Natural killer cells with regulated expression of anti-cancer related genes and uses thereof

Abstract

The present invention relates to a method for culturing highly pure and highly active natural killer cells in which the expression of anti-cancer-related genes is regulated, and to the use of the same in anti-cancer immunotherapy. According to one embodiment, the natural killer cells exhibit a significant increase or decrease in the expression of anti-cancer-related genes before or after culture, and exhibit significantly higher activity and proliferation rates while retaining the inherent properties of natural killer cells, enabling effective anti-cancer immunotherapy.

Description

[Technical field]

[0001] This application claims priority to Korean Patent Application No. 10-2020-0134623, filed on October 16, 2020, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a method for culturing highly pure and highly active natural killer cells in which the expression of anti-cancer-related genes is regulated, and to the use of the same in anti-cancer immunotherapy. [Background technology]

[0003] It is currently the anti-cancer immune cell therapy and T cell therapy that is the most actively undergoing clinical research worldwide. T cells are the most abundant immune cells and are the main immune cells with high anti-cancer immune function, but when allogenic T cells provided by another person are used, they can induce severe graft-versus host disease (GvHD) due to major histocompatibility complex (MHC) restrictions.

[0004] Meanwhile, natural killer cells (also called NK cells) are cells that morphologically have large granules in their cytoplasm, and account for approximately 5-15% of lymphocytes in the blood. Natural killer cells are immune cells that represent the primary defense mechanism (natural immune response) in the body, and compared to T cell-based therapies that form immune memory in the body and remain for a long time, they have fewer side effects such as cytokine storm, do not induce graft-versus-host disease, can be used as allogeneic cell therapy, can be developed as an off-the-shelf form of therapy, and can also induce antibody-dependent cell-mediated cytotoxicity (ADCC). Summary of the Invention [Problem to be solved by the invention]

[0005] However, despite the therapeutic benefits and safety of natural killer cells themselves, natural killer cells are known to be difficult to culture, unlike T cells, and in particular, for allogeneic use, highly pure natural killer cells must be cultured without contamination by T cells, but there is a limitation in that mass proliferation is difficult. Recently, a method for mass culturing natural killer cells using feeder cells has been developed, but when applied to therapeutic drugs, the feeder cells may be contaminated into the final product, causing safety issues. [Means for solving the problem]

[0006] The present inventors were developing a method for culturing highly pure and highly active natural killer cells, and confirmed that when CD3 negative and CD56 positive natural killer cells were extracted using leukapheresis and a closed system, and then cultured using IL-2, anti-NKp46 antibody, and donor plasma without using a feeder, the cells had higher proliferation ability and killing ability than conventional methods for producing natural killer cells, and highly pure natural killer cells could be produced. This led to the completion of the present invention.

[0007] One embodiment includes the steps of: 1) isolating monocytic cells from human peripheral blood via leukapheresis; 2) obtaining CD3-CD56+ natural killer cells from the isolated monocytic cells; 3) to provide a method for culturing natural killer cells, the method comprising the steps of treating the obtained CD3-CD56+ natural killer cells with IL-2, an anti-NKp46 antibody, and plasma, and culturing the cells in a culture medium that does not contain feeder cells.

[0008] Another aspect is to provide a natural killer cell or a population of cells cultured by said method.

[0009] Another embodiment is a composition for natural killer cell culture comprising IL-2, an anti-NKp46 antibody, and plasma, The composition is characterized in that it does not contain feeder cells.

[0010] Another aspect is to provide a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, natural killer cells cultured by the above method or a population of such cells.

[0011] Another aspect provides a method for preventing or treating cancer comprising the step of administering to an individual the pharmaceutical composition.

[0012] Another aspect provides the use of said composition for natural killer cell culture for the manufacture of a medicament for the prevention or treatment of cancer.

[0013] One embodiment includes the steps of: 1) isolating monocytic cells from human peripheral blood via leukapheresis; 2) obtaining CD3-CD56+ natural killer cells from the isolated monocytic cells; 3) treating the obtained CD3-CD56+ natural killer cells with IL-2, an anti-NKp46 antibody, and plasma, and culturing the cells in a culture medium that does not contain feeder cells.

[0014] One aspect is to provide a natural killer cell or a population of cells cultured by said method.

[0015] Unlike other known natural killer cell culture methods, the natural killer cells cultured by the natural killer cell culture method of the present specification exhibit a high natural killer cell purity of 99% or more, a proliferation rate of 300 to 350 times, and high cancer cell killing ability, and the expression of anti-cancer-related genes is significantly increased or decreased before and after culture.

[0016] The term "natural killer cells" or "NK cells" as used herein refers to cytotoxic lymphocytes that constitute a major component of the innate immune system, and are defined as large granular lymphocytes (LGLs), which constitute a third type of cells differentiated from common lymphoid progenitor (CLP)-producing B and T lymphocytes. The term "natural killer cells" or "NK cells" includes natural killer cells without additional modification derived from any tissue source, and may include not only mature natural killer cells, but also cellular precursors of natural killer cells. The natural killer cells are activated in response to interferon or macrophage-derived cytokines, and natural killer cells contain two types of surface receptors that control the cytotoxic activity of the cells, labeled with "activating receptors" and "inhibitory receptors".

[0017] The natural killer cells may be commercially purchased or may be obtained from a human or animal source, preferably from a human in need of natural killer cell therapy.

[0018] Natural killer cells can be produced from any source, such as hematopoietic cells from placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc., such as hematopoietic stem or precursor cells, placenta- or umbilical cord-derived stem cells, induced pluripotent stem cells, or cells differentiated therefrom. Furthermore, the natural killer cells can be supplied from any tissue source in vivo. For example, the natural killer cells can be contained in blood collected from a living body. The blood can be used as a source of natural killer cells for application to the culture method of the present specification, as long as it contains natural killer cells, and can be, for example, whole blood, umbilical cord blood, bone marrow, or peripheral blood.

[0019] The natural killer cells can be derived, for example, from peripheral blood mononuclear cells. The peripheral blood mononuclear cells can be obtained from the individual in need of treatment, i.e., autologous peripheral blood mononuclear cells, or can be allogenic. Thus, the natural killer cells can be autologous or allogenic natural killer cells.

[0020] The term "peripheral blood mononuclear cells (PBMCs)" as used herein refers to mononuclear cells isolated from peripheral blood of a mammal, preferably a human, and mainly includes immune cells such as B cells, T cells, and natural killer cells, and granulocytes such as basophils, eosinophils, and neutrophils. The PBMCs can be isolated from peripheral blood collected from a living body using leukapheresis or a density gradient centrifugation method using Ficoll, preferably using leukapheresis, but is not limited thereto.

[0021] The term "leukapheresis" as used herein refers to a method of selectively separating leukocytes from collected blood, and then returning the remaining components to the donor's blood, which has the advantage of being able to obtain a large amount of mononuclear leukocytes. Leukocytes separated by the above method can be used in the present invention without a separate method such as the Ficoll-Hypaque density gradient method. Methods and devices for performing leukapheresis are known in the art. Examples of leukapheresis devices include the COBESpectra manufactured by GAMBRO BCT and the CS3000 Plus Blood Cell Separator manufactured by Baxter Fenwal.

[0022] In one embodiment, the PBMCs herein may be isolated by performing leukapheresis. In one aspect, the culture method separates monocytes through leukapheresis, and then obtains and cultures CD3-CD56+ natural killer cells therefrom, thereby allowing the culture of natural killer cells at a higher concentration than when natural killer cells isolated from whole blood are cultured. Thus, the leukapheresis may affect the purity, activity, and expression profile of anti-cancer-related genes of natural killer cells.

[0023] The term "closed system" as used herein refers to any closed system that reduces the risk of contamination in cell culture during the culture process, such as the introduction of new substances, and during the cell culture steps, such as cell proliferation, differentiation, activation and / or isolation. Such a system works under GMP or GMP-like conditions ("sterile") and provides clinically applicable cell compositions. As an example of a closed system, CliniMACS Prodigy® (Mailenyi Biotec GmbH, Germany) is used herein. This system is disclosed in WO2009 / 072003. However, the use of the method of the present invention is not limited to CliniMACS Prodigy®.

[0024] In one embodiment, the CD3-CD56+ natural killer cells herein may be obtained using the CliniMACS Prodigy® system on PBMCs isolated by leukapheresis.

[0025] The term "positive or +" herein can mean, with respect to a cell label, that the label is present in greater amounts or at a higher concentration when compared to other cells in a reference. That is, a cell is positive for a label if the label is present inside or on the cell and can be used to distinguish the cell from one or more other cell types. It can also mean that the cell has enough of the label to produce a signal, e.g., a signal from a cell measurement device, at a value greater than background. For example, a cell can be detectably labeled with an NKp44-specific antibody, and if the signal from the antibody is greater than a control group (e.g., background value) and detectable, the cell is "NKp44+". The term "negative or -" herein means that the label cannot be detected using an antibody specific for a particular cell surface label compared to background values.

[0026] In one embodiment, the natural killer cells cultured according to the method may be natural killer cells in which one or more activities selected from the group consisting of immune activation receptors, apoptosis inducers (cancer cells), anti-apoptosis inducers (natural killer cells), chemokines, chemokine receptors, immune enhancing factors, and anti-cancer cofactors are increased, and one or more activities selected from the group consisting of immune inactivation receptors, immune suppressing factors, and apoptosis inducers (natural killer cells) are decreased. As used herein, the term "anti-cancer-related gene" refers to a gene encoding the anti-cancer-related factors listed above.

[0027] In one embodiment, the natural killer cells may refer to cells in which the cytotoxicity or inherent immunoregulatory ability of natural killer cells is activated or the expression of anti-cancer-related factors as described above is regulated, as compared with parent cells, such as hematopoietic cells, or cell precursors of natural killer cells. In a specific embodiment, the natural killer cells are CD3-CD56+.

[0028] In other embodiments, the expression of the immune activating receptor may be expression of one or more selected from the group consisting of NKG2D, NKp30, NKp44, and NKp46.

[0029] In other embodiments, the expression of the immune inactivation receptor may be expression of one or more selected from the group consisting of KIR3DL2 and Siglec9.

[0030] In another embodiment, the expression of the apoptosis inducer may be the expression of FASL and / or TRAIL (TNFSF10).

[0031] In another embodiment, the expression of the apoptosis inducer may be the expression of one or more selected from the group consisting of Granzyme A, Granzyme K, and Perforin.

[0032] In other embodiments, the chemokine and / or chemokine receptor expression may be expression of CCL1 and / or CCR5.

[0033] In another embodiment, the anti-cancer cofactor may be expression of DAP10 (HCST) and / or IL-2RA.

[0034] In another embodiment, the immune enhancing factor may be expression of GM-CSF. In another embodiment, the immunosuppressive factor may be expression of IL-8 (CXCL8) and / or IL-10.

[0035] In another embodiment, the apoptosis inducer (natural killer cells) may be expression of Noxa.

[0036] In another embodiment, the anti-apoptosis inducer (natural killer cells) may be expression of BCL-2.

[0037] The natural killer cells cultured by the culture method according to one embodiment may have one or more properties selected from the following (a) to (e) compared to the natural killer cells before culture.

[0038] (a) increased expression of one or more selected from the group consisting of NKG2D, NKp30, NKp44, and NKp46; (b) decreased expression of one or more selected from the group consisting of KIR3DL2, and Siglec9; (c) increased expression of one or more selected from the group consisting of FASL and TRAIL (TNFSF10); (d) increased expression of at least one protein selected from the group consisting of Granzyme A, Granzyme K, and Perforin; and (e) increased expression of one or more selected from the group consisting of CCL1 and CCR5.

[0039] Furthermore, the natural killer cells cultured by the above method may further have one or more properties selected from the following (f) to (j) compared to the natural killer cells before culture.

[0040] (f) increased expression of one or more selected from the group consisting of DAP10 (HCST), and IL-2RA; (g) Increased expression of GM-CSF (CSF2), (h) decreased expression of one or more selected from the group consisting of IL-8 (CXCL8) and IL-10; (i) decreased expression of Noxa (PMAIP1), and (j) Increased expression of BCL-2.

[0041] In one embodiment, the natural killer cells cultured by the method may be characterized by a significant increase in expression of activating receptors and a significant decrease in expression of inactivating receptors, as well as increased purity, cell proliferation fold, and cancer cell killing ability compared to the natural killer cells before culture.

[0042] As used herein, the term "activation" or "activating receptor" refers to a molecule on the surface of a natural killer cell that, upon stimulation, causes a measurable increase in any property or activity known in the relevant art to be associated with natural killer cell activity, such as cytokine (e.g., IFN-γ and TNF-α) production, an increase in intracellular free calcium, the ability to lyse target cells, or the ability to stimulate proliferation of natural killer cells.

[0043] Examples of the natural killer cell activating receptor include, but are not limited to, NKG2D, NKp30, NKp44, NKp46, IL-12R, IL-15R, IL-18R, and IL-21R.

[0044] Natural killer cell-activating receptors recognize ligands whose expression increases primarily when target cells are in an abnormal state, and induce cytotoxicity to eliminate the target cells.

[0045] NKG2D mediates cytotoxic activity by detecting UL16 binding proteins (ULBPs), MICA / B, RAE1, H60, and MULT1, which are intracellular molecules whose expression is increased during DNA damage, cancer development, and viral infection.

[0046] NKp30 is a receptor that is activated by the binding of extracellular ligands, including BAG6, NCR3LG1, and B7-H6, and stimulates cytotoxicity upon binding to these ligands.

[0047] NKp44 recognizes cell surface glycoproteins and proteoglycans, nuclear proteins that may be exposed outside the cell, and molecules that are released into the extracellular space or transported into extracellular vesicles as ligands. Recently, it has been reported that NKp44 recognizes soluble plasma proteins such as extracellular matrix (ECM)-derived glycoproteins or growth factors (e.g., PDGF-DD) (Cell. 2018 January 25; 172(3): 534-548.e19., Front Immunol. 2019; 10: 719.).

[0048] The term "inactivation" or "inactivating receptor" as used herein refers to any property well known in the relevant art associated with natural killer cell activity upon stimulation, such as cytokine (e.g., IFN-γ and TNF-α) production, an increase in intracellular free calcium, the ability to, for example, lyse target cells, or a molecule on the surface of a natural killer cell that measurably reduces activity.

[0049] Examples of natural killer cell inactivating receptors include, but are not limited to, KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E, Sigleg9 (sialic acid-binding Ig-like lectin 9), or ILRB5.

[0050] FASL and TRAIL are factors involved in the cytotoxicity and immune activation of natural killer cells, and are representative proteins that bind to cell surface receptors to induce apoptosis of target cells.

[0051] In one embodiment, natural killer cells have a significant increase in NKp44 and NKp30 expression of 1064-fold and 5.2-fold, respectively, a significant decrease in KIR3DL2 and Siglec9 expression, and a significant increase in FAS-L (FASLG) and TRAIL (TNFSF10) expression of at least 6.1-fold to at most 24.5-fold, and are expected to be useful as an efficient cell therapy drug with high anti-cancer effects.

[0052] T cells are mainly divided into CD8 positive cytotoxic T cells (Tc) and CD4 positive helper T cells (Th), and the cytokines secreted by the Th cells are divided into Th1 and Th2 types. Th1 cells are mainly involved in intracellular defense by phagocytosis, and mainly produce cytokines such as IL-2, IL-12, INF-γ, or TNF-α.

[0053] On the other hand, Th2 cells mainly produce cytokines such as IL-4, IL-6, IL-8, and TGF-β, suppressing the activity of T cells while suppressing the production of Th1-type cytokines. Therefore, the activation of Th1 cells and the suppression of Th2 cells may have an important effect on anti-cancer immune function.

[0054] Among them, IL-10 is produced by Th2 cells and inhibits cytokine production by Th1 cells.

[0055] IL-8 (CXCL8) is a pro-inflammatory cytokine that mainly mediates inflammatory responses, and is a representative chemokine that attracts neutrophils to the site of inflammation, where it binds to CXCR1 / 2 on tumors to promote the proliferation of cancer cells and the growth of new blood vessels (neoangiogenesis).

[0056] In one embodiment, the expression of IL-10 and IL-8, which suppress anti-cancer immune function, is significantly reduced to 1 / 100 and 1 / 1000, respectively, and it is expected that the natural killer cells can be used as an efficient cell therapy drug.

[0057] As used herein, the term "chemokine" refers to a substance secreted by activated natural killer cells, which can recruit (or migrate) various inflammatory cells to target organs. Chemokines are classified into four groups (C, CC, CXC, and CX3X groups) according to the sequence structure of the first two N-terminal cysteines in the amino acid base sequence, and more than 40 types of chemokines and more than 20 types of chemokine receptors are known to date. In particular, in the tumor microenvironment, which consists of tumors and host cells surrounding the tumor, tumor-associated host cells and cancer cells secrete various chemokines, thus recruiting and activating various types of cells that mediate the balance between anti-tumor and tumor-promoting responses. Furthermore, in addition to their role in chemotaxis, chemokines are also involved in other tumor-related processes, including tumor cell growth, angiogenesis, and metastasis.

[0058] In one embodiment, the chemokine receptor may be a CCR or a CXCR, and the CCR may be CCR1, CCR2, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10, preferably CCR2, CCR5 or CCR6. Further, the chemokine may be CCL1, CCL3, CCL4, CCL5, CCL22 or CXCL8, preferably CCL1.

[0059] As used herein, the term "CD3-CD56+ natural killer cells" may be used interchangeably with "CD3-CD56+ NK cells" or "CD3- / CD56+ NK cells."

[0060] The CD3-CD56+ natural killer cells may be in a thawed state after cryopreservation. In one embodiment, the peripheral blood mononuclear cells are isolated from blood, frozen, and then thawed again for use in natural killer cell culture. The freezing can be performed by a conventionally known method, and preferably by a freezing method comprising a first freezing step in the range of 4 to -42°C, a second freezing step in the range of -42 to -15°C, and a third freezing step in the range of -15 to -120°C. In this case, each freezing step can be performed by sequentially maintaining the sample at various discontinuous temperatures within the temperature range for a certain period of time, thereby increasing or decreasing the temperature to the upper or lower limit of the temperature range of each freezing step. In one embodiment, the first freezing step may be performed under conditions of freezing at 0° C. for 10 to 15 minutes, at −12° C. for 5 to 10 minutes, and at −42° C. for 0.5 to 1 minute, the second freezing step may be performed under conditions of freezing at −25° C. for 1 to 3 minutes and at −15° C. for 1 to 3 minutes after the first freezing step, and the third freezing step may be performed under conditions of freezing at −42° C. for 20 to 40 minutes and at −120° C. for 20 to 50 minutes after the second freezing step. In another embodiment, the first freezing step may be performed at 0.5 to 5° C. / m in the range of 4 to −40° C., the second freezing step may be performed at 1 to 10° C. / m in the range of −40 to −90° C. after the first freezing step, and the third freezing step may be performed at 1 to 10° C. / m in the range of −90 to −120° C. after the second freezing step. Each freezing step of the freezing method can be performed using a CRF (controlled rate freezer). When freezing the peripheral blood mononuclear cells of the present specification, it is preferable to freeze the isolated cells after suspending them in a mixture of cryostor CS10 or ALyS505NK-EX and Albumin + DMSO to adjust the cell number to an appropriate level. The cryopreserved peripheral blood mononuclear cells can be used for the culture and proliferation of natural killer cells described herein after thawing, and cell culture can be performed at a required time by going through the freezing process. The present specification provides a method for freezing and thawing peripheral blood mononuclear cells that maintains a high survival rate and can be effectively applied to the culture method of activated natural killer cells.

[0061] In one embodiment of the method for culturing natural killer cells, CD3-CD56+ natural killer cells can be cultured in the presence of gamma globulin (IgG) and fibronectin. The culture in the presence of gamma globulin and fibronectin can be, for example, a culture solution containing gamma globulin and fibronectin, or cells can be cultured in a culture vessel whose culture surface (the surface of the culture vessel that can contact cells) is coated with gamma globulin and fibronectin. Preferably, natural killer cells can be cultured in a culture vessel coated with gamma globulin and fibronectin. The culture vessel coated with gamma globulin and fibronectin can be prepared by adding a solution containing gamma globulin and fibronectin to coat the culture vessel. Also, a known adhesive protein can be used instead of the fibronectin, for example, collagen can be used. In one embodiment, a solution containing gamma globulin and fibronectin is added to a culture vessel (e.g., a T75 flask), and then the culture vessel can be coated with gamma globulin and fibronectin by incubating at a low temperature (e.g., 2 to 4°C).

[0062] The concentration of the gamma globulin can be 0.1 to 1 ng / ml, 1 to 10 ng / ml, 10 to 100 ng / ml, or 1 to 100 ng / ml. Gamma globulin can stimulate FcγRIII of natural killer cells to activate the natural killer cells.

[0063] The concentration of the fibronectin may be 0.1 to 50 μg / ml, 1 to 50 μg / ml, 5 to 50 μg / ml, or 10 to 50 μg / ml. Fibronectin can promote the migration of natural killer cells and facilitate cell-cell interactions.

[0064] The culture may be a culture of peripheral blood mononuclear cells to culture natural killer cells.

[0065] The peripheral blood mononuclear cells can be cultured under normal cell culture conditions, i.e., at about 37°C in a CO2 incubator, and can be continuously cultured by adding culture medium every 2 or 3 days. The concentration of PBMCs added to the medium is 4 × 10 5 ~5×10 7 The culture period may be, but is not limited to, 3 to 28 days, 5 to 25 days, 7 to 23 days, 10 to 18 days, or 10 to 14 days, and may be set appropriately within or outside the above culture period to obtain a desired number of natural killer cells. Culture vessels may include commercially available dishes, flasks, plates, multi-well plates, and culture bags.

[0066] Meanwhile, in order to maximize the proliferation of natural killer cells, the culture can be performed at each stage. In this case, each culture step may have the same or different culture medium components, culture vessels, and culture periods, and natural killer cells can be ultimately proliferated by finding the optimal culture period for each step. In addition, the present specification is characterized in that the culture composition (or culture medium) is added to the flask at intervals of 2 to 3 days during the culture period, which helps to make the culture of natural killer cells more effective.

[0067] Another embodiment is a composition for natural killer cell culture comprising IL-2, an anti-NKp46 antibody, and plasma, The composition is characterized in that it does not contain feeder cells.

[0068] In one embodiment of the method for culturing natural killer cells, CD3-CD56+ natural killer cells are treated with IL-2, anti-NKp46 antibody, and plasma, and then cultured in a culture medium that does not contain feeder cells. The culture medium may be a normal immune cell culture medium that contains IL-2, anti-NKp46 antibody, and plasma. Examples of immune cell culture media that can be used include, but are not limited to, ALyS505NK-EX (Cell Science & Technology Inst. Inc., Japan), RPMI1640 (Life technologies, USA), x-vivo10 (Lonza, USA), CellGro SCGM (CellGenix, Germany), and KBM (Kohjin bio, Japan).

[0069] The concentration of IL-2 contained in the culture medium may be, but is not limited to, 500 to 5,000 IU / ml, 600 to 4,000 IU / ml, 700 to 3,000 IU / ml, 800 to 2,000 IU / ml, 900 to 1,500 IU / ml, 900 to 1,200 IU / ml, or 1,000 to 1,200 IU / ml. The IL-2 can promote the growth of natural killer cells.

[0070] The concentration of the anti-NKp46 antibody contained in the culture medium may be, but is not limited to, 0.1 to 10 μg / ml, 0.5 to 10 μg / ml, 1 to 10 μg / ml, or 5 to 10 μg / ml. NKp46 is an activating receptor for natural killer cells, and therefore the anti-NKp46 antibody can stimulate NKp46 to activate natural killer cells.

[0071] The plasma contained in the culture medium can be obtained from peripheral blood from which peripheral blood mononuclear cells used in the culture of natural killer cells have been isolated. Serum can be used instead of the plasma.

[0072] The concentration of the plasma may be, but is not limited to, 1 to 20 v / v%, 1 to 15 v / v%, 2 to 15 v / v%, 5 to 15 v / v%, or 5 to 10 v / v% relative to the total culture medium.

[0073] A composition for natural killer cell culture according to one embodiment is characterized in that it does not contain feeder cells. In conventional natural killer cell culture, a cancer cell line is used as a feeder cell to improve the proliferation rate and cytotoxicity, but when a cancer cell line is used as a feeder cell, it is difficult to ensure important safety during clinical application, and the cultured natural killer cells have the limitation that they are natural killer cells that have been given priming specificity for specific cancer cells. In addition, the process of verifying that the feeder cells have been completely removed after culture can be eliminated, and the possibility of feeder cell contamination can be eliminated to further improve safety.

[0074] In one embodiment, the composition according to one aspect does not use feeder cells and contains only IL-2, anti-NKp46 antibody, and donor plasma. This allows stable culture of CD3-CD56+ natural killer cells with significantly high cell proliferation and cell viability even when cultured in vitro for a long period of time. Furthermore, the natural killer cells exhibit significantly increased cancer cell killing ability, do not contain feeder cells, and are clinically safer than existing natural killer cells.

[0075] The culture medium for each culture step may contain appropriate proteins, cytokines, antibodies, compounds and other ingredients, provided that they do not impair the effect of increasing the purity, proliferation rate and cancer cell killing ability of natural killer cells.

[0076] In one embodiment, the natural killer cells cultured with the composition may have one or more characteristics selected from the following (a) to (e) compared to the natural killer cells before culture.

[0077] (a) increased expression of one or more selected from the group consisting of NKG2D, NKp30, NKp44, and NKp46; (b) decreased expression of one or more selected from the group consisting of KIR3DL2, and Siglec9; (c) increased expression of one or more selected from the group consisting of FASL and TRAIL (TNFSF10); (d) increased expression of one or more proteins selected from the group consisting of Granzyme A, Granzyme K, and Perforin; and (e) increased expression of one or more selected from the group consisting of CCL1 and CCR5.

[0078] Furthermore, the natural killer cells cultured by the above method may further have one or more properties selected from the following (f) to (j) compared to the natural killer cells before culture.

[0079] (f) increased expression of one or more selected from the group consisting of DAP10 (HCST), and IL-2RA; (g) Increased expression of GM-CSF (CSF2), (h) decreased expression of one or more selected from the group consisting of IL-8 (CXCL8) and IL-10; (i) decreased expression of Noxa (PMAIP1), and (j) Increased expression of BCL-2.

[0080] In one embodiment, the plasma may be isolated from human peripheral blood. Additionally, in one embodiment, the composition may further comprise gamma globulin and fibronectin.

[0081] As used herein, the expression may be that the natural killer cells or cell population of natural killer cells expresses more or less of the anti-cancer-related genes or factors listed above after culture compared to the natural killer cells or cell population of natural killer cells before culture. Also, for example, the cultured natural killer cells or population of natural killer cells may express at least 70% or more, at least 80% or more, at least 90% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 99% or more, or 100% of the factors in the whole cells or the whole cell population. The "% expression" or "expression rate" may refer to the proportion of natural killer cells expressing one or more of the factors relative to the total number of cells in a sample. The expression or expression rate of the factors in the cells or cell population may be determined by the value detected using a flow cytometer.

[0082] In another embodiment, the present specification may provide a manufacturing method further comprising a step of preserving the natural killer cells by suspending the natural killer cells cultured by the natural killer cell culture method in a solution containing dextran and albumin.

[0083] In one example, natural killer cells cultured by the natural killer cell culture method of the present specification showed a high purity of 99% or more, a proliferation rate of 300 to 350 times, and a high cancer cell killing potency compared to before culture, and showed a significant increase in the expression of factors highly correlated with cell killing ability, a significant increase in the expression of chemokine receptors that mobilize inflammatory effector cells, a significant decrease in the expression of inactivating receptors that inactivate natural killer cells when bound to a ligand, a significant increase in genes that suppress apoptosis of natural killer cells, and a significant decrease in genes that promote it (see Figure 6 and Table 1).

[0084] As used herein, the natural killer cells may be cultured or genetically engineered to express the anti-cancer-related factors mentioned above, or to increase or decrease their expression or activity.

[0085] As used herein, the culture may mean that the hematopoietic cells, e.g., hematopoietic stem or progenitor cells, from any source, e.g., placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc., are cultured such that the expression or activity of anti-cancer-related factors as mentioned above is increased or decreased.

[0086] As used herein, "genetic engineering" or "genetically engineered" refers to the act of introducing one or more genetic modifications into a cell, or the cell produced thereby.

[0087] The term "increase in activity" or "increased activity" as used herein can refer to a detectable increase in the activity of a protein or enzyme. "Increase in activity" or "increased activity" refers to a higher level of protein or enzyme activity compared to a given parent cell or wild-type cell or pre-cultured cell (e.g., PBMC) cell.

[0088] Another aspect provides a cellular therapeutic agent or pharmaceutical composition comprising, as an active ingredient, the natural killer cells cultured by the above method or a population of such cells.

[0089] The term "cell therapy" in this specification refers to a drug used for therapeutic, diagnostic and preventive purposes through a series of actions such as in vitro proliferation, selection or other methods of changing the biological properties of living autologous, allogenic or xenogenic cells to restore the functions of cells and tissues. The United States has been managing cell therapy drugs as drugs since 1993, and Korea since 2002. Such cell therapy drugs can be broadly classified into two categories, the first being stem cell therapy drugs for tissue regeneration or organ function recovery, and the second being immune cell therapy drugs for immune response regulation, such as suppressing or enhancing in vivo immune responses.

[0090] The cellular therapy agent or pharmaceutical composition may be for the prevention or treatment of cancer or an infectious disease.

[0091] Another aspect provides the use of said natural killer cells or cell populations thereof for the manufacture of a medicament.

[0092] Another aspect provides a method of treating a disease comprising administering to an individual said natural killer cells or population of cells thereof.

[0093] The term "disease" herein may refer to a pathological condition, in particular cancer, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and transplant rejection.

[0094] The term "infectious disease" as used herein may refer to infectious diseases caused by organisms such as bacteria, viruses, and fungi. For example, in the case of viral infections such as HIV (Human Immunodeficiency Virus), EBV (Epstein-Barr Virus), HHV (Human Herpes Virus), and IAV (Influenza A virus), activation receptors such as NKp46, NKp44, and NKp30, whose expression is increased in natural killer cells according to one embodiment, can be activated by binding to viral glycoproteins, thereby inducing the death of infected cells. In addition, in the case of viral infections such as CMV (Cytomegalovirus), EMCV (Encephalomyocarditis virus), and the like, whose expression is increased in natural killer cells according to one embodiment, death of infected cells can be induced in combination with FASL, TRAIL, and the like.

[0095] The term "treatment" as used herein refers to or includes the alleviation, inhibition of progression, or prevention of a disease, disorder, or condition, or one or more symptoms thereof, and "active ingredient" or "pharmaceutical effective amount" can mean any amount of a composition used in the course of practicing the inventions provided herein sufficient to alleviate, inhibit the progression, or prevent a disease, disorder, or condition, or one or more symptoms thereof.

[0096] The terms "administer", "introduce" and "implant" herein are used interchangeably and can refer to the placement of a composition according to an embodiment into an individual by a method or route that results in at least partial localization of the composition according to an embodiment at a desired site. At least a portion of the cells or cellular components of a composition according to an embodiment can be administered by any suitable route that delivers the cells to a desired location within a viable individual. The survival period of the cells after administration to an individual can be as short as a few hours, e.g., 24 hours to several days or as long as several years.

[0097] Said administration can be combined with additional anti-cancer drugs or cytokine preparations.Examples of additional anti-cancer drugs can include alkylating agents, metabolic antagonists, spindle inhibitors, plant alkaloids, cytotoxic / anti-tumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers and kinase inhibitors.Examples of said anti-cancer drugs can include compounds used in targeting therapy and conventional chemotherapy. Examples of the antibody include alemtuzumab, apolizumab, acelizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidofusituzumab, cidofuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motavizumab, natalizumab, nimotuzumab, norovirus, and the like. These may include vizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecupsituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab, tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab, tucotuzumab-celmoleukin, tucotuzumab, umavizumab, urtoxazumab, and vicilizumab.

[0098] As used herein, the term "isolated cells", such as "isolated natural killer cells", refers to cells that have been substantially isolated from the tissue from which they are derived, such as peripheral blood.

[0099] The composition according to one embodiment can be used to treat or prevent tumors or cancers derived from neoplasms. The neoplasms can be malignant or benign, the cancers can be primary or metastatic, and the neoplasms or cancers can be early or late stage. Non-limiting examples of neoplasms or cancers that can be treated include one or more selected from the group consisting of glioma, gastrointestinal stromal tumor, leukemia, breast cancer, uterine cancer, cervical cancer, gastric cancer, colon cancer, prostate cancer, ovarian cancer, lung cancer, laryngeal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, fat cancer, fibrocytic cancer, blood cancer, lymphoma, and multiple myeloma, and preferably includes one or more selected from the group consisting of glioma, gastrointestinal stromal tumor, leukemia, breast cancer, uterine cancer, cervical cancer, gastric cancer, colon cancer, prostate cancer, and ovarian cancer. The glioma can preferably be glioma, and the leukemia can preferably be chronic myelomonocytic leukemia.

[0100] In one embodiment, the natural killer cells according to one aspect are expected to be more effective in carcinomas with high expression of NKp44 ligands and NKp30 ligands, such as MML5, HSPG, BAG6, and B6-H6. Tumors with increased PDGF include glioma, gastro-intestinal stromal tumor, chronic myelomonocytic leukemia, and breast cancer, and PDGFα in particular has been reported to be highly associated with lymph node metastasis, high histologic grade, ER / PR negativity, and triple-negative breast subtype of breast cancer (Breast Cancer Res Treat. 2018; 169(2):231-241.). Therefore, the natural killer cells according to one aspect may be more effective against these tumors.

[0101] Furthermore, in one embodiment, the natural killer cells according to one aspect have a significant increase in CCL1 (318-fold) among chemokines and CCR5 (49.9-fold) among chemokine receptors. Immune cells with increased expression of these are more advantageous in killing tumor cells by recruiting other effector cells such as monocytes, B cells, and dendritic cells, and in particular, increased CCR5 expression has affinity for CCL3 / CCL4 / CCL5+ tumor cells, making it more advantageous in killing these tumors. CCL5 expression is highly increased in metastatic / advanced breast cancer, cervical cancer, gastric cancer, colon cancer, prostate cancer, ovarian cancer (Mediators of Inflammation Volume 2014, Article ID 292376), and the natural killer cells according to one aspect with increased CCR5 may be more effective against the tumors.

[0102] The method of administration of the pharmaceutical composition in one embodiment is not particularly limited, and can be administered parenterally, such as intravenously, subcutaneously, intraperitoneally, by inhalation or topical application, or orally, depending on the desired method. The dosage ranges depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. A daily dose refers to an amount of a therapeutic substance according to one embodiment that is sufficient to treat a disease state alleviated by administration to an individual in need of treatment. The effective amount of a therapeutic substance varies depending on the specific compound, the disease state and its severity, and the individual in need of treatment, and can be routinely determined by one of ordinary skill in the art. As a non-limiting example, the dosage of a composition according to one embodiment to a human body can vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity. Based on an adult patient weighing 70 kg, for example, the dosage may be approximately 1,000 to 10,000 cells / dose, 1,000 to 100,000 cells / dose, 1,000 to 1,000,000 cells / dose, 1,000 to 10,000,000 cells / dose, 1,000 to 1,000,000,000 cells / dose, 1,000 to 10,000,000,000 cells / dose, or 1,000 to 100,000,000,000 cells / dose, and may be administered once to several times a day at regular intervals, or may be administered several times at regular intervals.

[0103] By "individual" is meant a subject in need of treatment for a disease, and more specifically means a mammal, such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0104] The pharmaceutical composition according to one embodiment may include a pharma- ceutically acceptable carrier and / or additive. For example, it may include sterilized water, physiological saline, a conventional buffer (such as phosphoric acid, citric acid, other organic acids, etc.), a stabilizer, a salt, an antioxidant (such as ascorbic acid), a surfactant, a suspending agent, an isotonicity agent, or a preservative. For local administration, it may also include combination with an organic material such as a biopolymer, an inorganic material such as hydroxyapatite, specifically a collagen matrix, a polylactic acid polymer or copolymer, a polyethylene glycol polymer or copolymer, and chemical derivatives thereof. When the pharmaceutical composition according to one embodiment is prepared into a formulation suitable for injection, the substance that increases immune cells or their activity may be dissolved in a pharma- ceutical acceptable carrier or frozen in a solution state in which it is dissolved.

[0105] The pharmaceutical composition according to one embodiment may contain a suspending agent, a solubilizing agent, a stabilizing agent, an isotonicity agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc., as necessary depending on the administration method or formulation. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature "Remington's Pharmaceutical Sciences, 19th ed., 1995". The pharmaceutical composition according to one embodiment may be prepared in a unit dose form by formulating with a pharma- ceutical acceptable carrier and / or excipient, or by placing in a multi-volume container, according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or in the form of a powder, granules, tablets or capsules.

[0106] Another aspect provides a use of the natural killer cells or cell populations cultured by the method.

[0107] The present invention also provides uses of pharmaceutical compositions containing, as an active ingredient, natural killer cells cultured by the above method or a cell population thereof.

[0108] Another aspect provides the use of said composition for natural killer cell culture for use in the manufacture of a pharmaceutical composition or formulation for the prevention or treatment of cancer.

[0109] Another aspect provides the use of said composition for natural killer cell culture for the manufacture of a medicament for the prevention or treatment of cancer.

[0110] The natural killer cells, pharmaceutical compositions, cancer, prevention, treatment, etc. are as described above. Effect of the Invention

[0111] According to one embodiment of the natural killer cells, the expression of anti-cancer-related genes significantly increases or decreases before or after culture, and while retaining the inherent characteristics of natural killer cells, the activity and proliferation fold is significantly high, making it possible to perform effective anti-cancer immunotherapy. [Brief description of the drawings]

[0112] [Figure 1A] FIG. 1A shows the results of FACS analysis of the phenotype of natural killer cells from Donor 1 on day 21 of culture. [Figure 1B] FIG. 1B shows the results of FACS analysis of the phenotype of natural killer cells from Donor 2 on day 21 of culture. [Diagram 2] FIG. 2 is a diagram showing changes in cell number over the culture period of natural killer cells according to one embodiment. [Diagram 3] FIG. 3 is a diagram showing the change in fold proliferation of natural killer cells after 21 days of culture according to one embodiment. [Figure 4] FIG. 4 is a diagram showing changes in survival rate of natural killer cells over the culture period according to one embodiment. [Diagram 5] FIG. 5 is a graph showing changes in cell killing ability depending on the E:T ratio of natural killer cells according to one embodiment. [Figure 6A] FIG. 6A is a heat map showing clustering changes due to changes in natural killer cell receptor-related genes in natural killer cells according to one embodiment. [Figure 6B] FIG. 6B is a heat map showing clustering changes due to cytokine / chemokine gene changes in natural killer cells according to one embodiment. [Figure 6C] FIG. 6C is a heat map showing clustering changes due to changes in cell killing-related genes of natural killer cells according to one embodiment. [Figure 7]FIG. 7 is a diagram comparing the change in cell killing ability depending on the E:T ratio between natural killer cells according to one embodiment and a representative human NK cell line. [Figure 8] FIG. 8 is a diagram showing an administration schedule for confirming the anti-cancer effect of administration of natural killer cells according to one embodiment. [Figure 9] FIG. 9 shows the results of confirming the anti-cancer activity of natural killer cells according to one embodiment in a mouse model of ovarian cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0113] The present invention will be described in more detail with reference to the following examples. However, these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0114] Example: Production of highly pure and highly active natural killer cells with regulated expression of anti-cancer related genes To produce highly pure and highly active natural killer cells in which the expression of anti-cancer-related genes was regulated, natural killer cells were cultured as follows.

[0115] 1. Production of natural killer cells 1.1. Isolation of CD3-CD56+ Natural Killer Cells Peripheral blood mononuclear cells (PBMCs) and plasma (300 ml) from three healthy donors (Donor 1, Donor 2, Donor 3) were isolated by leukapheresis, and 2–5 × 10 10 From the isolated monocytes (cells), CD3-CD56+ natural killer cells were obtained using the CliniMACS Prodigy® system.

[0116] Specifically, leukocyte apheresis was performed using a Spectra-Optia (Ver. 11) component collection device, and 14,000 to 16,000 mL of blood was circulated using 3000 U heparin / 500 mL of acid citrate dextrose (ACD). The final collected volume was 150 to 250 mL, and the total number of collected leukocytes was 2 to 5 × 10 10 (cells). Next, CD3-CD56+ natural killer cells were produced using a closed system, CliniMACS Prodigy (registered trademark). The T cell fraction (%) was calculated using the white blood cells in the collected blood as a starting material, and the number of T cells was calculated in the total volume. 9 The microbeads (7.5mL / 95mL buffer) bound to anti-CD3 monoclonal antibodies were placed in the cell culture chamber, and after 4-5 hours of reaction, CD3-negative cells were obtained (0.9-1×10 10 (cells)), and the CD3-negative cells were positively selected using anti-CD56-bound microbeads (7.5 mL / 95 mL buffer), and 0.3 to 3 × 10 CD3-CD56+ natural killer cells were isolated. 9 (Cells).

[0117] 1.2. Freezing and thawing CD3-CD56+ natural killer cells 3×10 CD3-CD56+ natural killer cells isolated in Example 1.1 8 The cells were frozen per vial, and a total of three vials were transferred to an LN2 tank for storage (below -130°C), and thawed for use in subsequent experiments when required.

[0118] Specifically, all cell suspensions obtained in Example 1.1 were centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed. Natural killer cells obtained in a Cryostor CS10 or ALyS505NK-EX+Albumin+DMSO mixture stored at 2-8°C were suspended, and the cell count was adjusted to 1-100μ10. 6The suspension cells were dispensed into 2 ml cryogenic vials at 1 ml each, and then the first stage was frozen using CRFs (controlled rate freezers) at 0°C for 10-15 minutes, at -12°C for 5-10 minutes, and at -42°C for 0.5-1 minute. After the first stage, the cells were frozen at -25°C for 1-3 minutes and at -15°C for 1-3 minutes, and after the second stage, the cells were frozen at -42°C for 20-40 minutes and at -120°C for 20-50 minutes. Alternatively, the first stage was frozen at 4-40°C at 3°C / m, after the first stage, the second stage was frozen at -40-90°C at 5°C / m, and after the second stage, the cells were frozen at -90-120°C at 5°C / m. The frozen cells were transferred to an LN2 tank and stored (below -130°C).

[0119] During thawing, the heat block was set to 37°C, and the culture medium supplemented with 10% plasma was added to the T flask. Depending on the cell concentration, the amount of culture medium may be adjusted to various amounts, for example, 4ml, 6ml, 8ml, or 10ml. The frozen vial was placed in a heat block to thaw the frozen cells. When the frozen cells were half thawed, they were transferred to a T flask containing culture medium. They were then placed in a 37°C, 5% CO2 incubator and cultured for one day. The cultured cells were collected in a tube, and Ca / Mg-free DPBS was added, centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed. The cells separated by centrifugation were suspended in a small amount of culture medium, and the cell count was measured.

[0120] 2. Cultivation of Natural Killer Cells 2.1. Preparation of fibronectin and gamma globulin coated culture flasks 0.01 mL fibronectin (#FC-010, Millipore) and 0.121 mL gamma globulin (#020A1004, Green Cross) solutions were placed in a 15 mL tube, and then 9.859 mL of Ca / Mg glass DPBS was added. The prepared coating solution was placed in a T75 flask (#156499, Nunc) using a pipette and reacted at 2-8 °C for more than 16 hours. The remaining coating solution before cell culture was washed with Ca / Mg-free DPBS and then removed.

[0121] 2.2. Primary culture of natural killer cells A cell suspension of CD3-CD56+ natural killer cells frozen and thawed in Example 1.2 was inoculated into the coated flask prepared in Example 2.1. IL-2, anti-NKp46 antibody and donor plasma were added without using feeder cells, and the cells were cultured at 37°C and 5% CO2 for 3 to 5 days.

[0122] 2.3. Secondary culture of natural killer cells After the primary culture in Example 2.2, the T75 flask in which the cells had been cultured in the incubator was removed and the cells were collected, and the cells were then added to the remaining donor plasma or FBS to prepare a cell suspension, which was then added to a 2L CO2-permeable culture bag containing culture medium. After the culture medium and cell suspension were thoroughly mixed, the bag was placed in a 37°C, 5% CO2 incubator and cultured for 7 to 14 days to allow the cells to grow. After the culture was completed, the final culture medium was placed in a centrifuge tube and the cells were collected by centrifugation several times.

[0123] Experimental Example 1. Analysis of purity and proliferation of natural killer cells 2×10 CD3-CD56+ natural killer cells from two donors cultured according to the above example 8 The number of cells was determined 8, 14, and 21 days after the culture, and the cells were stained with CD3 and CD56 antibodies, and the proportion of CD3-CD56+ natural killer cells was analyzed by FACS analysis using a known method.

[0124] As a result, the proportion of CD3-CD56+ natural killer cells was significantly high from the early stage of culture (culture day 0: 96.0%), and the proportion of CD3-CD56+ natural killer cells continued to increase throughout the culture period (average ratio of CD3-CD56+ natural killer cells in Donor 1 and Donor 2; culture day 8: 96.4%, culture day 14: 98.3%).

[0125] In particular, on the 21st day after culture, CD3-CD56+ NK cells accounted for 99.2% and 99.6%, respectively, confirming that the distribution was of higher purity natural killer cells compared to before culture (Figure 1).

[0126] In addition, CD3-CD56+ natural killer cells from two donors expanded by an average of 113 ± 27-fold on day 14 of culture, and by day 21 of culture, the number of natural killer cells was 6.9 × 10 10 , 4.9×10 10 It was confirmed that the cells proliferated in the culture medium (cells) and increased remarkably, with an average proliferation factor of 300 to 350 times (Figures 2 and 3). Furthermore, it was confirmed that all of the cells showed a high survival rate of 90% or more on average during the 21-day culture period (Figure 4).

[0127] From the above results, it was found that the natural killer cells according to one embodiment can be stably cultured with a cell number remarkably increased and a survival rate reaching 90% even when cultured in vitro for a long period of time. It was also confirmed that by performing leukocyte apheresis and selection of CD3-CD56+ natural killer cells and culturing them, natural killer cells with high purity containing fewer other impurities can be obtained compared to density gradient centrifugation.

[0128] Experimental Example 2. Analysis of the ability of natural killer cells to kill cancer cells To evaluate the cancer cell-killing ability of the natural killer cells cultured by the culture method according to the above embodiment, natural killer cells on the 21st day of culture were used and the cell-killing ability was evaluated using the blood cancer cell line K562, which is highly sensitive to natural killer cells, as target cells.

[0129] First, the target cancer cells (K562) were collected and centrifuged at 1,500 rpm for 5 minutes, after which the supernatant was removed. They were then diluted with DPBS and washed, and the cell pellets after washing were suspended in a culture medium containing phenol red-free RPMI medium supplemented with 10% FBS. 1 × 10 per condition 5 The cells were prepared and stained with CFSE (Life technologies) at a concentration of 5 μM for 10 minutes in an incubator under 5% CO2 conditions. After washing twice with DPBS, the cells were diluted with culture medium containing 10% FBS in RPMI medium without phenol red. Activated natural killer cells were prepared according to the E:T ratio with the target cells (1:1, 1.25:1, 2.5:1, 5:1, 10:1, 20:1) and mixed with the target cells on a 24-well plate. The reaction was allowed to proceed for 4 hours, and 7AAD (7-AminoactinomycinD) was treated 20 minutes before the end of the reaction. After the reaction was completed, the cells were collected in a 5 mL FACS tube and the cell killing ability was analyzed via a flow cytometer.

[0130] As a result, it was confirmed that natural killer cells cultured using one embodiment of the culture method showed a significantly increased cell killing ability of more than 90% at E:T ratios of 10:1 and 20:1, and in particular, possessed a significant killing ability of more than 80% even when the E:T (effective cells:target cells) ratio was 1:1 to 1.25:1 (Figure 5).

[0131] Taking the above results together, it was confirmed that the natural killer cells cultured using the culture method of one embodiment are highly pure natural killer cells whose purity increased to a level of more than 99% through the culture process according to the above embodiment, and that they not only exhibit high proliferation efficiency but also high cancer cell killing ability.

[0132] Experimental Example 3. Analysis of anti-cancer related gene expression in natural killer cells 3.1. Analysis of mRNA expression profile of anti-cancer related genes in natural killer cells The mRNA expression patterns of anti-cancer-related genes in natural killer cells derived from three donors cultured using the culture method according to the above example were analyzed immediately after isolation of the natural killer cells (D0) and after 14 days of culture (D14).

[0133] Specifically, RNA was extracted from the cells before and after culture using Trizol isolation, and total RNA sequencing was performed to confirm the mRNA expression profile (fold change, D14 natural killer cells / D0 natural killer cells). The expression profile was shown as the average value of three donors, and statistical significance was observed when the q-value, which is the corrected p-value, was 0.001 or higher. The results are shown in Figure 6 and Table 1 below.

[0134] [Table 1]

[0135] As a result, we confirmed an increase in the activating receptors NKG2D, NKp30, NKp44, and NKp46, with the most notable increases being NKp44 (1064-fold) and NKp30 (5.2-fold).We also confirmed that high levels of CD16, which mediates antibody-dependent cell-mediated cutotoxicity (ADCC) of target cells, were maintained even after culturing and freezing.

[0136] NKp44 of natural killer cells binds to cancer cell ligands MLL5 (mixed-lineage leukemia protein-5), HSPG (heparin sulfate proteoglycans), and PDGF (platelet-derived growth factor) to activate natural killer cells. NKp30 binds to cancer cell ligands BAG6 and B6-H6 to activate natural killer cells. Therefore, the natural killer cells according to one embodiment are expected to be more effective in cancers with high expression of NKp44 ligands and NKp30 ligands, such as MLL5, HSPG, BAG6, and B6-H6. Tumors with increased PDGF include glioma, gastro-intestinal stromal tumor, chronic myelomonocytic leukemia, breast cancer, etc., and it has been reported that PDGFα in particular is highly associated with lymph node metastasis, high histologic grade, ER / PR negativity, and triple-negative breast subtype of breast cancer (Breast Cancer Res Treat. 2018; 169(2):231-241.). Therefore, it is expected that natural killer cells according to one embodiment will be effective against these tumors.

[0137] Meanwhile, CCL1 (318-fold) among chemokines and CCR5 (49.9-fold) among chemokine receptors were significantly increased. Immune cells with increased expression of these are more advantageous in killing tumor cells by recruiting other effector cells such as monocytes, B cells, and dendritic cells, and in particular, increased CCR5 expression has affinity for CCL5+ tumor cells, which is more advantageous in killing these tumors. CCL5 expression is highly increased in metastatic / advanced breast cancer, cervical cancer, gastric cancer, colon cancer, prostate cancer, ovarian cancer (Mediators of Inflammation Volume 2014, Article ID 292376), etc., and natural killer cells with increased CCR5 in one embodiment are expected to have greater affinity for the tumors.

[0138] As shown in Table 1, the expression of NKp44 and NKp30, which are factors related to the activation of natural killer cells, as well as CCL1, a chemokine that mobilizes different effector cells, and CCR5, a chemokine receptor, increased by at least 5.2-fold and at most 1064-fold, while the expression of inactivating receptors KIR3DL2 and Siglec9, which inactivate natural killer cells when binding to ligands, was significantly decreased by 0.5-fold and 0.05-fold, respectively. When KIR3DL2 binds to cancer cell ligands HLAA3, A11, B27 homodimer, etc., it reduces the INF-γ production and cancer cell killing ability of natural killer cells. When Siglec9 binds to sialic acid on cancer cells, it induces ITIM phosphorylation in natural killer cells, recruiting SHP-1 and SHP-2 to inhibit cell killing ability. Therefore, it is expected that natural killer cells according to one embodiment, in which expression of such inactivating receptors is significantly reduced, can further increase cancer cell death.

[0139] In addition, the cell killing ability of natural killer cells is achieved through two major mechanisms: one is the secretion of granule substances such as granzyme and perforin in the cytoplasm to kill cancer cells, and the other is the progression of apoptosis of cancer cells by binding with ligands that recognize Fas or TNFR (tumor necrosis factor receptor), i.e., FASL and / or TRAIL, which are death receptors of cancer cells. In the case of natural killer cells according to one embodiment, the expression of granzyme A, granzyme K, and perforin, which are factors related to anti-cancer activity (cell killing ability), was also increased by at least 2.1 times to at most 10.7 times, and the expression of FAS-L (FASLG) and TRAIL (TNFSF10), which induce an increase in apoptosis, was significantly increased by at least 6.1 times to at most 24.5 times.

[0140] Furthermore, the expression of BCL-2, which delays natural killer cell apoptosis, was significantly increased by 10.5-fold, while the expression of Noxa, which promotes apoptosis, was significantly decreased by 0.03-fold.

[0141] 3.2. Differences in expression of anti-cancer-related genes with and without selection of CD3-CD56+ natural killer cells The expression profile of anti-cancer-related genes was analyzed between natural killer cells cultured after leukocyte apheresis and CD3-CD56+ selection using the CliniMACS Prodigy® system and natural killer cells cultured without the above-mentioned separation process. In the same manner as in Experimental Example 3, RNA was extracted from the cells before and after culture using a Trizol separation method, and total RNA sequencing was performed to confirm the mRNA expression profile (fold change, D14 natural killer cells / D0 natural killer cells). The expression profile was shown as the average value of three donors, and statistical significance was observed when the q-value, which is the corrected p-value, was 0.001 or more. The results are shown in Table 2 below.

[0142] [Table 2]

[0143] As a result, it was confirmed that the natural killer cells cultured after leukocyte apheresis and CD3-CD56+ selection using the CliniMACS Prodigy® system in Example 1 have the following characteristics compared to those not cultured.

[0144] a) There is a significant increase in NKp44 expression in activating receptors (1064-fold vs. 7.2-fold). b) In the case of cell killing, the expression of granzyme K (10.7-fold) and TNFSF10 (24.5-fold) is significantly increased, whereas in the absence of CD3-CD56+ separation, the expression of granzyme A is significantly increased (41.9-fold).

[0145] c) The expression of IL-8 (1 / 100 fold) and IL-10 (1 / 1000 fold) is significantly decreased.

[0146] d) There is a significant increase in the expression of CCR5, a chemokine receptor that recruits other effector cells (49.9 vs. 16.75-fold).

[0147] e) There was a clear increase in the expression of BCL-2 (10.5-fold), which delays apoptosis of natural killer cells, and a clear decrease in the expression of Noxa (0.03-fold), which promotes apoptosis.

[0148] 3.3. Comparison of activating receptor expression profiles between natural killer cells and human NK cell lines according to one embodiment The expression pattern of activating receptors of natural killer cells (pNK) cultured by the culture method according to one embodiment was compared with that of representative human NK cell lines, NK92mi and NK92. Human NK cell lines NK92mi and NK92 were purchased from ATCC (American Type Culture Collection), and the expression levels of representative activating receptors of natural killer cells, NKG2D, NKp46, NKp44, NKp30, DNAM-1, and CD16, were analyzed using a flow cytometer. The results are shown in Table 3 below.

[0149] [Table 3]

[0150] As a result, it was confirmed that the natural killer cells according to one embodiment express representative activating receptors NKG2D (80.13%), NKp44 (95.43%), NKp30 (93.20%), DNAM-1 (99.27%), and CD16 (90.60%) at high rates of 80 to 99%, and that NKp46 also has a high expression rate of about 55%. Meanwhile, in the case of human NK cell lines NK92mi and NK92, it was confirmed that the expression rates of activating receptors were significantly lower compared to the natural killer cells according to one embodiment.

[0151] Experimental Example 4. Cytotoxicity test of natural killer cells 4.1. Cytotoxicity testing using flow cytometry The cytotoxicity of natural killer cells (pNK) cultured by the culture method according to one embodiment was confirmed by flow cytometry. The cytotoxicity was confirmed by the 7AAD-CFSE method, which is useful for cytotoxicity testing using floating cells such as K562 cells without using radioisotopes. As target cells, K562, a chronic myelogenous leukemia cell line, A2780, a cisplatin-sensitive cell, and A2780cis, a cisplatin-resistant cell, as ovarian cancer cell lines, and MCF7, SKBR3, and T47D, breast cancer cell lines, were used after being cultured from the day before the test. As effector cells, NK92mi, a representative human NK cell line, was used as a comparison group along with the natural killer cells (pNK) according to one embodiment.

[0152] As a result, it was confirmed that the natural killer cells cultured by the culture method according to one embodiment have significantly increased cancer cell elimination ability depending on the E:T ratio in not only the blood cancer cell line K562 but also various solid cancers such as ovarian cancer and breast cancer compared to a representative human NK cell line (Figure 7).

[0153] From the above results, it was confirmed that the natural killer cells cultured by the culture method according to one embodiment have an increased cancer cell killing effect as the expression levels of anti-cancer related genes and the expression of activating receptors are significantly increased.

[0154] 4.2. Evaluation of anticancer activity in ovarian cancer mouse models The effectiveness of natural killer cells (pNK) cultured by one embodiment of the culture method as an immunological anticancer agent in suppressing ovarian cancer cell proliferation was evaluated. Mouse ovarian cancer cell line A2780cis was subcutaneously inoculated at 2×10 6A2780cis xenograft mouse models were created by injecting the ovarian cancer cell line in an amount of 1×10 (cells). After injection, mice in which tumors had formed on day 3 were randomly grouped into a group receiving intraperitoneal injections of cisplatin at 1 mg / kg once a week, and a group receiving intraperitoneal injections of natural killer cells according to one embodiment at 1×10 7 The mice were divided into a group that received intravenous injections of (cells) twice a week, and a control group that received PBS injections (FIG. 8). After injection of the ovarian cancer cell lines, tumor size and body weight were measured every day.

[0155] As a result, it was confirmed that when natural killer cells cultured by the culture method according to one embodiment were administered alone, a significant cancer cell proliferation suppression effect of 80% or more was observed compared to the control group. On the other hand, when cisplatin, an existing anticancer drug, was injected, a cancer cell proliferation suppression effect of about 55% was observed (Figure 9, A). Meanwhile, no significant change in body weight was observed in either the natural killer cell according to one embodiment and cisplatin administration group (Figure 9, B). It was confirmed that the weight of the tumor extracted after the mice were sacrificed was reduced by an average of 70% or more compared to the control group, and reduced by an average of 50% or more compared to the cisplatin administration group (Figure 9, C).

[0156] From the above results, it was confirmed that the natural killer cells cultured by the culture method according to one embodiment have increased anti-cancer activity and cell activity, inhibited apoptosis of natural killer cells, and increased in vivo survival rate. It was confirmed that such characteristics were clearly observed when the natural killer cells were cultured after leukocyte apheresis and CD3-CD56+ separation. In addition, as can be seen from the results of the animal model, it was confirmed that the natural killer cells cultured by the culture method according to one embodiment exhibited excellent anti-cancer effect of significantly reducing tumor size.

[0157] Therefore, the natural killer cells according to one embodiment are isolated and cultured in large quantities from healthy donors, and are cultured at a higher purity than existing cultures using peripheral blood mononuclear cells, and are therefore safer and have an increased cancer cell killing effect, making them usable as a cell therapy for efficient cancer treatment. Furthermore, since no feeder cells are used during culture, clinical safety is guaranteed, and the natural killer cells according to one embodiment are expected to be useful as a new allogeneic immune cell therapy with enhanced functions.

Claims

1. 1) isolating peripheral blood mononuclear cells or leukocytes from human peripheral blood via leukapheresis; 2) obtaining CD3-CD56+ natural killer cells from the isolated peripheral blood mononuclear cells or leukocytes via a closed system; 3) treating the obtained CD3-CD56+ natural killer cells with IL-2, an anti-NKp46 antibody, and plasma, and culturing the cells in a culture medium not containing feeder cells, The present invention relates to a method for culturing natural killer cells, wherein the natural killer cells cultured according to the method have one or more characteristics selected from the following (a) to (c) compared to the natural killer cells before culture. (a) decreased expression of one or more genes selected from the group consisting of KIR3DL2, and Siglec9; (b) increased expression of one or more selected from the group consisting of CCL1 and CCR5; (c) increased expression of one or more selected from the group consisting of NKG2D, NKp30, NKp44, and NKp46.

2. The method according to claim 1, wherein the natural killer cells cultured according to the method have one or more characteristics selected from (d) and (e) below, compared to the natural killer cells before culture. (d) increased expression of one or more selected from the group consisting of FASL and TRAIL (TNFSF10); and (e) increased expression of one or more proteins selected from the group consisting of Granzyme A, Granzyme K, and Perforin.

3. The method according to claim 2, wherein the natural killer cells cultured according to the method further have one or more characteristics selected from the following (f) to (j) compared to the natural killer cells before culture. (f) increased expression of one or more selected from the group consisting of DAP10 (HCST), and IL-2RA; (g) increased expression of GM-CSF (CSF2); (h) decreased expression of one or more selected from the group consisting of IL-8 (CXCL8), and IL-10; (i) decreased expression of Noxa (PMAIP1); and (j) BCL-2 expression is increased.

4. The method according to claim 1, wherein the natural killer cells cultured according to the method have increased purity, cell proliferation rate, and cancer cell killing ability compared to the natural killer cells before culture.

5. The method of claim 1, wherein step 1) is performed by leukapheresis.

6. The method according to claim 1, wherein the CD3-CD56+ natural killer cells in step 3) have been cryopreserved and then thawed.

7. The method according to claim 1, wherein the plasma in step 3) is isolated from human peripheral blood.

8. The method according to claim 1, wherein the culturing in step 3) is carried out in the presence of gamma globulin and fibronectin.

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