Method for producing natural killer cells and car-NK cells, natural killer cells and car-NK cells produced thereby, and use thereof

Abstract

The present invention relates to a method for producing natural killer cells and CAR-NK cells. In addition, the present invention relates to the use of natural killer cells and CAR-natural killer cells in drugs for treating cancer and autoimmune diseases. A method for producing hematopoietic stem cell-derived natural killer cells and / or CAR-NK cells according to the present invention can produce natural killer cells and / or CAR-NK cells at a high cell growth rate while using a simple combination of cytokines, and the natural killer cells and / or CAR-NK cells produced according to the production method of the present invention have higher cytotoxicity than those produced by conventional methods.

Description

Method for producing natural killer cells and CAR-NK cells, natural killer cells and CAR-NK cells produced thereby, and uses thereof

[0001] The present invention relates to a method for producing natural killer cells and CAR-NK cells. Furthermore, the present invention relates to the use of natural killer cells and CAR-NK cells in drugs for the treatment of cancer and autoimmune diseases.

[0002] Numerous previous studies and clinical trials have shown that cytotoxic T cells are effective in treating blood cancers. However, using cytotoxic T cells for cell therapy requires harvesting large quantities of the cells from the patient's blood. Furthermore, patients have suffered from serious side effects, such as cytokine storm, during the process of treating cancer with personalized T cell therapy through in vitro mass culture. In contrast, using natural killer cells (NK cells) offers the following advantages, as cells can be harvested from healthy donors rather than patients (Judge et al. Characterizing the Dysfunctional NK Cell: Assessing the Clinical Relevance of Exhaustion, Anergy, and Senescence, 2020).

[0003] - There is no need to obtain a large number of natural killer cells from patients. The cells can be obtained from healthy donors, cultured in large quantities, and then administered to patients.

[0004] - Since the MHC class between the donor and the patient is not identical, the mechanism by which the patient's cancer cells avoid natural killer cells is inhibited, resulting in a more effective anticancer effect.

[0005] - The biggest advantage of natural killer cells is that, unlike T cells, side effects are minimized because the immune response progresses through the innate immune response.

[0006] - Natural killer cells are immune cells that can present antigens and can also induce activation of T cells, an adaptive immune system, through their antigen presentation function.

[0007] However, since these natural killer cells only exist in about 5 to 20% of lymphocytes in the blood, the amount is too small to be separated and used from peripheral blood, and since most of the natural killer cells contained in peripheral blood are differentiated cells, there is a limit to their proliferation in the body after transplantation. Therefore, recently, various methods for producing large quantities of natural killer cells by in vitro differentiation of cells isolated from umbilical cord blood have been studied. According to the methods known so far, a number of complex cytokine combinations are used in the step of culturing hematopoietic stem cells isolated from umbilical cord blood and / or the step of differentiating them into natural killer cells (Shokouhifar et al., Cancer Cell International, Vol. 21, 298, (2021); Spanholtz et al., PLoS ONE5(2): e9221, (2010); Spanholtz et al., PLoS ONE6(6): e20740, (2011); Satwani et al., Cytotherapy, 13, 730-738, (2011)), or special culture media are used (Spanholtz et al., PLoS ONE5(2): e9221, (2010); Spanholtz et al., PLoS ONE6(6): e20740, (2011); Satwani et al.,Cytotherapy, 13, 730-738, (2011)), using specific chemicals (Shokouhifar et al.,Cancer Cell International,Vol. 21, 298, (2021); Spanholtz et al.,PLoS ONE5(2): e9221, (2010); Spanholtz et al.,PLoS ONE6(6): e20740, (2011)), using additional antibodies to grow and develop cells (Satwani et al.,Cytotherapy, 13, 730-738, (2011)), or using feeder cells (Denman et al.,PLoS ONE7(1): e30264, (2012)), but are not satisfactory in terms of cell growth rate, high cost and large manpower required for manufacturing, and the cytotoxicity of the manufactured natural killer cells also has a lot of room for improvement. In particular, since feeder cells are substances that should not be present in the final natural killer cells, a method for manufacturing natural killer cells with a high cell growth rate using a simplified cytokine combination without using feeder cells is required.

[0008] Furthermore, research on cell therapy is actively underway, focusing on CAR-T, which can treat various diseases by introducing a Chimeric Antigen Receptor (CAR) into T cells. Drugs such as Kymriah are already being used for blood cancers. However, limitations include the complex manufacturing method of autologous CAR-T and the high risk of adverse effects such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and graft-versus-host disease (GVHD).

[0009] The present invention aims to provide a method for producing natural killer cells, wherein the method has a high cell growth rate without using a complex combination of cytokines in the step of culturing and proliferating hematopoietic stem cells and / or the step of differentiating them into natural killer cells.

[0010] In addition, the present invention aims to provide a method for producing natural killer cells, which does not substantially use culture auxiliary cells in the step of culturing and proliferating hematopoietic stem cells and / or the step of differentiating them into natural killer cells.

[0011] Another aspect of the present invention is to provide a medium composition that increases the cell growth rate of natural killer cells for use in producing natural killer cells.

[0012] In addition, another aspect of the present invention is to provide natural killer cells with improved cytotoxicity, manufactured according to the above-described manufacturing method.

[0013] Another aspect of the present invention is to provide a pharmaceutical composition comprising the natural killer cells.

[0014] The purpose of the present invention is to provide a manufacturing method that does not use cultured auxiliary cells in the step of proliferating hematopoietic stem cells, the step of introducing a CAR gene, and the step of differentiating the hematopoietic stem cells into which the CAR has been introduced into natural killer cells.

[0015] The purpose of the present invention is to provide a medium composition that increases the cell growth rate of CAR-NK cells.

[0016] The purpose of the present invention is to provide a CAR-NK cell having target cell-specific cytotoxicity, manufactured according to the manufacturing method described above.

[0017] The present invention aims to provide a method for preventing or treating cancer, which comprises a step of administering natural killer cells and / or CAR-NK cells manufactured according to the above-described manufacturing method to an individual in need thereof.

[0018] The present invention aims to provide a method for preventing or treating an autoimmune disease, which comprises a step of administering natural killer cells and / or CAR-NK cells manufactured according to the above-described manufacturing method to an individual in need thereof.

[0019] The purpose of the present invention is to provide a use for using natural killer cells and / or CAR-NK cells manufactured according to the above-described manufacturing method in the manufacture of a composition for preventing or treating cancer.

[0020] The purpose of the present invention is to provide a use for using natural killer cells and / or CAR-NK cells manufactured according to the above-described manufacturing method in the manufacture of a composition for preventing or treating autoimmune diseases.

[0021] One aspect provides a method for producing natural killer cells, comprising the steps of (a) culturing hematopoietic stem cells; (b) inducing differentiation of the cultured hematopoietic stem cells into lymphoid progenitor cells; and (c) differentiating the lymphoid progenitor cells into natural killer cells in a culture medium containing Flt-3L, IL-2, IL-15, and IL-21.

[0022] The above hematopoietic stem cells may be isolated from bone marrow, umbilical cord blood, peripheral blood, or induced pluripotent stem cells. In one specific example, the above hematopoietic stem cells may be isolated from umbilical cord blood.

[0023] In this specification, the term “hematopoietic stem cell (HSC)” may be used interchangeably with hematopoietic stem cell, hematopoietic stem cell, or hematopoietic progenitor cell. In addition, the hematopoietic stem cell may be a CD34+ stem cell. Hematopoietic stem cell refers to a pluripotent stem cell that produces all blood cell types, including myeloid (monocytes and macrophages, granulocytes (neutrophils, basophils, eosinophils, and mast cells), erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells) cells (see [Doulatov et al., 2012; Notta et al., 2015]). The hematopoietic stem cell may express CD34 and CD133, and may be negative for CD38 expression. Hematopoietic stem cells may include CD34+ / CD45+ hematopoietic stem cells and CD34+ / CD45+ / CD43+ hematopoietic stem cells. Samples containing hematopoietic stem cells and / or progenitor cells may be obtained in any possible manner, such as, for example, ex vivo generation from a cell source containing hematopoietic stem cells and / or progenitor cells, such as bone marrow, umbilical cord blood, placental material, peripheral blood, embryonic stem cells, or any variant thereof, using cell culture steps. Hematopoietic stem cells and / or progenitor cells may be further purified from such a cell source containing hematopoietic stem cells and / or progenitor cells using an affinity purification method.

[0024] The above hematopoietic stem cells are 500 to 2Х10 6 Seed in a container, such as a plate, flask, or bag, at a concentration in the range of cells / mL. Specifically, 500 to 2Х10 6 Cells / mL, 500 to 1Х10 6 Cells / mL, 500 to 2Х10 5 Cells / mL, 500 to 1Х10 5 cells / mL, 1Х10 3 Inland 2X10 6cells / mL, 1Х10 3 1X10 inland 6 cells / mL, 1Х10 3 2X10 5 cells / mL, or 1Х10 3 1Х10 5 cells / mL. In one specific example, the hematopoietic stem cells are seeded at a concentration of 1Х10 5 Seeded at step (a) at cells / ml.

[0025] The culture medium of steps (a) and (b) may include at least one selected from the group consisting of SCF (Stem cell factor), Flt-3L (FMS-like tyrosine kinase 3 ligand), TPO (Thrombopoietin), IL-2 (Interleukin-2), IL-3 (Interleukin-3), IL-6 (Interleukin-6), IL-7 (Interleukin-7), IL-15 (Interleukin-15), and IL-21 (Interleukin-21). In one specific example, the culture medium of step (a) may include SCF, Flt-3L, TPO, IL-3, and IL-6. In addition, in one specific example, the culture medium of step (b) may include SCF, Flt-3L, IL-3, IL-7, and IL-15.

[0026] In steps (a) and (b), the SCF (stem cell factor) concentration is about 0.1 to 300 ng / mL, about 0.1 to 200 ng / mL, or about 0.1 to 150 ng / mL, the Flt-3L concentration is about 0.1 to 300 ng / mL, about 0.1 to 200 ng / mL, or about 0.1 to 150 ng / mL, the TPO (Thrombopoietin) concentration is about 1 to 300 ng / mL, about 10 to 200 ng / mL, or about 10 to 150 ng / mL, the IL-3 concentration is about 0.1 to 300 ng / mL, about 0.1 to 200 ng / mL, or about 0.1 to 100 ng / mL, and the IL-6 concentration is about 1 to The concentration of IL-7 may be about 0.1 to 200 ng / mL, about 0.1 to 100 ng / mL, or about 0.1 to 40 ng / mL, and the concentration of IL-15 may be about 0.1 to 200 ng / mL, about 0.1 to 100 ng / mL, or about 0.1 to 40 ng / mL.

[0027] Specifically, in the step (a), the SCF is 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 150 ng / mL, 1 to 100 ng / mL, 10 to 300 ng / mL, 10 to 200 ng / mL, 10 to 150 ng / mL, 10 to 100 ng / mL, 50 to 300 ng / mL, 50 to 200 ng / mL, 50 to 150 ng / mL, or 50 to 100 ng / mL, and the Flt-3L is 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 150 ng / mL, 1 to 100 ng / mL, 10 to 300 ng / mL, 10 to 200 ng / mL, 10 to 150 ng / mL, 10 to 100 ng / mL, 50 to 300 ng / mL, 50 to 200 ng / mL, 50 to 150 ng / mL, or 50 to 100 ng / mL, wherein the TPO is 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 150 ng / mL, 1 to 100 ng / mL, 10 to 300 ng / mL, 10 to 200 ng / mL, 10 to 150 ng / mL, 10 to 100 ng / mL, 50 to 300 ng / mL, 50 to 200 ng / mL, 50 to 150 ng / mL, or 50 to 100 ng / mL, IL-3 is 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 150 ng / mL, 1 to 100 ng / mL, 10 to 300 ng / mL, 10 to 200 ng / mL, 10 to 150 ng / mL, 10 to 100 ng / mL, 50 to 300 ng / mL, 50 to 200 ng / mL, 50 to 150 ng / mL, or 50 to 100 ng / mL, and IL-6 is 1 to 250 ng / mL, 1 to 150 ng / mL, 1 to 100 ng / mL, 10 to 250 ng / mL, 10 to 150 ng / mL, 10 to 100 ng / mL, 50 to 250 ng / mL,50 to 150 ng / mL, or 50 to 100 ng / mL, may be included in the hematopoietic stem cell culture medium and added or replaced once every 1 to 5 days.

[0028] Also specifically, in the step (b), the SCF is 0.1 to 300 ng / mL, 0.1 to 200 ng / mL, 0.1 to 100 ng / mL, 0.1 to 50 ng / mL, 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 10 to 300 ng / mL, 10 to 200 ng / mL, 10 to 100 ng / mL, or 10 to 50 ng / mL, and the Flt-3L is 0.1 to 300 ng / mL, 0.1 to 200 ng / mL, 0.1 to 100 ng / mL, 0.1 to 50 ng / mL, 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 5 to 300 ng / mL, 5 to 200 ng / mL, 5 to 100 ng / mL, 5 to 50 ng / mL, 10 to 200 ng / mL, 10 to 100 ng / mL, or 10 to 50 ng / mL, wherein the IL-3 is 0.1 to 250 ng / mL, 0.1 to 200 ng / mL, 0.1 to 150 ng / mL, 0.1 to 100 ng / mL, 0.1 to 50 ng / mL, 0.1 to 30 ng / mL, 0.1 to 10 ng / mL, 1 to 250 ng / mL, 1 to 200 ng / mL, 1 to 150 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 1 to 30 ng / mL, 1 to 10 ng / mL, 3 to 250 ng / mL, 3 to 200 ng / mL, 3 to 150 ng / mL, 3 to 100 ng / mL, 3 to 50 ng / mL, 3 to 30 ng / mL, or 3 to 10 ng / mL, wherein the IL-7 is 0.1 to 200 ng / mL, 0.1 to 100 ng / mL, 0.1 to 50 ng / mL, 0.1 to 40 ng / mL, 1 to 200 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 10 to 200 ng / mL, 10 to 100 ng / mL, 10 to 50 ng / mL, or 10 to 40 ng / mL, wherein the IL-15 is 0.1 to 200 ng / mL, 0.1 to 100 ng / mL, 0.1 to 50 ng / mL, 0.1 to 40 ng / mL, 1 to 200 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 5 to 200 ng / mL, 5 to 100 ng / mL, 5 to 50 ng / mL, 5 to 40 ng / mL, 5 to 20 ng / mL, 10 to 200 ng / mL, 10 to 100 ng / mL, or 10 to 50 ng / mL may be added or replaced once every 1 to 5 days by including the culture medium for differentiating hematopoietic stem cells into lymphoid progenitor cells.

[0029] In the step (c), the concentration of Flt-3L (FMS-like tyrosine kinase 3 ligand) may be about 1 to 250 ng / mL, about 10 to 150 ng / mL, or about 10 to 50 ng / mL, the concentration of IL-2 may be about 1 to 3000 IU / mL, about 10 to 1500 IU / mL, or about 10 to 500 IU / mL, the concentration of IL-15 may be about 1 to 200 ng / mL, about 10 to 100 ng / mL, or about 10 to 40 ng / mL, and the concentration of IL-21 may be about 1 to 300 ng / mL, about 10 to 200 ng / mL, or about 10 to 60 ng / mL.

[0030] Specifically, in the step (c), until the lymphocyte precursor cells differentiate into natural killer cells, the Flt-3L is 1 to 250 ng / mL, 1 to 150 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 1 to 30 ng / mL, 10 to 250 ng / mL, 10 to 150 ng / mL, 10 to 100 ng / mL, 10 to 50 ng / mL, or 10 to 30 ng / mL, and the IL-2 is 1 to 3000 IU / mL, 1 to 1500 IU / mL, 1 to 1000 IU / mL, 1 to 800 IU / mL, 1 to 500 IU / mL, 10 to 3000 IU / mL, 10 to 1500 IU / mL, 10 to 1000 IU / mL, 10 to 800 IU / mL, 10 to 500 IU / mL, 100 to 3000 IU / mL, 100 to 1500 IU / mL, 100 to 1000 IU / mL, 100 to 800 IU / mL, 100 to 500 IU / mL, 200 to 3000 IU / mL, 200 to 1500 IU / mL, 200 to 1000 IU / mL, 200 to 800 IU / mL, or 200 to 500 IU / mL, wherein the IL-15 is 1 to 200 ng / mL, 1 to 100 ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 5 to 200 ng / mL, 5 to 100 ng / mL, 5 to 50 ng / mL, 5 to 40 ng / mL, 10 to 200 ng / mL, 10 to 100 ng / mL, 10 to 50 ng / mL, or 10 to 40 ng / mL, wherein the IL-21 is 1 to 300 ng / mL, 1 to 200 ng / mL, 1 to 100 ng / mL, 1 to 60 ng / mL, 1 to 50 ng / mL, 10 to 300 ng / mL, 10 to 200 ng / mL, 10 to 100 ng / mL, 10 to 60 ng / mL,Alternatively, 10 to 50 ng / mL may be included in the culture medium and added or replaced once every 1 to 5 days or 1 to 3 days.

[0031] The above culture medium may be a mixture of the above cytokines according to each of steps (a), (b), and (c) in a conventional animal cell culture medium such as AIM-V media, RPMI1640, CellGro SCGM, CellGenix GMP SCGM, X-VIVO20, IMDM, DMEM, CTS NK-Xpander, or NK MACS.

[0032] The culture period of step (a) may be 1 to 15 days, the culture period of step (b) may be 5 to 30 days, and the culture period of step (c) may be 5 to 30 days. Specifically, the culture period of step (a) may be 1 to 15 days, 1 to 13 days, 1 to 10 days, 1 to 8 days, 3 to 15 days, 3 to 13 days, 3 to 10 days, 3 to 8 days, 5 to 15 days, 5 to 13 days, 5 to 10 days, or 5 to 8 days, the culture period of step (b) may be 5 to 30 days, 5 to 25 days, 5 to 20 days, 5 to 15 days, 10 to 30 days, 10 to 25 days, or 10 to 15 days, and the culture period of step (c) may be 5 to 30 days, 5 to 25 days, 5 to 20 days, 5 to 15 days, 10 to 30 days, 10 to 25 days, or 10 to 15 days.

[0033] The above lymphoid progenitor cells may be lymphoid progenitor cells, for example, multipotent progenitor cells, common lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T progenitor cells, or NK progenitor cells.

[0034] The above lymphocyte precursor cells may be differentiated into natural killer cells by expressing CD56 without growing into CD3, CD14 and / or CD19 cells under the above culture conditions.

[0035] The above culture medium may be characterized in that it does not contain feeder cells.

[0036] In this specification, the term "feeder cell" refers to a cell whose division and proliferation are generally inhibited through mitomycin C treatment or X-ray irradiation, and which is a cell that cannot divide or proliferate but has metabolic activity and produces various metabolites, thereby helping the proliferation of target cells. For example, a feeder cell refers to a cell that provides an extracellular matrix so that target cells can grow stably, a cell that helps the cell activation of target cells, a cell that produces various growth substances for target cells and thereby helps the proliferation of target cells, or a cell that presents a specific antibody through genetic manipulation. The above-mentioned culture auxiliary cells include all cells that are commonly used, for example, cells overexpressing human Delta-like ligand 4 (DLL-4), artificial antigen-presenting cells (aAPC) expressing mbIL21, and stromal cell lines such as OP9 cells, EL08-1D2 cells, and AFT024, but are not limited thereto.

[0037] The aAPC may be capable of activating and expanding natural killer cells. The aAPC may be engineered to express any gene for the expansion of natural killer cells. The aAPC may be a bead, a cell, a protein, an antibody, a cytokine, or any combination thereof. Since IL-21 activates natural killer cells, the aAPC may express membrane-bound IL-21 (mbIL21).

[0038] Another aspect provides natural killer cells manufactured by the above manufacturing method.

[0039] As used herein, the term "natural killer cell (NK cell)" refers to a lymphocyte cell that accounts for approximately 15% of peripheral blood lymphocytes and plays a crucial role in the innate immune response. Natural killer cells activate dendritic cells and induce cytotoxic T lymphocytes (CTLs) to specifically react with tumors, thereby eliminating tumor cells. Natural killer cells directly kill malignant tumors such as sarcoma, myeloma, carcinoma, lymphoma, and leukemia. Most natural killer cells present in the body of a normal person exist in an inactive state and are activated in response to interferon or macrophage-derived cytokines. NK cells can be detected in humans by specific surface markers such as CD16 and CD56.

[0040] In one specific example, the natural killer cells may express at least one selected from the group consisting of CD45+, CD56+, CD34-, CD3-, CD14-, CD19-, and combinations thereof. The CD45+, CD56+, CD34-, CD3-, CD14-, and CD19- are indicators on the surface of NK cells, and in one specific example, the expression of CD45+, CD56+, CD34-, CD3-, CD14-, CD19-, and combinations thereof was analyzed using flow cytometry to confirm that NK cells were produced.

[0041] Another aspect provides a method for producing CAR-NK cells, comprising additionally introducing a CAR gene into hematopoietic stem cells in step (a).

[0042] In one specific example, the CAR gene may be introduced once into hematopoietic stem cells. Specifically, the natural killer cells may be genetically modified to include a nucleic acid encoding a chimeric antigen receptor (CAR). The nucleic acid encoding the CAR can be introduced into the source cell by methods known in the art, such as γ-retroviral or lentiviral transduction, CRISPR-Cas9, TALEN, or ZFN-mediated gene editing (Themeli et al., Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy, Nat Biotechnol, 2013, 31(10): 928-33; Sadelain et al., Therapeutic T cell engineering, 2017, Nature, 545: 423-431; Eyquem et al., Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumor rejection, 2017, Nature, 543: 113-17). In addition, according to the CAR-NK cell manufacturing method according to the daily aspect, it was confirmed that even if CAR is introduced into hematopoietic stem cells only once, CAR is highly expressed and the yield of CAR-NK is high, so it is possible to mass-produce CAR-NK while reducing the CAR-NK process steps.

[0043] In the present specification, the term "chimeric antigen receptor" or "CAR" may have the following structures: (a) a first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain, (b) a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains, (c) a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains, and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful signaling of the CAR.

[0044] In one embodiment, the CAR comprises a CAR comprising an antigen binding domain, a transmembrane domain, and one or more signaling domains.

[0045] In one embodiment, the CAR further comprises one or more linkers. The format of an scFv is typically two variable domains joined by a flexible peptide sequence or 'linker' in a VH-linker-VL or VL-linker VH orientation.

[0046] In one embodiment, the antigen binding domain is selected from the group comprising antibodies, antigen binding fragments or fragments thereof, scFvs, and Fabs. In one embodiment, the antigen binding domain can bind to a ligand on a B cell. In one embodiment, the antigen binding domain can bind to CD19.

[0047] In one embodiment, the transmembrane domain comprises one selected from the group comprising a transmembrane region of TCRα, TCRβ, TCRζ, CD3ε, CD3γ, CD3δ, CD3ζ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40, CD40L / CD154, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD154, FcεRIγ, VEGFR2, FAS, FGFR2B, and functional variants thereof.

[0048] In one embodiment, the signaling domain(s) of the CAR comprise co-stimulatory domain(s). For example, the signaling domain may comprise a co-stimulatory domain. Alternatively, the signaling domain may comprise one or more co-stimulatory domains. In a specific embodiment, the signaling domain comprises a co-stimulatory domain. In some cases, when the CAR comprises two or more co-stimulatory domains, the two co-stimulatory domains may not be identical.

[0049] In one embodiment, the CAR comprises a CD3 zeta (CD3ζ) domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In one embodiment, the CAR can comprise (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain, or a 4-1BB domain, or a functional variant thereof. In one embodiment, the CAR comprises (i) an anti-CD19 scFv, (ii) a CD8α hinge and transmembrane domain or a functional variant thereof, (iii) a CD28 costimulatory domain or a functional variant thereof, and (iv) a CD3ζ signaling domain or a functional variant thereof.

[0050] In one embodiment, the CAR is a CD19 CAR, and in one embodiment, the polycistronic vector can comprise an expression cassette containing a nucleotide sequence encoding a CD19 CAR. In one embodiment, the CD19 CAR can comprise a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and / or an intracellular signaling domain in tandem.

[0051] In one embodiment, the signal peptide of the CD19 CAR can comprise a CD8α signal peptide. In one embodiment, the CD8α signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 1.

[0052] In one embodiment, the extracellular binding domain of the CD19 CAR is specific for CD19, e.g., human CD19. The extracellular binding domain of the CD19 CAR may be codon-optimized for expression in a host cell, or may be codon-optimized to include mutated sequences to enhance the function of the extracellular binding domain. In one embodiment, the extracellular binding domain comprises an immunogenic portion of an immunoglobulin molecule, e.g., scFv.

[0053] In one embodiment, the CD19-specific scFv can comprise a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 2. The light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 3.

[0054] In one embodiment, the hinge domain of the CD19 CAR can comprise a CD8α hinge domain, e.g., a human CD8α hinge domain. In one embodiment, the CD8α hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 4.

[0055] In one embodiment, the transmembrane domain of the CD19 CAR can comprise a CD8α transmembrane domain, e.g., a human CD8α transmembrane domain. In one embodiment, the CD8α transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 5.

[0056] In one embodiment, the intracellular costimulatory domain of the CD19 CAR can comprise a CD28 costimulatory domain, e.g., a human CD28 costimulatory domain. In one embodiment, the CD28 costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 6.

[0057] In one embodiment, the intracellular signaling domain of the CD19 CAR can comprise a CD3 zeta (ζ) signaling domain, e.g., a human CD3ζ signaling domain. In one embodiment, the CD3ζ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 7.

[0058] In one embodiment, the polycistronic vector can comprise an expression cassette containing a nucleotide sequence encoding a CD19 CAR, including a CD19 CAR comprising, for example, a CD19-specific scFv having a sequence set forth in SEQ ID NO: 2 and / or SEQ ID NO: 3, a CD8α hinge domain of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 5, a CD28 costimulatory domain of SEQ ID NO: 6, a CD3ζ signaling domain of SEQ ID NO: 7, and / or a variant thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, to the disclosed sequences). In any of these embodiments, the CD19 CAR may further comprise a signal peptide as described (e.g., a CD8α signal peptide, specifically a CD8α signal peptide of SEQ ID NO: 1).

[0059] In one embodiment, the CAR can target a tumor antigen. Exemplary tumor antigens include, but are not limited to, CD19, CD20, CD22, CD33 / IL3Ra, ROR1, mesothelin, c-Met, PSMA, PSCA, folate receptor alpha, folate receptor beta, EGFRvIII, GPC2, Tn-MUC1, GDNF family receptor alpha-4 (GFRa4), fibroblast activation protein (FAP), and IL13Ra2. In some cases, the tumor antigen can include CD19, CD20, CD22, BCMA, CD37, mesothelin, PSMA, PSCA, Tn-MUC1, EGFR, EGFRvIII, c-Met, HER1, HER2, CD33, CD133, GD2, GPC2, GPC3, NKG2D, KRAS, or WT1.

[0060] Another aspect provides a CAR-NK cell manufactured by the above CAR-NK cell manufacturing method.

[0061] In one specific example, the CAR-NK cell may express at least one selected from the group consisting of CD45+, CD56+, CD34-, CD3-, CD14-, CD19-, and combinations thereof. The CD45+, CD56+, CD34-, CD3-, CD14-, and CD19- are markers on the surface of NK cells, and in one specific example, the expression of CD45+, CD56+, CD34-, CD3-, CD14-, CD19-, and combinations thereof was analyzed using flow cytometry to confirm that NK cells were produced.

[0062] Another aspect provides a composition comprising the natural killer cells and / or the CAR-NK cells.

[0063] Another aspect provides a composition comprising a lentiviral vector comprising the CAR.

[0064] In one embodiment, the composition may further comprise one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In one embodiment, the composition may be used in a medicament used to treat a disease described herein (e.g., a cancer involving cells or tissues expressing a tumor antigen described herein, any malignancy, or an autoimmune disease). In one embodiment, the composition may be used in a method for treating a disease described herein (e.g., a cancer involving cells or tissues expressing a tumor antigen described herein, any malignancy, or an autoimmune disease). In one embodiment, the composition may be used in a medicament used to treat a disease associated with CD19 expression. In one embodiment, the composition may be used in a method for treating a disease associated with CD19 expression. In one embodiment, a pharmaceutical composition comprising natural killer cells and / or CAR-NK cells produced by the manufacturing process described herein is provided.

[0065] In one specific embodiment, the disease or condition may be selected from cancer, any malignancy, or an autoimmune disease comprising cells or tissues expressing a tumor antigen.

[0066] In this specification, the term "cancer" is a general term for a disease caused by cells that have aggressive characteristics in which cells divide and proliferate while ignoring normal growth limits, invasive characteristics in which cells infiltrate surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body, and is used with the same meaning as a malignant tumor.

[0067] The cancer may be selected from the group consisting of liver cancer, thyroid cancer, testicular cancer, bone cancer, glioblastoma, oral cancer, ovarian cancer, brain tumor, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, head and neck cancer, lymphoma, bladder cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, breast cancer, cervical cancer, prostate cancer, rectal cancer, spinal tumor, pancreatic cancer, salivary gland cancer, lung cancer, skin cancer, laryngeal cancer, and melanoma. The cancer may be a hematological cancer and / or a solid cancer.

[0068] In one specific example, the cancer is a hematological cancer selected from the group consisting of acute leukemia, chronic leukemia, hematological pathology, and combinations thereof. The cancer may also be, but is not limited to, B-cell acute lymphocytic leukemia ("BALL"), T-cell acute lymphocytic leukemia ("TALL"), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasma hairy lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, ineffective production (or dysplasia) of myeloid blood cells, and combinations thereof.

[0069] As used herein, the term “autoimmune disease” or “inflammatory disease” refers to any disease or disorder in which a subject initiates an immune response against its own tissues and / or cells. Autoimmune diseases can affect virtually any organ system of a subject (e.g., a human), including but not limited to diseases of the skin and other connective tissues, eyes, blood and blood vessels, as well as the nervous, gastrointestinal, and endocrine systems. Examples of autoimmune diseases include, but are not limited to, thyroiditis, systemic lupus erythematosus (SLE), Graves' disease, Sjogren's syndrome, systemic sclerosis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

[0070] In one embodiment, the autoimmune or inflammatory disorder is arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, deforming arthritis, chronic primaris polyarthritis, reactive arthritis, and ankylosing spondylitis), scleroderma (including systemic scleroderma), sclerosis (e.g., systemic sclerosis, multiple sclerosis (MS), MS associated with Epstein-Barr virus (EBV) infection, spino-optic MS, primary progressive MS (PPMS), relapsing-remitting MS (RRMS), progressive relapsing MS, secondary progressive MS (SPMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis and amacrine sclerosis), inflammatory bowel disease (IBD) including Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis and transmural colitis, and autoimmune inflammatory bowel disease), asthma (e.g., asthma bronchiale, bronchial asthma, and autoimmune asthma), lupus (lupus nephritis, lupus encephalitis, juvenile lupus, nonrenal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus [SLE], cutaneous SLE or subacute cutaneous SLE, neonatal neonatal lupus syndrome (NLE),and disseminated lupus erythematosus, type I diabetes, type II diabetes, and adult autoimmune diabetes (or type 1.5 diabetes), childhood-onset (type I) diabetes including insulin-dependent diabetes mellitus (IDDM), adult-onset diabetes (type II diabetes), idiopathic diabetes, back pain, diabetic retinopathy, diabetic neuropathy, and diabetic aortic disorders, vasculitis (including vasculitis, large-vessel vasculitis, polymyalgia rheumatica and large-cell (Takayasu) arteritis, medium-vessel vasculitis, Kawasaki disease, and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, such as systemic necrotizing vasculitis, and ANCA-associated vasculitis (e.g., Churg-Strauss vasculitis or syndrome (CSS) and ANCA associated small vessel vasculitis), CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes (e.g., secondary to sepsis, trauma, or hemorrhage), autoimmune endocrine disorders including primary hypothyroidism, hypoparathyroidism, thyroiditis, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis or subacute thyroiditis), autoimmune thyroid disease, idiopathic hypothyroidism, or Graves' disease.

[0071] In one embodiment, the term "disease associated with CD19 expression" may include any of the diseases listed above. For example, a proliferative disease such as a cancer or malignancy, or a precancerous disease such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or a disease associated with CD19 expression or a condition associated with cells expressing CD19, including a non-cancer-related indication associated with cells expressing CD19. In one embodiment, the cancer associated with CD19 expression is a hematological cancer. In one embodiment, the hematological cancer is leukemia or lymphoma. Additional diseases associated with CD19 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD19 expression. Non-cancer indications associated with CD19 expression may include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (e.g., allergies and asthma), and transplantation.

[0072] The above "treatment" refers to any action that improves or benefits the symptoms of cancer and / or autoimmune disease by administering the composition. In the case of cancer, it refers to inhibiting or preventing the growth of cancer cells or tissues, and this also includes reducing the growth and metastasis of cancer and reducing resistance to anticancer drugs to enhance the therapeutic effect compared to when no treatment or treatment is performed. The above cancer metastasis refers to the process in which tumor (cancer) cells spread to distant parts of the body, and "resistance to anticancer drugs" or "anticancer drug resistance" refers to the absence of a therapeutic effect from the beginning of treatment when treating a cancer patient using an anticancer drug, or the cancer treatment effect is lost during the course of continued treatment although the cancer treatment effect is initially effective. "Prevention" refers to any action that inhibits or delays the occurrence of cancer and / or autoimmune disease by administering the composition.

[0073] The above pharmaceutical composition can be used as a cell therapy agent.

[0074] In this specification, the term "cell therapy product" means a drug (as defined by the US FDA) used for the purposes of treatment, diagnosis, and prevention by isolating, culturing, and manufacturing cells and tissues from an individual through special manipulation, and by performing a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or by changing the biological characteristics of cells through other methods.

[0075] The above cell therapy composition may include natural killer cells manufactured according to a method for manufacturing natural killer cells according to one aspect and / or CAR-NK cells based on the manufactured natural killer cells, and may have a preventive or therapeutic effect on cancer and / or autoimmune disease.

[0076] Natural killer cells and / or CAR-NK cells, which are the active ingredients of the pharmaceutical composition, may be included in any amount (effective amount) depending on the intended use, formulation, compounding purpose, etc., as long as they can exhibit preventive and / or therapeutic activity against cancer and / or autoimmune disease. Here, the "effective amount" refers to the amount of the active ingredient that can induce an effect. Such an effective amount can be experimentally determined within the scope of a person skilled in the art. The pharmaceutical composition of the present invention may contain natural killer cells and / or CAR-NK cells as the active ingredient in an amount of about 0.1 wt% to about 90 wt%, specifically about 0.5 wt% to about 75 wt%, and more specifically about 1 wt% to about 50 wt%, based on the total weight of the composition.

[0077] Pharmacokinetic parameters, such as bioavailability, and underlying parameters, such as clearance rate, can also influence efficacy. Therefore, "enhanced efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and enhanced efficacy, and can be measured by comparing parameters such as clearance rate and the treatment or improvement of cancer and / or autoimmune diseases in test animals or human subjects.

[0078] The above pharmaceutical composition may comprise a conventional, non-toxic, pharmaceutically acceptable carrier formulated into a formulation according to a conventional method.

[0079] The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Examples of carriers include distilled water, alcohol, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0080] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as sweeteners, solubilizers, wetting agents, emulsifiers, isotonic agents, absorbents, antioxidants, preservatives, lubricants, fillers, buffers, and bacteriostatic agents may be added. More specifically, acceptable carriers, excipients or stabilizers are non-toxic to the subject at the dosages and concentrations employed and include buffers, such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers, such as polyvinylpyrrolidone, amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine, Monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as polysorbates (TWEEN™), poloxamers (PLURONICS™) or polyethylene glycol (PEG).In some embodiments, the pharmaceutical composition may include a pharmaceutically acceptable buffer (e.g., neutral buffered saline or phosphate buffered saline).

[0081] In one embodiment, the pharmaceutical composition may comprise one or more electrolyte alkaline solutions selected from the group consisting of Lactated Cryostar®, Ringer's solution, Plasma Lite A™, Iscove's modified Dulbecco's medium, Normosol-R™, Veen-D™, Polysal®, and Hank's balanced salt solution (phenol red free). These alkaline solutions closely resemble the composition of extracellular mammalian physiological fluids.

[0082] In one embodiment, the pharmaceutical composition may comprise one or more cryoprotectants selected from the group consisting of arabinogalactan, glycerol, polyvinylpyrrolidone (PVP), dextrose, dextran, trehalose, sucrose, raffinose, hydroxyethyl starch (HES), propylene glycol, human serum albumin (HSA), and dimethyl sulfoxide (DMSO).

[0083] The pharmaceutical composition described above can be prepared in various dosage forms for parenteral administration (e.g., intramuscular, intravenous, or subcutaneous injection). When the pharmaceutical composition is prepared in a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier and a method known in the art. Injectable preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be used, such as withepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. Meanwhile, injections can include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0084] Formulation of pharmaceutical compositions is well known in the art, and reference can be made to references such as Remington's Pharmaceutical Sciences (19th ed., 1995), which are incorporated herein by reference.

[0085] The pharmaceutical composition may be administered to a subject in a therapeutically effective amount or a pharmaceutically effective amount.

[0086] As used herein, the term "administration" means introducing a given substance into an individual by an appropriate method, and the route of administration of the composition may be any common route as long as it can reach the target tissue. Examples of such routes include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, and rectal administration.

[0087] The above "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and does not cause side effects. The level of the above effective amount may be determined based on factors including the patient's health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route and excretion rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. Specifically, the above therapeutically effective amount refers to an amount of a drug that is effective in treating cancer.

[0088] Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, sex, and weight. Specifically, the natural killer cells and / or CAR-NK cells may be administered in an amount of about 1×10 1 About 1×10 2 cells, approximately 1×10 1 About 1×10 3 cells, approximately 1×10 1 About 1×10 4 cells, approximately 1×10 1 About 1×10 5 cells, approximately 1×10 1 About 1×10 6 cells, approximately 1×10 1 About 1×10 7 cells, approximately 1×10 1 About 1×10 8 cells or about 1×10 1 About 1×10 9The cells may be administered daily or every other day, or divided into one to three times a day. However, the dosage may increase or decrease depending on the route of administration, severity of the disease, gender, weight, age, etc., and thus the scope of the present invention is not limited thereto. In one specific embodiment, the pharmaceutical composition may have a volume of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 ml. In one embodiment, a therapeutically effective or clinically effective amount of the pharmaceutical composition is about 1 x 10 in a volume of about 5 ml to 500 ml. 5 About 1 x 10 9 It may contain dog cells.

[0089] The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, administered sequentially or simultaneously with a conventional therapeutic agent, or administered singly or in multiple doses. In this case, the other therapeutic agent may be any compound, antibody, or natural extract known to be safe and active for enhancing or enhancing the preventive or therapeutic activity of cancer and / or autoimmune diseases. Taking all of the above factors into consideration, it is important to administer an amount that achieves maximum effect with minimal or no side effects, a dosage that can be readily determined by those skilled in the art.

[0090] The antibodies include Ocrelizumab, Olaratumab, Erlotinib, Panitumumab, Trastuzumab, Trastuzumab emtansine, Pertuzumab, Cetuximab, Rituximab, Bevacizumab, Axitinib, Lenvatinib, Ramucirumab, Aflibercept, Obinutuzumab, Daratumumab, Denosumab, Ibrutinib, Dasatinib, Radotinib, Nilotinib, Imatinib, Bosutinib, Galunisertib, Vactosertib, Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Regorafenib, Masitinib, Semaxanib, Ceritinib, Tivozanib, Brigatinib, Vandetanib, Pazopanib, Trametinib, Temsirolimus, Dabrafenib, Dacomitinib, Afatinib Lapatinib, Neratinib, Lenalidomide, Osimertinib, Ixazomib, Olmutinib, Everolimus, Ruxolitinib, Lestaurtinib, Pacritinib,Cobimetinib, Selumetinib, Binimetinib, Bortezomib, Alectinib, Crizotinib, Venetoclax, Bemcentinib, Gilteritinib, Selpercatinib, Pralsetinib, Vemurafenib, Olaparib, Talazoparib, Niraparib, Rucaparib, Azacitidine, Decitabine, Guadecitabine, Gefitinib, Abemaciclib It may be any one selected from the group consisting of Ribociclib, Palbociclib and DMXAA.

[0091] The above "subject" may be, but is not limited to, a mammal that has developed or may develop cancer and / or an autoimmune disease. Preferably, it may be a human.

[0092] Another aspect provides the use of a pharmaceutical composition comprising natural killer cells and / or CAR-NK cells as an active ingredient for preparing a medicament for the prevention or treatment of cancer.

[0093] Another aspect provides the use of a pharmaceutical composition comprising natural killer cells and / or CAR-NK cells as an active ingredient for preparing a medicament for the prevention or treatment of an autoimmune disease.

[0094] Natural killer cells, CAR-NK cells, pharmaceutical compositions, cancer, autoimmune diseases, prevention and treatment are the same as described above.

[0095] Another aspect provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition comprising natural killer cells and / or CAR-NK cells as active ingredients.

[0096] Another aspect provides a method for preventing or treating an autoimmune disease, comprising administering to a subject a pharmaceutical composition comprising natural killer cells and / or CAR-NK cells as an active ingredient.

[0097] Natural killer cells, CAR-NK cells, pharmaceutical compositions, cancer, autoimmune diseases, prevention, treatment, and administration are the same as described above.

[0098] Another aspect provides a kit for preventing or treating cancer comprising natural killer cells and / or CAR-NK cells.

[0099] Another aspect provides a kit for preventing or treating autoimmune diseases comprising natural killer cells and / or CAR-NK cells.

[0100] Natural killer cells, CAR-NK cells, cancer, autoimmune diseases, prevention and treatment are the same as described above.

[0101] The above kit is not particularly limited in type, and a kit of a type commonly used in the relevant technical field can be used.

[0102] The above kit may be packaged with natural killer cells and / or CAR-NK cells contained in individual containers, or in a single container divided into one or more compartments. Additionally, the natural killer cells and / or CAR-NK cells may be packaged in a unit dose form for a single administration, but is not limited thereto.

[0103] [Definition of Terms]

[0104] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of certain embodiments, suitable methods and materials are described herein. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0106] Implementation according to one embodiment uses conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art, unless otherwise indicated.

[0107] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one aspect, the term "about" or "approximately" refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0108] As used herein, the terms "substantially" or "essentially" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more as compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one aspect, the terms "essentially the same" or "substantially the same" refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0109] The method for producing hematopoietic stem cell-derived natural killer cells and / or CAR-NK cells according to the present invention can produce natural killer cells and / or CAR-NK cells with a high cell growth rate using a simple combination of cytokines, and natural killer cells and / or CAR-NK cells produced according to the production method of the present invention have higher cytotoxicity compared to those produced by conventional methods.

[0110] In addition, the method for producing natural killer cells and / or CAR-NK cells derived from hematopoietic stem cells according to the present invention exhibits a high cell growth rate without using cultured feeder cells. Since cultured feeder cells are impurities that should not be included in the final natural killer cell therapeutic agent, it is very advantageous in that there is no need for a separate process for removing cultured feeder cells.

[0111] Natural killer cells and / or CAR-NK cells manufactured according to the manufacturing method of the present invention exhibit high cytotoxicity and thus exhibit superior cancer and / or autoimmune disease prevention and / or treatment effects.

[0112] Figure 1 is a schematic diagram showing a method for producing natural killer cells (NK cells) derived from hematopoietic stem cells (HSCs) and NK cells introduced with CARs.

[0113] Figure 2 is a photograph taken with an optical microscope of cells at the HSC proliferation and common lymphoid progenitor (CLP) expansion stages during the process of differentiating HSCs into NK cells.

[0114] Figure 3 is a graph showing the growth rate (a), survival rate (b), and number of CD56 positive cells (c) of NK cells during differentiation of non-transduced NK cells (Non-Transduction (NT) NK cells).

[0115] Figure 4 shows raw data (a) and a graph (b) of the distribution of CD45+CD56+ cells confirmed through flow cytometry to confirm the purity of differentiated NK cells.

[0116] Figure 5 shows raw data (a) and a graph (b) of the distribution of CD45+CD34+, CD45+CD3+, CD45+CD14+, and CD45+CD19+ cells confirmed through flow cytometry to confirm the purity of differentiated NK cells.

[0117] Figure 6 shows raw data (a) and graph (b) confirming the expression of NK cell receptors through flow cytometry after completion of NK cell differentiation.

[0118] Figure 7 shows FACS raw data (a), a table showing the numbers (b), and a graph (c) that confirm the cytotoxicity of differentiated NK cells against K562 cells, a blood cancer cell line, according to the E:T ratio.

[0119] Figure 8 shows FACS raw data (a), a table showing the numbers (b), and a graph (c) that confirm the cytotoxicity of differentiated NK cells against MDA-MB-453 cells, a HER2-positive breast cancer cell line, according to the E:T ratio.

[0120] Figure 9 is a bar graph measuring the change in body weight of NSG mice by group during the tumor size observation period.

[0121] Figure 10 is a graph showing tumor size observed by group from the time of tumor transplantation.

[0122] Figure 11 shows photos of tumors formed by group.

[0123] Figure 12 is a graph showing the measured values ​​of tumor weights formed by each group.

[0124] Figure 13 shows FACS raw data (a), a table showing the numbers (b), and a graph (c) analyzing the cell death induction ability according to the E:T ratio after co-treatment with the monoclonal anti-CD20 antibody Rituximab on the Raji cell line of differentiated NK cells.

[0125] Figure 14 shows FACS raw data (a), a table (b) showing the numbers, and a graph (c) analyzing the apoptosis induction ability according to the E:T ratio after co-treatment with the monoclonal anti-CD20 antibody Rituximab on a cell group from which CD56 and CD16 positive cells were removed from peripheral blood mononuclear cells (SLE PBMCs) derived from systemic lupus erythematosus (SLE) patients with differentiated NK cells.

[0126] Figure 15 shows the structure of CAR introduced into NK cells. Signal peptide (S) - scFv (V H -G4S Linker-V L ) - CD8 Hinge - CD8 Transmembrane (TM) - CD28 co-stimulatory (CS) - CD3ζ signaling

[0127] Figure 16 is an optical microscope photograph of cells at the HSC proliferation and common lymphoid progenitor (CLP) expansion stages during the process of differentiating HSCs into anti-CD19 CAR-NK cells.

[0128] Figure 17 is a graph showing the growth rate, survival rate, and number of CD56 positive cells of CAR-NK cells during differentiation of anti-CD19 CAR-NK cells.

[0129] Figure 18 is a graph and raw data showing the distribution of CD45+CD56+ cells through flow cytometry analysis to confirm the purity of differentiated anti-CD19 CAR-NK cells.

[0130] Figure 19 is a graph and raw data showing the distribution of CD45+CD34+, CD45+CD3+, CD45+CD14+, and CD45+CD19+ cells through flow cytometry analysis to confirm the purity of differentiated anti-CD19 CAR-NK cells.

[0131] Figure 20 shows raw data (a) and graph (b) confirming the expression of CAR-NK cell receptors through flow cytometry after completion of anti-CD19 CAR-NK cell differentiation.

[0132] Figure 21 shows flow cytometry raw data (a) and graph (b) confirming CD19 CAR expression during anti-CD19 CAR-NK cell differentiation.

[0133] Figure 22 shows FACS raw data (a), a table (b) showing the numbers, and a graph (c) confirming the cytotoxicity of differentiated anti-CD19 CAR-NK cells against CD19 positive Raji cell lines according to the E:T ratio.

[0134] Figure 23 shows FACS raw data (a), a table showing the numbers (b), and a graph (c) confirming the cytotoxicity of differentiated anti-CD19 CAR-NK cells against peripheral blood mononuclear cells (PBMCs) derived from systemic lupus erythematosus (SLE) patients according to the E:T ratio.

[0135] Figure 24 is FACS raw data (a) and graph (b) confirming the cytotoxicity of differentiated anti-CD19 CAR-NK cells against various immune cells according to the E:T ratio.

[0136] The present invention will be described in more detail below through examples. These examples are merely illustrative examples to more specifically illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0137]

[0138] Example 1. Production of natural killer cells (NK cells) and CAR-NK cells

[0139] 1-1. Isolation and flow cytometric analysis of CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood.

[0140] To isolate CD34+ hepatocyte stem cells (HSCs) from umbilical cord blood, the Ficoll-Paque method using SepMate™ (Stemcell) tubes was used. CD34+ HSCs were isolated from the separated buffy coat using the MACSprep kit (Miltenyi). Cells isolated from the kit were analyzed by flow cytometry using anti-CD34-PE (Biolegend), confirming a CD34+ HSC isolation rate of over 90%.

[0141] 1-2. Differentiation of HSCs into NK cells

[0142] The culture medium used in steps (a), (b), and (c) below can be a conventional animal cell culture medium such as AIM-V media, RPMI1640, CellGro SCGM, CellGenix GMP SCGM, X-VIVO20, IMDM, DMEM, CTS NK-Xpander, and NK MACS, and cytokines can be added thereto. A glutamine supplement may be used as needed, and may include glutamine or a stabilized derivative thereof, such as a dipeptide glutamine substitute such as alanyl glutamine or glycyl glutamine, and these materials may be provided as commercial products, but are not limited thereto. The culture medium of the present invention may include various blood-derived supplements, and may include, for example, human serum derived from autologous or allogeneic donors, platelet lysate, human plasma, fibrinogen-depleted serum, conditioned serum, or a pharmaceutically acceptable combination thereof.

[0143] (a) Day 0~7 (Lentivirus transduction): 1 X 10 were cultured in cell culture vessels including well plates and flasks using CellGenix GMP SCGM medium and cytokines SCF, TPO, Flt-3L, IL-3, and IL-6. 5 Cells were added at a concentration of 1 x 10 cells / ml and cultured for 4 days. On the 4th day, cells were harvested and the cell concentration was adjusted to 1 x 10 5Cells were reseeded to 10 cells / ml. When producing normal natural killer cells, nothing was processed other than the culture medium, and when producing CAR-NK cells, a mock or anti-CD19 CAR gene-containing lentivirus was introduced once. Gene transduction enhancers can be used for gene introduction, and include, but are not limited to, polybrene, retronectin, and lentiboost. The cells were then cultured for 3 days, and the cell growth rate and viability were measured on days 4 and 7 when the cells were obtained. When producing CAR-NK cells, CAR expression was confirmed on day 7. The concentrations of each cytokine added during culture were as follows: SCF 100 ng / ml, TPO 100 ng / ml, Flt-3L 100 ng / ml, IL-3 100 ng / ml, and IL-6 100 ng / ml.

[0144] (b) Day 7~21: This period is the proliferation period of Common Lymphoid Progenitor (CLP), and the cells were cultured in CellGenix GMP SCGM medium containing cytokines SCF, Flt-3L, IL-3, IL-7, and IL-15. The coating mixture (DLL4 + Fibronectin) was first added to cell culture vessels including well plates and flasks, and incubated at room temperature for 2 hours. After 2 hours, the cells were washed with DPBS and 1.95 X 10 5 Cells were cultured for 14 days in a cell / flask configuration. Cytokines were supplemented by half media change or medium addition on days 10, 14, and 17 of culture. The concentrations of each cytokine added during culture were as follows: SCF 20 ng / ml, Flt-3L 10 ng / ml, IL-3 5 ng / ml, IL-7 20 ng / ml, and IL-15 10 ng / ml.

[0145] (c) Day 21~35: This period is the period for NK cell differentiation, and the cells were cultured in NK MACS medium containing Flt-3L, IL-2, IL-15, and IL-21 cytokines. On day 21, cells were harvested, washed with DPBS, and then harvested using CTS TrypLE and placed in a 50 mL conical tube. The solution containing all cells obtained through the process was centrifuged at 400 g for 5 minutes. Afterwards, the supernatant was removed and cultured in T flak (25T ~175T) using the above culture medium. Half media was changed or medium was added on days 25, 28, and 32 of culture, and cytokines were added on days 23 and 30 of culture. On day 35, cells were harvested and used as intended or cryopreserved. The growth rate and viability of cells were measured on days 21 and 35 of cell harvesting. CAR expression was confirmed on day 35 during CAR-NK production. The concentrations of each cytokine added during culture were as follows: Flt-3L 25 ng / ml, IL-2 500 IU / ml, IL-15 20 ng / ml, and IL-21 30 ng / ml.

[0146] Cell photographs were taken on days 4, 7, 10, 14, 17, and 21 of culture during the HSC proliferation and CLP expansion period and are shown in Fig. 2. In addition, cell growth rate, viability, and CD56-positive cell count were measured on days 7, 21, and 35 during the differentiation period from HSC to NK cells and are shown in Figs. 3a, 3b, and 3c, respectively. Upon completion of NK differentiation, NK cells were confirmed to have proliferated more than 80,000-fold, cell viability was confirmed to be 83%, and the number of CD56-positive cells was 4 X 10 10 It was confirmed that there were more than one dog.

[0147]

[0148] Experimental Example 1. Confirmation of marker expression at the differentiation stage into natural killer cells.

[0149] Differentiation into natural killer cells (NK cells) was induced from hematopoietic stem cells (HSCs) for 35 days, and the results were analyzed using FACSLyric using antibodies that can confirm the purity and anti-human CD45 (eFluor™ 450, IgG1, κ, Invitrogen), anti-human CD3 (PerCP-Cy5.5, IgG1, κ, BD), anti-human CD14 (APC-eFluor™ 780, IgG1, κ, Invitrogen), anti-human CD19 (APC, IgG1, κ, BD), anti-human CD34 (FITC, IgG1, κ, BD), anti-human CD56 (Alexa488, IgG1, κ, BD), anti-CD16 (PE-Cy7, IgG1, κ, BD) at 21 and 35 days after the start of differentiation. TM It was confirmed with a Flow Cytometer (BD). In addition, the values ​​of each indicator that were nonspecifically bound to cells were excluded and analyzed by comparing them using appropriate isotype antibodies (eFluor™ 450, Mouse IgG1, κ Isotype Control, Invitrogen), (PerCP-Cy5.5, Mouse IgG1, κ Isotype Control, BD), (APC-eFluor™ 780, Mouse IgG1, κ Isotype Control, Invitrogen), (APC, Mouse IgG1, κ Isotype Control, BD), (PE, Mouse IgG1, κ, BD), (FITC, Mouse IgG1, κ Isotype Control, BD), (Alexa Fluor 488, Mouse IgG1, κ Isotype Control, BD), (PE-Cy7, Mouse IgG1, κ Isotype Control, BD) and Fixable Viability Dye eFluor™ 506.

[0150] The results are shown in Figs. 4 and 5. On the 21st day of culture, the CD45+CD56+ cell ratio was 51.7%, and after differentiation was completed (on the 35th day of culture), the CD45+CD56+ cell ratio was confirmed to be over 99% (Fig. 4). In addition, the ratios of CD34, a hematopoietic stem cell (HSC) marker, CD3, a T cell marker, CD14, a macrophage marker, and CD19, a B cell marker, were confirmed to be less than 1% on the 21st and 35th days of culture, respectively (Fig. 5). This indicates that NK cells were differentiated with high purity.

[0151]

[0152] Experimental Example 2. Confirmation of receptor expression after differentiation into natural killer cells.

[0153] To analyze the receptor expression rate of differentiated natural killer cells, anti-CD56 (Alexa Fluor488; BD), anti-CD45 (eFluor 450, eBioscience), anti-DNAM1 (APC, Biolegend), anti-NKp44 (PE, Biolegend), anti-NKp46 (APC, Biolegend), anti-NKp30 (PE, BD), anti-2B4 (APC, Biolegend), anti-NKG2D (PE-Cy7, BD), anti-CD16 (PE-Cy7, BD), anti-NKG2A (APC, Biolegend), and fixed viability dye (eFluor 506, eBioscience) antibody staining was performed. Nonspecific binding was excluded using the isotype corresponding to each fluorescence (eFluor™ 450, Mouse IgG1, κ Isotype Control, Invitrogen), (Alexa Fluor 488, Mouse IgG1, κ Isotype Control, BD), (APC, Mouse IgG1, κ Isotype Control, BD), (PE, Mouse IgG1, κ, BD), (PE-Cy7, Mouse IgG1, κ Isotype Control, BD).

[0154] The results are shown in Figure 6, and the expression rates of NK activating receptors CD56, DNAM-1, NKp44, NKp46, NKp30, 2B4, NKG2D, CD16 and NK inhibitory receptor NKG2A were confirmed to be 99.2%, 95.2%, 28%, 93.4%, 87.9%, 99.6%, 28.1%, 28.3% and 32.3%, respectively.

[0155]

[0156] Experimental Example 3. Confirmation of the cytotoxicity of natural killer cells against cancer cells.

[0157] Cancer cell lines (K562, MDA-MB-453: ATCC) as target cells were seeded at 4 x 10 6 Prepare cells / 5 mL and stain with CFSE (Carboxyfluorescein succinimidyl ester) (Invitrogen) for 15 minutes at 37°C in a 5% CO2 incubator. After staining, the cells were washed and diluted with 10 mL of RPMI (Gibco) to obtain a cancer cell line (K562, MDA-MB-453) cell concentration of 4 x 10 5 cells / mL. Natural killer cells derived from hematopoietic stem cells (HSCs) were recovered, and 2 x 10 for each hematologic malignancy cell line (K562) and breast cancer cell line (MDA-MB-453) were added to achieve an E:T ratio of 5:1 and 10:1, respectively. 6 cells, 4 x 10 6 The cells were transferred to a 50 mL conical tube. The supernatant was removed by centrifugation and RPMI (Gibco) medium was added. Additional medium was added to prepare the cell number corresponding to the required E:T ratio. Cancer cells (Target) and natural killer cells (Effector) were dispensed together in a 96-well plate (Corning) at a ratio corresponding to each E:T ratio, excluding the fraction showing spontaneous killing of cancer cells (Target) over time. The 96-well plate was reacted for 2 hours in a 37°C, 5% CO2 incubator. The 96-well plate after the reaction was completed was stained with 7-AAD (7-aminoactinomycin D) (BD) for 5 minutes at room temperature.

[0158] FACS data and normalized cytotoxicity results analyzing the anticancer effect through cytotoxicity at ratios of 1:1, 2:1, and 5:1 in natural killer cells and blood cancer cell line (K562) and at ratios of 1:1, 2:1, 5:1, and 10:1 in breast cancer cell line (MDA-MB-453) are shown in Figures 7 and 8 (Normalized cytotoxicity = FACS raw cytotoxicity - Target spontaneous).

[0159] The cytotoxicity of NK cells against K562 was confirmed to be 45% at 1:1, 64% at 2:1, and 80% at 5:1, depending on the E:T ratio (Fig. 7), and the cytotoxicity of NK cells against MDA-MB-453 was confirmed to be 45% at 1:1, 60% at 2:1, 68% at 5:1, and 84% at 10:1, depending on the E:T ratio (Fig. 8). Through this, it was confirmed that NK cells manufactured through the process according to one embodiment exhibited high cytotoxicity.

[0160]

[0161] Experimental Example 4. In vivo efficacy evaluation of hematopoietic stem cell-derived natural killer cells in a tumor xenograft animal model derived from a breast cancer cell line (MDA-MB-453)

[0162] Mouse care and observation were conducted in accordance with the Animal Ethics Committee guidelines, and in vivo efficacy evaluation was conducted after preparing an animal experiment plan and receiving approval from the Animal Experiment Ethics Committee (EUMC-IACUC-24-001-1 and EUMC-IACUC-24-001-2).

[0163] 5 x 10 HER2-expressing breast cancer cell line MDA-MB-453 6 Prepare cells, mix 4:1 with Matrigel (Corning 354234) and inject a total of 0.2 mL into 6-week-old NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ, The Jackson Laboratory) was injected subcutaneously into the right flank using a 1 mL syringe. Trastuzumab (Selleckchem) and IL-2 (peprotech) were prepared according to the manufacturer's instructions. Tumors measuring 100 mm 3 150 mm in diameter 3 At the time, intraperitoneal administration of trastuzumab at a concentration of 0.01 mg / kg twice a week and IL-2 at a concentration of 20,000 IU three times a week was started. In the case of hematopoietic stem cell-derived natural killer cells, 1x10 6 The cells were administered intravenously twice a week for a total of 5 weeks. After tumor transplantation, the long and short axes of each tumor were measured three times a week using a caliper (Mitutoyo 500-180-30), and the tumor size was calculated using the Ellipsoidal formula [long axis x (short axis)] 2 / 2] was calculated. Statistically, analysis was conducted by performing a t-test on the mean and standard deviation for all evaluation items per experimental group, and a p value less than 0.05 was determined to be a statistically significant difference.

[0164] As a result, as shown in Figures 9 to 12, not only was there no change in body weight between the experimental groups, but in the case of single administration of hematopoietic stem cell-derived natural killer cells, the tumor size was significantly reduced by about 44% compared to the vehicle, and when combined with trastuzumab, the tumor size was significantly reduced by about 60% compared to the vehicle, confirming a synergistic effect.

[0165]

[0166] Experimental Example 5. Confirmation of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of Natural Killer Cells Against Raji Cell Lines

[0167] Raji cells (Target (T)) were stained with Violet proliferation dye 450 (BD) after serum removal, and treated with Control IgG and anti-CD20 antibody Rituximab at a concentration of 5 μg / mL each. For each condition, cells were dispensed into FACS tubes at 8 × 10⁴ cells / 100 μL and pretreated for 1 hour in a 37°C, 5% CO₂ incubator. Effector (E) cells were hematopoietic stem cell (HSC)-derived NK cells differentiated for 35 days, and the cell concentration was 4 × 10⁴ using Complete medium (Gibco) at an E:T ratio of 5:1. 6 cells / mL. After that, NK cells were serially diluted with E:T ratios of 2:1, 1:1, and 0.5:1 using the medium, and 100 μL of each NK cell and 100 μL of target cells were mixed and co-cultured for 24 hours. After the culture was completed, cells were collected and 7-AAD staining was performed, and the degree of apoptosis was quantified by measuring the ratio of 7-AAD-positive cells within the Violet dye-positive group using a FACS LyricTM Flow Cytometer (BD). The analysis program was FlowJo. TM Software (BD) was used, and experiments were conducted in triplicate for each group. Statistical significance was compared between means (P<0.05) using an unpaired Student's t-test, and data were expressed as mean ± standard deviation (mean ± SD).

[0168] FACS data and normalized cytotoxicity results analyzing the anticancer effect through antibody-dependent cytotoxicity at ratios of 0.5:1, 1:1, and 2:1 of natural killer cells and Raji cells are shown in Figure 13 (Normalized cytotoxicity = FACS raw cytotoxicity - Target spontaneous).

[0169] In both rituximab combination groups, the apoptosis induction rate significantly increased in proportion to the E:T ratio compared to the IgG control group, and in particular, under the condition of E:T = 2:1, the rituximab combination group induced an average of 58.7% apoptosis, which was approximately 22% higher than the control group, confirming that hematopoietic stem cell-derived NK cells exhibit potent antibody-dependent cytotoxicity (ADCC) activity against Raji cells.

[0170]

[0171] Experimental Example 6. Confirmation of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of Natural Killer Cells on Peripheral Blood Mononuclear Cells (NK-Depleted SLE PBMCs) from Patients with CD56+ / CD16+ Absence

[0172] Isolation buffer (0.1% BSA) was prepared using MACS rinsing solution (Miltenyi biotec) and BSA solution (Miltenyi biotec). Dynabeads® Pan Mouse IgG were washed and prepared using a magnet (Invitrogen) and the prepared isolation buffer. SLE PBMC (STEMCELL) isolated for use as target cells were purified using anti-human CD56 (Clone MY31, Cat. No. 347740, BD Biosciences) and LEAF™ purified anti-human CD16 (Clone 3G8, Cat. No. 302057, Biolegend) at a density of 1 x 10 SLE PBMC. 7 5 μg per cell was stained and reacted with the prepared Dynabeads® Pan Mouse IgG. After the reaction was completed, a magnet was used to detect CD56 + / CD16 +SLE PBMCs (NK depleted SLE PBMCs) were collected, and the serum remaining in the medium was removed. Target cells were divided into three groups after staining with Violet proliferation dye 450 (BD), and treated with Control IgG and Rituximab at a final concentration of 5 μg / mL each. Hematopoietic stem cell (HSC)-derived natural killer cells were collected for use as effector cells, and the cell concentration was 4 x 10 at an E:T ratio of 5:1 in Serum Free Medium (Gibco). 6 / mL and diluted sequentially to 5:1, 1:1, and 0:1 based on the E:T ratio. Then, 100 μL of the prepared target cells and effector cells were dispensed and co-cultured for 24 hours in a 5% CO2 incubator. To evaluate antibody-dependent cytotoxicity, co-cultured cells were collected and stained with anti-human CD19 (APC, IgG1, κ, BD) and analyzed by FACSLyric TM The CD19 positivity rate was measured in the Violet proliferation 450 positive group using a Flow Cytometer (BD). The analysis program was FlowJo. TM Software (BD) was used, and experiments were conducted in triplicate for each group. Statistical significance was compared between means (P<0.05) using an unpaired Student's t-test, and data were expressed as mean ± standard deviation (mean ± SD).

[0173] FACS data and normalized cytotoxicity results analyzing the anticancer effect through antibody-dependent cytotoxicity at ratios of 0:1, 1:1, and 5:1 of natural killer cells and systemic lupus erythematosus (SLE) patient-derived peripheral blood mononuclear cells (SLE PBMC) cells are shown in Figure 14 (Normalized cytotoxicity = FACS raw cytotoxicity - Target spontaneous).

[0174] Compared to the control group (IgG), the rituximab combination group showed a gradual depletion of B cells as the E:T ratio increased (0:1, 1:1, 5:1). In particular, at an E:T ratio of 5:1, the rituximab combination group showed a decrease of approximately 37%, confirming that hematopoietic stem cell-derived NK cells exhibit potent antibody-dependent cellular cytotoxicity (ADCC) activity against PBMCs derived from SLE patients.

[0175]

[0176] Experimental Example 7. Confirmation of anti-CD19 CAR-NK cell differentiation, NK cell marker, receptor expression, and CAR expression.

[0177] 7-1. Confirmation of anti-CD19 CAR-NK cell differentiation

[0178] The CAR structure of the anti-CD19 CAR-NK is shown in Figure 15, and the sequence of the CAR is shown in SEQ ID NOs: 1 to 7.

[0179] Cell photographs were taken on days 4, 7, 10, 14, 17, and 21 of culture during the HSC proliferation and CLP expansion period during the production of anti-CD19 CAR-NK, and are shown in Figure 16. It was confirmed that there was no difference in cell shape between each group.

[0180] In addition, the cell growth rate, viability, and number of CD56-positive cells were measured on days 7, 21, and 35 during the differentiation period from HSC to anti-CD19 CAR-NK cells, and are shown in Fig. 17. Upon completion of differentiation, Mock-NK cells were confirmed to have proliferated more than 43,000-fold, and anti-CD19 CAR-NK cells were confirmed to have proliferated more than 53,000-fold. The cell viability was confirmed to be more than 80% for Mock-NK and more than 76% for anti-CD19 CAR-NK, and the number of CD56-positive cells was 4 X 10 for Mock-NK. 9 More than 5 x 10 anti-CD19 CAR-NKs 9 It was confirmed that there were more than one dog.

[0181] Through this, it was confirmed that there was no effect on natural killer cell differentiation ability due to CAR introduction.

[0182] 7-2. Confirmation of NK cell marker expression

[0183] The NK cell markers of differentiated anti-CD19 CAR-NK cells were confirmed by the same experiment as in Experimental Example 1.

[0184] As a result, as shown in Fig. 18, it was confirmed that the proportion of CD45+CD56+ cells was 90% or more in all groups after culture completion, and as shown in Fig. 19, it was confirmed that the proportion of CD34, a hematopoietic stem cell (HSC) marker, CD3, a T cell marker, CD14, a macrophage marker, and CD19, a B cell marker, was 1% or less in all groups after culture completion. Through these results, it was confirmed that the introduction of anti-CD19 CAR did not affect the purity of NK cells.

[0185] 7-3. Confirmation of NK cell receptor expression

[0186] The NK cell markers of differentiated anti-CD19 CAR-NK cells were confirmed by the same experiment as in Experimental Example 2.

[0187] As a result, as shown in Figure 20, DNAM-1, an NK activating receptor, was 95.4% (Mock), 94.8% (anti-CD19 CAR), NKp44 was 79.6% (Mock), 79.7% (anti-CD19 CAR), NKp46 was 93% (Mock), 94.6% (anti-CD19 CAR), NKp30 was 50.8% (Mock), 59% (anti-CD19 CAR), 2B4 was 98.7% (Mock), 98.6% (anti-CD19 CAR), NKG2D was 24.2% (Mock), 24.6% (anti-CD19 CAR), CD16 was 41% (Mock), 44% (anti-CD19 CAR), and NKG2A, an NK inhibitory receptor, was 66.6% (Mock), 61.9% (anti-CD19 CAR). The expression ratio of CD19 CAR was shown. Through these results, it was confirmed that there was no significant effect on NK receptor expression due to CAR introduction.

[0188] 7-4. Confirmation of CAR expression

[0189] To confirm anti-CD19 CAR expression, anti-FMC63 antibody (PE, Acrobiosystems) was used, and nonspecific binding was excluded using the corresponding isotype. After staining, FACSLyric TM Flow cytometry was performed using a Flow Cytometer (BD).

[0190] As a result, as shown in Figure 21, it was confirmed that the anti-CD19 CAR expression rate was 35.6% on Day 3 of hematopoietic stem cell proliferation after CAR introduction (7 days after total hematopoietic stem cell proliferation), and the anti-CD19 CAR expression rate was 26.5% upon completion of NK differentiation (Day 35).

[0191]

[0192] Experimental Example 8. Cytotoxicity of anti-CD19 CAR-NK cells against Raji cell lines

[0193] Raji cell line was prepared as target and stained with Violet proliferation dye 450 (BD). Mock, anti-CD19 CAR, and hematopoietic stem cell (HSC)-derived natural killer (NK) cells were collected for use as effector cells and cultured in Complete Medium (Gibco) at an E:T ratio of 5:1 and a cell concentration of 4 x 10 6 / mL and diluted sequentially to 5:1, 2:1, 1:1, 0.1:1, and 0:1 based on the E:T ratio. 100 μL of the prepared target and effector cells were dispensed and co-cultured for 4 hours in a 5% CO2 incubator. To evaluate cytotoxicity, co-cultured cells were collected and stained with 7-AAD. FACS Lyric TM The 7-AAD positivity rate was measured in the Violet proliferation 450 positive group using a Flow Cytometer (BD). The analysis program was FlowJo. TM Software (BD) was used, and experiments were conducted in triplicate for each group. Statistical significance was compared between means (P<0.05) using an unpaired Student's t-test, and data were expressed as mean ± standard deviation (mean ± SD).

[0194] As a result, as shown in Fig. 22, under the condition of an E:T ratio of 5:1 in Mock NK cells, the average cytotoxicity was approximately 55%, and under the same condition, anti-CD19 CAR-NK cells induced 65.6% cell death, demonstrating higher cytotoxicity than Mock NK cells. This means that anti-CD19 CAR-NK cells according to one embodiment exhibit high cytotoxic activity against the Raji cell line.

[0195]

[0196] Experimental Example 9. Cytotoxicity of anti-CD19 CAR natural killer cells against peripheral blood mononuclear cells (SLE PBMCs) derived from systemic lupus erythematosus (SLE) patients.

[0197] Peripheral blood mononuclear cells (PBMCs) derived from systemic lupus erythematosus (SLE) patients were isolated from the blood of SLE patients donated by Konkuk University Hospital (IRB approval number: 2024-04-017) for use as target cells and stained with Violet proliferation dye 450 (BD). Mock, anti-CD19 CAR hematopoietic stem cell (HSC)-derived natural killer (NK) cells were collected for use as effector cells and cultured in Complete Medium (Gibco) at an E:T ratio of 5:1 with a cell concentration of 4 x 10 6 / mL and diluted sequentially to 5:1, 2:1, 1:1, 0.5:1, 0.1:1, 0.02:1, 0.01:1, 0.005:1, and 0:1 based on the E:T ratio. 100 μL of the prepared target cells and effector cells were dispensed and co-cultured for 2 hours in a 5% CO2 incubator. To evaluate cytotoxicity, co-cultured cells were collected and stained with anti-human CD19 (APC, IgG1, κ, BD). FACSLyric TM The CD19 positivity rate was measured in the Violet proliferation 450 positive group using a Flow Cytometer (BD). The analysis program was FlowJo. TM Software (BD) was used, and experiments were conducted in triplicate for each group. Statistical significance was compared between means (P<0.05) using an unpaired Student's t-test, and data were expressed as mean ± standard deviation (mean ± SD).

[0198] As a result, as shown in Figure 23, only in the anti-CD19 CAR-NK cell group, the CD19-positive cell group showed a gradual decrease as the E:T ratio increased. After the E:T ratio of 0.1:1, the degree of depletion of CD19-positive B cells was significantly higher than that of Mock NK, and in particular, it was confirmed that more than about 97% of cells were depleted under the condition of E:T = 5:1. This means that the anti-CD19 CAR-NK cells according to one embodiment exhibit potent cytotoxic activity against PBMCs derived from SLE patients.

[0199]

[0200] Experimental Example 10. Confirmation of the marker-specific cytotoxicity of anti-CD19 CAR natural killer cells against peripheral blood mononuclear cells (SLE PBMCs) derived from systemic lupus erythematosus (SLE) patients.

[0201] Peripheral blood mononuclear cells (PBMCs) derived from systemic lupus erythematosus (SLE) patients were isolated from the blood of SLE patients donated by Konkuk University Hospital (IRB approval number: 2024-04-017) for use as target cells and stained with Violet proliferation dye 450 (BD). Mock, anti-CD19 CAR hematopoietic stem cell (HSC)-derived natural killer (NK) cells were collected for use as effector cells and cultured in Complete Medium (Gibco) at an E:T ratio of 5:1 with a cell concentration of 4 x 10 6 / mL. 100 μL each of the prepared target cells and effector cells were dispensed and co-cultured for 2 hours in a 5% CO2 incubator. To evaluate the cytotoxicity specificity of each marker, co-cultured cells were collected and stained with anti-human CD19 (APC, IgG1, κ, BD), anti-human CD3 (PerCP-Cy5.5, IgG1, κ, BD), anti-human CD14 (APC-eFluor™ 780, IgG1, κ, Invitrogen), and anti-human CD56 (Alexa488, IgG1, κ, BD). FACSLyric TM The positive rates of CD19, CD3, CD14, and CD56 were measured in the Violet proliferation 450 positive group using a Flow Cytometer (BD). The analysis program was FlowJo. TM Software (BD) was used, and experiments were conducted in triplicate for each group. Statistical significance was compared between means (P<0.05) using an unpaired Student's t-test, and data were expressed as mean ± standard deviation (mean ± SD).

[0202] As a result, as shown in Fig. 24, it was confirmed that only CD19-positive B cells in peripheral blood mononuclear cells (SLE PBMCs) derived from systemic lupus erythematosus patients were selectively depleted in the anti-CD19 CAR-NK cell group at an E:T ratio of 5:1, and other immune cells were not affected. This indicates that the anti-CD19 CAR-NK cells according to one embodiment possess a precise cytotoxicity mechanism that acts specifically on B cell antigens in an autoimmune disease environment without affecting other cell lineages.

Claims

1. (a) Step of culturing hematopoietic stem cells; (b) a step of inducing differentiation of the cultured hematopoietic stem cells into lymphocyte progenitor cells; and (c) A method for producing natural killer cells (NK cells), comprising a step of differentiating the above lymphocyte precursor cells into natural killer cells in a culture medium containing Flt-3L, IL-2, IL-15, and IL-21.

2. A method for producing natural killer cells, wherein the hematopoietic stem cells in paragraph 1 are isolated from bone marrow, umbilical cord blood, peripheral blood, or induced pluripotent stem cells.

3. A method for producing natural killer cells, wherein the hematopoietic stem cells in the second paragraph are isolated from umbilical cord blood.

4. A method for producing natural killer cells in the first paragraph, wherein the culture medium of steps (a) and (b) comprises at least one selected from the group consisting of SCF, Flt-3L, TPO, IL-3, IL-6, IL-7, and IL-15.

5. A method for producing natural killer cells in the fourth paragraph, wherein the culture medium of step (a) contains SCF, Flt-3L, TPO, IL-3, and IL-6.

6. A method for producing natural killer cells in the fourth paragraph, wherein the culture medium of step (b) contains SCF, Flt-3L, IL-3, IL-7, and IL-15.

7. In the fourth paragraph, the SCF concentration is about 0.1 to 300 ng / mL, about 0.1 to 200 ng / mL, or about 0.1 to 150 ng / mL, the Flt-3L concentration is about 0.1 to 300 ng / mL, about 0.1 to 200 ng / mL, or about 0.1 to 150 ng / mL, the TPO concentration is about 1 to 300 ng / mL, about 10 to 200 ng / mL, or about 10 to 150 ng / mL, the IL-3 concentration is about 0.1 to 300 ng / mL, about 0.1 to 200 ng / mL, or about 0.1 to 100 ng / mL, and the IL-6 concentration is about 1 to 250 ng / mL, about 10 to A method for producing natural killer cells, wherein the concentration of IL-7 is about 0.1 to 200 ng / mL, about 0.1 to 100 ng / mL, or about 0.1 to 40 ng / mL, and the concentration of IL-15 is about 0.1 to 200 ng / mL, about 0.1 to 100 ng / mL, or about 0.1 to 40 ng / mL.

8. A method for producing natural killer cells, characterized in that in paragraph 1, the Flt-3L concentration is about 1 to 250 ng / mL, about 10 to 150 ng / mL, or about 10 to 50 ng / mL, the IL-2 concentration is about 1 to 3000 IU / mL, about 10 to 1500 IU / mL, or about 10 to 500 IU / mL, the IL-15 concentration is about 1 to 200 ng / mL, about 10 to 100 ng / mL, or about 10 to 40 ng / mL, and the IL-21 concentration is about 1 to 300 ng / mL, about 10 to 200 ng / mL, or about 10 to 60 ng / mL.

9. A method for producing natural killer cells, wherein in the first paragraph, the culture period of step (a) is 1 to 15 days, the culture period of step (b) is 5 to 30 days, and the culture period of step (c) is 5 to 30 days.

10. A method for producing natural killer cells, characterized in that the culture medium in paragraph 1 does not contain feeder cells.

11. A method for producing natural killer cells according to claim 10, wherein the cultured auxiliary cells are cells overexpressing human Delta-like ligand 4 (DLL-4), artificial antigen-presenting cells (aAPC) expressing mbIL21, or stromal cell lines.

12. Natural killer cells manufactured by the manufacturing method according to Articles 1 to 11.

13. In the 12th paragraph, the natural killer cell is a natural killer cell that expresses at least one selected from the group consisting of CD45+, CD56+, CD34-, CD3-, CD14-, CD19-, and combinations thereof.

14. A method for producing CAR-NK cells, comprising additionally introducing a CAR gene into hematopoietic stem cells in step (a) of the method of paragraph 1.

15. A method for producing CAR-NK cells, wherein the CAR gene is introduced once into hematopoietic stem cells in the 14th paragraph.

16. A method for producing a CAR-NK cell according to claim 14, wherein the CAR gene comprises a signal peptide, scFv, CD8 hinge, CD8 membrane-penetrating domain, CD28 co-stimulatory domain, and CD3ζ signaling domain.

17. CAR-NK cells manufactured by the manufacturing method according to Articles 14 to 16.

18. A composition comprising a cell according to claim 12 or claim 17.

19. A pharmaceutical composition for preventing or treating cancer according to claim 18.

20. A pharmaceutical composition for preventing or treating cancer, wherein the cancer in claim 19 is selected from the group consisting of liver cancer, thyroid cancer, testicular cancer, bone cancer, glioblastoma, oral cancer, ovarian cancer, brain tumor, multiple myeloma, gallbladder cancer, bile duct cancer, colon cancer, head and neck cancer, lymphoma, bladder cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, breast cancer, cervical cancer, prostate cancer, rectal cancer, spinal cord tumor, pancreatic cancer, salivary gland cancer, lung cancer, skin cancer, laryngeal cancer, and melanoma.

21. A pharmaceutical composition for preventing or treating an autoimmune disease according to claim 18.

22. A pharmaceutical composition for preventing or treating an autoimmune disease according to claim 21, wherein the autoimmune disease is selected from the group consisting of thyroiditis, systemic lupus erythematosus (SLE), Grave's disease, Sjogren's syndrome, systemic sclerosis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

23. A pharmaceutical composition according to claim 19 or 21, which is administered in combination with an antibody.

24. A method for preventing or treating cancer, comprising administering a cell according to claim 12 or claim 17 to a subject in need thereof.

25. A method for preventing or treating an autoimmune disease, comprising administering a cell according to claim 12 or claim 17 to a subject in need thereof.

26. Use of the cells according to Article 12 or 17 for the manufacture of a composition for preventing or treating cancer.

27. Use of the cells according to Article 12 or 17 for the manufacture of a composition for preventing or treating autoimmune diseases.

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