after transduction, the CD34+ cells are washed and then step (a) of the method of the invention is carried out.

[0167] In one embodiment, the exogenous nucleic acid sequence introduced into the cell encodes a chimeric antigen receptor (CAR), which is a cell surface protein that recognizes an antigen, such as a cell surface protein specifically expressed by a target cell (e.g., expressed by a cancer cell or an infected cell).

[0168] In one embodiment, the exogenous nucleic acid sequence introduced into the cell encodes a protein selected from the group including or consisting of a cytokine or cytokine receptor or a variant thereof (e.g., a cytokine or cytokine receptor variant with enhanced stability, etc.) In one embodiment, the exogenous nucleic acid sequence encodes a chimeric cytokine receptor or an orthogonal cytokine-receptor pair.

[0169] In one embodiment, the exogenous nucleic acid sequence encodes IL-15 or a variant thereof.

[0170] In one embodiment, the exogenous nucleic acid sequence introduced into the cell encodes CD16 or a variant thereof, such as a cleavage-resistant variant of CD16.

[0171] In one embodiment, the gene modification step(s) is a gene disruption step, which aims to reduce or eliminate the expression of a specific gene. Examples of genes that can be removed include, but are not limited to, genes from the group including or consisting of PD1, TIGIT, LAG-3, TIM-3, cytokine-induced STAT inhibitor (CIS) and signal regulatory protein alpha (SIRPα).

[0172] In one embodiment, the gene modification step(s) is a gene disruption step aimed at reducing or eliminating the expression of a specific gene. Examples of genes that can be removed include, but are not limited to, genes from the group including or consisting of PD1, TIGIT, LAG-3, TIM-3, TGFB2, cytokine-induced STAT inhibitor (CIS) and signal regulatory protein alpha (SIRPα).

[0173] Another object of the present invention is a population of NK cells likely to be obtained or obtained by the in vitro method of the present invention.

[0174] In one embodiment, the NK cells of the present invention are CD3-CD56+ NK cells. In one embodiment, the NK cell population of the present invention comprises CD3-CD56+ NK cells.

[0175] In one embodiment, the CD3-CD56+ NK cells generated by the methods of the invention have a purity of at least about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95%, i.e., greater than 60% of the cells recovered at the end of step b) are CD3-CD56+ NK cells.

[0176] NK cells recognize tumor cells and infected cells by receptors expressed on their cell surface membrane, which include activating and inhibitory receptors.

[0177] In one embodiment, the NK cells likely to be obtained or obtained by the methods of the invention express at least one activating receptor selected from the group including or consisting of KIRDS1 / S2, KIR2DS4, KIR2DL4, CD94 / NKG2C, KIR3DL2, CD16, NKG2D, NCRs, DNAM-1, 2B4, NTBA and NKp80.

[0178] In one embodiment, the NK cells likely to be obtained or obtained by the method of the invention express at least one molecule selected from the group consisting of CD161 and activating receptors selected from the group consisting of NKp30, NKp44, NKp46, DNAM1 and NKG2D. In one embodiment, the NK cells likely to be obtained or obtained by the method of the invention are or comprise CD3-CD56+ cells, wherein at least 80%, preferably at least 85% (e.g., at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of said CD3-CD56+ cells express at least one molecule selected from the group consisting of CD161 and activating receptors selected from the group consisting of NKp30, NKp44, NKp46, DNAM1 and NKG2D.

[0179] In one embodiment, the NK cells likely to be obtained or obtained by the method of the invention express CD161, NKp30, NKp44, NKp46, DNAM1 and NKG2D. In one embodiment, the NK cells likely to be obtained or obtained by the method of the invention are or comprise CD3-CD56+ cells, wherein at least 80%, preferably at least 85% (e.g., at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of said CD3-CD56+ cells express CD161, NKp30, NKp44, NKp46, DNAM1 and NKG2D.

[0180] In one embodiment, NK cells likely to be obtained or obtained by the method of the invention express at least one molecule selected from the group comprising or consisting of CD161, CD62L, and activating receptors selected from the group consisting of NKp30, NKp44, NKp46, DNAM1, and NKG2D. In one embodiment, NK cells likely to be obtained or obtained by the method of the invention express CD161, NKp30, NKp44, NKp46, DNAM1, NKG2D, and CD62L.

[0181] In one embodiment, at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the NK cells likely to be obtained or obtained by the methods of the invention express at least one molecule selected from an activating receptor selected from the group comprising or consisting of CD161 and NKp30, NKp44, NKp46, DNAM1, and NKG2D. In one embodiment, at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the NK cells likely to be obtained or obtained by the methods of the invention express CD161, NKp30, NKp44, NKp46, DNAM1, and NKG2D.

[0182] In one embodiment, at least about 50, 55, 60, 65, or 75% of the NK cells likely to be obtained or obtained by the methods of the invention express at least one molecule selected from an activating receptor selected from the group comprising or consisting of CD161 and NKp30, NKp44, NKp46, DNAM1, and NKG2D. In one embodiment, at least about 50, 55, 60, 65, or 75% of the NK cells likely to be obtained or obtained by the methods of the invention express CD161, NKp30, NKp44, NKp46, DNAM1, and NKG2D.

[0183] In one embodiment, the NK cells likely to be obtained or obtained by the methods of the invention express CD62L, hi one embodiment, at least 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, or 25% of the NK cells express CD62L.

[0184] In one embodiment, the NK cells likely to be obtained or obtained by the methods of the invention express CCR5, hi one embodiment, at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95% of the NK cells express CCR5.

[0185] In one embodiment, the NK cells likely to be obtained or obtained by the methods of the present invention do not express at least one inhibitory receptor selected from the group including or consisting of KIR2DL1 / 2 / 3, KIR3DL1, KIR3DL2, CD94 / NKG2A, LIR-1, KLRG-1, CEACAM1, TIGIT, Siglec-3, -7, -9, LAIR-1 and CD300A.

[0186] In one embodiment, the NK cells likely to be obtained or obtained by the method of the invention do not express at least one inhibitory receptor selected from the group comprising or consisting of KIR2DL1 / 2 / 3, KIR3DL1, KIR3DL2, and KLRG-1. In one embodiment, the NK cells likely to be obtained or obtained by the method of the invention do not express KIR2DL1 / 2 / 3, KIR3DL1, KIR3DL2, or KLRG-1.

[0187] In one embodiment, less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% of the NK cells likely to be obtained or obtained by the methods of the present invention express at least one inhibitory receptor selected from the group including or consisting of KIR2DL1 / 2 / 3, KIR3DL1, KIR3DL2, and KLRG-1.

[0188] In one embodiment, less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% of the NK cells likely to be obtained or obtained by the methods of the invention express CD16.

[0189] In one embodiment, less than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55% of the NK cells likely to be obtained or obtained by the methods of the invention express CD16.

[0190] In one embodiment, at least about 35, 40, or 45% of the NK cells likely to be obtained or obtained by the methods of the invention express CD94 and / or NKG2A. In one embodiment, at least about 15, 20, 25, or 30% of the NK cells likely to be obtained or obtained by the methods of the invention express CD94 and / or NKG2A. In one embodiment, at least about 50, 55, 60, 65, 70, or 75% of the NK cells likely to be obtained or obtained by the methods of the invention express CD94 and / or NKG2A.

[0191] In one embodiment, the NK cells likely to be obtained or obtained by the methods of the invention are (or include) immature NK cells, e.g., lacking expression of KIR receptors and / or CD16.

[0192] In one embodiment, the NK cells likely to be obtained or obtained by the methods of the invention are mature NK cells. Mature NK cells may, for example, express CD16 and / or KIR receptors.

[0193] In one embodiment, the NK cells likely to be obtained or obtained by the methods of the invention are (or include) mature NK cells, such as, for example, memory-like NK cells.

[0194] In one embodiment, NK cells likely to be obtained or obtained by the methods of the present invention include immature NK cells and mature NK cells (eg, memory-like NK cells).

[0195] The present invention further relates to a population of NK cells, wherein the NK cells express CD161, DNAM1, NKp30, NKp44, NKp46 and NKG2D, and do not express KIR3DL1 / DL2, KIR3DL2 / DL3, KLRG1. In one embodiment, said population of NK cells is isolated.

[0196] The present invention further relates to a population of NK cells, wherein the NK cells are or comprise CD3-CD56+ cells, wherein at least 80%, preferably at least 85% (e.g., at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of said CD3-CD56+ cells express at least one molecule selected from the group comprising or consisting of CD161 and NKp30, NKp44, NKp46, DNAM1, and NKG2D.

[0197] In one embodiment, the NK cells express CD62L.

[0198] The present invention further relates to NK cells that express CD161, DNAM1, NKp30, NKp44, NKp46 and NKG2D, and do not express KIR3DL1 / DL2, KIR3DL2 / DL3, KLRG-1. In one embodiment, said NK cells are isolated.

[0199] In one embodiment, at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the cells of the NK cell population of the invention express CD161 and the activating receptors NKp30, NKp44, NKp46, DNAM1, and NKG2D. In one embodiment, at least about 50, 55, 60, 65, or 75% of the cells of the NK cell population of the invention express CD161 and the activating receptors NKp30, NKp44, NKp46, DNAM1, and NKG2D.

[0200] In one embodiment, the NK cells of the present invention express CCR5. In one embodiment, at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95% of the NK cells of the population express CCR5.

[0201] In one embodiment, up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%, more preferably up to about 2%, of the NK cells of the population express the inhibitory receptors KIR2DL1 / 2 / 3, KIR3DL1, KIR3DL2 and KLRG-1.

[0202] In one embodiment, up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% of the NK cells of the population of the invention express CD16.

[0203] In one embodiment, less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% of the NK cells of the population of the invention express CD16.

[0204] In one embodiment, less than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40% of the NK cells of the population of the invention express CD16.

[0205] In one embodiment, less than about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55% of the NK cells of the population of the invention express CD16.

[0206] In one embodiment, at least about 35, 40, or 45% of the NK cells of the population express CD94. In one embodiment, at least about 15, 20, 25, or 30% of the NK cells of the population of the invention express CD94.

[0207] In one embodiment, at least about 35, 40, or 45% of the NK cells of the population express NKG2A. In one embodiment, at least about 15, 20, 25, or 30% of the NK cells of the population of the invention express NKG2A. In one embodiment, at least about 50, 55, 60, 65, 70, and 75% of the NK cells of the population of the invention express NKG2A.

[0208] In one embodiment, less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% of the NK cells of the population of the invention express NKG2A.

[0209] In one embodiment, at least about 15, 20, 25, 30, 35, 40, or 45% of the NK cells of the population express NKG2A and CD94.

[0210] In one embodiment, the NK cells of the present invention are immature NK cells. In one embodiment, the NK cell population of the present invention comprises immature NK cells. In one embodiment, the NK cells of the present invention are mature NK cells (e.g., memory-like NK cells). In one embodiment, the NK cell population of the present invention comprises mature NK cells (e.g., memory-like NK cells). In one embodiment, the NK cell population of the present invention comprises immature NK cells and mature NK cells (e.g., memory-like NK cells).

[0211] In one embodiment, the NK cells of the present invention (eg, obtainable by the methods of the present invention) are functional, ie, capable of cytotoxic activity.

[0212] The function of NK cells can be verified by conventional methods known in the art. Examples of such methods include, but are not limited to, cytotoxicity assays, measuring the secretion of IFNγ or TNF-α, and measuring the production (and degranulation ability) of perforin and granzymes. Examples of such methods are presented in the experimental section.

[0213] In one embodiment, the NK cells are cytotoxic in vitro under the conditions of Test A.

[0214] In practice, Test A is a flow cytometry-based cytotoxicity assay, which is performed as follows: target cells (e.g., K562 or THP1 cells) are labeled with CellTrace Violet dye by incubating with 1 μM of the dye for 10 min at 37° C. to distinguish target cells from effector NK cells. The labeled target cells are then incubated with NK cells at different effector-to-target ratios in RPMI medium supplemented with 10% FBS and 30 IU / ml hIL-2 for 5 h at 37° C. in a CO2 incubator. After 5 h of incubation, cells are stained with 7-AAD to distinguish target cells killed by NK cells. The effect of spontaneous target cell death is normalized by including one condition in which only target cells are incubated without NK cells (by subtracting the percentage of spontaneous target cell death without effector cells from the percentage of target cell death in the presence of effector cells). In one embodiment, in Test A, the positive control condition corresponds to a condition in which target cells are exposed to a detergent such as, for example, Tween 20 diluted to about 0.2% in PBS, thereby measuring total target cell death.

[0215] In one embodiment, the cells are cytotoxic under the conditions of Test A if the percentage of dead cells in the target cells is at least about 10% in at least one effector to target cell ratio and the following criteria are met: the percentage of spontaneous target cell death is about 5% or less, the percentage of total target cell death is about 99% or more; and the coefficient of variation between replicates is about 20% or less.

[0216] In one embodiment, the cells are cytotoxic under the conditions of Test A if a cytotoxicity rate of at least about 5, 10 or 15% is measured with a dose-dependent response at an effector:target ratio of 1.25:1, meaning that the more critical the effector:target cell ratio, the more cytotoxic the NK cells are.

[0217] In one embodiment, the cells are cytotoxic in the conditions of Test A if a cytotoxicity rate of at least about 20% is measured with a dose-dependent response at an effector:target ratio of 1.25:1, meaning that the more critical the effector:target cell ratio, the more cytotoxic the NK cells are.

[0218] In one embodiment, the NK cells of the invention express a CAR. In one embodiment, the NK cells of the invention express a protein selected from the group including or consisting of a cytokine or cytokine receptor or a variant thereof (such as a cytokine or cytokine receptor variant with increased stability). In one embodiment, the NK cells of the invention express a chimeric cytokine receptor or an orthogonal cytokine-receptor pair. In one embodiment, the NK cells of the invention express IL-15 or a variant thereof. In one embodiment, the NK cells of the invention express CD16 or a variant thereof (such as a cleavage-resistant variant of CD16). In one embodiment, the NK cells of the invention do not express at least one gene selected from the group including or consisting of PD1, TIGIT, LAG-3, TIM-3, cytokine-induced STAT inhibitor (CIS), and signal regulatory protein alpha (SIRPα). In one embodiment, the NK cells of the present invention do not express at least one gene selected from the group including or consisting of PD1, TIGIT, LAG-3, TIM-3, TGFB2, cytokine-induced STAT inhibitor (CIS), and signal regulatory protein alpha (SIRPα).

[0219] The present invention further relates to a kit for carrying out the method of the present invention comprising TNF-α or a fragment thereof, Notch ligand or a fragment thereof, and at least three (e.g., three, four or five, preferably five) cytokines selected from the group comprising or consisting of interleukin-7 (preferably hIL-7), stem cell factor (preferably hSCF), interleukin-15 (preferably hIL-15), interleukin-2 (preferably hIL-2) and Flt3 ligand (preferably hFLT3L). In one embodiment, the kit for carrying out the method of the present invention may comprise IL-7 (preferably hIL-7), SCF (preferably hSCF), IL-15 (preferably hIL-15) and FLT3-L (preferably hFLT3L). In one embodiment, the kit further comprises fibronectin or a fragment thereof. In one embodiment, the kit further comprises IL-12 (preferably hIL-12) and / or IL-18 (preferably hIL-18).

[0220] Such kits are particularly adapted and designed for carrying out the methods disclosed herein.

[0221] Another object of the present invention is a population of NK cells as described herein for use in increasing or increasing the number of NK cells in a subject in need thereof.

[0222] Another object of the present invention is the NK cell population described herein for use in a therapy, e.g., an "off-the-shelf" therapy.

[0223] Another object of the present invention is a population of NK cells as described herein for use as a medicament.

[0224] The present invention further relates to the use of the NK cell populations described herein for the manufacture of a medicament for increasing the number of NK cells in a subject.

[0225] The present invention further relates to methods for increasing the number of NK cells in a subject in need thereof comprising administering to the subject a population of NK cells as described herein (particularly a therapeutically effective amount of the NK cells as described herein).

[0226] The present invention further relates to a composition comprising, consisting essentially of, or consisting of a population of NK cells according to the present invention.

[0227] In one embodiment, the composition is a pharmaceutical composition and further comprises at least one pharma- ceutical acceptable excipient.Accordingly, the present invention further relates to a pharmaceutical composition.

[0228] In one embodiment, the pharmaceutical composition comprises, consists essentially of, or consists of a population of NK cells according to the present invention and at least one pharma- ceutically acceptable excipient.

[0229] Pharmaceutically acceptable excipients that may be used in the pharmaceutical composition of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fats.

[0230] In one embodiment, the composition of the invention is a medicament or is for use as a medicament. Thus, the present invention further relates to a medicament.

[0231] In one embodiment the medicament comprises, comprises, consists essentially of or consists of a population of NK cells according to the invention.

[0232] As used herein, the term "consisting essentially of" with respect to a composition, pharmaceutical composition or medicament means that the NK cells of the present invention are the only therapeutic or agent having biological activity in said composition, pharmaceutical composition or medicament.

[0233] In one embodiment, the NK cell population, composition, pharmaceutical composition or medicament of the invention is for treating cancer or an infectious disease.

[0234] The present invention therefore relates to NK cells, NK cell populations, compositions, pharmaceutical compositions or medicaments for treating cancer or an infectious disease.

[0235] The present invention further relates to the use of an NK cell or NK cell population disclosed herein for the manufacture of a medicament for the treatment of cancer or an infectious disease.

[0236] The present invention further relates to methods for treating cancer or an infectious disease in a subject in need thereof comprising administering to the subject a population of NK cells as described herein (particularly a therapeutically effective amount of the NK cells as described herein).

[0237] In one embodiment, the subject is a human.

[0238] In one embodiment, the subject is afflicted with, preferably diagnosed with, cancer.

[0239] Examples of cancer include, but are not limited to, leukemia (i.e., acute myeloid leukemia), lymphoma (e.g., B lymphoma), non-Hodgkin's lymphoma, multiple myeloma, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, thyroid cancer, melanoma, uterine cancer, kidney cancer, sarcoma, carcinoma, non-small cell lung cancer, oral and oropharyngeal cancer, methylcholanthrene-induced sarcoma, and colorectal cancer.

[0240] Examples of cancer include leukemia (e.g., acute myeloid leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia), lymphoma (e.g., B lymphoma, peripheral T-cell lymphoma), non-Hodgkin's lymphoma, neuroblastoma, multiple myeloma, cervical cancer, breast cancer (e.g., triple-negative breast cancer), ovarian cancer, bladder cancer, prostate cancer, pancreatic cancer, gastric cancer, thyroid cancer, melanoma, uterine cancer, kidney cancer, liver cancer (e.g., hepatocellular carcinoma), sarcoma, carcinoma (e.g., renal cell carcinoma, breast cancer), small cell lung cancer, non-small cell lung cancer, pediatric solid tumors, CD133+ cancer stem cells, NKGDL+ cancer cells, PD-L1+ cancer cells, oral and oropharyngeal cancer (e.g., tongue cancer, esophageal cancer, laryngeal cancer, Cancers that are caused by or have been caused by certain pathogenic or toxic substances include, but are not limited to, pulmonary cancer, pharyngeal cancer, methylcholanthrene-induced sarcoma, and colorectal cancer.

[0241] In one embodiment, the subject suffers from, and preferably has been diagnosed with, an infectious disease.

[0242] In one embodiment, the subject suffers from, and preferably is diagnosed with, a persistent viral infection caused by a virus selected from the group including or consisting of human immunodeficiency virus (HIV), herpes virus (e.g., herpes simplex virus-1, cytomegalovirus (CMV)), influenza, retrovirus, human papilloma virus (HPV), enterovirus (e.g., Coxsackie B3 virus).

[0243] In one embodiment, the subject suffers from a parasitic infection, preferably diagnosed with a parasitic infection. Examples of parasitic infections include, but are not limited to, toxoplasmosis, trypanosomiasis, leishmaniasis and malaria.

[0244] In one embodiment of the invention, the subject is administered at least one therapeutically effective amount of the composition described herein above.

[0245] In one embodiment, a single dose of the NK cells of the present invention is administered to a subject, hi another embodiment, multiple doses of the NK cells of the present invention are administered over a period of time.

[0246] In one embodiment, a therapeutically effective amount of NK cells is administered, is to be administered, or will be administered to a subject. In one embodiment, a therapeutically effective amount is about 0.5×10 7 ~Approx. 3×10 7 cells / kg body weight range.

[0247] In one embodiment, the number of NK cells that can be infused into a subject is about 0.5×10 7 ~Approx. 3×10 7 cells / kg body weight range.

[0248] In one embodiment, the NK cells, NK cell populations, compositions, pharmaceutical compositions or medicaments of the invention are (or will be or are intended to be) administered intravesically, intravaginally, intraosseously, intraperitoneally, intrauterinely, intraocularly, intradermally, intraarterially, intracerebrally, intranasally, enterally, buccally, intranasally, orally, rectally, or by inhalation.

[0249] In one embodiment, the NK cells, NK cell populations, compositions, pharmaceutical compositions or medicaments of the invention are (or will be or are intended to be) administered by injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0250] Examples of forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms, such as powders, suitable for use in preparing a solution or suspension upon the addition of liquid prior to use, liposomal forms, and the like. [Brief description of the drawings]

[0251] [Figure 1]Figure 1 is a combination of dot plots and histograms showing the generation of CD3-CD56+ NK cells from CB or mPB CD34+ HSPCs using the DL-4 / TNF-α culture system. (A) Figure 1A is a combination of dot plots depicting the phenotype of NK cells generated after 7 days of culture in DL-4 / TNF-α medium followed by DL-4 / NK cell differentiation medium without TNF-α at the indicated time points. (B, C) Figure 1B and Figure 1C are a combination of graphs showing the mean frequency (B) and number per CD34+ cell (C) of CD3-CD56+ NK cells at day 14 (light grey) and day 21 (dark grey) (mean ± SEM, n=5). [Diagram 2] Figure 2 is a combination of histograms and graphs showing the expression of NK cell receptors and transcription factors by NK cells (CD3-CD56+) derived from CB or mPB HSPCs (exposed to DL-4 / TNF-α). (A, B) Figures 2A and 2B are graphical representations of the expression rates (mean ± SEM, n=2 or 3) of NK cell receptors (A) and transcription factors (Eomes, T-bet, and ID-2) (B) by CD3-CD56+ NK cells generated after 21 days of culture. Expression levels were compared to those of control overnight activated peripheral blood (PB)-NK cells (in the presence of hIL-2 and IL15). [Diagram 3] Figure 3 is a combination of dot plots showing the expression of cytotoxic molecules by CB or mPB NK cells (CD3-CD56+) and their cytokine expression after stimulation with K562 cells. (A) Figure 3A is a combination of representative dot plots showing the expression of cytotoxic granules (perforin and granzyme B) by generated CD3-CD56+ NK cells. (B, C) Figures 3B and 3C are a combination of representative dot plots showing degranulation (expression of CD107a on the cell surface) (B) and expression of TNF-α and IFNg (C) by CD3-CD56+ NK cells upon stimulation with K562 cells at an effector (NK):target (K562) ratio of 1:2 for 6 hours. [Figure 4]Figure 4 is a combination of dot plots and histograms showing the generation of transduced NK cells from CB or mPB CD34+ HSPCs using the DL-4 / TNF-α culture system. (A) Figure 4A is a combination of representative dot plots showing the generation of transduced (GFP+CD3-CD56+) NK cells. (B, C) Figures 4B and 4C are histograms showing the average frequency (B) and number per CD34+ cell (C) (mean ± SEM, n=5) of transduced GFP+CD3-CD56+ NK cells generated on days 14 (light grey) and 21 (dark grey) after transduced NK cell generation culture. [Diagram 5] FIG. 5 is a histogram showing NK cell receptor expression (mean±SEM, n=2 or 3) by generated transduced GFP+CD3-CD56+ NK cells. [Figure 6] Figure 6 is a combination of dot plots showing degranulation and cytokine expression of transduced CB or mPB NK cells after stimulation with K562 cells. Figures 6A and B are a combination of representative dot plots showing degranulation (expression of CD107a on the cell surface) (A), induction of TNF-α and IFNg expression (B), and secretion of cytotoxic granules (granzyme B and perforin). [Figure 7] Figure 7 is a composite of graphs showing the cytotoxicity of transduced NK cells generated in vitro. (A) Figure 7A is a graphical representation of the killing of K562 cells (target cells) by transduced CB or mPB NK cells (effector cells) after 5 hours of co-incubation at the indicated effector:target cell (E:T) ratios (one representative experiment). (B) Figure 7B is a graphical representation of the killing of THP1 cells (target cells) by transduced CB NK cells (effector cells) after 5 hours of co-incubation at the indicated effector:target cell (E:T) ratios (one representative experiment). [Figure 8]FIG. 8 is a graph showing the expression of GATA3 and BCL11B by CD7+ cells obtained after culturing CD34+ cells in the presence of TNFα and a Notch ligand according to the first step of the method of the invention. [Figure 9] Figure 9 is a combination of graphs showing the generation of functionally promising CAR NK cells from CB CD34+HSPCs using the DL-4 / TNFα culture system. (A) Figure 9A is a representative FACS plot showing the generation of CAR (CAR+CD56+) NK cells after NK cell generation culture including transduction with lentivirus encoding CD19-CAR during a first step of DL-4 / TNFα culture for 7 days and a second step of NK cell differentiation for 8 days without feeder cells or DL-4 / TNFα. (B) Figure 9B is a graphical representation of the killing of target cells (NALM-6) by CD19-CAR NK cells (effector cells) after 5 hours of co-incubation at the indicated effector:target cell (E:T) ratios.

[0252] Working Example The present invention is further illustrated by the following examples.

[0253] Example 1: Generation of functional CD3-CD56+ NK cells Materials and Methods CD34+ cells Cord blood (CB) samples were collected by ethically approved procedures from donors at Saint Louis Hospital (Paris, France) after providing informed consent. Mobilized peripheral blood (mPB) samples used were unused fractions of grafts from healthy donors mobilized with granulocyte colony-stimulating factor and providing informed consent for research use. mPB samples were part of a collection approved by the French Ministry of Research (reference: DC-2014-2272, dated March 23, 2015). Cord blood or mPB CD34+ hematopoietic stem and progenitor cells (HSPCs) were magnetically enriched from CB and mPB samples using the Indirect CD34 MicroBead Kit, human (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The purity of the isolated fraction of CD34+ cells used was >94%.

[0254] In vitro NK cell differentiation assay In vitro generation of NK cells was carried out in two steps for a total of 21 days.

[0255] The first step consisted of culturing human CD34+ cells in the DL-4 / TNF-α culture system for 7 days. CB or mPB CD34+ HSPCs were cultured in wells coated with DL-4-Fc fusion protein (5 μg / ml) and RetroNectin® (25 μg / ml) in α-MEM medium (Thermo Fischer, MA, USA) supplemented with 20% FBS (Hyclone, GE Healthcare Life Sciences), 100 ng / ml hSCF, 100 ng / ml hTPO, 100 ng / ml hFlt3-L, and 100 ng / ml hIL-7 in the presence of TNF-α (10 ng / ml) for 7 days.

[0256] The second step consisted of culturing the precursor cells (whole population without selection of specific cell populations) obtained from the first step in a feeder-free culture system containing a human cytokine cocktail but without DL-4 and TNF-α for 14 days to generate NK cells. The precursor cells from the first step were cultured in RPMI Glutamax medium (Thermo Fischer, MA, USA) supplemented with 10% FBS (Hyclone, GE Healthcare Life Sciences), 50 ng / ml hSCF, 50 ng / ml hFlt3-L, 20 ng / ml hIL-7, 20 ng / ml hIL-15 (Peprotech), 500 IU / ml hIL-2 (Novartis) and in the absence of TNF-α for 14 days in non-coated wells (without DL-4) to obtain the NK cell product.

[0257] Flow cytometry Human CD56-APCVio770 / PEVio770 (clone REA196), CD161-PEVio770 (clone 191B8), NKG2C (CD159c)-APC (clone REA205), KLRG1-PE (clone REA261), CD158e / k (KIR3DL1 / DL2)-PEVio770 (clone REA970), CD158b (KIR2DL2 / DL3)-APC (clone DX27), NKG2D (CD314)-APC (clone REA797), NKG2A (CD159a)-PEVio770 (clone REA110), CD94-PE (clone REA113), NKp44 (CD336) Antibodies against NKp30 (CD337)-APC (clone REA823), CD16-Vioblue (clone REA423), DNAM-1 (CD226)-PEVio770 (clone REA1040), T-bet-APC (clone REA102), CD107a-PEVio770 (clone REA792), TNF-α-PE (clone REA656), IFNg-APC (clone 45-15), perforin-PE (clone REA1061), granzyme B-APC (clone REA226) and 7-aminoactinomycin D (7-AAD) were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany). Anti-human CD7-PE (clone MT701) and NKp46-PE (clone 9E2 / NKp46(9-E2)) were obtained from BD Biosciences (San Jose, CA). Anti-human CD3-BV421(UCHT1) was purchased from Sony Biotechnology (San Jose, CA). Antibody against human CCR5(CD195)-APC (clone J418FI) was obtained from Biolegend (San Diego, CA). Anti-human Eomes-PE (clone WD1928) and ID2-PECy7 (clone ILCID2) were obtained from eBioscience (San Diego, CA).

[0258] For surface staining, cells were incubated with the appropriate antibody for 15 min on ice, washed, and then resuspended in FACS buffer.

[0259] For intracellular staining, cells were pre-stained for surface markers, fixed and permeabilized using either a Fixation / Permeabilization Solution Kit (BD Biosciences) or a Foxp3 / Transcription Factor Staining Buffer Set (eBioscience), and then incubated with the appropriate antibody for 30 min at room temperature. Cells were then washed and resuspended in FACS buffer prior to analysis.

[0260] All flow cytometry data were acquired on a Gallios flow cytometer (Beckman Coulter, Krefeld, Germany) and data were analyzed using FlowJo software (version 10.2, Treestar, Ashland, OR). All gating during FACS analysis was performed on live cells (as measured by exclusion of 7-AAD dye).

[0261] Degranulation and cytokine induction assays The resulting NK cells derived from CB or mPB CD34+ HSPCs (CB HSPC-NK or mPB HSPC-NK) were stimulated with K562 cells (chronic myeloid leukemia cell line) by incubating together at 1:2 effector (E) to target (T) ratio at 37°C in a CO2 incubator without cytokines. Anti-human CD 107a antibody was added (20 μl / ml) during incubation and then incubated for 1 h. After 1 h, Brefeldin (GolgiStop) (4 μl / ml) and Monensin (GolgiPlug) (1 μl / ml) were added and further incubated for 5 h. After 5 h incubation, cells were harvested, washed and surface stained for CD3 and CD56. They were further stained intracellularly for TNF-α and IFNg. Stained cells were acquired on a Gallios flow cytometer.

[0262] Cytotoxicity assay Flow cytometry-based cytotoxicity assays were performed by using K562 cells as target cells. Target cells (K562 or THP1) were labeled with CellTrace Violet dye by incubating them with 1 μM of the dye for 10 min at 37° C. to distinguish target cells from effector NK cells. The labeled target cells were then incubated with effector NK cells at different effector-to-target ratios in RPMI medium supplemented with 10% FBS and 30 IU / ml hIL-2 for 5 h at 37° C. in a CO2 incubator. After 5 h of incubation, cells were stained with 7-AAD to distinguish target cells killed by effector NK cells. The effect of spontaneous target cell death was normalized by including one condition of incubating only target cells without effector cells (by subtracting the percentage of spontaneous target cell death without effector cells from the percentage of target cell death in the presence of effector cells). Peripheral blood derived NK cells were used as positive control effector cells in parallel with in vitro generated NK cells.

[0263] For all experiments, two controls were used: CB CD34+ cells treated with second step medium (CB CD34+, no DL-4 / TNF-α) and peripheral blood-derived NK alone activated overnight with IL-2 and IL-15 (PB-NK).

[0264] result Generation of NK CD3-CD56+ cells To test the capacity for NK cell generation using DL-4 / TNF-α cultures from CB or mPB CD34+ HSPCs, CD34+ cells were cultured for 7 days on DL-4 and retronectin coated wells in the presence or absence of TNF-α (10 ng / ml), followed by NK cell differentiation cultures of cells obtained after the first 7 days of culture (CB CD34+(+DL-4 / +TNF-α) or mPB CD34+(+DL-4 / +TNF-α)) without DL-4 / TNF-α. In parallel, CB CD34+ HSPCs not exposed to DL-4 / TNF-α were also cultured in NK cell differentiation medium without DL-4 / TNF-α. Cultures were analyzed for their differentiation into NK cells (identified as CD3-CD56+ cells) after 14 and 21 days of total culture. While differentiation of NK cells (CD3-CD56+ cells) was very low (max 3%) for CB CD34+ (no DL-4 / no TNF-α), HSPCs exposed to DL-4, CB CD34+(+DL-4) or mPB CD34+(+DL-4) could efficiently differentiate into CD3-CD56+ NK cells without T cell contamination within a short period of 14 and 21 days for both TNF-α-exposed and non-exposed conditions. Interestingly, the percentage of NK cells reached up to 90% after day 14 in the presence of TNF-α (Figure 1A and B). The purity of generated NK cells was more homogenous in the TNF-α-exposed condition, especially for adult mPB cells (mPB CD34+(+DL-4)) (Figure 1B). Importantly, analysis of total NK cell yields showed that addition of TNF-α during the first 7 days of DL-4 exposure of CB or mPB CD34+ HSPCs increased the number of NK cells obtained per CD34+ HSPC by 8-fold and 18-fold for CB and mPB, respectively, after 21 days of culture (Figure 1C). However, the number of NK cells obtained from CB CD34+ cells not exposed to DL-4 / TNF-α remained significantly lower, as shown in Figure 1C.Furthermore, analysis of NK cell generation using +DL-4 / +TNF-α cultures from different CD34+HSPC donors showed that a single CB CD34+HSPC was able to give rise to an average of 2488 ± 1832 (mean ± SEM) NK cells for CB and an average of 879 ± 805 (mean ± SEM) NK cells for mPB on day 14 under TNF-α exposure conditions, and an average of 6389 ± 3723 (mean ± SEM) NK cells for CB and an average of 1881 ± 1458 (mean ± SEM) NK cells for mPB on day 21 (Figure 1C). These data demonstrate that the combination of a first culture step for 7 days in the presence of +DL-4 / +TNF-α and a second culture step in DL-4 / TNF-α-free medium can generate a high percentage and number of NK cells during a short culture period of 14 to 21 days.

[0265] Characterization of generated NK cells Phenotypic characterization of NK cells from generated CB or mPB CD34+HSPCs (+DL-4 / +TNF-α exposed) showed that NK cells expressed the activating receptors NKG2D, NKp46, NKp44, NKp30, DNAM1, and CD161, but not the inhibitory receptors KLRG-1, KIR2DL2 / DL3, and KIR3DL1 / DL2 (Figure 2A and Table 1). Importantly, they also expressed the chemokine receptor CCR5 at even higher levels than activated PB-NK cells. However, only a small low percentage of these cells (ranging from about 5 to about 8%) showed CD16 expression, and about 50% of them expressed NKG2A (expressing percentages ranging from about 45% to about 63%) and CD94 (expressing percentages ranging from about 50% to about 66%) (Figure 2A and Table 1). NK derived from CD34+ (not exposed to DL-4 / TNF-α) expressed lower levels of activating receptors compared to those of CB or mPB NK cells (+DL-4 / +TNF-α). Activated PB-NK cells showed some level of expression of KLRG1, an exhaustion marker of NK cells. Thus, generated CB or mPB NK cells were phenotypically distinct from activated PB-NK cells with respect to expression of CD16, KIR, and KLRG1, which were expressed at very low levels or not expressed in CB or mPB NK cells (+DL-4 / +TNF-α) (Figure 2A and Table 1). Notably, NKp44 was not well expressed by activated PB-NK cells, whereas CB or mPB NK cells (+DL-4 / +TNF-α) highly expressed NKp44 (Figure 2A and Table 1). The lack of expression of KIR and KLRG1, combined with the uniform expression of CD56 (Figure 1A), and the very low expression of CD16 (Figure 2A and Table 1) suggested an immature phenotype of CB or mPB NK cells (+DL-4 / +TNF-α). Interestingly, similar to activated PB-NK cells, CB or mPB NK (+DL-4 / +TNF-α) cells also express the transcription factors Eomes, T-bet, and ID2 (Figure 2B), which are known to be essential for NK cell differentiation and function.

[0266] Table 1: Comparison of CB-NK cells, mPB-NK cells and PB-NK phenotypes (mean ± SEM; n = 2 or n = 3) [Table 1]

[0267] Generated NK cells are functional Degranulation and expression of perforin and granzymes To determine the functionality of the generated CB or mPB NK cells, we first analyzed them for expression of the cytotoxic molecules perforin and granzyme B, which are known to be constitutively expressed by NK cells and are important for inducing target cell killing. Similar to activated PB-NK cells, CB or mPB NK cells (not exposed to TNF-α or exposed to TNF-α) expressed both perforin and granzyme B (Figure 3A), reflecting their ability to be cytotoxic to their target cells. However, CB CD34+-NK (no DL-4 / no TNF-α) cells expressed lower levels of perforin and granzyme B than CB or mPB NK (+DL-4 / with or without TNF-α) and PB-NK cells (Figure 3A).

[0268] Next, NK cells were evaluated for their ability to undergo degranulation upon stimulation with their target cells, which is a key process of secreting cytotoxic molecules to kill target cells, and to induce the expression of interferon gamma (IFNg) and TNF-α, which can mediate target cell killing by inducing apoptosis. Upon stimulation with myeloid leukemia cell line K562 cells, CB or mPB NK (+DL-4 / TNF-α or not) cells showed degranulation, indicated by detection of CD107 on their cell surface, which was comparable to that of activated PB-NK cells (Figure 3B). The level of degranulation was higher in TNF-α-exposed conditions compared to their non-exposed conditions, especially for mPB. In addition to lower perforin and granzyme B expression (as described above), degranulation levels were also lower in CB CD34+-NK cells (no DL-4 / no TNF-α) compared to CB or mPB NK cells (+DL-4 / with or without TNF-α) (Figure 3B). IFNg and TNF-α were induced upon stimulation with K562 cells under all conditions except CB CD34+-NK cells (no DL-4 / no TNF-α) (Figure 3C). Importantly, however, CB or mPB NK cells derived from TNF-α-exposed conditions showed higher TNF-α induction than their TNFα-unexposed counterparts. Furthermore, especially for mPB, IFNg induction was lower in conditions without TNFα exposure (Figure 3C). Taken together, these data suggest that NK cells generated by the methods of the present invention are functional.

[0269] Cytotoxicity The cytotoxic activity of CB or mPB NK cells (+DL-4 / +TNF-α) was tested by incubating the NK cells with K562 cells used as target cells. CB and mPB NK cells (+DL-4 / +TNF-α) NK cells were able to efficiently kill K562 cells (data not shown). These data suggest that the NK cells generated by the method of the present invention possess cytotoxicity and can kill target cells in vitro.

[0270] Example 2: CD34+ cells can be efficiently transduced and differentiated into CD3-CD56+ NK cells Materials and Methods Transduction protocol During the first culture step of 7 days, 1 × 10 cells were cultured in X-vivo 20 medium (Lonza) in the presence of human (h) cytokines - 300 ng / ml hSCF, 100 ng / ml hTPO, 300 ng / ml hFlt3-L, 100 ng / ml hIL-7, 20 ng / ml hIL-3 (Peprotech) and in the absence of TNF-α (R and D Systems). 6 CB or mPB CD34+ HSPCs at a cell concentration of 100 cells / ml were preactivated overnight on wells coated with DL-4-Fc fusion protein (5 μg / ml) and RetroNectin® (25 μg / ml). Preactivated cells were then transduced at a multiplicity of infection (MOI) of 100 with VSV-G pseudotyped lentivirus encoding a GFP reporter protein in the presence of 4 μg / ml protamine sulfate in the same preactivation medium for 6 h. Six hours after transduction, the transduced cells were washed with α-MEM medium (Thermo Fischer, MA, USA), and the transduction medium was replaced with α-MEM medium supplemented with 20% FBS (Hyclone, GE Healthcare Life Sciences), 100 ng / ml hSCF, 100 ng / ml hTPO, 100 ng / ml hFlt3-L, and 100 ng / ml hIL-7 in the presence of TNF-α (10 ng / ml), and further cultured in DL-4- and retronectin-coated wells for up to 7 days.

[0271] The second step consisted of culturing the precursor cells obtained from the first step (whole population without any selection of specific cell populations) in a feeder-free culture system containing a human cytokine cocktail but without DL-4 and TNF-α for 14 days to generate NK cells. The precursor cells from the first step were cultured for 14 days in RPMI Glutamax medium (Thermo Fischer, MA, USA) supplemented with 10% FBS (Hyclone, GE Healthcare Life Sciences), 50 ng / ml hSCF, 50 ng / ml hFlt3-L, 20 ng / ml hIL-7, 20 ng / ml hIL-15 (Peprotech), 500 IU / ml hIL-2 (Novartis) in the absence of TNF-α in non-coated wells (without DL-4) to obtain the NK cell product.

[0272] result Generation of transduced NK CD3-CD56+ cells To test whether transduced NK cells could be generated by using this method, CB or mPB CD34+ cells were preactivated overnight in DL-4 and Retronectin coated wells in the transduction cytokine cocktail and then transduced with GFP-expressing VSV-G pseudotyped lentivirus for 6 hours. After transduction, the transduced cells were further cultured in DL-4 and Retronectin coated wells in the culture cytokine cocktail in the presence of TNF-α (10 ng / ml) for up to 7 days. This was followed by a DL-4 / TNF-α-free NK cell differentiation culture of the cells obtained after the first 7 days of culture. The cultures were analyzed for their differentiation into transduced NK cells (identified as CD3-GFP+CD56+ cells) after 14 and 21 days of culture. As shown in Figure 4A and B, transduced GFP+CD3-CD56+NK cells were observed within a short culture period of 14 and 21 days, with an average frequency of 58±4.5 (mean±SEM) for CB and 33±7.5 (mean±SEM) for mPB on day 21. Analysis of the total yield of transduced GFP+CD3-CD56+NK cells showed that a single CB CD34+HSPC was able to generate an average of 1600±1442 (mean±SEM) transduced NK cells for CB and 318±365 (mean±SEM) transduced NK cells for mPB on day 14, and an average of 3857±2537 (mean±SEM) transduced NK cells for CB and 770±664 (mean±SEM) transduced NK cells for mPB on day 21 (Figure 4C). These data demonstrate that this culture method can generate large numbers of transduced NK cells within a short culture period of 14 to 21 days.

[0273] Characterization of generated NK cells Similar to the non-transduced NK cells generated by the method of the present invention (Figure 2A), the transduced GFP+CD3-CD56+ CB or mPB NK cells (+DL-4 / +TNF-α) express the activating receptors NKG2D, NKp46, NKp44, NKp30, DNAM1, and CD161, but not the inhibitory receptors KLRG-1, KIR2DL2 / DL3, and KIR3DL1 / DL2 (Figure 5). Importantly, they also express the chemokine receptor CCR5. They express very low CD16, and NKG2A and CD94 were expressed by only 50% of the cells (Figure 5). This data suggests that the transduction conditions did not affect the expression of NK receptors on the transduced NK cells.

[0274] Generated NK cells are functional Degranulation and expression of perforin and granzymes To evaluate the functional capacity of the generated transduced NK cells, degranulation and cytokine induction assays were performed by stimulating transduced CB or mPB NK cells (with or without +DL-4 / TNF-α) with K562 cells. Upon stimulation, transduced CB or mPB NK cells (with or without +DL-4 / TNF-α) showed degranulation as indicated by detection of CD107 on their cell surface (Figure 6A). The level of degranulation was higher in TNF-α exposed conditions compared to their non-exposed counterparts, especially for mPB (Figure 6A). Expression of IFNg and TNF-α was also induced in transduced CB or mPB NK cells (with or without +DL-4 / TNF-α) upon stimulation with K562 cells (Figure 6B). Interestingly, transduced CB or mPB NK cells from the TNF-α-exposed condition showed higher TNF-α induction than their counterparts not exposed to TNF-α. Furthermore, IFNg induction was lower in the condition not exposed to TNFα, especially for mPB (Figure 6B). Finally, granzyme B and perforin expression were also induced in transduced CB or mPB NK cells (with +DL-4 / TNF-α) upon stimulation with K562 cells (data not shown). These data suggest that transduced CB or mPB NK cells still retain their functional capabilities.

[0275] Cytotoxicity The cytotoxic activity of transduced CB or mPB NK cells (+DL-4 / +TNF-α) was tested by incubating the NK cells with K562 cells or THP1 cells as target cells. As shown in FIG. 7A, the transduced CB or mPB NK cells (+DL-4 / +TNF-α) obtained by the method of the present invention could efficiently kill K562 cells at a level similar to that of non-transduced (mock) conditions or PB-NK, suggesting that transduction did not affect the cytotoxic ability of the transduced NK cells. Furthermore, the transduced CB NK cells (+DL-4 / +TNF-α) obtained by the method of the present invention could also efficiently kill THP1 cells (FIG. 7B). These data suggest that the transduced NK cells generated by the method of the present invention can efficiently kill their target cells in vitro.

[0276] Example 3: Generation of functionally promising CAR NK cells from CB CD34+ HSPCs Materials and Methods Transduction protocol During the first culture step of 7 days, CB CD34+ HSPCs were cultured at 1 × 10 in X-vivo 20 medium (Lonza) in the presence of human (h) cytokines: 300 ng / ml hSCF, 100 ng / ml hTPO, 300 ng / ml hFlt3-L, and 100 ng / ml hIL-7 (Peprotech) and in the absence of TNF-α (R and D Systems). 6Cells were preactivated overnight on wells coated with DL-4-Fc fusion protein (5 μg / ml) and RetroNectin® (25 μg / ml) at a cell concentration of 1000 cells / ml. Preactivated cells were then transduced with VSV-G pseudotyped lentivirus encoding a CD19-targeting CAR at a multiplicity of infection (MOI) of 100 in the presence of 4 μg / ml protamine sulfate in the same preactivation medium for 6 hours. Six hours after transduction, the transduced cells were washed with α-MEM medium (Gibco) and the transduction medium was replaced with α-MEM medium supplemented with 20% FBS (Hyclone, GE Healthcare Life Sciences), 100 ng / ml hSCF, 100 ng / ml hTPO, 100 ng / ml hFlt3-L and 100 ng / ml hIL-7 in the presence of TNF-α (10 ng / ml) and further cultured in DL-4 and retronectin coated wells for up to 7 days.

[0277] The second step consisted of culturing the precursor cells obtained from the first step (total population without any selection of specific cell populations) in a feeder-free culture system containing a human cytokine cocktail but without DL-4 and TNF-α for 8 days to generate NK cells. The precursor cells obtained after the first culture step were cultured in RPMI Glutamax medium (Gibco) supplemented with 10% FBS (Hyclone, GE Healthcare Life Sciences), 50 ng / ml hSCF, 50 ng / ml hFlt3-L, 20 ng / ml hIL-7, 20 ng / ml hIL-15 (Peprotech), 500 IU / ml hIL-2 (Novartis) in the absence of TNF-α for 8 days in non-coated wells (without DL-4) to obtain the NK cell product.

[0278] Cytotoxicity assay Flow cytometry-based cytotoxicity assays were performed by using NALM-6 (B cell precursor leukemia cell line) cells as target cells. NALM-6 target cells were labeled with CellTrace Violet dye by incubating them with 1 μM of the dye for 10 min at 37° C. to distinguish target cells from effector NK cells. The labeled target cells were then incubated with effector NK cells at different effector-to-target ratios in RPMI medium supplemented with 10% FBS and 30 IU / ml hIL-2 for 5 h at 37° C. in a CO2 incubator. After 5 h of incubation, cells were stained with 7-AAD to distinguish target cells killed by effector NK cells. The effect of spontaneous target cell death was normalized by including one condition of incubation of only target cells without effector cells (by subtracting the percentage of spontaneous target cell death without effector cells from the percentage of target cell death in the presence of effector cells).

[0279] result To test whether chimeric antigen receptor (CAR) expressing NK cells could be generated by using this method, CB CD34+ cells were preactivated overnight on DL-4 and retronectin coated wells in a transduction cytokine cocktail and then transduced with a VSV-G pseudotyped lentivirus encoding a CAR targeting CD19 for 6 hours. After transduction, the transduced cells were further cultured in DL-4 and retronectin coated wells in a DL-4 culture cytokine cocktail in the presence of TNF-α (10 ng / ml) for up to 7 days. This was followed by a DL-4 / TNF-α-free NK cell differentiation culture of feeder cells and precursor cells obtained after the first 7 days of culture. Cultures were analyzed after 8 days for their differentiation into CAR-expressing NK cells (identified as CD3-CAR+CD56+ cells). As shown in Figure 9A, CAR+CD56+ NK cells were observed within a short culture period of 15 days at a frequency of 46.5%. These data demonstrate that this culture method can generate CAR NK cells within a short culture period of 15 days.

[0280] The cytotoxic activity of the generated CD19-targeting CAR NK cells was tested by incubating the NK cells with cells of NALM-6 (a B-cell precursor leukemia cell line expressing CD19) as target cells. As shown in Figure 9B, the CD19-CAR NK cells were able to efficiently kill NALM-6 cells, in contrast to mock NK cells, which were unable to kill the target NALM-6 cells. These data suggest that the CAR NK cells generated in this manner can efficiently kill specific target cells in vitro.

[0281] Example 4: Phenotypic characterization of CD3-CD56+ NK cells and CAR-NK cells generated with or without IL-12 and IL18 Materials and Methods In vitro NK cell differentiation assay In vitro generation of NK cells was performed in two steps over a total of 14 days.

[0282] The first step consisted of culturing human CD34+ cells in the DL-4 / TNF-α culture system for 7 days. CB CD34+ HSPCs were cultured in wells coated with DL-4-Fc fusion protein (5 μg / ml) and RetroNectin® (25 μg / ml) in α-MEM medium (Thermo Fischer, MA, USA) supplemented with 20% FBS (Hyclone, GE Healthcare Life Sciences), 100 ng / ml hSCF, 100 ng / ml hTPO, 100 ng / ml hFlt3-L, and 100 ng / ml hIL-7 in the presence of TNF-α (10 ng / ml) for 7 days.

[0283] The second step consisted of culturing the precursor cells obtained from the first step (total population without any selection of specific cell populations) for 14 days in a feeder-free culture system containing a human cytokine cocktail but without DL-4 and TNF-α to generate NK cells. The progenitor cells from the first step were cultured in RPMI Glutamax medium (Thermo Fischer, MA, USA) supplemented with 10% FBS (Hyclone, GE Healthcare Life Sciences), 50 ng / ml hSCF, 50 ng / ml hFlt3-L, 20 ng / ml hIL-7, 20 ng / ml hIL-15 (Peprotech), 500 IU / ml hIL-2 (Novartis) with or without 10 ng / ml hIL-12 (Peprotech) and 100 ng / ml hIL-18 (MBL International Corporation) for 14 days and in non-coated wells (without DL-4) in the absence of TNF-α for 7 days to obtain NK cell products.

[0284] Flow cytometry Human CD56-APC Vio770 / PEVio770 (clone REA196), NKG2C (CD159c)-APC (clone REA205), KLRG1-PE (clone REA261), CD158e / k (KIR3DL1 / DL2)-PEVio770 (clone REA970), CD158b (KIR2DL2 / DL3)-APC (clone DX27), NKG2D (CD314)- Antibodies against APC (clone REA797), NKG2A (CD159a)-PEVio770 (clone REA110), CD94-PE (clone REA113), NKp44 (CD336)-PEVio770 (clone REA1163), DNAM-1 (CD226)-PEVio770 (clone REA1040), and 7-aminoactinomycin D (7-AAD) were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany). Anti-human CD7-PE (clone MT701), NKp46-PE (clone 9E2 / NKp46(9-E2)), and CD62L-BV421 (clone M-T701) were obtained from BD Biosciences (San Jose, CA). Anti-human CD3-BV421 / BV510 (UCHT1) was purchased from Sony Biotechnology (San Jose, Calif.). Anti-human NKp30 (CD337)-BV421 (clone DREG-56) and CD16-BV510 (3G8) were obtained from Biolegend (San Diego, Calif.).

[0285] For surface staining, cells were incubated with the appropriate antibody for 15 min on ice, washed, and then resuspended in FACS buffer.

[0286] All flow cytometry data were acquired on a Gallios flow cytometer (Beckman Coulter, Krefeld, Germany) and data were analyzed using FlowJo software (version 10.2, Treestar, Ashland, OR). During FACS analysis, all gating was performed on live cells (as measured by exclusion of 7-AAD dye).

[0287] Transduction protocol During the first culture step of 7 days, CB CD34+ HSPCs were cultured at 1 × 10 in X-vivo 20 medium (Lonza) in the presence of human (h) cytokines: 300 ng / ml hSCF, 100 ng / ml hTPO, 300 ng / ml hFlt3-L, and 100 ng / ml hIL-7 (Peprotech) and in the absence of TNF-α (R&D Systems). 6 Cells were preactivated overnight on wells coated with DL-4-Fc fusion protein (5 μg / ml) and RetroNectin® (25 μg / ml) at a cell concentration of 100. Preactivated cells were then transduced with VSV-G pseudotyped lentivirus encoding the ZsG reporter protein at a multiplicity of infection (MOI) of 100 in the presence of 4 μg / ml protamine sulfate in the same preactivation medium for 6 h. Six hours after transduction, the transduced cells were washed with α-MEM medium (Gibco) and the transduction medium was replaced with α-MEM medium supplemented with 20% FBS (Hyclone, GE Healthcare Life Sciences), 100 ng / ml hSCF, 100 ng / ml hTPO, 100 ng / ml hFlt3-L and 100 ng / ml hIL-7 in the presence of TNF-α (10 ng / ml) and further cultured in DL-4 and retronectin coated wells for up to 7 days.

[0288] The second step consisted of culturing the precursor cells obtained from the first step (total population without any selection of specific cell populations) in a feeder-free culture system containing a human cytokine cocktail but without DL-4 and TNFα for 8 days to generate NK cells. The precursor cells obtained after the first culture step were cultured in non-coated wells (without DL-4) in RPMI Glutamax medium (Gibco) supplemented with 10% FBS (Hyclone, GE Healthcare Life Sciences), 50 ng / ml hSCF, 50 ng / ml hFlt3-L, 20 ng / ml hIL-7, 20 ng / ml hIL-15 (Peprotech), 500 IU / ml hIL-2 (Novartis) with or without 10 ng / ml hIL-12 and 100 ng / ml hIL-18 in the absence of TNF-α for 8 days to obtain the NK cell product.

[0289] result Phenotypic characterization of CD34+HSPC (+DL-4 / +TNF-α exposed)-derived NK cells and CD34+HSPC (+DL-4 / +TNF-α exposed)-derived CAR-NK cells generated with or without IL-12 and IL18 showed that NK cells and CAR NK cells express the activating receptors NKG2D, NKp46, NKp44, NKp30, DNAM1, and CD62L, but not the inhibitory receptors KLRG1, KIR2DL2 / DL3, and KIR3DL1 / DL2 (Table 2). NK cells and CAR NK cells also express the inhibitory receptors CD96 and NKG2A. Furthermore, NK cells and CAR NK cells generated in the presence of hIL-12 and hIL18 express a higher percentage of activating receptors CD62L (16 and 19%, respectively, versus 4%) and CD16 (50 and 44%, respectively, versus 22 and 15%), and a higher percentage of inhibitory receptors CD94 (26 and 30%, respectively, versus 16 and 13%) and NKG2A (24 and 28%, respectively, versus 11 and 10%), compared to NK cells and CAR NK cells generated without hIL-12 and hIL-18 (Table 2).

[0290] Table 2: Comparison of phenotypes of NK and CAR-NK cells cultured with or without hIL-12 and hIL18 on day 14 (mean ± SEM; n = 3). [Table 2]

Claims

1. 1. An in vitro method for generating NK cells, comprising: a) culturing CD34+ cells in the presence of TNF-α or a fragment thereof and a Notch ligand or a fragment thereof, thereby obtaining a first population of cells; and b) culturing the population of cells obtained in step a) in a cytokine-containing medium; Including, The cytokine-containing medium does not contain TNF-α or a fragment thereof or a Notch ligand or a fragment thereof. In vitro methods.

2. 2. The in vitro method of claim 1, wherein in step a) the cells are cultured in the presence of TNF-α or a fragment thereof and a Notch ligand or a fragment thereof for more than 5 days and less than 9 days, preferably for about 7 days.

3. 2. The in vitro method of claim 1, wherein the Notch ligand is a Delta-like 4 ligand or a fragment thereof, preferably a soluble domain of a Delta-like 4 ligand.

4. 2. The in vitro method of claim 1, wherein in step a) the cells are also exposed to a fibronectin fragment comprising RGDS, linking segment 1 (CS-1) and / or a heparin-binding domain, preferably the fibronectin fragment is CH-296.

5. 2. The in vitro method of claim 1, wherein the CD34+ cells are isolated from an adult donor or from umbilical cord blood cells.

6. 2. The in vitro method according to claim 1, wherein the cytokine-containing medium of step (b) contains at least three, preferably five cytokines selected from the group consisting of interleukin-7 (IL-7), stem cell factor (SCF), interleukin-15 (IL-15), interleukin-2 (IL-2), and Flt3 ligand (FLT3L).

7. 2. The in vitro method of claim 1, wherein in step b) the cells are cultured in a cytokine-containing medium for more than 7 days and less than 21 days.

8. 2. The in vitro method of claim 1, which comprises the additional step of transducing the cells with a vector, preferably during or before step (a).

9. 9. The in vitro method of claim 8, wherein the vector encodes a chimeric antigen receptor (CAR).

10. A NK cell population likely to be obtained by the in vitro method of any one of claims 1 to 9, wherein more than 60% of the cells are CD3-CD56+.

11. The NK cell population of claim 10, wherein the cells are CD3-CD56+ cells and do not express at least one inhibitory receptor selected from the group consisting of KIR3DL1 / DL2, KIR2DL2 / DL3, and KLRG1.

12. The NK cell population of claim 10, wherein the cells are CD3-CD56+ cells and express at least one molecule selected from CD161 and an activating receptor selected from the group consisting of NKp30, NKp44, NKp46, DNAM-1, and NKG2D.

13. An NK cell population that expresses CD161, NKp30, NKp44, NKp46, DNAM-1, and NKG2D, but does not express KIR3DL1 / DL2, KIR3DL2 / DL3, or KLRG1.

14. 11. The NK cell population according to claim 10 for use as a medicine, in particular for increasing the number of NK cells in a subject in need thereof.

15. The NK cell population of claim 10 for treating cancer, persistent viral infections, and parasitic diseases.

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