Non-viral dual plasmid vector capable of highly efficient gene expression, stem cell expressing same, and differentiated hematopoietic system cell

Abstract

The present invention relates to: a non-viral dual plasmid vector capable of highly efficient gene expression; a stem cell expressing same; and a differentiated hematopoietic system cell. It has been confirmed that when introducing a dual plasmid vector expressing a CAR gene and a CD24 or IL-15 gene together, according to the present invention, into induced pluripotent stem cells and differentiating the induced pluripotent stem cells into T cells, hematopoietic stem cells, or NK cells, cytotoxicity to cancer cells is high in cells expressing CAR, CD24, or IL -15. Accordingly, T cells, hematopoietic stem cells, NK cells, or the like differentiated from induced pluripotent stem cells into which the dual plasmid vector according to the present invention is introduced are expected to be useful in the preparation of a composition for preventing or treating cancer.

Description

Nonviral dual plasmid vector capable of high-efficiency gene expression and stem cells and differentiated hematopoietic cells expressing the same

[0001] The present invention relates to a non-viral dual plasmid vector capable of highly efficient gene expression and stem cells and differentiated hematopoietic cells expressing the same.

[0002] This invention claims the benefit of priority from Korean Patent Application No. 10-2023-0103184, filed August 7, 2023, and Korean Patent Application No. 10-2024-0104051, filed August 5, 2024, the entire contents of which are incorporated herein by reference.

[0003]

[0004] CAR-T (Chimeric antigen receptor T cells; also referred to as “CAR-T cells”) cancer treatment is a cancer treatment that isolates T cells from the human body, manufactures them to directly recognize antigens on the surface of cancer cells and attack them, and administers the engineered cells to patients. CAR-T cell therapy has shown a groundbreaking effect in patients with blood cancers, including leukemia and lymphoma, who are no longer responding to existing anticancer drugs, and six products have been approved by 2023. Based on these effects, it is being developed as a treatment for various intractable solid cancers for which there are no alternatives.

[0005] Meanwhile, natural killer cells (NK cells), among the cells that make up the immune system, recognize abnormally altered cell membranes, such as virus-infected cells or cancer cells. NK cells play a crucial role in the early stages of viral infection or tumor formation, before activated cytotoxic T lymphocytes are produced in large numbers. Histologically, NK cells are large granular lymphocytes. Intracellular granules contain molecules with pre-formed, potent biological functions that are secreted when NK cells come into contact with target cells. Some of these molecules form pores in the target cell membrane, lysing the cell or invading the target cell, increasing nuclear DNA fragmentation and inducing apoptosis or programmed cell death.

[0006] Natural killer (NK) cells are known to have the ability to nonspecifically kill cancer cells. This killing ability is being utilized in the treatment of solid tumors using lymphokine-activated killer cells (LAK) and tumor-infiltrating lymphocytes (TILs), and in immunotherapy using donor lymphocyte infusions to prevent rejection during bone marrow or organ transplantation, a novel cell therapy approach.

[0007] In addition, defects in the differentiation and activity of NK cells have been reported to be associated with various cancers, including breast cancer, melanoma, and lung cancer, and NK cell therapy is emerging to treat these diseases.

[0008] Effective use of NK cells as anticancer immunotherapy requires securing a large number of NK cells. However, NK cells account for 10-15% of lymphocytes in the blood, and cancer patients often have reduced numbers, differentiation, and function, making securing sufficient numbers of NK cells challenging. Therefore, mass production of NK cells through proliferation or differentiation is required for their application as NK cell therapy.

[0009] Accordingly, the inventors of the present invention attempted to utilize the vector containing the CAR gene in stem cells such as induced pluripotent stem cells and differentiate them into hematopoietic cells such as T cells, hematopoietic stem cells, or NK cells that express the CAR, thereby preventing or treating cancer.

[0010]

[0011] The present inventors produced a dual plasmid vector that co-expresses a CAR gene and a CD24 or IL-15 gene, and introduced this into induced pluripotent stem cells to differentiate them into T cells, hematopoietic stem cells, or NK cells. They confirmed that cells with high expression of CAR, CD24, or IL-15 exhibited high cytotoxicity toward cancer cells, and completed the present invention based on this.

[0012] Accordingly, the purpose of the present invention is to provide a chimeric antigen receptor (CAR); and

[0013] A non-viral dual plasmid vector comprising one or more genes selected from the group consisting of CD24 and IL-15 is provided.

[0014] Another object of the present invention is to provide a cell transfected with the non-viral dual plasmid vector.

[0015] Another object of the present invention is to provide hematopoietic cells differentiated from stem cells transfected with a non-viral dual plasmid vector.

[0016] Another object of the present invention is to provide a composition for differentiating stem cells into hematopoietic cells, comprising a non-viral dual plasmid vector.

[0017] Another object of the present invention is to provide a kit for differentiating stem cells into hematopoietic cells, comprising a non-viral dual plasmid vector and a differentiation medium.

[0018] Another object of the present invention is to provide a method for producing a stem cell comprising the steps of: transfecting a non-viral dual plasmid vector into an isolated stem cell; and

[0019] A method for differentiating stem cells into hematopoietic cells is provided, comprising a step of culturing the above-mentioned transformed stem cells in a differentiation medium.

[0020] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0021]

[0022] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0023]

[0024] To achieve the above purpose, the present invention provides a chimeric antigen receptor (CAR); and

[0025] A non-viral dual plasmid vector comprising one or more genes selected from the group consisting of CD24 and IL-15 is provided.

[0026] As one embodiment of the present invention, the CAR gene may include one or more base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, but is not limited thereto.

[0027] As another embodiment of the present invention, the CD24 gene may be, but is not limited to, a Don't eat me signal gene.

[0028] As another embodiment of the present invention, the CD24 gene may include, but is not limited to, the base sequence of SEQ ID NO: 6.

[0029] As another embodiment of the present invention, the IL-15 gene may include, but is not limited to, the base sequence of SEQ ID NO: 8.

[0030] As another embodiment of the present invention, the vector comprises mEF1α (mouse elongation factor 1α), Neo r (neomycin resistance gene), and rEF1α (Rat elongation factor 1α), but is not limited thereto.

[0031] As another embodiment of the present invention, the CD24 and IL-15 genes may be linked to the encoding base sequence of a 2A peptide, but are not limited thereto.

[0032] In addition, the present invention provides a cell transfected with the non-viral dual plasmid vector.

[0033] In one embodiment of the present invention, the cell may be at least one selected from the group consisting of human induced pluripotent stem cells (iPSC), human embryonic stem cells (ESC), blood-derived T cells, hematopoietic stem cells, NK cells, and macrophages, but is not limited thereto.

[0034] In another embodiment of the present invention, the cell comprises a CAR; and

[0035] One or more genes selected from the group consisting of CD24 and IL-15 may be co-expressed, but is not limited thereto.

[0036] In addition, the present invention provides hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0037] As one embodiment of the present invention, the stem cells may be at least one selected from the group consisting of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (ESC), but are not limited thereto.

[0038] As another embodiment of the present invention, the hematopoietic cells may be at least one selected from the group consisting of blood-derived T cells, hematopoietic stem cells, NK cells, and macrophages, but are not limited thereto.

[0039] In another embodiment of the present invention, the hematopoietic cell comprises a CAR; and

[0040] It may be, but is not limited to, co-expressing one or more genes selected from the group consisting of CD24 and IL-15.

[0041] In addition, the present invention provides a composition for differentiating stem cells into hematopoietic cells, comprising the non-viral dual plasmid vector,

[0042] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0043] A composition is provided, characterized in that it expresses one or more genes selected from the group consisting of CD24 and IL-15.

[0044] In addition, the present invention provides a kit for differentiating stem cells into hematopoietic cells, comprising the non-viral dual plasmid vector and a differentiation medium.

[0045] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0046] A kit is provided, characterized in that it expresses one or more genes selected from the group consisting of CD24 and IL-15.

[0047] As one embodiment of the present invention, the differentiation medium may include, but is not limited to, one or more media selected from the group consisting of Essential 8, Essential 6, stemline, and Stempro34.

[0048] In another embodiment of the present invention, the differentiation medium comprises CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15), FMS-like tyrosine kinase 3 ligand (FLT3-L), beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, insulin-transferrin-selenium (ITS-G), It may further include, but is not limited to, one or more selected from the group consisting of thrombopoietin (TPO), stromal cell-derived factor-1a (SDF-1a), SB203580, interleukin-2 (IL-2), dexamethasone, CD3, interleukin-21 (IL-21), interleukin-12 (IL-12), interleukin-18 (IL-18), tumor necrosis factor (TNF)-like ligand 1A (TL-1A), Z-VAD, and human serum.

[0049] In addition, the present invention comprises a step of transfecting the non-viral dual plasmid vector into an isolated stem cell; and

[0050] A method for differentiating stem cells into hematopoietic cells, comprising a step of culturing the above-mentioned transformed stem cells in a differentiation medium,

[0051] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0052] A method is provided, characterized in that it expresses one or more genes selected from the group consisting of CD24 and IL-15.

[0053] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0054] In one embodiment of the present invention, the cancer may be at least one selected from the group consisting of blood cancer, colon cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, glioblastoma, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, gastrointestinal cancer, glioblastoma, endometrial cancer, salivary gland cancer, vulvar cancer, and head and neck cancer, but is not limited thereto.

[0055] In addition, the present invention provides a pharmaceutical composition comprising, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0056] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector as an active ingredient.

[0057] In addition, the present invention provides a use for preventing or treating cancer of a composition comprising, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0058] In addition, the present invention provides a use for the preparation of a preparation for preventing or treating cancer of hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0059] In addition, the present invention relates to the use of a composition comprising the non-viral dual plasmid vector for differentiating stem cells into hematopoietic cells,

[0060] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0061] Provided is a use characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

[0062] In addition, the present invention relates to the use of the non-viral dual plasmid vector for the preparation of a preparation for differentiating stem cells into hematopoietic cells,

[0063] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0064] Provided is a use characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

[0065]

[0066] When a dual plasmid vector expressing both the CAR gene and the CD24 or IL-15 gene according to the present invention is introduced into induced pluripotent stem cells and differentiated into T cells, hematopoietic stem cells, or NK cells, it was confirmed that cells expressing CAR, CD24, or IL-15 exhibit high cytotoxicity toward cancer cells. Therefore, it is expected that T cells, hematopoietic stem cells, or NK cells differentiated from induced pluripotent stem cells into which the dual plasmid vector according to the present invention has been introduced will be useful in the manufacture of compositions for preventing or treating cancer.

[0067]

[0068] FIG. 1A and 1B are drawings showing the structure of a dual plasmid vector and the structure of an inserted CAR according to one embodiment of the present invention, wherein FIG. 1A shows a vector including CAR #11, 13, 14, 15, and 16 genes, and FIG. 1B shows a #22 vector including CAR #13 gene and additionally including CD24 and IL15 genes.

[0069] FIG. 2a is a diagram schematically illustrating a selection process for human induced pluripotent stem cells (iPSCs) having dual plasmids according to one embodiment of the present invention.

[0070] FIG. 2b is a diagram showing the introduction of four types of vectors (#11, 13, 15, 16) containing CARs #11, 13, 15, and 16 according to one embodiment of the present invention into iPSCs, and comparing the CAR expression levels before and after G418 selection.

[0071] Figure 2c is a diagram showing the CAR expression level before G418 selection after introducing four types of vectors (#11, 13, 15, 16) containing CARs #11, 13, 15, and 16 according to one embodiment of the present invention into iPSCs.

[0072] Figures 2d to 2i are drawings showing the CAR expression levels after introducing four types of vectors (#11, 13, 15, 16) containing CARs #11, 13, 15, and 16 according to one embodiment of the present invention into iPSCs and G418 selection.

[0073] Figure 2j is a drawing showing the CAR expression level before G418 selection after introducing a vector (#22) containing CAR #13 and CD24-IL-15 genes according to one embodiment of the present invention into iPSCs.

[0074] Figure 2k is a drawing showing the CAR expression level after introducing a vector (#22) containing CAR #13 and CD24-IL-15 genes according to one embodiment of the present invention into iPSCs and G418 selection.

[0075] Figure 2l is a drawing showing the results of introducing a vector (#22) containing CAR #13 and CD24-IL-15 genes according to one embodiment of the present invention into iPSCs and measuring the amount of IL-15 secretion after G418 selection.

[0076] FIG. 3a is a diagram schematically illustrating the differentiation process into natural killer cells from spheroids manufactured and cultured using iPSCs having dual plasmids according to one embodiment of the present invention.

[0077] Figure 3b is a drawing showing the results of confirming the expression ratio of CAR and CD24 (Don't eat me signal gene) in NK cells obtained by differentiating iPSCs according to one embodiment of the present invention.

[0078] Figure 3c is a diagram schematically showing cells seeded in a 96-well plate in a cytotoxicity test to verify differentiation of human induced pluripotent stem cells into natural killer cells according to one embodiment of the present invention.

[0079] Figure 3d is a drawing showing the results of confirming cytotoxicity against Nalm6 and Raji in NK cells obtained by differentiating iPSCs according to one embodiment of the present invention.

[0080] FIG. 4a is a diagram showing the results of confirming the expression ratio of CAR and CD24 (Don't eat me signal gene) in hematopoietic stem cells (HPCs) obtained by differentiating iPSCs for 14 days according to one embodiment of the present invention.

[0081] Figure 4b is a drawing showing the results of confirming the CAR expression ratio in T cells obtained by differentiating iPSCs for 42 days according to one embodiment of the present invention.

[0082] Figure 4c is a diagram schematically showing cells seeded in a 96-well plate in a cytotoxicity test to verify differentiation of iPSCs into T cells according to one embodiment of the present invention.

[0083] Figure 4d is a drawing showing the results of confirming cytotoxicity toward Nalm6 in T cells obtained by differentiating iPSCs according to one embodiment of the present invention.

[0084]

[0085] In one embodiment of the present invention, a dual plasmid vector was prepared to express chimeric antigen receptor (CAR) #11, 13, 14, 15, or 16 and CD24, or CAR #13 and CD24 and IL-15, and when this was introduced into human induced pluripotent stem cells, it was confirmed that the expression level of CAR or IL-15 was high (see Example 3).

[0086] In another embodiment of the present invention, when human induced pluripotent stem cells into which the dual plasmid vector was introduced were differentiated into NK cells, it was confirmed that cytotoxicity against cancer cells was exhibited in NK cells and T cells expressing CAR and CD24 (see Examples 4 and 5).

[0087]

[0088] Hereinafter, the present invention will be described in detail.

[0089]

[0090] The present invention relates to a chimeric antigen receptor (CAR); and

[0091] A non-viral dual plasmid vector comprising one or more genes selected from the group consisting of CD24 and IL-15 is provided.

[0092] In the present invention, "chimeric antigen receptor (CAR)" means a synthetic receptor capable of targeting a specific antigen, and the CAR according to the present invention may target the human CD19 antigen.

[0093] In the present invention, the CAR may be #11, 13, 14, 15, or 16, and each of these may be encoded by a gene consisting of or including a base sequence having a sequence homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, in that order, but is not limited thereto.

[0094] In the present invention, the CAR #11 is CD8sp-V L -(GGGGS)3-V H - It can have the structure of CD8h-CD8TM-41BB-CD3z (14aa: K), and CAR #13 is CD8sp-V L -(GGGGS)3-V H- It can have the structure of CD8h-CD8TM-41BB-CD3z (14aa: Q), and CAR #14 is CD8sp-V H -(GGGGS)3-V L - It can have the structure of CD8h-CD8TM-41BB-CD3z (14aa: K), and CAR #15 is CSF2RAsp-V L -whitlow linker-V H - It can have the structure of CD28h-CD28TM-CD28cyto-CD3z, and CAR #16 is GMCSFRsp-V L -whitlow linker-V H -It may have a structure of IgG4h-CD28TM-41BB-CD3z. In each CAR, each configuration may be encoded by a gene consisting of or including the base sequence shown in Table 2, but is not limited thereto.

[0095] In the present invention, the CAR may include, but is not limited to, one or more selected from the group consisting of a signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain (TM), and an intracellular signaling domain (cytoplasmic domain or cytoplasmic signaling domain).

[0096] In the present invention, a "signal peptide" may be located in front of the antigen binding domain, and in one embodiment of the present invention, the signal peptide may be CD8sp, CSF2RAsp, or GMCSFRsp. In the present invention, the CD8sp may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 9, CSF2Rasp may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 17, and GMCSFRsp may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 23, but is not limited thereto.

[0097] In the present invention, "antigen-binding domain" refers to an antibody, protein, or peptide domain capable of specifically recognizing and binding to a target antigen. In the present invention, "antigen" refers to a polypeptide, compound, or other substance capable of specifically binding to a humoral immune mediator such as an antibody or a cellular immune mediator such as a T cell receptor. In the present invention, the target antigen may be the human CD19 antigen.

[0098] The antigen binding domain may be an antigen-specific antibody, an antigen-specific protein, an antigen-specific peptide, or a fragment of an antibody (preferably, an antigen-binding fragment).

[0099] In the present invention, "antibody" means an immunoglobulin molecule that immunologically reacts with a specific antigen (epitope), and includes polyclonal antibodies, monoclonal antibodies, and functional fragments thereof. In addition, the term may include forms produced by genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous antibodies (e.g., bispecific antibodies). The antibody comprises a variable region of a heavy chain and / or a light chain (V H , heavy chain variable region; V L , light chain variable region). The variable region includes a portion forming an antigen-binding site of an antibody molecule as a primary structure, and the antibody of the present invention may be composed of a fragment including the variable region. A linker may be positioned between the light chain and heavy chain variable regions of the antibody, protein, peptide, or antigen-binding fragment of the antibody. The "linker" refers to a polypeptide that connects the light chain variable region and the heavy chain variable region to each other without damaging their original antigen-binding properties.

[0100] In one embodiment of the present invention, the antigen binding domain is V L -(GGGGS)3-V H , V H -(GGGGS)3-V L , or V L -whitlow linker-V H It may include. In the present invention, the V L -(GGGGS)3-V H may be encoded by a gene consisting of or including the base sequence of sequence number 10, and V H -(GGGGS)3-V Lmay be encoded by a gene consisting of or including the base sequence of sequence number 16, and V L -whitlow linker-V H may be encoded by a gene consisting of or including the base sequence of sequence number 18 or 24, but is not limited thereto.

[0101] In the present invention, the "hinge region" refers to a region located between the antigen binding domain and the transmembrane domain and functions as a flexible linker. The hinge region allows the antigen binding domain to be appropriately positioned when bound to an antigen, thereby forming a stable binding with the antigen. For example, the hinge region has the purpose of extending the antigen binding domain from the cell membrane of a cell expressing a CAR. In one embodiment of the present invention, the hinge region may include a hinge region of CD8 (CD8h), CD28 (CD28h), or IgG4 (IgG4h). In the present invention, the CD8h may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 11, CD28h may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 19, and IgG4h may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 25, but is not limited thereto.

[0102] In the present invention, a "transmembrane domain (TM)" refers to any polypeptide or oligopeptide that functions to cross a cell membrane and connect extracellular and intracellular signaling domains. The transmembrane domain can penetrate the cell membrane so that the antigen binding domain of the chimeric antigen receptor can be located on the cell surface (outside the cell) and the intracellular signaling domain can be located within the cell. That is, the transmembrane domain serves as a support for the chimeric antigen receptor while simultaneously connecting the antigen binding domain (or hinge domain) and the intracellular signaling domain. In one embodiment of the present invention, the transmembrane domain may include a transmembrane domain of CD8 (CD8TM) or CD28 (CD28TM). In the present invention, the CD8TM may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 12, and CD28TM may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 20 or 26, but is not limited thereto.

[0103] In the present invention, the "intracellular signaling domain (cytoplasmic domain or cytoplasmic signaling domain)" refers to a polypeptide or oligopeptide located inside the cell membrane. The intracellular signaling domain receives a signal transmitted by the antigen binding domain and transmits the signal to the inside of a cell expressing the CAR. The signal is transmitted inside the cell and causes activation or inhibition of a biological process. The intracellular signaling domain is not particularly limited in type as long as it transmits a signal capable of inducing cell activation when the antigen binding domain binds to an antigen. In the present invention, the intracellular signaling domain may include one or more intracellular co-stimulatory domains. The co-stimulatory domain is located in the extracellular or intracellular portion of the CAR and serves to transmit a signal to a cell expressing the CAR. That is, the co-stimulatory domain contributes to inducing a sufficient response (activation) of a T cell following binding of a target antigen. The co-stimulatory domain may be selected from, but is not limited to, domains derived from CD27, CD28, 41BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1 (lymphocyte function-associated antigen-1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3. In one embodiment of the present invention, the intracellular signaling domain may comprise one or more intracellular signaling domains selected from the group consisting of 41BB, CD28cyto, and CD3ζ.In the present invention, the 41BB may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 13, CD28cyto may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 21, and CD3z may be encoded by a gene consisting of or including the base sequence of SEQ ID NO: 14, 15, 22, or 27, but is not limited thereto.

[0104] In the present invention, the genes encoding the components included in the CAR may be composed of or include a base sequence having a sequence homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% to the base sequence indicated by the corresponding sequence number, but are not limited thereto.

[0105] In the present invention, “dual plasmid vector” means a vector manufactured by inserting two or more genes into one plasmid.

[0106] In the present invention, a "vector" refers to any medium for cloning and / or transferring nucleotides into a host cell. A vector may be a replicating unit (replicon) capable of binding to another DNA fragment and causing replication of the bound fragment. A "replicating unit" refers to any genetic unit (e.g., a plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., is capable of replicating under its own control. The term "vector" encompasses viral and non-viral vectors for introducing nucleotides into a host cell in vitro, ex vivo, or in vivo.

[0107] In the present invention, the vector may include a #11 vector comprising the CAR #11 and CD24 genes and consisting of or including the base sequence of SEQ ID NO: 31, a #13 vector comprising the CAR #13 and CD24 genes and consisting of or including the base sequence of SEQ ID NO: 32, a #14 vector comprising the CAR #14 and CD24 genes and consisting of or including the base sequence of SEQ ID NO: 33, a #15 vector comprising the CAR #15 and CD24 genes and consisting of or including the base sequence of SEQ ID NO: 34, a #16 vector comprising the CAR #16 and CD24 genes and consisting of or including the base sequence of SEQ ID NO: 35, and a #22 vector comprising the CAR #13, CD24, and IL-15 genes and consisting of or including the base sequence of SEQ ID NO: 36.

[0108] In the present invention, "CD24" is a sialoglycoprotein expressed on the surface of most B lymphocytes and differentiating neuroblasts, and may be a "Don't eat me" signal gene. It has been reported that CD24 acts as an immune checkpoint molecule that avoids phagocytosis by interacting with sialic acid-binding Ig-like lectin 10 (Siglec-10), a receptor expressed on tumor-associated macrophages (TAMs). If the HLA gene is knocked out to eliminate immune rejection, the cell will be attacked by innate immune cells. To prevent this, CD24, which is a "Don't eat me" gene, is inserted as a pair with the HLA gene knockout. Since the platform of the vector according to the present invention is a platform for knocking out the HLA gene, CD24 was inserted together. In the present invention, the CD24 gene may be composed of or include a base sequence having a sequence homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% to the base sequence of SEQ ID NO: 6, but is not limited thereto.

[0109] In the present invention, "IL-15 (interleukin-15)" is secreted by mononuclear phagocytes (and some other cells) after viral infection, and this cytokine induces cell proliferation of natural killer cells and increases the persistence of natural killer cells. In the present invention, the IL-15 gene may be composed of or include a base sequence having a sequence homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% to the base sequence of SEQ ID NO: 8, but is not limited thereto.

[0110] In the present invention, the vector is mEF1α (Mouse elongation factor 1α), Neo r (neomycin resistance gene), and rEF1α (Rat elongation factor 1α), but is not limited thereto. In the present invention, the mEF1α gene may be composed of or include the base sequence of SEQ ID NO: 28, the rEF1α gene may be composed of or include the base sequence of SEQ ID NO: 29, and Neo rThe gene may be composed of or include a base sequence of SEQ ID NO: 30, but is not limited thereto. The genes may be composed of or include a base sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% with the base sequence of SEQ ID NO: 28, 29, or 30, but is not limited thereto.

[0111] In the present invention, the CD24 and IL-15 genes may be linked to the encoding base sequence of a 2A peptide, and the encoding base sequence of the 2A peptide may be composed of or include a base sequence having a sequence homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% with the base sequence of SEQ ID NO: 7, but is not limited thereto.

[0112] In the present invention, the vector may have the structure of Figures 1a and 1b. As one embodiment of the present invention, the vector may have the structure of mEF1α->CD24->rEF1α->CAR->Neo. r or mEF1α->CAR->rEF1α->CD24->2A->IL-15->Neo r It may be progressing in the direction of .

[0113]

[0114] In addition, the present invention provides a cell transfected with the non-viral dual plasmid vector.

[0115] In the present invention, "transfection" is also called transfection and is a compound word of "trans" and "infection" and refers to injecting nucleic acid into a eukaryotic cell. In the present invention, the method of transfection may use known methods such as nucleofection, transient transfection, microinjection, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, and electroporation, but is not limited thereto.

[0116] In the present invention, the cell may be at least one selected from the group consisting of human induced pluripotent stem cells (iPSC), human embryonic stem cells (ESC), blood-derived T cells, hematopoietic stem cells, NK cells, and macrophages, but is not limited thereto.

[0117] In the present invention, the cell is CAR; and

[0118] One or more genes selected from the group consisting of CD24 and IL-15 may be co-expressed, for example, expressing CAR and CD24, or expressing CAR, CD24, and IL-15, but is not limited thereto.

[0119]

[0120] In addition, the present invention provides hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0121] In the present invention, the stem cells may be at least one selected from the group consisting of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (ESC), but are not limited thereto.

[0122] In the present invention, "induced pluripotent stem cells (iPSCs)" refer to cells that have been artificially induced to possess pluripotency through a process of artificially reprogramming differentiated cells. Since iPSCs can be obtained by reprogramming differentiated cells using reprogramming factors, they enable the generation of patient-immune pluripotent cell lines without somatic cell nuclear transfer. Therefore, iPSCs can be derived from patient cells, thereby avoiding immune rejection when applied clinically. Furthermore, since iPSCs do not use eggs or embryos, they have the advantage of being free of bioethical controversies or religious criticism.

[0123] In the present invention, the hematopoietic cell is a hematopoietic stem cell and a cell group differentiated from the hematopoietic stem cell, and may be, for example, one or more selected from the group consisting of blood-derived T cells, hematopoietic stem cells, NK cells, and macrophages, but is not limited thereto.

[0124] In the present invention, the hematopoietic cells are CAR; and

[0125] One or more genes selected from the group consisting of CD24 and IL-15 may be co-expressed, for example, expressing CAR and CD24, or expressing CAR, CD24, and IL-15, but is not limited thereto.

[0126]

[0127] In addition, the present invention provides a composition for differentiating stem cells into hematopoietic cells, comprising the non-viral dual plasmid vector,

[0128] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0129] A composition is provided, characterized in that it expresses one or more genes selected from the group consisting of CD24 and IL-15.

[0130] In addition, the present invention provides a kit for differentiating stem cells into hematopoietic cells, comprising the non-viral dual plasmid vector and a differentiation medium.

[0131] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0132] A kit is provided, characterized in that it expresses one or more genes selected from the group consisting of CD24 and IL-15.

[0133] In the present invention, the term "kit" refers to a tool that enables differentiation of stem cells into hematopoietic cells by including the vector and differentiation medium. In addition to the vector and differentiation medium, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are typically required for differentiation of stem cells into hematopoietic cells. In this case, the vector and differentiation medium may be applied one or more times without limitation, and there is no restriction on the order in which each substance is applied, and the application of each substance may be performed simultaneously or microscopically.

[0134] In the present invention, the kit may include a container; an instruction manual; and the vector and differentiation medium. The container may serve to package the vector and differentiation medium, and may also serve to store and fix the vector and differentiation medium. The container may be made of a material such as a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., and may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0135] In the present invention, the differentiation medium may include one or more media selected from the group consisting of Essential 8, Essential 6, stemline, and Stempro34, but is not limited thereto.

[0136] In the present invention, the differentiation medium comprises CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15), FMS-like tyrosine kinase 3 ligand (FLT3-L), beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, insulin-transferrin-selenium (ITS-G), It may further include, but is not limited to, one or more selected from the group consisting of thrombopoietin (TPO), stromal cell-derived factor-1a (SDF-1a), SB203580, interleukin-2 (IL-2), dexamethasone, CD3, interleukin-21 (IL-21), interleukin-12 (IL-12), interleukin-18 (IL-18), tumor necrosis factor (TNF)-like ligand 1A (TL-1A), Z-VAD, and human serum.In the present invention, the SCF, TPO, IL-7, SDF-1a, IL-2, IL-15, IL-21, IL-12, IL-18, or TL-1A may include, but is not limited to, recombinant human (rh) SCF, TPO, IL-7, SDF-1a, IL-2, IL-15, IL-21, IL-12, IL-18, or TL-1A.

[0137]

[0138] In addition, the present invention comprises a step of transfecting a non-viral dual plasmid vector into an isolated stem cell; and

[0139] A method for differentiating stem cells into hematopoietic cells, comprising a step of culturing the above-mentioned transformed stem cells in a differentiation medium,

[0140] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0141] A method is provided, characterized in that it expresses one or more genes selected from the group consisting of CD24 and IL-15.

[0142] In the present invention, after the step of transfection, the method may further include a step of culturing stem cells in a selective medium containing G418 (Geneticin) to select stem cells transfected with a non-viral dual plasmid vector; and a step of culturing the selected stem cells into spheroids, but is not limited thereto.

[0143] In the present invention, the step of culturing the transfected stem cells in a differentiation medium may include, but is not limited to, a step of culturing in differentiation media 1, a step of culturing in differentiation media 2, a step of culturing in differentiation media 3, and a step of culturing in differentiation media 4. The differentiation media 1 may be a medium including at least one selected from the group consisting of Essential 8, CHIR-99021, BMP4, and VEGF, the differentiation media 2 may be a medium including at least one selected from the group consisting of Essential 6, SB431542, SCF, and VEGF, the differentiation media 3 may be a medium including at least one selected from the group consisting of stemline, SCF, and FLT3-L, and the differentiation media 4 may be a medium including at least one selected from the group consisting of stemline, SCF, IL-7, IL-15, and FLT3-L, but is not limited thereto. According to one embodiment of the present invention, the differentiation media 1 may be a medium comprising CHIR-99021, BMP4, and VEGF in Essential 8, the differentiation media 2 may be a medium comprising SB431542, SCF, and VEGF in Essential 6, the differentiation media 3 may be a medium comprising SCF and FLT3-L in stemline, and the differentiation media 4 may be a medium comprising SCF, IL-7, IL-15, and FLT3-L in stemline.

[0144] According to one embodiment of the present invention, induced pluripotent stem cells can be differentiated into natural killer cells through the step of culturing in the differentiation media 1 to 4.

[0145] In the present invention, CHIR-99021 and SB431542 may be included in the differentiation media at, but not limited to, 0.1 to 5 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.3 to 5 μM, 0.3 to 3 μM, 0.3 to 2 μM, 0.5 to 5 μM, 0.5 to 3 μM, or 0.5 to 2 μM.

[0146] In the present invention, the BMP4 and VEGF may be included in the differentiation media at, but not limited to, 10 to 200 ng / ml, 10 to 150 ng / ml, 10 to 100 ng / ml, 50 to 200 ng / ml, 50 to 150 ng / ml, 50 to 100 ng / ml, 60 to 200 ng / ml, 60 to 150 ng / ml, or 60 to 100 ng / ml.

[0147] In the present invention, the SCF, FLT3-L, and IL-7 may be included in the differentiation media at 1 to 100 ng / ml, 1 to 70 ng / ml, 1 to 50 ng / ml, 10 to 100 ng / ml, 10 to 70 ng / ml, 10 to 50 ng / ml, 20 to 100 ng / ml, 20 to 70 ng / ml, or 20 to 50 ng / ml, but is not limited thereto.

[0148] In the present invention, the step of culturing the transfected stem cells in a differentiation medium may include, but is not limited to, a step of culturing in a hematopoietic progenitor cell (HPC) differentiation medium, a step of culturing in a double-positive (DP) T cell differentiation medium, a step of culturing in a single-positive (SP) T cell differentiation medium, and a step of culturing in a single-positive (SP) T cell expansion medium.

[0149] In the present invention, in each of the above steps, the culturing may be performed for 1 to 50 days, 1 to 45 days, 1 to 40 days, 1 to 30 days, 1 to 20 days, 1 to 10 days, 2 to 50 days, 2 to 45 days, 2 to 40 days, 2 to 30 days, 2 to 20 days, 2 to 10 days, 5 to 50 days, 5 to 45 days, 5 to 40 days, 5 to 30 days, 5 to 20 days, or 5 to 10 days, but is not limited thereto.

[0150] The above HPC differentiation medium is a medium for differentiating into hematopoietic progenitor cells, and may include the differentiation media 1 or the differentiation media 2. Or, it may include a medium including at least one selected from the group consisting of rhSCF, FLT3L, b-mercaptoethanol, sodium selenite, ethanolamine, and ascorbic acid in Stempro34. According to one embodiment of the present invention, the HPC differentiation medium may include differentiation media 1; differentiation media 2; and a medium including rhSCF, FLT3L, b-mercaptoethanol, sodium selenite, ethanolamine, and ascorbic acid in Stempro34.

[0151] In the present invention, the step of culturing in the HPC differentiation medium may include a step of culturing in differentiation medium 1; a step of culturing in differentiation medium 2; and a step of culturing in a medium containing at least one selected from the group consisting of rhSCF, FLT3L, b-mercaptoethanol, sodium selenite, ethanolamine, and ascorbic acid in Stempro34.

[0152] The above DP T cell differentiation medium is a medium for differentiating into double positive T cells, and may include a medium comprising at least one selected from the group consisting of Stempro34, ITS-G, b-mercaptoethanol, Ascorbic acid, rhSCF, rhTPO, rhIL-7, FLT3L, rhSDF-1a, and SB203580. According to one embodiment of the present invention, the DP T cell differentiation medium may include a medium comprising Stempro34, ITS-G, b-mercaptoethanol, Ascorbic acid, rhSCF, rhTPO, rhIL-7, FLT3L, rhSDF-1a, and SB203580.

[0153] The above SP T cell differentiation medium is a medium for differentiating into single positive T cells, and may include a medium comprising Stempro34, at least one selected from the group consisting of ITS-G, Ascorbic acid, rhIL-7, rhIL-2, dexamethasone, and CD3; or a medium comprising Stempro34, at least one selected from the group consisting of ITS-G, Ascorbic acid, rhIL-7, rhIL-2, and dexamethasone. According to one embodiment of the present invention, the SP T cell differentiation medium may include a medium comprising Stempro34, ITS-G, Ascorbic acid, rhIL-7, rhIL-2, dexamethasone, and CD3; and a medium comprising Stempro34, ITS-G, Ascorbic acid, rhIL-7, rhIL-2, and dexamethasone.

[0154] In the present invention, the step of culturing in the single positive (SP) T cell differentiation medium may include a step of culturing in a medium including at least one selected from the group consisting of ITS-G, Ascorbic acid, rhIL-7, rhIL-2, dexamethasone, and CD3 in Stempro34; and a step of culturing in a medium including at least one selected from the group consisting of ITS-G, Ascorbic acid, rhIL-7, rhIL-2, and dexamethasone in Stempro34.

[0155] The SP T cell expansion medium may include a medium comprising Stempro34, at least one selected from the group consisting of ITS-G, Ascorbic acid, rhIL-7, rhIL-15, rhIL-21, rhIL-12, rhIL-18, rhTL-1A, and Z-VAD; or a medium comprising Stempro34, at least one selected from the group consisting of human serum, ITS-G, Ascorbic acid, rhIL-7, and rhIL-15. According to one embodiment of the present invention, the SP T cell expansion medium may include a medium comprising Stempro34, ITS-G, Ascorbic acid, rhIL-7, rhIL-15, rhIL-21, rhIL-12, rhIL-18, rhTL-1A, and Z-VAD; and Stempro34 may include a medium containing human serum, ITS-G, Ascorbic acid, rhIL-7, and rhIL-15.

[0156] In the present invention, the step of culturing in the single positive (SP) T cell expansion medium may include a step of culturing in a medium including at least one selected from the group consisting of ITS-G, Ascorbic acid, rhIL-7, rhIL-15, rhIL-21, rhIL-12, rhIL-18, rhTL-1A, and Z-VAD in Stempro34; and a step of culturing in a medium including at least one selected from the group consisting of human serum, ITS-G, Ascorbic acid, rhIL-7, and rhIL-15 in Stempro34.

[0157] In the present invention, the ITS-G may be 1x ITS-G, but is not limited thereto.

[0158] In the present invention, the ascorbic acid may be included in the medium at 1 to 70 μg / ml, 1 to 60 μg / ml, 1 to 50 μg / ml, 10 to 70 μg / ml, 10 to 60 μg / ml, 10 to 50 μg / ml, 20 to 70 μg / ml, 20 to 60 μg / ml, or 20 to 50 μg / ml, but is not limited thereto.

[0159] In the present invention, the b-mercaptoethanol is 0.1 to 120 μM, 0.1 to 110 μM, 0.1 to 100 μM, 0.1 to 10 μM, 0.1 to 7 μM, 0.1 to 5 μM, 0.3 to 120 μM, 0.3 to 110 μM, 0.3 to 100 μM, 0.3 to 10 μM, 0.3 to 7 μM, 0.3 to 5 μM, 0.5 to 120 μM, 0.5 to 110 μM, 0.5 to 100 μM, 0.5 to 10 μM, 0.5 to 7 μM, 0.5 to 5 μM, 5 to 120 μM, 5 to 110 μM, 5 to 100 μM, It may be included in the medium at, but is not limited to, 5 to 10 μM, or 5 to 7 μM.

[0160] In the present invention, the rhSCF, FLT3-L, rhIL-12, rhIL-18, and rhTL-1A may be included in the medium at 1 to 100 ng / ml, 1 to 70 ng / ml, 1 to 50 ng / ml, 10 to 100 ng / ml, 10 to 70 ng / ml, 10 to 50 ng / ml, 20 to 100 ng / ml, 20 to 70 ng / ml, or 20 to 50 ng / ml, but is not limited thereto.

[0161] In the present invention, the sodium selenite may be included in the medium at 0.1 to 40 ng / ml, 0.1 to 30 ng / ml, 0.1 to 20 ng / ml, 0.5 to 40 ng / ml, 0.5 to 30 ng / ml, 0.5 to 20 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, or 1 to 20 ng / ml, but is not limited thereto.

[0162] In the present invention, the ethanolamine may be included in the medium at 1 to 120 μM, 1 to 110 μM, 1 to 100 μM, 10 to 120 μM, 10 to 110 μM, 10 to 100 μM, 20 to 120 μM, 20 to 110 μM, or 20 to 100 μM, but is not limited thereto.

[0163] In the present invention, the rhTPO may be included in the medium at 30 to 120 ng / ml, 30 to 110 ng / ml, 30 to 100 ng / ml, 40 to 120 ng / ml, 40 to 110 ng / ml, 40 to 100 ng / ml, 50 to 120 ng / ml, 50 to 110 ng / ml, or 50 to 100 ng / ml, but is not limited thereto.

[0164] In the present invention, the rhIL-7 may be included in the medium at 1 to 70 ng / ml, 1 to 60 ng / ml, 1 to 50 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 1 to 10 ng / ml, 5 to 70 ng / ml, 5 to 60 ng / ml, 5 to 50 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, 5 to 10 ng / ml, 10 to 70 ng / ml, 10 to 60 ng / ml, 10 to 50 ng / ml, 20 to 70 ng / ml, 20 to 60 ng / ml, or 20 to 50 ng / ml, but is not limited thereto.

[0165] In the present invention, the rhSDF-1a may be included in the medium at 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 5 to 50 nM, 5 to 40 nM, 5 to 30 nM, 10 to 50 nM, 10 to 40 nM, or 10 to 30 nM, but is not limited thereto.

[0166] In the present invention, the SB203580 and Z-VAD may be included in the medium at 1 to 30 μM, 1 to 20 μM, 1 to 15 μM, 1 to 10 μM, 5 to 30 μM, 5 to 20 μM, 5 to 15 μM, or 5 to 10 μM, but are not limited thereto.

[0167] In the present invention, the rhIL-2 and rhIL-15 may be included in the medium at 1 to 20 ng / ml, 1 to 15 ng / ml, 1 to 10 ng / ml, 5 to 20 ng / ml, 5 to 15 ng / ml, or 5 to 10 ng / ml, but are not limited thereto.

[0168] In the present invention, the dexamethasone may be included in the medium at 1 to 20 nM, 1 to 15 nM, 1 to 10 nM, 5 to 20 nM, 5 to 15 nM, or 5 to 10 nM, but is not limited thereto.

[0169] In the present invention, the CD3 may be included in the medium at 100 to 700 ng / ml, 100 to 600 ng / ml, 100 to 500 ng / ml, 200 to 700 ng / ml, 200 to 600 ng / ml, or 200 to 500 ng / ml, but is not limited thereto.

[0170] In the present invention, the rhIL-21 may be included in the medium at 1 to 40 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 5 to 40 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, 10 to 40 ng / ml, 10 to 30 ng / ml, or 10 to 20 ng / ml, but is not limited thereto.

[0171] According to one embodiment of the present invention, induced pluripotent stem cells can be differentiated into hematopoietic stem cells through the step of culturing in the HPC differentiation medium, and induced pluripotent stem cells can be differentiated into T cells through the step of culturing in the DP T cell differentiation medium, SP T cell differentiation medium, and SP T cell expansion medium.

[0172] In the present invention, the culture in each of the above steps may be performed for 5 to 25 days, 5 to 23 days, 5 to 21 days, 7 to 25 days, 7 to 23 days, or 7 to 21 days, but is not limited thereto. For example, in the step of culturing in the HPC differentiation medium, the culturing may be performed for 10 to 18 days, 10 to 16 days, 10 to 14 days, 12 to 18 days, 12 to 16 days, 12 to 14 days, 14 to 18 days, 14 to 16 days, or 14 days, and in the step of culturing in the DP T cell differentiation medium, the culturing may be performed for 17 to 25 days, 17 to 23 days, 17 to 21 days, 19 to 25 days, 19 to 23 days, 19 to 21 days, 21 to 25 days, 21 to 23 days, 19 days, or 21 days, and in the step of culturing in the SP T cell differentiation medium, the culturing may be performed for 5 to 10 days, 5 to 9 days, 5 to 8 days, 6 to 10 days, 6 to 9 days, The culturing may be performed for 6 to 8 days, 7 to 10 days, 7 to 9 days, 7 to 8 days, or 7 days, and in the step of culturing in the SP T cell expansion medium, the culturing may be performed for 6 to 14 days, 6 to 12 days, 6 to 10 days, 8 to 14 days, 8 to 12 days, 8 to 10 days, 10 to 14 days, 10 to 12 days, or 10 days, but is not limited thereto.

[0173]

[0174] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0175] In the present invention, the cancer may be at least one selected from the group consisting of blood cancer, colon cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, glioblastoma, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, gastrointestinal cancer, glioblastoma, endometrial cancer, salivary gland cancer, vulvar cancer, and head and neck cancer, but is not limited thereto.

[0176] In addition, the present invention provides a pharmaceutical composition comprising, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0177] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0178] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0179] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0180] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0181] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0182] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0183] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0184] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0185] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0186] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0187] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0188] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0189] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0190] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0191] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0192] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0193] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0194]

[0195] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector as an active ingredient.

[0196] In addition, the present invention provides a use for preventing or treating cancer of a composition comprising, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0197] In addition, the present invention provides a use for the preparation of a therapeutic agent for preventing or treating cancer using hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector.

[0198] In addition, the present invention relates to the use of a composition comprising the non-viral dual plasmid vector for differentiating stem cells into hematopoietic cells,

[0199] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0200] Provided is a use characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

[0201] In addition, the present invention relates to the use of the non-viral dual plasmid vector for the preparation of a preparation for differentiating stem cells into hematopoietic cells,

[0202] The above hematopoietic cells are chimeric antigen receptors (CAR); and

[0203] Provided is a use characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

[0204] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0205] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0206] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, and “treatment” means any action that improves or beneficially changes the target disease and its accompanying metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention.

[0207]

[0208] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0209]

[0210] [Example]

[0211] Example 1. Dual plasmid vector cloning

[0212] For gene cloning, gene synthesis for chimeric antigen receptors (CARs) #11, 13, 14, 15, 16 and CD24 and IL-15 was performed at Azenta (US). The base sequences of the CARs #11, 13, 14, 15, 16 are indicated by sequence numbers 1 to 5, respectively, and are shown in Table 1 below. In addition, the sequences according to the composition of the CARs are shown in Table 2 below.

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225] After gene synthesis as described above, the synthesized genes (CAR, CD24, or IL-15) were inserted into the MCS1 and MSC2 regions of the pVITRO-neo-mcs vector (Invivogen). The structures of the manufactured dual plasmid vectors and the structures of the inserted CARs are shown in Figs. 1a and 1b. Vectors #11, 13, 14, 15, and 16 were manufactured by inserting CARs #11, 13, 14, 15, and 16 and the CD24 gene, respectively (see Fig. 1a), and vector #22 was manufactured by inserting CAR #13 together with the CD24 and IL-15 genes (see Fig. 1b).

[0226] Among these, vector #22 was constructed as a polycistronic vector by linking CD24 and IL-15 with a 2A peptide sequence.

[0227] At this time, the sequences of inserted CD24, 2A, and IL-15 are shown in Table 3 below.

[0228]

[0229]

[0230] In addition, the vector contains mEF1α, and Neo in addition to the CAR, CD24-2A-IL-15 genes. r Additional gene sequences were included, and the sequences of these genes are shown in Table 4 below.

[0231]

[0232]

[0233]

[0234]

[0235] Additionally, the entire sequences of vectors #11, 13, 14, 15, 16, and 22 manufactured by the above method are shown in Table 5 below.

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252] Example 2. Human induced pluripotent stem cell culture

[0253] 2-1. Experimental Preparation

[0254] First, for culture vessel coating, 1.5 ml of DPBS containing 9.6 μl of iMatrix-511 per well was added to a 6-well plate and coated in an incubator at 37°C for 1 hour.

[0255] The culture medium was melted at room temperature using StemFit Basic04, 1 ml of the antibiotic Primosin was added to the medium, and then mixed to prepare a complete medium.

[0256] To prepare a Y-27632 solution, Y-27632 was added to distilled water (DW) to make a 10 mM solution. This was then aliquoted into EP tubes and stored at -20°C.

[0257]

[0258] 2-2. Human induced pluripotent stem cell culture

[0259] The complete medium prepared in Example 2-1 above was prepared by warming it at room temperature for more than 30 minutes.

[0260]

[0261] 2-2-1. General cultivation

[0262] Induced pluripotent stem cells were seeded at 1.5x10 per well in a 6-well plate. 4Cells were seeded and cultured in an incubator at 37°C and 5% CO2. After 24 hours, the complete medium was removed and replaced with 2.5 ml of fresh complete medium, and cultured until the next subculture.

[0263]

[0264] 2-2-2. Subculture

[0265] For subculture, 10 mM Y-27632 prepared in Example 2-1 was dissolved at room temperature and added 1000x to the complete medium warmed at room temperature, and the culture medium of the plate containing the cells to be subcultured was removed. Then, 1 mL of DPBS was added per well of a 6-well plate and washed. 1 ml of TrypLE reagent was added and incubated for 15 minutes at 37°C, 5% CO2 conditions. After confirming that the cells had detached, 1 ml of the reacted TrypLE reagent was added to a 15 ml tube containing 8 ml of complete medium. The medium was added once more to the existing 15 ml tube (8 ml of complete medium + TrypLE reagent), and the cells were collected again and placed into the 15 ml tube. Then, this was centrifuged at 300 g for 5 minutes, and the medium was removed after leaving only the cells. Afterwards, 3 ml of medium + Y-27632 was added, and cells were counted and seeded.

[0266]

[0267] Example 3. Introduction of dual plasmids into human induced pluripotent stem cells.

[0268] 3-1. Experimental Preparation

[0269] First, for culture vessel coating, 56.7 μl of iMatrix-511 (175 μg) was added to 6 ml of DPBS in a 100 mm dish and coated in an incubator for 1 hour.

[0270] The culture medium was melted at room temperature using StemFit Basic04, 1 ml of the antibiotic Primosin was added to the medium, and then mixed to prepare a proliferation complete medium.

[0271] To prepare a Y-27632 solution, Y-27632 was added to distilled water (DW) to make a 10 mM solution. This was then aliquoted into EP tubes and stored at -20°C.

[0272]

[0273] 3-2. Harvesting human induced pluripotent stem cells

[0274] The proliferation complete medium prepared in Example 3-1 above was prepared by warming it at room temperature for more than 30 minutes.

[0275] To harvest human induced pluripotent stem cells, the culture medium of the plate containing the human induced pluripotent stem cells cultured in Example 2 was removed, and 1 mL of DPBS was added per well of a 6-well plate and washed. 1 ml of TrypLE reagent was added and incubated for 15 minutes at 37°C, 5% CO2 conditions. After confirming that the cells had detached, 1 ml of the reacted TrypLE reagent was added to a 15 ml tube containing 8 ml of complete medium, medium was added once more, and the cells were collected again and placed in the 15 ml tube. Then, this was centrifuged at 300 g for 5 minutes, and the medium was removed after leaving only the cells. Afterwards, 3 ml of medium + Y-27632 was added, and the cells were counted.

[0276]

[0277] 3-3. Electroporation

[0278] 4 ml of E buffer was added to the Neon electroporation kit tube and then mounted on the machine.

[0279] 5x10 human induced pluripotent stem cells harvested in Example 3-2 above 5 After placing the cells in an EP tube, centrifugation was performed at 300 g for 5 minutes, washing was performed with 1 ml of DPBS, and centrifugation was performed again at 300 g for 5 minutes. The supernatant was removed, leaving only the cells.

[0280] Then, 5 μg of the dual plasmid DNA prepared in Example 1 was added to the tube containing the cells, and resuspended in 100 μl of R buffer from the Neon electroporation kit.

[0281] After inserting a 100 μl tip into the pipette of the Neon electroporation kit, the cells and DNA in the R buffer were added, placed into a tube containing E buffer, and then transfected by electrical stimulation.

[0282] Afterwards, the seeds were seeded in the 100 mm dish coated in Example 3-1 and cultured for 3 days in the same manner as in Example 2-2-1.

[0283]

[0284] 3-4. Selection of human induced pluripotent stem cells with dual plasmids

[0285] Human induced pluripotent stem cells having dual plasmids transfected by the method of Example 3-3 above were selected through the following process, and this process is schematically shown in Figure 2a.

[0286] Day 0: Selective medium 1 was made by adding 100 μg / ml G418 (50 μg / ml Geneticin) to StemFit Basic04 medium. In Example 3-3, the growth medium (Stemfit Basic04) was suctioned out of the culture medium cultured in a 100 mm dish after transfection into the induced pluripotent stem cells, and 10 ml of selective medium 1 was added and cultured. After 24 hours, selective medium 1 was removed and replaced with selective medium 1, and this process was repeated until day 3.

[0287] Day 3: Selective medium 2 was prepared by adding 200 μg / ml G418 (50 μg / ml geneticin) to StemFit Basic04 medium. Selective medium 1 was removed and replaced with 10 ml of Selective medium 2. After 24 hours, the process of removing Selective medium 2 and replacing it with Selective medium 2 was repeated until day 6.

[0288] Day 6: Selective medium 3 was made by adding 400 μg / ml G418 (50 μg / ml geneticin) to StemFit Basic04 medium. Selective medium 2 was removed and replaced with 10 ml of selective medium 3. After 24 hours, selective medium 3 was removed and replaced with selective medium 3. This process was repeated until day 9-10.

[0289] Day 9~10: 4.8 μl of iMatrix-511 (175 μg) was added to each well of a 12-well plate and coated in a coating incubator for 1 hour. Then, the DPBS in the 12-well plate was removed, and 500 μl of StemFit Basic04 medium containing 200 μg / ml G418 and 10 mM Y-27632 was added.

[0290] The tip of a Pasteur pipette was heated with an alcohol lamp to create a ring shape, and then cultured repeatedly by replacing it with selective medium 3 until Day 9-10. One colony of human induced pluripotent stem cells selected from the culture (Day 6 to Day 9-10) was broken into clumps and transferred to a 12-month plate coated with the medium using a 20 μl pipette. Then, general culture was performed for 7 days using selective medium 3 in the same manner as in Example 2-2-1.

[0291] Day 16~17: 10 mM Y-27632 was dissolved at room temperature and added 1000x to the complete medium warmed at room temperature, and 400 μg / ml G418 was added.

[0292] Afterwards, the culture medium of the plate containing the cells to be subcultured (cells cultured normally from Day 9-10 to Day 16-17) was removed. Then, 1 mL of DPBS was added per well of a 6-well plate and washed. 1 ml of TrypLE reagent was added and cultured for 15 minutes at 37°C, 5% CO2 conditions. After confirming that the cells had detached, 1 ml of the reacted TrypLE reagent was added to a 15 ml tube containing 8 ml of complete medium, the medium was added once more, and the cells were collected again and placed in a 15 ml tube. Then, it was centrifuged at 300 g for 5 minutes, and the medium was removed after leaving only the cells. After that, 3 ml of selective medium 3 + Y-27632 was added and the cells were counted, and 3 x 10 4 Cells were seeded at 2.5 ml / well and cultured normally using selective medium 3 for 7 days.

[0293] Day 23~24: Selection was completed and cells were harvested for cell analysis.

[0294]

[0295] 3-5. Verification of human induced pluripotent stem cells with dual plasmids using flow cytometry (FACS)

[0296] After harvesting the cells selected through the process of Example 3-4 above, they were centrifuged at 300 g for 5 minutes. Then, the medium was removed, 10 ml of FACS buffer was added, and washed by centrifugation at 300 g for 5 minutes, and 1x10 5 Cells were added. Then, 1 μl of each antibody and isotype was added to the tube and incubated on ice for 1 hour. The antibodies and isotypes used were as follows: PE-SSEA-4 (Cat No. 560128), isotype: PE-IgG3k (Cat No. 556659), APC-Labeled Human CD19 (20-291) Protein (Cat No. CD9-HA2H5).

[0297] Afterwards, 1 ml of FACS buffer was added and centrifuged at 300 g for 5 minutes to wash, and the process of adding 1 ml of FACS buffer and centrifuging at 300 g for 5 minutes was repeated 3 times. Then, measurements were made using a FACS device.

[0298]

[0299] Four vectors (#11, 13, 15, 16) containing CARs #11, 13, 15, and 16 were introduced into iPSCs by electroporation, G418 selection was performed, and colonies were picked. As shown in Figures 2b to 2i, it was confirmed that the CAR expression level was higher than before G418 selection.

[0300] In Figure 2b, #11-3 refers to the 3rd iPSC colony picked after introducing the CAR #11 vector into iPSC, and the numerical value refers to the % value when CAR was detected by FACS.

[0301] In addition, as a result of introducing a vector (#22) containing CAR #13 and CD24-IL-15 genes into iPSCs, performing G418 selection, and then picking colonies, it was confirmed that iPSCs introducing #22 also showed a higher CAR expression level of 90% compared to before G418 selection (50%), as shown in Figures 2j and 2k.

[0302] The above results showed that the dual plasmid of the present invention was introduced into human induced pluripotent stem cells using electroporation.

[0303]

[0304] 3-6. Verification of human induced pluripotent stem cells with dual plasmids using ELISA

[0305] After harvesting the cells selected through the process of Examples 3-4 above, they were centrifuged at 300 g for 5 minutes. Then, the medium was removed, and ELISA was performed according to the manufacturer's instructions for the ELISA kit (Cat No. D1500), and IL-15 was quantified by measuring the absorbance.

[0306]

[0307] As a result, as shown in Fig. 2l, when a vector (#22) containing CAR #13 and CD24-IL-15 genes was introduced into iPSCs and the amount of IL-15 secreted was measured after G418 selection, it was confirmed that it was secreted at a high level.

[0308]

[0309] Example 4. Differentiation of human induced pluripotent stem cells into natural killer cells.

[0310] 4-1. Experimental Preparation

[0311] First, for culture vessel coating, 75 μl of iMatrix-511 (175 μg) was added to 15 ml of DPBS in a T-75 flask and coated for 1 hour in a coating incubator.

[0312] The culture medium was melted at room temperature using StemFit Basic04, 1 ml of the antibiotic Primosin was added to the medium, and then mixed to prepare a complete proliferation medium.

[0313] To prepare a Y-27632 solution, Y-27632 was added to distilled water (DW) to make a 10 mM solution. This was then aliquoted into EP tubes and stored at -20°C.

[0314]

[0315] 4-2. Differentiation of human induced pluripotent stem cells into natural killer cells

[0316] The proliferation complete medium prepared in Example 4-1 above was prepared by warming it at room temperature for more than 30 minutes.

[0317]

[0318] 4-2-1. Spheroid manufacturing and culture

[0319] After seeding human induced pluripotent stem cells with the dual plasmid selected in Example 3-4 on a SPHERICAL PLATE 5D plate, spheroids were produced by culturing them for 24 hours at 37°C and 5% CO2 conditions.

[0320] Then, the manufactured spheroids were seeded into T-75 flasks coated in Example 4-1, and 15 ml of the complete proliferation medium prepared in Example 4-1 and warmed at room temperature for more than 30 minutes was added to each T-75 flask, and seeded into 7 flasks. Thereafter, general culture was performed in the same manner as Example 2-2-1 for 3 to 5 days until the colony size reached 750 μm to 1000 μm.

[0321]

[0322] 4-2-2. Differentiation process into natural killer cells

[0323] In the above Example 4-2-1, spheroids manufactured and cultured using human induced pluripotent stem cells with dual plasmids were differentiated into natural killer cells through the following process, and this process is schematically shown in Figure 3a.

[0324] Day 0: After culturing spheroids in a T-75 flask in Example 4-2-1, the growth medium (Stemfit Basic04) was removed by suction, washed with Essential 8 medium, and cultured by adding 15 ml of differentiation media 1 (Essential 8 + 0.5-2 μM CHIR-99021 + 60-100 ng / ml BMP4 + 60-100 ng / ml VEGF).

[0325] Day 2~3: After aspirating and removing differentiation media 1, 15 ml of differentiation media 2 (Essential 6 + 0.5~2 μM SB431542 + 20~50 ng / ml SCF + 60~100 ng / ml VEGF) was added and cultured.

[0326] Day 4~5: After aspirating and removing differentiation media 2, 15 ml of differentiation media 3 (stemline + 20~50 ng / ml SCF + 20~50 ng / ml FLT-3L) was added and cultured.

[0327] Day 8~9: Add 15 ml of differentiation media 3 (total 30 ml).

[0328] Day 10~14: Harvest the medium in the T-75 flask during differentiation (30 ml / T-75).

[0329] Group 1: The harvested medium was centrifuged at 300 g for 5 minutes, and then 15 ml of differentiation media 4 (stemline + 20–50 ng / ml SCF + 20–50 ng / ml IL-7 + 10–50 ng / ml IL-15 + 10–50 ng / ml FLT-3L) was added (15 ml / T-75).

[0330] Group 2 / 3: Using the FACS sorting function, cells harvested were divided into Group 2 containing cells expressing APC (Allophycocyanin) and Group 3 containing cells not expressing APC, and then 15 ml of differentiation media 4 was added and seeded in the original flask.

[0331] Day 12~16: Add an additional 15 ml of Differentiation Media 4 (total 30 ml).

[0332] Days 18–48: Half the medium was changed every four days. Then, 15 ml per T-75 flask was placed in a 50-ml tube and centrifuged at 300 g for 5 minutes. The medium was removed, and 15 ml of fresh Differentiation Media 4 was added to suspend the cells and placed in the flasks.

[0333] Days 46–52: Differentiation was completed, and cells were harvested for various analyses. The medium in the flask was collected and transferred to a 50-ml tube. The tube was centrifuged at 300 g for 5 minutes, then Differentiation Media 4 was added. Cells were counted (using a cell counter, not manual counting). The cell counts for each analysis were then determined.

[0334]

[0335] 4-3. Verification of differentiation of human induced pluripotent stem cells into natural killer cells using flow cytometry (FACS).

[0336] After harvesting the differentiating cells at each stage, they were centrifuged at 300 g for 5 minutes. Afterwards, the medium was removed, 10 ml of FACS buffer was added, and the cells were washed by centrifugation at 300 g for 5 minutes. Then, 1x10 5Cells were added, and 1 μl of antibodies and isotypes for each marker in the differentiation verification process into natural killer cells shown in Table 6 below were added, and the cells were incubated on ice for 1 hour.

[0337] Afterwards, 1 ml of FACS buffer was added and centrifuged at 300 g for 5 minutes to wash, and the process of adding 1 ml of FACS buffer and centrifuging at 300 g for 5 minutes was repeated 3 times. Then, measurements were made using a FACS device.

[0338]

[0339]

[0340] As a result, as shown in Fig. 3b, the expression rates of CAR and CD24 (Don't eat me signal gene) in NK cells obtained by differentiating iPSCs were 10% for both CAR and CD24 when vector #13-9 was introduced, and 70% for CAR and 90% for CD24 when vector #11-3 was introduced.

[0341]

[0342] 4-4. Verification of differentiation of human induced pluripotent stem cells into natural killer cells using cell cytotoxicity tests.

[0343] The target cell number was 1x10 for Nalm6 and Raji, respectively. 4 Cells / well, effector cells are 1x10 iNK cells 5 The E:T (effector:target) ratio was 10:1 using cells / well.

[0344] The medium used was stempro-34 medium supplemented with 50 ng / ml of SCF, FLT3-L, IL7, and IL15, respectively.

[0345] Cells were seeded in a U-bottom 96-well plate as shown in Figure 3c and cultured for 3 hours and 15 minutes. Then, the cultured plate was taken out, lysis buffer was added at 10 μl each (maintaining a constant pipetting frequency), cultured for 45 minutes, and centrifuged at 300 g for 5 minutes. After centrifugation, 50 μl each was transferred to a new flat 96-well plate, and 50 μl of pre-dissolved substrate solution was added. After 30 minutes of culture, 50 μl of stop solution was added, and measurement was performed at 490 nm using a microplate reader. At this time, air bubbles in the wells were removed.

[0346]

[0347] As a result of the in vitro cytotoxicity test analysis on B-cell blood cancer cells, Nalm6 and Raji, as shown in Fig. 3d, the cytotoxicity against Nalm6 and Raji in cells introduced with vector #13-9 was high at approximately 67% and 33%, respectively, and it was found that this result was similar to the expression ratio of CAR confirmed in Example 4-3 above.

[0348]

[0349] Example 5. Differentiation of human induced pluripotent stem cells into T cells.

[0350] 5-1. Differentiation process into T cells

[0351] In Example 4-2-1 above, spheroids prepared and cultured using human induced pluripotent stem cells with dual plasmids were differentiated into T cells through the following process.

[0352] Day -4: Embryoid bodies (EBs) were formed uniformly and reproducibly using a spheroid cell culture plate in the same manner as in Example 4-2-1.

[0353] Day -3: EB was harvested and seeded.

[0354] Day -2, Day -1: The entire medium was replaced with iPSC medium (proliferation complete medium prepared in Example 4-1 above).

[0355] Day 0: The entire medium was replaced with hematopoietic progenitor cells (HPC) differentiation medium (Differentiation Media 1: Essential 8 + 0.5–2 μM CHIR-99021 + 60–100 ng / ml rhBMP4 + 60–100 ng / ml rhVEGF).

[0356] Day 2: The entire medium was replaced with HPC differentiation medium (Differentiation medium 2: Essential 6 + 0.5–2 μM SB431542 + 20–50 ng / ml rhSCF + 60–100 ng / ml rhVEGF).

[0357] Day 4: The entire medium was replaced with HPC differentiation medium (Stempro34 + 20–50 ng / ml rhSCF + 20–50 ng / ml FLT3L + 0.5–5 μM b-mercaptoethanol + 1–20 ng / ml Sodium selenite + 20–100 μM Ethanolamine + 20–50 ng / ml Ascorbic acid).

[0358] Day 6: HPC differentiation medium (Stempro34 + 20–50 ng / ml rhSCF + 20–50 ng / ml FLT3L + 0.5–5 μM b-mercaptoethanol + 1–20 ng / ml Sodium selenite + 20–100 μM Ethanolamine + 20–50 ng / ml Ascorbic acid) was added.

[0359] Day 10: Half of the HPC differentiation medium was replaced with HPC differentiation medium (Stempro34 + 20–50 ng / ml rhSCF + 20–50 ng / ml FLT3L + 0.5–5 μM b-mercaptoethanol + 1–20 ng / ml Sodium selenite + 20–100 μM Ethanolamine + 20–50 ng / ml Ascorbic acid).

[0360] Day 14: HPCs were harvested and completely replaced with double positive (DP) differentiation medium (Stempro34 + 1x ITS-G (Insulin-Transferrin-Selenium) + 5–100 μM b-mercaptoethanol + 20–50 μg / ml Ascorbic acid + 20–50 ng / ml rhSCF + 50–100 ng / ml rhTPO (thrombopoietin) + 20–50 ng / ml rhIL-7 + 20–50 ng / ml FLT3L + 10–30 nM rhSDF-1a + 5–15 μM SB203580).

[0361] Day 16~35: DP stage T cells were prepared by replacing 80% of the medium with DP differentiation medium (changing the medium every two days) (Day 14~35).

[0362] Day 35~42: On Day 35, the single positive (SP) differentiation medium-CD3 activation medium (Stempro34 + 1x ITS-G + 20~50 μg / ml Ascorbic acid + 5~10 ng / ml rhIL-7 + 5~10 ng / ml rhIL-2 + 5~10 nM dexamethasone + 200~500 ng / ml CD3) was completely replaced with the single positive (SP) differentiation medium-CD3 activation medium (Stempro34 + 1x ITS-G + 20~50 μg / ml Ascorbic acid + 5~10 ng / ml rhIL-7 + 5~10 ng / ml rhIL-2 + 5~10 nM dexamethasone) to prepare SP stage T cells from DP.

[0363] Day 42~52: On Day 42, SP T cells were harvested and the entire medium was replaced with SP T cell expansion medium (Stempro34 + 1x ITS-G + 10~50 μg / ml Ascorbic acid + 5~10 ng / ml rhIL-7 + 5~10 ng / ml rhIL-15 + 10~20 ng / ml rhIL-21 + 20~50 ng / ml rhIL-12 + 20~50 ng / ml rhIL-18 + 20~50 ng / ml rhTL-1A (Tumor necrosis factor (TNF)-like ligand 1A) + 5~10 μM Z-VAD). On Day 45, Stempro34 + 20% human serum + 1x ITS-G + 20~50 μg / ml Ascorbic acid + 5~10 The medium was completely replaced with medium containing 5–10 ng / ml rhIL-7 + 5–10 ng / ml rhIL-15, cultured until Day 52, and SP T cells were harvested.

[0364]

[0365] 5-2. Verification of T cell differentiation from human induced pluripotent stem cells using flow cytometry (FACS)

[0366] In the differentiation verification step, differentiated cells were obtained through the process of Example 5-1 and centrifuged at 300 g for 5 minutes. The supernatant was then removed, FACS buffer was added, and the cells were washed by centrifugation at 300 g for 5 minutes. The supernatant was then removed, resuspended in FACS buffer, and cells were counted.

[0367] 1x10 of the above cells 5 Prepared as 100 μl and placed in a FACS tube, 1 μl of antibodies against each marker in the T cell differentiation verification process shown in Table 7 below were added to the FACS tube and incubated on ice for 1 hour. Afterwards, 1 ml of FACS buffer was added and centrifuged at 300 g for 5 minutes. Then, the supernatant was removed, 1 ml of FACS buffer was added, and centrifuged at 300 g for 5 minutes.

[0368] The above process was repeated once more and analyzed using a FACS device.

[0369]

[0370]

[0371] As a result, as shown in Fig. 4a, the expression rates of CAR and CD24 (Don't eat me signal gene) in hematopoietic stem cells (HPCs) obtained by differentiating iPSCs for 14 days were 70% for both CAR and CD24 when vector #13-9 was introduced, and 30% for CAR and 90% for CD24 when vector #11-3 was introduced.

[0372] In addition, as shown in 4b, it was confirmed that the CAR expression rate in T cells obtained by differentiating iPSCs for 42 days was as high as 99% when vector #13-9 was introduced.

[0373]

[0374] 5-3. Verification of T cell differentiation from human induced pluripotent stem cells using cell cytotoxicity testing.

[0375] Differentiation into T cells was confirmed through a cytotoxicity test by confirming the activity of lactate dehydrogenase (LDH).

[0376] For this purpose, target cells K562 and T cells as effector cells were prepared, and target cells K562 were 1x10 5 cells / ml, effector T cells are 1x10 6 Prepared as cells / 1 ml.

[0377] The culture medium was prepared by adding 10 ng / ml IL-7, 10 ng / ml IL-15, and 1x Glutamax to stempro-34 medium.

[0378] Cells were seeded in 96-well plates as shown in Figure 4c and cultured for 3 hours and 15 minutes. The cultured plates were then removed, 10 μl of lysis buffer was added, and the plates were cultured for another 45 minutes, followed by centrifugation at 300 g for 5 minutes. Fifty μl of the supernatant was then transferred to new 96-well plates, and 50 μl of substrate solution was added. The plates were then incubated for 30 minutes in a darkened environment, 50 μl of stop solution was added, and the spectrum was measured at 490 nm using a microplate reader.

[0379]

[0380] As a result of the in vitro cytotoxicity test analysis on Nalm6, a B-cell blood cancer cell, as shown in Figure 4d, it was confirmed that the cytotoxicity against Nalm6 in cells introduced with vector #13-9 was approximately 28%.

[0381]

[0382] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0383]

[0384] When a dual plasmid vector expressing the CAR gene and CD24 or IL-15 gene together according to the present invention is introduced into induced pluripotent stem cells and differentiated into T cells, hematopoietic stem cells, or NK cells, it was confirmed that cells expressing CAR, CD24, or IL-15 exhibit high cytotoxicity toward cancer cells. Therefore, T cells, hematopoietic stem cells, or NK cells differentiated from induced pluripotent stem cells into which the dual plasmid vector according to the present invention has been introduced can be usefully used in the manufacture of compositions for preventing or treating cancer, and thus have industrial applicability.

Claims

1. Chimeric antigen receptor (CAR); and A nonviral dual plasmid vector comprising one or more genes selected from the group consisting of CD24 and IL-15.

2. In paragraph 1, A non-viral dual plasmid vector, characterized in that the CAR gene comprises one or more base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

5.

3. In paragraph 1 or 2, A nonviral dual plasmid vector characterized in that the CD24 gene is a Don't eat me signal gene.

4. In any one of paragraphs 1 to 3, A non-viral dual plasmid vector, characterized in that the CD24 gene comprises the base sequence of sequence number 6.

5. In any one of paragraphs 1 to 4, A nonviral dual plasmid vector, characterized in that the IL-15 gene comprises the base sequence of SEQ ID NO:

8.

6. In any one of paragraphs 1 to 5, The above vector is mEF1α (Mouse elongation factor 1α), Neo r A non-viral dual plasmid vector characterized in that it further comprises one or more genes selected from the group consisting of (neomycin resistance gene), and rEF1α (Rat elongation factor 1α).

7. In any one of paragraphs 1 to 6, A nonviral dual plasmid vector characterized in that the CD24 and IL-15 genes are linked to the encoding base sequence of a 2A peptide.

8. A cell transfected with the non-viral dual plasmid vector of any one of claims 1 to 7.

9. In paragraph 8, A cell characterized in that the cell is at least one selected from the group consisting of human induced pluripotent stem cells (iPSC), human embryonic stem cells (ESC), blood-derived T cells, hematopoietic stem cells, NK cells, and macrophages.

10. In paragraph 8 or 9, The above cells are CAR; and A cell characterized by co-expressing one or more genes selected from the group consisting of CD24 and IL-15.

11. Hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector of any one of claims 1 to 7.

12. In paragraph 11, A hematopoietic cell, characterized in that the stem cell is at least one selected from the group consisting of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (ESC).

13. In paragraph 11 or 12, A hematopoietic cell, characterized in that the hematopoietic cell is at least one selected from the group consisting of blood-derived T cells, hematopoietic stem cells, NK cells, and macrophages.

14. In any one of paragraphs 11 to 13, The above hematopoietic cells are CAR; and A hematopoietic cell characterized by co-expressing one or more genes selected from the group consisting of CD24 and IL-15.

15. A composition for differentiating stem cells into hematopoietic cells, comprising a non-viral dual plasmid vector of any one of claims 1 to 7, The above hematopoietic cells are chimeric antigen receptors (CAR); and A composition characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

16. A kit for differentiating stem cells into hematopoietic cells, comprising a non-viral dual plasmid vector of any one of claims 1 to 7 and a differentiation medium, The above hematopoietic cells are chimeric antigen receptors (CAR); and A kit characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

17. In paragraph 16, A kit, characterized in that the differentiation medium comprises at least one medium selected from the group consisting of Essential 8, Essential 6, stemline, and Stempro34.

18. In paragraph 17, The differentiation medium contained CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15), FMS-like tyrosine kinase 3 ligand (FLT3-L), beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, insulin-transferrin-selenium (ITS-G), A kit characterized in that it further comprises at least one selected from the group consisting of thrombopoietin (TPO), stromal cell-derived factor-1a (SDF-1a), SB203580, interleukin-2 (IL-2), dexamethasone, CD3, interleukin-21 (IL-21), interleukin-12 (IL-12), interleukin-18 (IL-18), tumor necrosis factor (TNF)-like ligand 1A (TL-1A), Z-VAD, and human serum.

19. A step of transfecting a separated stem cell with a non-viral dual plasmid vector of any one of clauses 1 to 7; and A method for differentiating stem cells into hematopoietic cells, comprising a step of culturing the above-mentioned transformed stem cells in a differentiation medium, The above hematopoietic cells are chimeric antigen receptors (CAR); and A method characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

20. In paragraph 19, A method characterized in that the stem cells are at least one selected from the group consisting of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (ESC).

21. In paragraph 19 or 20, A method, characterized in that the hematopoietic cells are at least one selected from the group consisting of blood-derived T cells, hematopoietic stem cells, NK cells, and macrophages.

22. In any one of paragraphs 19 to 21, A method, characterized in that the differentiation medium comprises at least one medium selected from the group consisting of Essential 8, Essential 6, stemline, and Stempro34.

23. In paragraph 22, The differentiation medium contained CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15), FMS-like tyrosine kinase 3 ligand (FLT3-L), beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, insulin-transferrin-selenium (ITS-G), A method characterized in that it further comprises at least one selected from the group consisting of thrombopoietin (TPO), stromal cell-derived factor-1a (SDF-1a), SB203580, interleukin-2 (IL-2), dexamethasone, CD3, interleukin-21 (IL-21), interleukin-12 (IL-12), interleukin-18 (IL-18), tumor necrosis factor (TNF)-like ligand 1A (TL-1A), Z-VAD, and human serum.

24. A pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector of any one of claims 1 to 7.

25. In paragraph 24, A pharmaceutical composition, characterized in that the hematopoietic cells are at least one selected from the group consisting of blood-derived T cells, hematopoietic stem cells, NK cells, and macrophages.

26. In paragraph 24 or 25, The above hematopoietic cells are CAR; and A pharmaceutical composition characterized in that it co-expresses one or more genes selected from the group consisting of CD24 and IL-15.

27. In any one of paragraphs 24 to 26, A pharmaceutical composition characterized in that the cancer is at least one selected from the group consisting of blood cancer, colon cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, glioblastoma, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, gastrointestinal cancer, glioblastoma, endometrial cancer, salivary gland cancer, vulvar cancer, and head and neck cancer.

28. A pharmaceutical composition comprising, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector of any one of claims 1 to 7.

29. A method for preventing or treating cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising, as an active ingredient, hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector of any one of claims 1 to 7.

30. Use of a composition comprising hematopoietic cells differentiated from stem cells transfected with the non-viral dual plasmid vector of any one of claims 1 to 7 as an active ingredient for the prevention or treatment of cancer.

31. Use of the non-viral dual plasmid vector of any one of claims 1 to 7 for the manufacture of a preparation for the prevention or treatment of cancer of hematopoietic cells differentiated from transfected stem cells.

32. Use of a composition comprising a non-viral dual plasmid vector of any one of claims 1 to 7 for differentiating stem cells into hematopoietic cells, The above hematopoietic cells are chimeric antigen receptors (CAR); and A use characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

33. Use of a non-viral dual plasmid vector according to any one of claims 1 to 7 for the manufacture of a preparation for differentiating stem cells into hematopoietic cells, The above hematopoietic cells are chimeric antigen receptors (CAR); and A use characterized by expressing one or more genes selected from the group consisting of CD24 and IL-15.

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