NK progenitor cells and method for producing NK cells using same

Abstract

In order to identify NK progenitor cells and provide a method for producing mature NK cells or CAR-NK cells using said progenitor cells, it was found that CD56−CD34− cells are NK progenitor cells that are major sources in the production of CAR-NK cells.

Description

NK precursor cells and method for producing NK cells using the same

[0001] The present invention relates to natural killer (NK) precursor cells. The present invention also relates to a method for producing NK cells using the precursor cells. Furthermore, the present invention relates to a method for producing NK cells expressing a chimeric antigen receptor (CAR) using the precursor cells.

[0002] Autologous CAR-T cell therapy has shown remarkable efficacy in patients with B-cell leukemia / lymphoma and multiple myeloma, but it is very expensive and requires several weeks to generate CAR-T cells from autologous T cells.

[0003] On the other hand, natural killer (NK) cells, when infused into allogeneic recipients, do not induce graft-versus-host disease and therefore could serve as a universal source of cellular immunotherapy. This approach also allows for the preparation of ready-made products for use in allogeneic recipients. Furthermore, the expression of CARs in NK cells allows for the reprogramming of specific target specificity. NK cells from various sources, including induced pluripotent stem cells, umbilical cord blood (CB), and NK cell lines, have been used to generate CAR-NK cells. Furthermore, CB-derived CAR-NK cells targeting CD19 have been shown to be effective against B-cell malignancies in clinical trials. Furthermore, CB-derived CAR-NK cells targeting various other molecules are currently undergoing clinical trials.

[0004] Regarding the preparation of such NK cells, and further CAR-NK cells, purified CD56 ＋ It is known that NK cells can be expanded by stimulation with K562-based feeder cells and interleukin (IL)-2 (Non-Patent Documents 1 and 2). It has also been shown that human NK cells can be differentiated from hematopoietic progenitor cells in vitro (Non-Patent Documents 3 to 11). Furthermore, CB mononuclear cells (MNCs) depleted of CD3, CD14, and CD19 contain many immature hematopoietic cells and have been used to establish CAR-NK cells (Non-Patent Document 12).

[0005] However, it was unclear whether CAR-NK cells were derived from mature NK cells or NK precursor cells. Furthermore, it was not clear what kind of cells NK precursor cells are in the first place, making it difficult to efficiently produce CAR-NK cells and the like.

[0006] Liu E. et al., Leukemia, 2018, Vol. 32, No. 2, pp. 520-531; Chaudhry K. et al., Mol Ther Methods Clin Dev., 2022, Vol. 27, No. 415-430; Dolstra H. et al., Clin Cancer Res., 2017, Vol. 23, No. 15, pp. 4107-4118; Cany J. et al., Oncoimmunology., 2015, Vol. 4, No. 7, e1017701; Cany J. et al., PLoS One, 2013, Vol. 8, No. 6, e64384; Spanholtz J. et al., PLoS One, 2011, Vol. 6, No. 6, e20740. Spanholtz J. et al., PLoS One, 2010, Vol. 5, No. 2, e9221. Freud A. G. et al., J Exp Med., 2006, Vol. 203, No. 4, pp. 1033-1043. Zhao X. et al., Blood Adv., 2018, Vol. 2, No. 19, pp. 2452-2461. Herrera L. et al., Front Immunol., 2017, 8:755. Renoux V. M. Liu E. et al., Immunity, 2015, Vol. 43, No. 2, pp. 394-407; Liu E. et al., N Engl J Med., 2020, Vol. 382, ​​No. 6, pp. 545-553; Miller JS. et al., Blood, 1994, Vol. 83, No. 9, pp. 2594-2601; Hao QL. et al., Blood, 2001, Vol. 97, No. 12, pp. 3683-3690; Hernandez DC. et al., Immunity, 2021, Vol. 54, No. 10, pp. 2417-2432. e2415. Perez SA. et al., Int Immunol. , 2006, Vol. 18, No. 1, pp. 49-58

[0007] The present invention has been made in view of the problems associated with the prior art, and aims to clarify whether CAR-NK cells are derived from mature NK cells or NK precursor cells. Another object of the present invention is to identify NK precursor cells and provide a method for producing mature NK cells or CAR-NK cells using the precursor cells.

[0008] As a result of extensive research to achieve the above object, the present inventors have found that CAR-NK cells are mainly CD56 － We found that they are derived from NK precursor cells. － We found that a significant number of CAR-NK cells were generated from CB mononuclear cells after 2 weeks of in vitro culture. That is, CB-derived CAR-NK cells expressed CD56 ＋ Not mature NK cells, but mainly CD56 － It has been revealed that they are derived from NK precursor cells.

[0009] Also, CD56 － CD3 － CD14 － CD19 － Single-cell RNA sequencing analysis of CB mononuclear cells revealed that these cells could be subdivided into three subpopulations based on the expression of CD34 and HLA-DR. Of these, NK cells were mainly CD34 － HLA-DR － Furthermore, it was found that the CD34 － HLA-DR － Among the cells, CD7 ＋ The present inventors have shown that only cells differentiate into NK cells.

[0010] In previous studies, CD34 cells from human bone marrow or CB were cultured with various cytokines, including IL-15. ＋ It has been reported that NK cells differentiate from CD34 cells (Non-Patent Documents 11 and 13-15). ＋It has been reported that co-culture with stromal cells is necessary to generate NK cells from cells (Non-Patent Documents 3, 4, 7, and 16). However, these studies have shown that 3 to 5 weeks of culture is required to generate NK cells, and in most cases, the efficiency is low.

[0011] On the other hand, as mentioned above, the present inventors have investigated the CD34 － They found that the fraction contained committed NK cell precursors and demonstrated that it produced significant numbers of NK cells after just two weeks of culture.

[0012] Also, CD56 － CD7 ＋ CD34 － HLA-DR － Lineage marker (Lin (CD3 / CD14 / CD19)) － We found that CD122 (IL-2 / IL-15 receptor β chain), which constitutes the IL-15 receptor, is highly expressed in K562 cells. Based on this finding, we prepared two types of feeder cells: K562 cells overexpressing 4-1BB ligand and membrane-bound IL-21 (mbIL-21), and cells further transfected with membrane-bound IL-15 (mbIL-15), and compared their NK cell induction abilities. As a result, we found that the use of feeder cells expressing mbIL-15 in addition to 4-1BB / mbIL-21 resulted in significantly higher NK cell production.

[0013] From the above results, CD56 － CD7 ＋ CD34 － HLA-DR － Lin － The present inventors have found that human CB cells are a major source for producing human CB-derived CAR-NK cells, and have completed the present invention.

[0014] That is, the present invention provides the following aspects.

[0015] [1] Isolated natural killer (NK) precursor cells that are negative for CD56 and CD34.

[0016] [2] The NK precursor cells according to [1], which are further negative for HLA-DR and Lin and positive for CD7.

[0017] [3] An isolated cell population comprising NK precursor cells, wherein 60% or more of the total cells of the population are the NK precursor cells according to [1] or [2].

[0018] [4] A method for producing NK cells, comprising a step of culturing the NK precursor cells according to [1] or [2], or the cell population according to [3], in the presence of feeder cells and / or cytokines, wherein the NK cells are CD56-positive mature NK cells, and the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-21, IL-15, and IL-18.

[0019] [5] A method for producing NK cells expressing a chimeric antigen receptor (CAR), the method according to [4], further comprising introducing a nucleotide encoding the CAR into the NK precursor cells or cells derived therefrom according to [1] or [2] in the culture step.

[0020] [6] The method according to [4] or [5], wherein the cytokine is IL-2.

[0021] [7] The method according to any one of [4] to [6], wherein the feeder cells are artificial antigen-presenting cells.

[0022] [8] The production method according to any one of [4] to [6], wherein the feeder cells are K562 cells that express membrane-bound IL-15 and 4-1BBL.

[0023] [9] The production method according to any one of [4] to [6], wherein the feeder cells are K562 cells that express membrane-bound IL-21 and 4-1BBL.

[0024]

[10] The production method according to any one of [4] to [6], wherein the feeder cells are K562 cells that express membrane-bound IL-15, membrane-bound IL-21, and 4-1BBL.

[0025] According to the present invention, NK precursor cells are provided. Furthermore, mature NK cells or CAR-NK cells can be produced using the precursor cells.

[0026] FIG. 1 shows a protocol for generating chimeric antigen receptor-expressing natural killer cells (CAR-NK cells) from umbilical cord blood (CB) mononuclear cells (MNC). － CD56 ＋ This is a graph showing an increase in the number of NK cells (n=6). Error bars represent the standard error of the mean (s.e.m.). This is a diagram showing representative results of flow cytometry analysis of the expression of CD56, CD3, and CAR in CAR-NK cells. － CD56 ＋ Cells and CD3 － CD56 － The cells were selected and cultured separately for 14 days to obtain CD3 － CD56 ＋ This figure shows the results of analyzing NK cells. In the figure, the graph in the middle shows the results of NK cell analysis using 1 × 10 ５ The graph on the right shows the number of NK cells produced from 1 x 10 ５ CD56 produced from CB MNCs ＋ cells or CD56 － The number of NK cells derived from the cells is shown. 1 x 10 ５ CD56 produced from CB MNCs ＋ cells or CD56 － 1D is a graph showing the results of measuring the number of NK cells derived from CD3 cells. Note that the results are compiled using five independent CB samples, which are different from those in FIG. 1D. Error bars represent the standard error of the mean (s.e.m.). － CD56 ＋ cells or CD3 － CD56 － Raji cells (CD19 CAR-NK cells or non-transduced NK cells) produced from Raji cells (CD19 ＋ Specific lysis of B-cell lymphoma cells ５１1 is a graph showing the results of analysis using Cr release assay. Representative results from experiments with four different donors are shown. Error bars represent the standard error of the mean (s.e.m.). CD56 ＋ CB MNC or CD56 － 1 is a dot plot diagram showing the results of flow cytometry analysis of the expression of CAR or NK cell receptors in NK cells derived from CB MNC. － CD56 － CD14 － CD19 － The cells were selected and cultured for 7 or 14 days to obtain CD3 － CD56 ＋ This is a diagram showing the results of flow cytometry analysis of NK cells. － CD56 － CD14 － CD19 － CB MNCs (7254 cells from three CB samples) were analyzed by single-cell RNA sequencing and plotted using the uniform manifold approximation and projection method (UMAP). － CD56 － CD14 － CD19 － This is a violin plot showing the results of analyzing the expression of cell surface markers for five subpopulations differentiated from CB MNCs. In the figure, "HSPC" represents hematopoietic stem or progenitor cells, and "pDC" and "cDC" represent plasmacytoid dendritic cells and classical dendritic cells, respectively. CD3 － CD56 － CD14 － CD19 － From CB, CD34 － HLA-DR ＋ cells, CD34 ＋ HLA-DR ＋／－ Cells and CD34 － HLA-DR － The cells were selected and cultured separately for 14 days to obtain CD3 － CD56 ＋This figure shows the results of analyzing NK cells. Representative results of CB samples are shown. In the figure, the bar graph shows 1.5 × 10 ４ The total number of cells and the number of NK cells produced from each of the selected cells are shown. － CD56 － CD14 － CD19 － From CB MNC, CD34 － HLA-DR ＋ cells, CD34 ＋ HLA-DR ＋／－ Cells and CD34 － HLA-DR － The cells were sorted and 1.5 x 10 ４ CD3 cells were cultured separately for 14 days. － CD56 ＋ This is a graph showing the results of analyzing the number of NK cells. The results using three independent CB samples are summarized. The error bars represent the standard error of the mean (s.e.m.). CD34 － HLA-DR － CD56 － Lin － This figure shows the results of flow cytometry analysis of the expression of cell surface markers in CB MNCs. Representative results from three CB samples are shown. CD34 － HLA-DR － CD56 － Lin － From CB MNC, CD7 ＋ Cells and CD7 － The cells were selected and cultured separately for 14 days to obtain CD3 － CD56 ＋ This figure shows the results of analyzing NK cells. Representative results of CB samples are shown. In the figure, the bar graph shows 5 × 10 ３ The number of NK cells generated from each of the sorted cells is shown. － HLA-DR － CD56 － Lin － From CB MNC, CD7 ＋ Cells and CD7 － The cells were sorted and 5 × 10 ３CD3 cells were cultured separately for 14 days. － CD56 ＋ 1 is a graph showing the results of analyzing the number of NK cells. The results are summarized for three independent CB samples. Error bars represent the standard error of the mean (s.e.m.). NK cells were counted from CB MNCs. － CD7 － CD34 － HLA-DR － Lin － cells (non-NK progenitor population) and CD56 － CD7 ＋ CD34 － HLA-DR － Lin －

[0023] Figure 1 shows an outline of RNA-seq analysis performed on cells (NK precursor cell populations) sorted using a cell sorter. Figure 2 shows a dot plot diagram illustrating the process of sorting non-NK precursor cell populations and NK precursor cell populations from CB MNCs. Figure 3 shows a volcano plot diagram comparing the results of RNA-seq analysis of non-NK precursor cell populations and NK precursor cell populations. In the diagram, the right side shows genes with high expression levels in the NK precursor cell population, and the left side shows genes with low expression levels in the NK precursor cell population. The horizontal axis shows the ratio of variation (FC), and the vertical axis shows statistical significance (P). Figure 4 shows the results of flow cytometry analysis of the expression of CD122 (IL-2 / IL-15 receptor β chain) or CD132 (IL-2 receptor common γ chain) in non-NK precursor cell populations and NK precursor cell populations. In the figure, the peaks on the left side show the results of analysis using an isotype control, and the peaks on the right side show the results of analysis using an anti-CD122 antibody or an anti-CD132 antibody. This figure shows the results of flow cytometry analysis of the expression of 4-1BBL, IL-15, or IL-21 in K562 cells (K562-4-1BBL-mbIL-21) forced to express 4-1BB ligand (4-1BBL) and membrane-bound IL-21 (mbIL-21), and in K562 cells (K562-4-1BBL-mbIL-15-mbIL-21) forced to express 4-1BBL, mbIL-21, and membrane-bound IL-15 (mbIL-15). In the figure, the peaks on the left side show the results of analysis using an isotype control, and the peaks on the right side show the results of analysis using an anti-4-1BBL antibody, an anti-IL-15 antibody, or an anti-IL-21 antibody. CD3 obtained by co-culturing CB MNC with feeder cells (K562-4-1BBL-mbIL-21 or K562-4-1BBL-mbIL-15-mbIL-21) － CD56 ＋ 1 is a graph showing the results of analyzing the number of NK cells. In the figure, the vertical axis represents the CD56 ＋ CD3 －The relative values ​​of NK cell counts on day 14 after the start of culture are shown. Results from five independent CB samples are summarized. Error bars represent standard error of the mean (s.e.m.). Asterisks indicate P<0.05.

[0027] <Natural Killer Progenitor Cells> In the present invention, "natural killer (NK) cells" refer to a type of hematopoietic lymphocyte that has the ability to lyse cells infected with viruses, tumor cells, etc. (cytotoxic activity) and acts as a major factor in innate immunity. "Mature NK cells" are cells that are at least CD56-positive and CD3-negative. Mature NK cells according to the present invention are preferably cells that are positive for at least one cell marker (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) selected from the group consisting of CD16, NKG2D, NKp46, NKp30, NKp80, NKG2A, CD7, CD161, IL2RB, IL7R, and c-KIT in addition to CD56. In the present invention, "positive" and "negative" refer to the presence or absence of expression of each cell marker, respectively, and can be determined by one skilled in the art, for example, by flow cytometry, as shown in the Examples described below.

[0028] In the present invention, "natural killer (NK) precursor cells" refer to cells that develop from hematopoietic stem / progenitor cells and can differentiate into NK cells, and are cells that are negative for at least CD56 and CD34. The NK precursor cells of the present invention are cells that are negative for at least one cell marker (e.g., 1, 2, 3, 4, or 5) selected from the group consisting of HLA-DR and lineage markers (Lin (CD3, CD14, CD19, etc.)) in addition to CD56 and CD34, more preferably Lin- and HLA-DR-negative cells, and even more preferably CD3, CD14, CD19, and HLA-DR-negative cells. Furthermore, the NK precursor cells of the present invention are preferably CD7-positive cells in addition to being negative for each of the above cell markers. Furthermore, it is preferable that the NK precursor cells of the present invention express an IL-15 receptor (particularly CD122).

[0029] The NK precursor cells of the present invention can be prepared by isolation from hematopoietic cells or tissues containing hematopoietic cells. Examples of "hematopoietic cells or tissues containing hematopoietic cells" derived from living organisms include umbilical cord blood, placenta, peripheral blood, and bone marrow. In vitro differentiation can also be achieved by inducing differentiation of stem cells into NK cells. Examples of "stem cells" that can differentiate into NK cells in vitro include hematopoietic stem / progenitor cells and pluripotent stem cells (ES cells, iPS cells, etc.). Stem cell differentiation into NK cells can be induced by, for example, the culture method described in E. Mrozek et al., Blood. April 1, 1996, Vol. 87, No. 7, pp. 2632-2640, or the culture method described in Ye Li et al., Cell Stem Cell, August 2, 2018, Vol. 23, No. 2, pp. 181-192. The differentiation induction system includes cells prepared by such culture or cells in the course of such culture.

[0030] In the present invention, the "isolation" of NK precursor cells is not particularly limited, and can be performed by a person skilled in the art using known techniques as appropriate. For example, as shown in the Examples below, NK precursor cells can be isolated by cell sorting (FACS, etc.) or magnetic bead cell separation using antibodies against the various cell markers described above, using the presence or absence of expression of the cell markers described above as an indicator.

[0031] Furthermore, the present invention makes it possible to concentrate NK precursor cells by isolating them from the environment in which they normally exist (e.g., in vivo). Therefore, the present invention also provides an isolated cell population primarily comprising NK precursor cells. Here, "primarily" means 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more. By subjecting a cell population enriched in NK precursor cells to the culture method described below, it becomes possible to efficiently obtain highly purified mature NK cells and the like. Furthermore, the cell population of the present invention may be in the form of a cell suspension, suspended in, for example, a physiological salt solution (phosphate-buffered saline, Krebs-Ringer buffer, Hank's balanced salt solution, etc.) or a cell culture medium (RPMI 1640 medium, Dulbecco's modified Eagle's medium (DMEM), αMEM, Ham's F12 medium, etc.). Furthermore, the sample may be in the form of a frozen sample by containing a cryoprotective reagent (dimethyl sulfoxide (DMSO), methylcellulose, dextran, serum albumin, trehalose, glycerol, etc.).

[0032] <Method for Producing Mature Natural Killer Cells> As shown in the Examples below, identification and isolation of the above-mentioned NK precursor cells enables efficient production of mature NK cells with high purity. Thus, the present invention also provides a method for producing mature NK cells, which includes a step of culturing the above-mentioned NK precursor cells of the present invention in the presence of feeder cells and / or cytokines.

[0033] The "feeder cells" to be cultured together with the NK precursor cells of the present invention are not particularly limited as long as they can provide an environment in which the NK precursor cells or NK cells can at least survive, and examples thereof include artificial antigen-presenting cells (aAPCs). The "aAPCs" of the present invention include not only artificially produced antigen-presenting cells that present at least one antigen that can be recognized by NK cells and that can induce a cellular immune response in the cells, but also cells that have been artificially modified to forcibly express immune-related molecules (the conventional concept of aAPCs). Examples of "immune-related molecules" include costimulatory molecules (4-1BB ligand (4-1BBL) and the like) and interleukins (e.g., membrane-bound interleukins such as mbIL-15 and mbIL-21). In the feeder cells of the present invention, it is preferable that a costimulatory molecule and a membrane-bound interleukin are expressed, it is more preferable that 4-1BBL and mbIL-15 and / or mbIL-21 are expressed, and it is even more preferable that 4-1BBL, mbIL-15, and mbIL-21 are expressed. Examples of "cells" that express immune-related molecules include lymphoblastoid cells such as K562 cells and EBV-transformed lymphoblastoid cell lines (e.g., 721.221 cells). More specifically, such modified K562 cells (K562-based feeder cells) include K562 cells that express membrane-bound interleukin and 4-1BBL, preferably K562 cells that express mbIL-15 and / or mbIL-21 and 4-1BBL, more preferably K562 cells that express mbIL-15, mbIL-21, and 4-1BBL. Furthermore, in the present invention, it is desirable to suppress the proliferation of feeder cells by irradiation (e.g., γ-ray irradiation) or mitomycin C treatment when co-cultured with the NK precursor cells of the present invention.

[0034] In the present invention, the "cytokine" to be added to the culture of NK precursor cells includes, from the viewpoint of inducing differentiation of NK precursor cells into mature NK cells and expanding the NK cells, at least one cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-21, IL-15, and IL-18, preferably IL-2. The concentration of the cytokine to be added in the culture of NK precursor cells is not particularly limited as long as the induction of differentiation and expansion are possible, and may be, for example, 5 to 50 IU / mL, preferably 10 to 30 IU / mL, and more preferably 20 IU / mL.

[0035] In the present invention, the culture of NK precursor cells in the presence of the above-mentioned feeder cells and cytokines is usually carried out using a medium supplemented with these. The "medium" to which such feeder cells and cytokines are added is not particularly limited as long as the NK precursor cells, NK cells, and feeder cells can at least survive, and can be prepared based on a known basal medium for culturing hematopoietic cells. Examples of known basal media include RPMI 1640 medium, DMEM, αMEM, Ham's F12 medium, Williams E medium, KSOM medium, Eagle's MEM, Glasgow MEM medium, Ham's medium, Fisher's medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc Option Improved Medium, IMDM medium, Medium 199 medium, and any mixture thereof. The medium of the present invention may be a serum-containing medium or a serum-free medium. The serum contained in the serum-containing medium is not particularly limited, and an example thereof is fetal bovine serum. Serum-free medium refers to a medium that does not contain unprepared or unpurified serum, and examples thereof include media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors). The medium of the present invention may also contain a serum substitute. Examples of serum substitutes include commercially available products such as Knockout Serum Replacer (KSR, manufactured by Invitrogen), Chemically-defined Lipid Concentrated (manufactured by Gibco), and Glutamax (manufactured by Gibco). Furthermore, the medium according to the present invention may contain amino acids (e.g., L-glutamine, non-essential amino acids), hormones (e.g., progesterone, β-estradiol), growth factors (e.g., insulin, transferrin, selenite, ITS), reducing agents (e.g., β-mercaptoethanol), antibiotics (e.g., penicillin, streptomycin), organic acids (e.g., pyruvic acid, sodium pyruvate, lactic acid), fatty acids (e.g., linoleic acid) or lipids, sugars, vitamins, cytokines other than those mentioned above, antioxidants, buffers (e.g., HEPES), inorganic salts, and the like.

[0036] In the present invention, other conditions for inducing differentiation of NK precursor cells into mature NK cells can be appropriately selected and adjusted by a person skilled in the art in accordance with known culture conditions for hematopoietic cells. For example, the culture temperature is not particularly limited, but is usually about 30 to 40°C, preferably about 37°C. ２ The concentration of oxygen is usually about 1 to 10%, preferably about 2 to 5%. The oxygen concentration is usually 1 to 10%. The culture period may be any period required for differentiation into mature NK cells, for example, 3 to 20 days, preferably 7 to 18 days, and more preferably 10 to 14 days. Furthermore, during this culture period, the above-mentioned medium, cytokines, and feeder cells may be replaced with new ones as appropriate.

[0037] Furthermore, whether the cells obtained by such culture are mature NK cells can be determined by a person skilled in the art by performing flow cytometry using the expression of the above-mentioned cell markers as an indicator, for example, as shown in the Examples described below.

[0038] <Chimeric Antigen Receptor> In the present invention, NK cells expressing a chimeric antigen receptor (CAR) (CAR-NK cells) can also be produced by introducing a nucleotide encoding a chimeric antigen receptor (CAR) during the production of the above-mentioned mature NK cells.

[0039] In the present invention, the term "chimeric antigen receptor (CAR)" refers to a chimeric protein in which a region that binds to a target antigen (described below), a transmembrane region, and an intracellular signal region are arranged in this order from the N-terminus and linked directly or indirectly.

[0040] In the present invention, the "region that binds to a target antigen (antigen-binding region)" can be, for example, an antibody against the target antigen or a functional fragment thereof, but typically, an scFv against the target antigen is used for CAR. An "scFv" is a structure in which the light chain variable region and heavy chain variable region of a monoclonal antibody (immunoglobulin) are linked via a linker, and retains antigen-binding ability.

[0041] In the present invention, the "transmembrane region" is not particularly limited as long as it is a polypeptide that has the function of penetrating a cell membrane and can anchor a CAR to the cell membrane. Examples of proteins from which polypeptides carrying a transmembrane region are derived include CD28, CD3ζ, CD3ε, 4-1BB, CD45, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, and the α chain and β chain of a T cell receptor.

[0042] In the present invention, the "intracellular signaling region" refers to a region that is located intracellularly when the CAR is located on the cell membrane. The intracellular signaling region is capable of transmitting a signal required for immune cell effector function, i.e., a signal required for immune cell activation (a so-called primary signal) when the antigen-binding region binds to a target antigen. Examples of such intracellular signaling regions include those that have only an activation signaling domain such as a T cell receptor (TCR) and a CD3 complex (first-generation CARs), those that have an activation signaling domain plus a costimulatory signaling domain derived from a costimulatory molecule (e.g., the intracellular domain of CD28 or 4-1BB) (second-generation CARs), and those that have an activation signaling domain plus multiple costimulatory signaling domains (e.g., the intracellular domains of CD28 and 4-1BB) (third-generation CARs). Other examples include those that contain a CD79A intracellular region and a CD40 intracellular region (see Mol Ther 2021 Sep 1;29(9):2677-2690).

[0043] As described above, the "intracellular signaling region" may be any region capable of transmitting a primary signal, and an activation signaling domain of a protein involved in the signal can be used. Primary signaling is known to involve immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of proteins having ITAMs include CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12.

[0044] The CAR of the present invention may comprise other domains in addition to the above-mentioned antigen-binding region, transmembrane region, and intracellular signal region. Examples of other domains include, but are not limited to, a costimulatory signaling region, a spacer sequence, a signal peptide, etc.

[0045] It is known that costimulatory molecules expressed on T cells transmit costimulatory signals intracellularly upon binding to ligands specific to each costimulatory molecule expressed on antigen-presenting cells, thereby assisting the activation of immune cells (secondary signaling). In the present invention, a "costimulatory transmission region" refers to an intracellular domain involved in the costimulatory signal transmission of the above-mentioned costimulatory molecules. Examples of costimulatory molecules include CD28, 4-1BB (CD137), OX40 (CD134), ICOS, CD2, CD4, CD5, CD8α, CD8β, CD154, and the like. The CAR of the present invention can comprise the costimulatory transmission region of these costimulatory molecules.

[0046] The spacer sequence is not particularly limited, and examples of the spacer that can be used include the hinge region or a part thereof of IgG, preferably human IgG (e.g., subtypes IgG1 and IgG4), the hinge region and a part of CH2, or a part of a factor used in a transmembrane domain (e.g., CD28).

[0047] In the present invention, the term "signal peptide" refers to a peptide that promotes the secretion of a CAR or directs its localization to the cell membrane, and is also referred to as a leader sequence. The signal peptide can usually be directly or indirectly bound to the N-terminus of the antigen-binding region. Optionally, the signal peptide may be cleaved from the antigen-binding region during cellular processing and localization of the CAR to the cell membrane. Examples of such signal peptides include signal peptides of immunoglobulins (such as IgK), human GM-CSF receptor, the α and β chains of T-cell receptor, CD8α, CD8β, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, and the like.

[0048] Examples of each region, etc. in the CAR of the present invention have been described above. However, those skilled in the art can appropriately obtain specific amino acid sequences related to these regions, etc. by searching known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). Furthermore, the regions, etc. of the present invention are not limited to such typical amino acid sequences (e.g., NCBI reference sequences), and may also include variants and homologs of these typical amino acid sequences, so long as the function of the region is maintained. Furthermore, such variants and homologs usually have high homology or identity to the typical amino acid sequence. High homology or identity is usually 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

[0049] There have been several reports of experiments and clinical studies using CAR (e.g., Rossig C, et al. Mol Ther 10:5-18, 2004; Dotti G, et al. Hum Gene Ther 20:1229-1239, 2009; Ngo MC, et al. Hum Mol Genet 20(R1):R93-99, 2011; Ahmed N, et al. Mol Ther 17:1779-1787, 2009; Pulé MA, et al. Nat Med 14:1264-1270, 2008; Louis CU, et al. Blood 118:6050-6056, 2011; Kochenderfer JN, et al. Blood 116:4099-4102, 2010; Kochenderfer JN, et al. Blood 119:2709-2720, 2012; Porter DL, et al. N Engl J Med365:725-733, 2011; Kalos M, et al. Sci Transl Med 3:95ra73, 2011; Brentjens RJ, et al. Blood 118:4817-4828, 2011; Brentjens RJ, et al. SciTransl Med 5:177 ra38, 2013), the CAR according to the present invention can be constructed by referring to these reports.

[0050] In the present invention, CAR-NK cells can be produced by introducing a nucleotide encoding the above-mentioned CAR. Such a nucleotide may be in the form of a vector, such as a viral vector, a plasmid vector, an episomal vector, an artificial chromosome vector, or a transposon vector.

[0051] Examples of "viral vectors" include retroviral vectors such as lentivirus, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors. Examples of "plasmid vectors" include plasmid vectors for expression in animal cells, such as pcDNA3.1, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. An "episomal vector" is a vector capable of autonomous replication outside of a chromosome. Specific means for using episomal vectors are known (see Yu et al., Science, 2009, pp. 324, 797-801). Examples of episomal vectors include vectors that contain sequences necessary for autonomous replication derived from EBV, SV40, etc., as vector elements. Specific examples of vector elements necessary for autonomous replication include a replication origin and a gene encoding a protein that binds to the replication origin and controls replication, such as the replication origin oriP and EBNA-1 gene in EBV, and the replication origin ori and SV40LT gene in SV40. Examples of "artificial chromosome vectors" include YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, and PAC (P1-derived artificial chromosome) vectors.

[0052] Of these vectors, retroviral vectors are preferred from the viewpoint that they can easily transfer genes efficiently even into cells that grow slowly, and that stable expression strains can be easily established.

[0053] The vector of the present invention may include, in addition to the above-described polynucleotide encoding a CAR, an expression control sequence such as a promoter, an enhancer, a poly A addition signal, or a terminator, a nucleotide sequence encoding a replication origin or a protein that binds to the replication origin to control replication, nucleotides encoding other proteins, and the like.

[0054] The polynucleotide encoding the above-mentioned CAR and nucleotides encoding other proteins described below can be operably positioned downstream of a promoter to enable efficient transcription of each polynucleotide. Examples of such "promoters" include a CMV (cytomegalovirus) promoter, an SRα promoter, an SV40 early promoter, a retroviral LTR, an Rous sarcoma virus (RSV) promoter, an HSV-TK (herpes simplex virus thymidine kinase) promoter, an EF1α promoter, a metallothionein promoter, and a heat shock promoter.

[0055] Examples of "nucleotides encoding other proteins" include nucleotides encoding interleukin (IL-15, etc.). Other examples include marker genes such as fluorescent protein genes, reporter genes, and drug resistance genes. Further examples include nucleotides encoding EGFR (truncate EGFR, tEGFR) that does not have an intracellular domain. Furthermore, by using DNA encoding an IRES or a 2A peptide sequence (e.g., 2A peptide (T2A) derived from Thosea asigna), the other protein can be polycistronically expressed together with the above-mentioned CAR.

[0056] Furthermore, in order to appropriately induce apoptosis of the administered CAR-NK cells in the body of a subject to which the CAR-NK cells have been administered, the vector of the present invention may contain a suicide gene as a "nucleotide encoding another protein." Examples of suicide genes include herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9 (iCasp9). Examples of drugs that activate the functions of such genes include ganciclovir for HSV-TK and AP1903, a chemical induction of dimerization (CID) compound for iCasp9 (Cooper LJ. et al., Cytotherapy. 2006; 8(2): pp. 105-17; Jensen M.C. et al., Biol Blood Marrow Transplant. 2010 Sep; 16(9): pp. 1245-56; Jones B.S., Front Pharmacol. 2014 Nov 27; 5: 254; Minagawa K., Pharmaceuticals (Basel). 2015 May 8;8(2):230-49; Bole-Richard E., Front Pharmacol. 2015 Aug 25;6:174).

[0057] Methods for introducing the nucleotides of the present invention into cells include lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun technology. Furthermore, when the nucleotides of the present invention are used in a retroviral vector, appropriate packaging cells may be selected based on the LTR sequence and packaging signal sequence contained in the vector, and these may be used to prepare retroviral particles. Examples of packaging cells include PG13, PA317, GP+E-86, GP+envAm-12, and Psi-Crip. 293 cells and 293T cells, which have high transfection efficiency, can also be used as packaging cells. The timing for introducing the nucleotides of the present invention into cells is not particularly limited, but is preferably 3 to 10 days, preferably 5 to 8 days, and more preferably 7 days, after the initiation of culture in the presence of the above-mentioned feeder cells and / or cytokines. Furthermore, the nucleotides of the present invention are typically introduced after the differentiation of NK precursor cells has been induced. Therefore, the introduction of nucleotides includes not only NK precursor cells but also cells derived therefrom. "Cells derived from NK precursor cells" include not only mature NK cells but also cells in the process of differentiating or maturing them. After introducing the nucleotide according to the present invention into cells, the expression of CAR in the cells can be confirmed by known methods such as flow cytometry, RT-PCR, Northern blotting, Western blotting, ELISA, and fluorescent immunostaining.

[0058] In the present invention, the "target antigen" is not particularly limited, and examples thereof include disease-specific biomolecules targeted by CAR-NK cells. For example, in the case of targeting cancer, examples of the target antigen include cancer antigens such as biomolecules that are specifically expressed in cancer cells or their precursor cells (cancer stem cells, etc.) (tumor-specific antigens: TSAs) and biomolecules whose expression is enhanced in cancer cells, etc. compared to normal cells (tumor-associated antigens: TAAs).

[0059] Examples of "cancer antigens" include CD19, HER2 (ERBB2, NEU), BCMA, CD5, CD7, CD10, CD13, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD41, CD44, CD44v6, CD52, CD74, CD123, CD133, CD138, CD160, CD171, CS-1, CLL-1, CA19-9, CA72-4, CA125, CAM17.1, CA15-3, CA195, and CA242. , CA-50, CAM43, c-Met, A33, EGFR, EGFRvIII, VEGFR2, FOLR, GD2, GD3, PSCA, PSMA, ROR1, FLT3, FAP, TAG72, CEA, EPCAM, B7H3, KI T, IL-13Ra2, mesothelin, vimentin, IL-11Ra, PRSS21, LewisY, CD24, CD68, CD79a, CD79b, CD72, CD97, CD179a, CO-029, FGF-5, G250, TE M1 / CD248, CD300LF, PDGFR-β, SSEA-4, folate receptor α, folate receptor β, MUC1, MUC16, NCAM, prostase, PAP, ELF2M, ephrinB2, IGF-IR, CAIX, LMP2, gp100, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, TEM7R, CL DN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY -ESO-1, LAGE-1a, MAGE-1, MAGE-3, MAGE-A1, MAGE-4, MAGE-5, MAGE-6, BAGE, GAGE-1, GAGE-2, RAGE, p15, p16, legumain, EBVA, HPV E6,E7, ETV6-AML, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, Ras, K-ras, Ras mutant, prostein, survivin, WT1, telomerase, hTERT, PCTA-1 / galectin-8, melan-A / MART1, ML-IAP, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-1, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RU1, RU2, muthsp70-2, LAIR1, FCAR, LILRA2, CLEC12A, BST2 , EMR2, LY75, GPC3, FCRL5, IGLL1, TSP-180, nm-23H1, NuMa, Mum-1, 43-9F, 5T4, 791Tgp72, HTgp- 175, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TLP, and TPS. ,

[0060] Target antigens also include antigens specific to infectious diseases. Such antigens may be substances that are specifically expressed in viruses or bacteria that cause infectious diseases, and examples thereof include HIV-specific antigens (such as HIV gp120), HBV-specific antigens, EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lassa virus-specific antigens, influenza virus-specific antigens, and fungal-specific antigens.

[0061] Further, target antigens also include antigens specific to inflammatory diseases, such as AOC3 (VAP-1), CAM-3001, CCL11, CD125, CD147, CD154 (CD40L), CD2, CD20, CD23, CD25, CD3, CD4, CD5, IFNα, IFNγ, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-6, IL-6 receptor, integrin a4, integrin α4β7, Lama glama, LFA-1 (CD1 la), MEDI-528, myostatin, OX-40, and rhuMAb. β7, scleroscin, SOST, TGFβ1, TNF-α, and VEGF-A.

[0062] <Kit for producing mature NK cells or NK cells expressing a CAR> The present invention provides a kit used for producing the above-described mature NK cells or NK cells expressing a CAR. The kit of the present invention may contain at least the above-described NK precursor cells and / or feeder cells of the present invention, but may also contain the above-described cytokines, medium, etc. Furthermore, when the kit is used for producing NK cells expressing a CAR, it may further contain nucleotides encoding the above-described CAR.

[0063] Furthermore, the kit of the present invention can include, in addition to components for producing mature NK cells, etc., reagents for confirming that the produced cells are mature NK cells, etc. Examples of such reagents include antibodies that recognize the above-mentioned cell markers, etc., and labeled secondary antibodies that recognize the antibodies. Furthermore, the kit of the present invention can include instructions for using the kit.

[0064] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. The reference numbers cited below in the description of these examples correspond to the reference numbers shown in the <References> section below. The examples were carried out using the materials and methods shown below.

[0065] <Materials and Methods> (Cells) After obtaining informed consent from the donors, umbilical cord blood (CB) cells were obtained from the Kinki Cord Blood Cell Bank and the Hyogo Prefectural Cord Blood Bank. This experiment was approved by the Institutional Review Boards of the Osaka University Graduate School of Medicine, the Kinki Cord Blood Association, and the Hyogo Prefectural Cord Blood Association. Raji cells were purchased from the JCRB Cell Bank. K562 cells expressing membrane-bound IL-15 and 4-1BBL (K562-mb15-41BBL cells, see Reference 10) were kindly provided by St. Jude Children's Research Hospital.

[0066] (Retrovirus Production) The pEQ-PAM3(-E) and pRDF plasmids were kindly provided by Toshio Kitamura (University of Tokyo) and Keiichiro Mihara (Fujita Health University), respectively. The cDNAs encoding the κ light chain variable region and heavy chain variable region of the anti-CD19 monoclonal antibody FMC63 (Reference 25) fused with CD28, CD3ζ, and T2A-IL-15 cDNAs were inserted into a retroviral vector. To prepare viral supernatants, the retroviral vectors, pEQ-PAM3(-E), and pRDF were cotransfected into 293T cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). The supernatants containing the retroviruses were then collected 48 and 72 hours later.

[0067] (Generation of CAR-NK cells) T cells were depleted from CB mononuclear cells (MNC) using CD3 microbeads (Miltenyi Biotec). T cell-depleted CB MNC (3.75 × 10 ５ / mL) was added to 100 Gy-irradiated K562-mb15-41BBL cells (2.5 × 10 ５The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 20 IU / mL IL-2, along with CD19 CAR-T2A-IL-15 (10 IU / mL). One week later, retrovirus carrying the CD19 CAR-T2A-IL-15 cDNA was transduced into CB-derived NK cells using RetroNectin (Takara Bio). Subsequently, the cells were stimulated again with 100 Gy-irradiated K562-mb15-41BBL cells, cultured for another week, and used in further experiments.

[0068] (Preparation of Feeder Cells) 4-1BBL cDNA (SEQ ID NO: 1) was synthesized and inserted into a retroviral vector using an InFusion cloning kit (Takara Bio Inc.) to prepare a 4-1BBL expression vector. SEQ ID NO: 2 shows the amino acid sequence encoded by the 4-1BBL cDNA.

[0069] A membrane-bound IL-21 cDNA (SEQ ID NO: 3) was synthesized by fusing cDNAs for IL-21, an IgG4 heavy chain constant region sequence, and a CD4 transmembrane region, and inserted into a retroviral vector using an InFusion cloning kit (Takara Bio Inc.) to prepare a membrane-bound IL-21 expression vector. SEQ ID NO: 4 shows the amino acid sequence encoded by the membrane-bound IL-21 cDNA. In this amino acid sequence, the sequence consisting of amino acids 1 to 17 represents a leader sequence, the sequence consisting of amino acids 18 to 150 represents IL-21 (positions 30 to 162), the sequence consisting of amino acids 151 to 379 represents the IgG4 heavy chain constant region sequence, and the sequence consisting of amino acids 380 to 401 represents the CD4 transmembrane region.

[0070] A membrane-bound IL-15 cDNA (SEQ ID NO: 5) was synthesized by fusing the cDNAs for the CD8α leader sequence, IL-15, and CD8α hinge / transmembrane region, and inserted into a retroviral vector using an InFusion cloning kit (Takara Bio Inc.) to prepare a membrane-bound IL-15 expression vector. SEQ ID NO: 6 shows the amino acid sequence encoded by the membrane-bound IL-15 cDNA. In this amino acid sequence, the sequence consisting of amino acids 1 to 21 represents the CD8α leader sequence, the sequence consisting of amino acids 22 to 135 represents IL-15, and the sequence consisting of amino acids 136 to 204 represents the CD8α hinge / transmembrane region.

[0071] Each retroviral vector DNA prepared in this manner was introduced into 293-derived producer cells using Lipofectamine 2000 (Thermo Fisher). After 48 hours of culture, the culture supernatant was collected and filtered to recover the retrovirus. K562 cells (CCL-243, ATCC) were then suspended in RPMI 1640 + 10% FBS medium supplemented with the collected retrovirus, and the cells were centrifuged at 32°C, 2000 x g, and 90 minutes to introduce each gene into the cells.

[0072] After introduction of 4-1BBL, the cells were expanded and stained with anti-4-1BBL antibody, and positive cells were single-cell sorted using a cell sorter (FACS ARIA II (Becton Dickinson)). After expansion, membrane-bound IL-21 was introduced into the 4-1BBL-expressing K562 cells, and after expansion, the cells were stained with anti-IL-21 antibody and single-cell sorted using a cell sorter, thereby preparing feeder cells in which 4-1BBL and membrane-bound IL-21 (mbIL-21) were forcibly expressed in K562 cells.

[0073] Membrane-bound IL-15 was further introduced into feeder cells in which 4-1BBL and mbIL-21 were forcedly expressed in K562 cells, and the cells were expanded. After that, the cells were stained with anti-IL-15 antibody and positive cells were single-cell sorted using a cell sorter to prepare feeder cells in which K562 cells expressed membrane-bound IL-15 (mbIL-15) in addition to 4-1BB ligand and mbIL-21.

[0074] (Flow cytometry and cell sorting) Single cell suspensions were stained with fluorochrome-conjugated monoclonal antibodies. The following antibodies were used for staining: anti-human CD56-PE (HCD56, BioLegend); anti-human CD3-PE Cy7, APC, FITC, or APC Cy7 (UCHT1, BioLegend); anti-human CD19-APC Cy7 or APC (HIB19, BioLegend); anti-human CD14-FITC, APC, or PE-Cy7 (M5E2, BioLegend); anti-human HLA-DR-APC (L243, BioLegend); anti-human CD34-BV421 (561, BioLegend); anti-human CD34-APC (8G12, BD Pharmigen); anti-human CD7-FITC (CD7-6B7, BioLegend); anti-human CD16-APC Cy7 (3G8, BioLegend); anti-human NKG2D-PE Cy7 (1D11, BioLegend); anti-human NKp46-PE Cy7 (9E2, BioLegend); anti-human NKp30-BV785 (P30-15, BioLegend); anti-human NKp80-APC (5D12, BioLegend); anti-human NKG2A-PE Cy7 (S19004C, BioLegend); anti-human CD161-BV785 (HP-3G10, BioLegend); anti-human CD122-BV421 (TU27, BioLegend); anti-human CD127-FITC (A019D5, BioLegend); anti-human CD117-APC Cy7 (104D2, BioLegend); anti-human CD20-APC (L27, BD Pharmigen); anti-CD19CARFMC63 idiotype antibody (Miltenyi anti-IL-15-PE (34559, R&D); anti-IL-21-Alexa Fluor 647 (3A3-N2, BioLegend); anti-4-1BBL antibody (5F4, BioLegend).Flow cytometry and cell sorting were performed using BD FACS Canto II or FACS Aria II (BD Biosciences), and analysis was performed using FlowJo software (BD Biosciences).

[0075] Cytotoxicity Assay The ability of NK cells to lyse target cells is determined by: ５１ Cr release assay. ５ Target cells were treated with 25 μCi of [ ５１ The cells were labeled with sodium chromate (PerkinElmer) at 37°C for 1.5 hours. The labeled target cells (1 x 10 ４ ) was incubated with effector cells for 4 hours. ５１ Cr release was measured in a gamma counter. ５１ The total and spontaneous Cr release was measured in 1% Triton X-100 or medium at 1 × 10 ４ The percentage of specific lysis was determined by incubating 1000 labeled target cells with 1000 labeled target cells. ５１ Specific release of Cr- ５１ Spontaneous release of Cr] / [ ５１ Total amount of Cr released - ５１ The amount of spontaneously released Cr was calculated as follows: [amount of spontaneously released Cr] × 100.

[0076] Single-cell RNA sequencing (RNA-seq) analysis: CB MNCs from three independent donors were stained for CD14, CD19, CD3, CD56, and one of three different TotalSeq-C anti-human hashtag antibodies (LNH-94; 2M2, Barcoded, BioLegend, 394661, 394663, and 394665). － CD3 － CD14 － CD19 －MNCs were sorted and analyzed by fluorescence-activated cell sorting (FACS). Single-cell RNA-seq libraries were constructed using the Chromium Next GEM Single Cell 5' Library and Gel Bead Kit v2 (10x Genomics). Libraries were sequenced in 28+90 base paired-end mode on a NovaSeq 6000 platform to obtain a minimum of 20,000 reads per cell for gene expression. Single-cell RNA-seq datasets were processed, explored, and visualized using the Cellenics® community instance (https: / / scp.bioimage.net / ), hosted by Bioimage (https: / / biomage.net / ).

[0077] The data referred to in this example are stored in the Gene Expression Omnibus of the National Center for Biotechnology Information (NCBI) (Reference 26) and are accessible via GEO series accession number GSE253575 (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE253575).

[0078] (RNA-seq analysis) RNA from each cell population separated by sorting was extracted using Trizol reagent (Thermo Fisher Scientific) and RNeasy Mini Kit (QIAGEN). Full-length cDNA was generated using the SMART-Seq HT Kit (Takara Bio). Each library was prepared using the Nextera XT DNA Library Prep Kit (Illumina). Whole-transcriptome sequencing was performed on the RNA samples in 100-base single-end mode using the Illumina HiSeq 3000 platform (Illumina). Sequencing reads were analyzed using TopHat v2.0.13 with Bowtie2 ver. 2.2.3 and SAMtools ver. The fragments were mapped to the human reference genome sequence (hg19) using Cufflinks ver. 0.1.19 in combination with the genomics library. The number of fragments per kilobase of exon per million mapped fragments was calculated using Cufflinks ver. 2.2.1.

[0079] Statistical Analysis: Unpaired two-tailed Student's t-test was used to determine statistically significant differences between samples. P<0.05 was considered to indicate a significant difference.

[0080] The results obtained using the above materials and methods are shown below.

[0081] (CAR-NK cells are primarily derived from human CB CD56 － CAR-NK cells, which target CD19 and secrete IL-15, were established using a previously reported method (References 7 and 10) with some modifications (Fig. 1A). After 2 weeks of culture, CD3 － CD56 ＋ The number of NK cells increased 78.7±17.7-fold (FIG. 1B), and it was confirmed that the CD19 CAR was efficiently transduced into NK cells (FIG. 1C).

[0082] Next, CD3 － CD56 ＋ cells or CD3 － CD56 －The cells were purified by FACS and cultured separately for two weeks to attempt to generate (or expand) NK cells (Figure 1D). － CD56 ＋ Not only cells, but also CD3 － CD56 － The production of NK cells was also observed from the cells (Fig. 1D). More specifically, after 2 weeks of culture in the CB sample, 1 × 10 ５ CD3 － CD56 ＋ cells or CD3 － CD56 － From cells, 3.9 × 10 ６ or 2.4 x 10 ６ CD56 ＋ CD3 － The production of NK cells was observed. ＋ CD3 － Cells and CD56 － CD3 － The abundance ratios of cells were 4.3% and 54.3%, respectively. ５ (=3.9 x 10 ６ × 0.043) CD56 ＋ NK cells derived from cells and 1.3 × 10 ６ (=2.4 x 10 ６ × 0.543) CD56 － NK cells derived from the cells were 1.0 × 10 ５ It was suggested that CB can be produced from MNCs (Fig. 1D).

[0083] In addition, analysis using other CB samples showed that CD3 － CD56 － The number of CAR-NK cells produced from the cells was determined by CD3 － CD56 ＋ The number of cells produced from CD56 cells was significantly higher than that from CD56 cells (n=5, Fig. 1E). ＋ Cell-derived NK cells and CD56 － Both cell-derived NK cells and Raji cells (CD19 ＋ When co-cultured with CD56 cells, the cells showed significant cytotoxicity (Fig. 1F). －The expression levels of receptor molecules such as CD16, NKG2D, NKp46, NKp30, NKp80, and NKG2A in the cell-derived CAR-NK cells were comparable to those in the CD56+ cell-derived CAR-NK cells (Figure 1G).

[0084] (CB-derived NK cells are mainly CD7 ＋ CD34 － HLA － Dr. － CD56 － Lineage marker (Lin) － CD56 derived from CD40 cells ＋ CD3 － CD56 may generate NK cells － CD3 － CD14 － CD19 － Single-cell RNA-seq analysis of CB mononuclear cells (Figure 2A) revealed that they were subdivided into five subpopulations (Figures 2B and 2C). ＋ The second and third subpopulations are enriched for cells expressing hematopoietic stem and progenitor cells, likely dendritic cells, that show high expression of HLA-DR but no expression of FCGR3A. The fourth subpopulation is enriched for cells expressing HLA-DR. ＋ FCGR3A ＋ The fifth subpopulation is enriched in cells that do not express either CD34 or HLA-DR. This subpopulation contains cells that express killer cell lectin-like receptor B1 (KLRB1), IL-2RB, or killer cell lectin-like receptor K1 (KLRK1), suggesting that it is enriched for cells committed to the NK cell lineage (Reference 23).

[0085] Consistent with the results of single-cell RNA-seq analysis, CD56 － CD3 － CD14 － CD19 － Flow cytometry analysis of CB MNCs revealed that CD34 － HLA-DR ＋ , CD34 ＋ HLA-DR ＋／－and CD34 － HLA-DR － These three populations were then purified and cultured separately to generate NK cells (Fig. 2D). After 14 days of culture, NK cells were almost exclusively CD34 － HLA-DR － It was revealed that CD3 is produced from CD4+ cells (Fig. 2D, 2E). － CD14 － CD19 － " Regarding " Lineage Marker (Lin) － " is also called.

[0086] As a result of analyzing the expression of several cell surface antigens that have been reported to be expressed on NK cells or NK precursor cells (Reference 23), the expression of CD7 was used as an index, and CD34 － HLA-DR － CD56 － Lin － The cells could be further subdivided into two distinct subpopulations (Figure 2F). These were then cultured separately to attempt to generate NK cells. As a result, NK cells were CD7 ＋ It was produced from CD7 cells. － It was not produced by the cells (Fig. 2G, 2H).

[0087] To summarize the above results, CD7 ＋ CD34 － HLA － Dr. － CD56 － Lin － These cells have been shown to be the major source of NK cells.

[0088] (IL-15 receptors are highly expressed on NK precursor cells) CB MNC to CD56 － CD7 － CD34 － HLA-DR － Lin (CD3 / CD14 / CD19) － cells (non-NK progenitor population) and CD56 － CD7 ＋ CD34 －HLA-DR － Lin (CD3 / CD14 / CD19) － The NK progenitor cell population was sorted using a cell sorter and subjected to RNA-seq analysis (Figures 3A and 3B). The results revealed that CD122 (IL-2 / IL-15 receptor β chain) and CD132 (IL-2 receptor common γ chain), which constitute IL-15 receptors, were highly expressed in the NK progenitor cell population (Figure 3C).

[0089] Furthermore, we analyzed the expression of CD122 and CD132 in each cell population using flow cytometry. As a result, CD132 expression was observed in both cell populations. However, CD122 expression was observed in all cells of the NK precursor cell population, whereas no expression was observed in the non-NK precursor cell population (Fig. 3D).

[0090] The IL-15 receptor is a heterodimer of IL2RG (IL-2 receptor common γ chain) and IL2RB (IL-2 / IL-15 receptor β chain). ＋ It is clear that it is expressed only in the NK precursor cell population.

[0091] (Feeder cells expressing mbIL-15 in addition to mbIL-21 efficiently induce the production of NK cells from umbilical cord blood NK precursor cells.) Based on the above results, we hypothesized that stimulating the IL-15 receptor expressed in the NK precursor cell population would result in the production of more mature NK cells. Therefore, we prepared feeder cells in which 4-1BB ligand and mbIL-21 were overexpressed in K562 cells, and feeder cells in which mbIL-15 was further transfected and expressed, and compared their NK cell induction abilities (Figure 4A). CB MNCs and irradiated feeder cells were co-cultured in medium supplemented with IL-2. The CD56 receptor expression increased 14 days after the start of culture compared to the initial level. ＋ CD3 － The number of NK cells increased by fold was compared.

[0092] As a result, the use of feeders expressing mbIL-15 in addition to 4-1BB / mbIL-21 induced significantly more NK cells (Fig. 4B), suggesting that K562-4-1BBL-mbIL-15-mbIL-21 has the ability to induce more CB-derived NK cells.

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[0094] As described above, the present invention provides NK precursor cells. Furthermore, mature NK cells or CAR-NK cells can be produced using the precursor cells. Therefore, the present invention is useful in the medical field for treating cancer, infectious diseases, and the like.

Claims

1. Isolated natural killer (NK) precursor cells that are CD56 and CD34 negative.

2. The NK precursor cells according to claim 1, which are further negative for HLA-DR and Lin and positive for CD7.

3. An isolated cell population comprising NK precursor cells, wherein 60% or more of the total cells in the population are the NK precursor cells according to claim 1 or 2.

4. A method for producing NK cells, comprising the step of culturing the NK precursor cells according to claim 1 in the presence of feeder cells and / or cytokines, wherein the NK cells are mature NK cells that are positive for CD56, and the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-21, IL-15, and IL-18.

5. A method for producing NK cells expressing a chimeric antigen receptor (CAR), comprising the step of culturing the NK precursor cells or cells derived therefrom according to claim 1, further comprising introducing a nucleotide encoding the CAR into the NK precursor cells or cells derived therefrom according to claim 4.

6. The method of claim 4 or 5, wherein the cytokine is IL-2.

7. The production method according to claim 4 or 5, wherein the feeder cells are K562 cells that express membrane-bound IL-15 and / or membrane-bound IL-21 and 4-1BBL.

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