Stem cells with suppressed FTO gene and immune cells differentiated from them

Abstract

The present invention relates to stem cells with suppressed FTO genes, immune cells differentiated therefrom, and their uses. The present invention confirmed that suppressing FTO gene expression in immune cells significantly enhances cytolytic activity, and by confirming that immune cells differentiated from stem cells with suppressed FTO gene expression possess excellent cytolytic activity against cancer cells, it resolved the limitations of existing immunotherapy drugs, such as immune incompatibility and limited cell counts. Furthermore, the present invention produced stem cells capable of expressing the CAR gene while deleting the FTO gene by introducing the CAR gene to the FTO gene site of stem cells, and confirmed that immune cells differentiated therefrom possess significantly excellent cytolytic activity against cancer cells. Therefore, the present invention can be effectively utilized as an immunotherapy for cancer.

Description

Technology Field

[0001] The present invention relates to stem cells in which the expression of the FTO gene is suppressed, immune cells differentiated therefrom, and uses thereof. Background Technology

[0003] Natural Killer (NK) cells are representative innate granular lymphocytes and are a type of immune cell that eliminates virus-infected cells or cancer cells. Immunotherapy utilizing these NK cells represents a cutting-edge treatment technique capable of addressing various issues and side effects in cancer therapy. Specifically, anticancer treatment using NK cells has the advantage of allowing hematopoietic stem cell transplantation because it prevents Graft-versus-Host Disease (GVHD). Furthermore, because it is free from side effects such as Cytokine Release Syndrome (CRS), it can be advantageously utilized in anticancer immunotherapy. Recently, various studies, including the development of CAR-NK (Chimeric antigen receptor-NK), are being conducted to enhance NK cell activity, and the necessity of such research is becoming increasingly prominent.

[0004] However, currently used immunotherapy agents have many drawbacks in that they involve obtaining immune cells from a patient's blood, proliferating and processing them, and then reapplying them to the patient. Consequently, there is a need for research on the development of anticancer immunotherapy agents using human pluripotent stem cells (hPSCs) to address issues such as immune incompatibility and the limited number of immune cells. Using hPSCs allows for the proliferation of patient-derived stem cells, which eliminates the side effects of immune incompatibility. Furthermore, since hPSCs enable differentiation into various immune cells and mass proliferation, they can overcome the limitations of insufficient cell numbers. Therefore, the production of these human pluripotent stem cells and the technology for differentiation into specific cell types are essential for the development of hPSC-derived cell therapies.

[0005] Meanwhile, N6-methyladenosine (m6A) is an RNA modification in which a methyl group is added to the nitrogen atom position of the 6th adenine base in RNA, and it is the most common RNA modification in eukaryotic mRNA or lncRNA. This m6A can regulate almost all RNA metabolism, such as regulating RNA stability or translation into protein. The FTO (Fat mass and obesity-associated protein) protein is a protein that removes this m6A and is known to regulate gene expression by controlling the characteristics of specific RNAs containing m6A.

[0006] Against this technical background, the present invention aims to provide a next-generation CAR-NK cell therapy by combining gene editing technology with the production of hPSCs and differentiation into NK cells. Specifically, high-efficiency, high-activity CAR-NK cells were produced by inserting a CAR gene into a specific gene site of hPSCs using the CRISPR-Cas9 system and inducing differentiation into CAR-NK cells. Furthermore, as it was confirmed that the anticancer activity of NK cells increases when the expression of the FTO gene in NK cells is suppressed, the present invention aims to provide a next-generation high-activity hPSC-derived NK cell therapy by inserting a CAR gene into the FTO gene site of NK cells, thereby simultaneously inhibiting FTO and expressing CAR. The problem to be solved

[0008] The present invention aims to provide stem cells capable of differentiating into immune cells.

[0009] In addition, the present invention aims to provide an anticancer immune cell therapeutic agent comprising the immune cells.

[0010] In addition, the present invention aims to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the immune cells. means of solving the problem

[0012] To achieve the above objective, one aspect of the present invention provides stem cells in which the expression of the FTO (fat mass and obesity-associated protein) gene is suppressed.

[0013] In addition, to achieve the above objective, another aspect of the present invention provides an immune cell in which the expression of the FTO (fat mass and obesity-associated protein) gene is suppressed.

[0014] In addition, to achieve the above objective, another aspect of the present invention provides a cell therapeutic agent comprising immune cells in which the expression of the FTO (fat mass and obesity-associated protein) gene is suppressed.

[0015] In addition, to achieve the above objective, another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising immune cells in which the expression of the FTO (fat mass and obesity-associated protein) gene is suppressed. Effects of the invention

[0017] The present invention confirmed that inhibiting FTO gene expression in immune cells significantly enhances cytolytic activity, and by confirming that immune cells differentiated from stem cells after inhibiting FTO gene expression in stem cells possess excellent cytolytic activity against cancer cells, it resolved the limitations of existing immunotherapy drugs, such as immune incompatibility and a limited number of cells. Furthermore, the present invention produced stem cells capable of expressing the CAR gene while deleting the FTO gene by introducing the CAR gene to the FTO gene site of stem cells, and confirmed that immune cells differentiated from these cells possess significantly excellent cytolytic activity against cancer cells. Therefore, the present invention can be effectively utilized as an immunotherapy drug.

[0018] However, the effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below. Brief explanation of the drawing

[0020] Figure 1 shows a schematic diagram of the process of producing an FTO-deleted mouse by deleting 76 nucleotides containing a start codon in the first exon of the FTO gene using the CRISPR / Cas9 system (pT7 plasmid). Figure 2 shows the m6A content in splenocytes and bone marrow cells extracted from normal mice and FTO-deficient mice. Figure 3 shows the results of measuring the extent of cancer cell metastasis to the lungs, tumor size, and mouse survival rate after intravenously injecting a B16F10 mouse melanoma cell line into the tails of normal mice and FTO-deficient mice. Figure 4 shows the results of confirming whether B16F10 metastatic nodules were formed after depleting NK cells by treating normal mice and FTO-deficient mice with anti-NK1.1 antibody. Figure 5 shows the results of confirming cytotoxicity by extracting NK cells from normal mice and FTO-deficient mice, activating the NK cells by treating them with IL-2 and IL-15, and then treating them with EL4 lymphoma cell lines and YAC1 lymphoma cell lines. Figure 6 shows the results of measuring the secretion and expression levels of IFNγ by extracting NK cells from normal mice and FTO-deficient mice. Figure 7 shows the results of measuring FTO expression levels after treating human NK92 cell lines with two types of siRNA. Figure 8 shows the results of measuring m6A content after treating human NK92 cell lines with two types of siRNA (NK92-siFTO). Figure 9 shows the results of measuring cytotoxicity and degranulation activity against K562 lymphoma cell lines after co-culturing normal NK92 cell lines or NK92-siFTO cell lines with K562 lymphoma cell lines. Figure 10 shows the results of measuring granzyme B protein expression and cytokine (TNFα, IFNγ) release after treating normal NK92 cell lines and NK92-siFTO cell lines with IL-2. Figure 11 shows the results of measuring cytotoxicity and IFNγ expression levels in K562 lymphoma cell lines after introducing the FTO gene into human NK92 cell lines to overexpress FTO gene expression (NK92+FTO). Figure 12 shows the results of measuring the expression levels of activating receptors (NKG2D, NKp30, NKp44, NKp46, CD16), IL-2 receptor (CD25), TNF receptor (CD27), and TGFβ receptor (CD105) in normal NK92 cell lines and NK92-siFTO cell lines. Figure 13 shows the results of confirming the expression profiles of the JAK / STAT signaling pathway in normal NK92 cell lines and NK92-siFTO cell lines. Figure 14 shows the results of confirming the expression profile of the JAK / STAT signaling pathway after introducing the FTO gene into human NK92 cell lines and overexpressing the FTO gene (NK92+FTO). Figure 15 shows the results of measuring SOCS family gene expression in normal NK92 cell lines and NK92-siFTO cell lines. Figure 16 shows the results of measuring m6A methylated mRNA (MeRIP) levels in normal NK92 cell lines, NK92-siFTO cell lines, and NK92+FTO cell lines. Figure 17 shows the results of 3'UTR reporter system analysis performed on SOCS3 and CISH genes. Figure 18 shows human umbilical cord blood-derived CD3 - CD56 + After inhibiting FTO expression in NK cell lines by treating them with siRNA (CD56 + This shows the results confirming the cytotoxicity of -siFTO) on target cells. Figure 19 shows the control group CD56 + NK cell line and CD3 - CD56 + This shows the results of measuring the mRNA expression levels of cytokines and effector molecules in NK cell lines. Figure 20 shows the control group CD56 + NK cell line and CD3 - CD56 + This shows the results of confirming the expression profile of the JAK / STAT signaling pathway in NK cell lines. Figure 21 shows the control group CD56 + NK cell line and CD3 - CD56 +This shows the results of measuring SOCS family gene expression in NK cell lines. Figure 22 shows the results of measuring the FTO expression level after infecting human NK92 cell lines with shRNA via a lentivirus to constantically suppress FTO gene expression (NK92-shFTO). Figure 23 shows the results of measuring cytotoxicity and degranulation activity against K562 lymphoma cell lines after treating K562 lymphoma cell lines with control NK92-TRC cell lines or NK92-shFTO cell lines. Figure 24 shows a schematic diagram of an experiment in which a luciferase-expressing K562 lymphoma cell line (K562-Luc) was injected into the tail vein of an NSG mouse, and a control NK92-TRC cell line or NK92-shFTO cell line was injected three times, followed by the measurement of luminescence signal and viability. Figure 25 shows the results of measuring luminescence signal and viability after injecting a luciferase-expressing K562 lymphoma cell line (K562-Luc) into the tail vein of an NSG mouse and injecting a control NK92-TRC cell line or NK92-shFTO cell line three times. Figure 26 shows a schematic diagram of the mechanism of action in which the cytolytic activity of NK cells with a deleted FTO gene is enhanced. Figure 27 illustrates the process of deleting the FTO gene by inserting a nucleotide sequence encoding mCherry fluorescent protein into the FTO gene site in human pluripotent stem cells using the CRISPR-Cas9 system. Figure 28 shows the results of measuring FTO gene expression by PCR in human pluripotent stem cell lines (FTO-KO hPSCs) in which the FTO gene is deleted. Figure 29 shows the results of analyzing mCherry protein fluorescence in FTO-KO hPSCs. Figure 30 shows the results of measuring the expression levels of pluripotency markers in FTO-KO hPSCs by performing immunocytochemical analysis and flow cytometry analysis. Figure 31 shows the results of confirming whether chromosomal abnormalities were detected in FTO-KO hPSCs by performing karyotype analysis. Figure 32 shows the results of measuring the expression levels of endoderm, mesoderm, and ectoderm markers in FTO-KO hPSCs. Figure 33 shows that FTO-KO hPSCs were treated with differentiation-specific conditions, and the FTO-KO hPSCs were each CD34 + CD45 + Hematopoietic stem cells and CD45 + CD56 + This shows the result of differentiation into NK cells. Figure 34 shows the results of measuring the expression of SOCS family genes, TNFα, Granzyme B, MYC, and Prf1 genes in control NK cell lines and NK cell lines differentiated from FTO-KO hPSCs (FTO-KO NK). Figure 35 shows the results of measuring the killing ability of control NK cell lines and FTO-KO NK cell lines against cancer cells. Figure 36 illustrates the process of deleting the FTO gene by introducing the CD5-CAR gene to the FTO gene site in human pluripotent stem cells using the CRISPR-Cas9 system. Figure 37 shows the results of measuring CD5-CAR gene expression by PCR in human pluripotent stem cell lines (FTO-CD5 hPSCs) in which the CD5-CAR gene was introduced to the FTO gene site. Figure 38 shows the results of confirming CD5-CAR protein expression in the cell membrane of FTO-CD5 hPSCs cell lines using Myc, a marker of CD5-CAR. Figure 39 shows the results of measuring the expression levels of pluripotency markers in FTO-CD5 hPSCs by performing immunocytochemical analysis and flow cytometry analysis. Figure 40 shows the results of confirming whether chromosomal abnormalities were detected in FTO-CD5 hPSCs by performing karyotype analysis. Figure 41 shows that FTO-CD5 hPSCs were treated with differentiation-specific conditions, and the FTO-CD5 hPSCs became CD45 + CD56 + This shows the result of differentiation into NK cells. Figure 42 shows the results of measuring the killing ability of NK cell lines (FTO-CD5 NK) differentiated from control CD5-CAR NK cell lines and FTO-CD5 hPSCs against cancer cells. Specific details for implementing the invention

[0021] The present invention will be described in detail below.

[0023] 1. Stem cells with suppressed FTO gene expression

[0024] One aspect of the present invention provides stem cells in which the expression of the FTO (fat mass and obesity-associated protein) gene is suppressed.

[0025] The above stem cells can differentiate into immune cells, and the immune cells differentiated in this way may have the expression of the FTO gene suppressed, just like the above stem cells.

[0026] The above "FTO (fat mass and obesity-associated protein)" is a protein also referred to as alpha-ketoglutarate-dependent deoxygenase, and refers to an mRNA demethylation enzyme that has the function of removing methyl groups from mRNA. FTO is known to have the activity of removing N6-methyladenosine (m6A), an RNA variant in which a methyl group is added to the nitrogen atom position of the adenine base 6 of RNA. Since m6A can regulate almost all RNA metabolism, such as RNA stability and protein translation, FTO can regulate various gene expressions by regulating m6A.

[0027] In the present invention, the stem cell may comprise siRNA, shRNA, miRNA, or an antisense oligonucleotide that binds complementarily to the mRNA of the FTO gene. The siRNA, shRNA, miRNA, or antisense oligonucleotide may inhibit the expression of the FTO gene in the stem cell by binding complementarily to the mRNA of the FTO gene and reducing the transcription, mRNA level / stability, and / or translation level of the FTO gene.

[0028] Additionally, in the present invention, the stem cell may have a deletion of part or all of the FTO gene, and said deletion may be performed by a CRISPR (Clustered regularly interspaced short palindromic repeats) / Cas system. In this case, the stem cell may have a foreign gene inserted at the FTO gene location within the chromosome.

[0029] In one embodiment of the present invention, stem cells in which the expression of the FTO gene is suppressed were produced by deleting the FTO gene through the insertion of a nucleotide sequence encoding mCherry fluorescent protein at the FTO gene site of human pluripotent stem cells using a CRISPR / Cas system.

[0030] The above "stem cell" refers to an undifferentiated cell capable of dividing for a long period through self-renewal and capable of differentiating into various types of cells upon treatment with specific conditions. In the present invention, the stem cell may be a pluripotent stem cell (PSC), an embryonic stem cell (ESC), or an induced pluripotent stem cell (iPSC), but may include any stem cell capable of differentiating into an immune cell without limitation.

[0031] The above "CRISPR / Cas system" or "CRISPR / Cas" refers to a gene editing system that utilizes a Cas protein (e.g., Cas9) as an effector protein. The system refers to a complex comprising a gene editing protein or a nuclease, such as an endonuclease, and a nucleic acid targeting molecule corresponding to the nuclease, which is capable of binding to a target nucleic acid or a target gene to cut or edit a target site of the target nucleic acid or gene. In this case, the cutting of the target site may cause a double-strand break (DSB) by cutting the outer part of the target nucleic acid or 1 to 5 bp inside the 3' end of the target nucleic acid. The system may be composed of various types depending on the gene editing protein and the guide RNA (gRNA), which is a nucleic acid targeting molecule corresponding to the protein, and the function and efficiency of the gene editing protein may vary depending on the type of nucleic acid targeting molecule. Gene editing proteins utilize nucleic acid targeting molecules to bind to target nucleic acids or target genes, and perform DNA double-strand or single-strand cuts or base corrections at those sites. The system does not require the generation of custom proteins to target specific sequences, and can produce a diverse repertoire of systems by modifying the spacer sequence, which is the target site binding sequence within the guide molecule. In the present invention, the CRISPR / Cas system may be an engineered or non-naturally occurring system.

[0032] The above "guide RNA" generally refers to an RNA molecule (or collectively a group of RNA molecules) capable of binding to a Cas protein and helping to target the Cas protein to a specific location within the target nucleic acid. The guide RNA may be any polynucleotide sequence having complementarity with the target gene sequence sufficient to hybridize with all or part of the sequence of the target nucleic acid and induce sequence-specific binding of the CRISPR complex to the target sequence.

[0033] The above "Cas protein" refers to an endonuclease capable of cleaving target nucleic acids by forming a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA). The above Cas protein may be a naturally occurring Cas protein or a functional derivative thereof. "Functional derivative" includes, but is not limited to, fragments of natural sequences and natural sequence polypeptides and derivatives of their fragments, provided that they refer to proteins having biological activity common to the corresponding natural sequence polypeptide. Meanwhile, the above Cas protein may be a Cas9 protein or a functional derivative thereof, such as inactivated Cas9 (dCas9) or Cas9 nickase. The inactivated Cas9 may be an RFN (RNA-guided FokI Nuclease) with a FokI nuclease domain attached to dCas9, or a transcription activator or repressor domain attached to dCas9, and the Cas9 nuclease may be D10A Cas9 or H840A Cas9, but is not limited thereto.

[0034] In the present invention, the Cas9 protein is Streptococcus piogenes ( Streptococcus pyogenes ), Francisela Novicida( Francisella novicida ), Streptococcus thermophilus( Streptococcus thermophilus), Legionella pneumophila( Legionella pneumophila ), Listeria Innoqua( Listeria innocua ), or Streptococcus mutans ( Streptococcus mutans It may be derived from, but is not limited to.

[0035] The "target nucleic acid" mentioned above refers to a nucleotide sequence present within a target gene, meaning a sequence recognized by the guide RNA of the CRISPR / Cas system or a target sequence modified by the CRISPR / Cas system. Specifically, the target nucleic acid refers to a sequence that is complementary to the guide sequence contained in the guide RNA or a sequence that binds complementarily to the guide sequence. The "target strand" refers to a strand containing the target sequence. If the target nucleic acid or target gene is single-stranded, that strand may be the target strand. Alternatively, if the target nucleic acid or target gene is double-stranded, one of the double strands may be the target strand, and a strand complementary to the target strand may exist. In this case, the strand complementary to the target strand is referred to as the "non-target strand." The non-target strand includes a PAM (Protospacer Adjacent Motif) sequence and a protospacer sequence. The PAM sequence is a sequence recognized by the Cas protein of the CRISPR / Cas system. The above protospacer sequence is a sequence located at the 5' or 3' end of a PAM sequence, which is a sequence that is complementary to the target sequence or binds complementarily to the target sequence. The correlation between the protospacer sequence and the target sequence is similar to the correlation between the target sequence and the guide sequence. Due to these characteristics, the protospacer sequence can generally be used when designing a guide sequence. That is, when designing a guide sequence that binds complementarily to the target sequence, the guide sequence can be designed as a nucleotide sequence having the same base sequence as the protospacer sequence. In this case, the guide sequence can be designed by replacing T in the base sequence of the protospacer sequence with U.

[0036] In the present invention, the CRISPR / Cas system may be composed of a plasmid. The plasmid may be composed of a 5' homologous arm, a promoter sequence, a gene sequence introduced at the FTO gene site (e.g., a nucleotide sequence encoding a fluorescent protein, or a nucleotide sequence encoding a CAR protein), a poly-A sequence, and a 3' homologous arm. The plasmid may be, for example, a pT7 plasmid, but may be used without limitation as long as the CRISPR / Cas system can function normally.

[0037] For the purposes of the present invention, the CRISPR / Cas system of the present invention may delete part or all of the FTO gene in the stem cell of the present invention, and furthermore, may insert a foreign gene at the location of the FTO gene within the chromosome of the stem cell of the present invention, and any gene editing technique capable of functioning as described above may be included without limitation.

[0038] Meanwhile, in the present invention, the foreign gene may be a gene encoding a chimeric antigen receptor (CAR), and in this case, the gene encoding the chimeric antigen receptor may be inserted at the FTO gene location within the chromosome of the stem cell of the present invention, and the chimeric antigen receptor may specifically bind to a tumor antigen.

[0039] The above "Chimeric antigen receptor (CAR)" is a synthetic complex designed to induce an immune response when it recognizes and binds to a target antigen and a cell expressing said antigen. The chimeric antigen receptor may include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The chimeric antigen receptor is expressed on the surface of immune cells and, through the antigen binding site included in the extracellular domain, recognizes and binds to a specific antigen, such as an antigen specifically expressed on the surface of a cancer cell, thereby causing signal transduction within the immune cell and altering the activity of the immune cell, thus enabling it to induce an immune response by targeting only a specific antigen.

[0040] When the above chimeric antigen receptor is expressed on the surface of an immune cell, if the receptor binds to a target antigen, signal transduction occurs within the immune cell, which may induce an enhancement of the cytotoxicity (or cytolytic activity) of the immune cell and / or promote cytokine secretion by the immune cell. The enhancement of cytotoxicity (or cytolytic activity) or promotion of cytokine secretion may involve exhibiting cytotoxicity (or cytolytic activity) or secreting cytokines at a higher level than that exhibited by immune cells in the absence of the antigen.

[0041] The above-mentioned extracellular domain comprises an antigen-binding site that specifically binds to a desired target antigen, and is not limited to any specific type. The above-mentioned extracellular domain may be applied without limitation as long as it has a structure capable of specifically recognizing and binding to biological molecules, such as cell surface receptors, tumor proteins, lipids, polysaccharides, or other cell surface target molecules, and may be, for example, a protein, polypeptide, or a variant thereof having such characteristics. "Specifically binding" may mean binding to another molecule with a binding affinity greater than background binding; for example, the above-mentioned extracellular domain may be about 10 for the target antigen -5 It may be binding to a target antigen with an affinity of M or greater or Ka (equilibrium dissociation constant of a specific binding interaction having units of 1 / M). The above affinity is such that the equilibrium dissociation constant (Kd) of a specific binding interaction having units of M is 10 -5 M to 10 -13 M or may be less than the above range.

[0042] The above target antigen may be specifically expressed in cancer cells. For example, the above target antigens are glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, cyclin BL, lectin-reactive AFP, Fos-associated antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUl, RU2, SSX2, AKAP-4, LCK, OY-TESl, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plurisial acid, PLAC1, RUl, RU2(AS), enteric carboxyesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumine, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), MLIAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG(TMPRSS2 ETS fusion gene), NA17, Neutrophil elastase, EWSR1-ATF1, NY-BR-1, ephnnB2, CD5, CD19, CD20, CD19 / CD20, CD22, CD24, CD30, CD33, CD38,CD44v6, CD44v7 / 8, CD97, CD138, CD171, CD179a, CD244, EBNA3C, EGP-2, EGP-40, FBP, GPA7, KDR, Androgen receptor, FAP, Insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, Folic acid receptor (FRa), Folic acid receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, Mezothelin, Tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5, p53, Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB3, c-met, nm-23H1, IL13Ra2, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCASl, SDCCAG1 6, TA-90 / Mac-2 binding protein / cyclophyllin C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM,IL6 and MET may be, but are not limited to, IL6 and MET.

[0043] The antigen-binding site may be an antibody or an antigen-binding fragment thereof. Specifically, the antigen-binding site may be an antibody or an antigen-binding fragment thereof that specifically binds to a target antigen specifically expressed in cancer cells, and more specifically, the antigen-binding site may be an antibody or an antigen-binding fragment thereof that specifically binds to CD5, but is not limited thereto.

[0044] The above antibody refers to an immunoglobulin molecule that is reactive by specifically binding to an epitope of an antigen immunologically. The above antibody may include monoclonal antibodies, polyclonal antibodies, antibodies having a full-length chain structure (full-length antibody), functional fragments having at least an antigen-binding function (antigen-binding fragments), and recombinant antibodies; specifically, the antibody of the present invention may be a monoclonal antibody or an antigen-binding fragment thereof. The above monoclonal antibody refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such monoclonal antibodies exhibit single-binding specificity and affinity for a specific epitope. The above full-length antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain may be connected to the heavy chain by a disulfide bond. The above antibody comprises a heavy chain (HC) and a light chain (LC) polypeptide, and the heavy chain and the light chain may include a variable region and a constant region.

[0045] The above-mentioned constant region is a region that mediates the binding of the antibody to various types of cells of the immune system (such as T-cells) and host tissues containing components of the complement system. The above-mentioned constant region performs the same function regardless of the type of antigen for antibodies of the same type originating from the same species, and the amino acid sequence constituting it is also identical or has a high degree of similarity for each antibody. The above-mentioned constant region can be divided into a heavy chain constant region (which may be abbreviated as CH) and a light chain constant region (which may be abbreviated as CL). The above-mentioned heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and / or epsilon (ε) types, and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and / or alpha 2 (α2) as subclasses. The light chain constant region has kappa (κ) and lambda (λ) types. IgG is a subtype that includes IgG1, IgG2, IgG3, and IgG4.

[0046] The variable region is an antibody region that has specificity for an antigen and can be divided into a variable region of the heavy chain (which may be abbreviated as VH) and a variable region of the light chain (which may be abbreviated as VL). The variable region may include three complementary-determining regions (CDRs) and four framework regions (FRs). The CDRs may be ring-shaped regions involved in antigen recognition, and specificity for the antigen may be determined according to the amino acid sequence of the CDRs. The CDRs may be referred to as CDR1, CDR2, and CDR3 according to their order, and depending on whether they are the CDRs of the heavy chain or light chain polypeptide, they may be referred to as CDR-H1, CDR-H2, and CDR-H3 for the heavy chain variable region, and CDR-L1, CDR-L2, and CDR-L3 for the light chain variable region. Likewise, FR can be referred to as FR-H1, FR-H2, FR-H3, and FR-H4 for the heavy chain variable region, and FR-L1, FR-L2, FR-L3, and FR-L4 for the light chain variable region.

[0047] The above-mentioned antigen-binding fragment refers to any fragment of an antibody that possesses the antigen-binding function of an antibody. The above-mentioned antigen-binding fragment may be referred to interchangeably with terms such as "fragment" or "antibody fragment," and the above-mentioned antigen-binding fragment may be Fab, Fab', F(ab')2, Fv, etc., but is not limited thereto.

[0048] The above Fab has a structure having variable regions of the light and heavy chains, a constant region of the light chain, and a first constant region of the heavy chain (CH1 domain), and has one antigen-binding site. The above Fab' differs from the above Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The above F(ab')2 is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. The above Fv refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In a two-chain variable fragment, the heavy chain variable region and the light chain variable region are connected by non-covalent bonds, while in a single-chain variable fragment (scFv), the heavy chain variable region and the light chain variable region are generally connected by covalent bonds via a peptide linker or directly at the C-terminus, so they can form a dimer-like structure similar to the two-chain variable fragment. The above antigen-binding fragment can be produced using a proteolytic enzyme (for example, if the whole antibody is restricted to papain, Fab can be obtained, and if it is restricted to pepsin, F(ab')2 fragment can be obtained), or through genetic recombination technology, but is not limited thereto.

[0049] The linker may be a peptide linker and may have a length of about 10 to 25 amino acids. For example, the linker may include hydrophilic amino acids such as glycine (G) and / or serine (S). The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (where n and m are each 1 to 10), for example (GnS)m (where n and m are each 1 to 10), but is not limited thereto.

[0050] The extracellular domain may further include at least one selected from the group consisting of a hinge domain and a spacer domain. The antigen binding site of the extracellular domain may be connected to a transmembrane domain through the hinge domain and / or the spacer domain.

[0051] The hinge domain is a portion that allows the antigen binding site to be physically separated from the surface of the immune cell expressing the chimeric antigen receptor, so as to enable appropriate cell / cell contact, appropriate antigen / antigen binding, and appropriate activation of the chimeric antigen receptor, and can play an important role in localization of the extracellular domain. The chimeric antigen receptor may include one or more hinge domains between the extracellular domain and the transmembrane domain. The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. The altered hinge region is (a) a naturally occurring hinge region having up to 30% amino acid change (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), (b) a portion of a naturally occurring hinge region of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) length having up to 30% amino acid change (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), or (c) a core hinge region (which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). It refers to a portion of a naturally occurring hinge region that includes (which may be of length). In certain embodiments, one or more cysteine ​​residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have another amino acid residue, such as a proline residue of the wild-type immunoglobulin hinge region substituted with cysteine.The hinge domain may be a hinge region derived from the extracellular domain of type 1 membrane proteins such as CD8, CD4, CD28, and CD7, but any capable of connecting the antigen binding site, the transmembrane domain, and the intracellular signaling domain across the cell membrane may be used without limitation. Additionally, it may be a wild-type hinge region from these molecules or may be modified.

[0052] The above spacer domain may be referred to as a connection domain, and may include, for example, a hinge domain derived from CD28 and / or a hinge domain derived from CD8, and may include all or part of the hinge domain derived from CD28 and / or a hinge domain derived from CD8.

[0053] The hinge domain and / or spacer domain may be at least one selected from the group consisting of a Myc epitope, a CD8 hinge domain and Fc, and specifically may include a Myc epitope and a CD8 hinge domain, but is not limited thereto.

[0054] The above transmembrane domain refers to a portion of the region that connects and fuses the extracellular domain and the intracellular signaling domain and serves to anchor a chimeric antigen receptor to the plasma membrane of an immune cell. The above transmembrane domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The above transmembrane domain may be any one selected from the group consisting of the alpha (α), beta (β), or zeta (ζ) chain of a T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, but is not limited thereto.

[0055] The above-mentioned transmembrane domain may be attached to the above-mentioned extracellular domain through a linker. For example, the linker may be a short oligopeptide or polypeptide linker of length 2 to 10 amino acids, such as a glycine (G)-serine (S) doublet, but is not limited thereto.

[0056] The intracellular signaling domain described above corresponds to a portion that serves to transmit signals generated by the binding of the chimeric antigen receptor and the antigen into the immune cell in order to induce the function of the immune cell (e.g., activation including the release of cytotoxic (or cytolytic activity) factors toward target cells bound to the chimeric antigen receptor and the antigen, cytokine production, proliferation and cytotoxic or cytolytic activity, or other cellular responses induced by said antigen binding). The intracellular signaling domain may be a part of a protein that transmits an actuating signal and directs the cell to perform a specific function.

[0057] The intracellular signaling domain mentioned above may be an intracellular signaling domain that was previously used in the development process of chimeric antigen receptors. Specifically, it may include only CD3ζ, which was used in first-generation CARs (chimeric antigen receptors). Also, as used in second-generation CARs, a form combining CD3ζ with a co-stimulating domain (CD28 or CD137 / 4-1BB) may be used to enhance responsiveness to immune cells. Furthermore, as used in third-generation CARs, two or more co-stimulating domains may be used, and in this case, to achieve expansion and persistence of immune cells containing the CAR in vivo, the co-stimulating domain may be combined with 4-1BB, CD28, or OX40, etc. Furthermore, as used in 4th generation CARs, additional genes encoding cytokines such as IL-12 or IL-15 may be included to allow for additional expression of cytokine-based immune proteins, and as used in 5th generation CARs, an interleukin receptor chain, e.g. IL-2Rβ, may be additionally included to enhance immune cells.

[0058] The intracellular signaling domain may include at least one selected from the group consisting of T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d.

[0059] More specifically, the activation of the immune cell by the intracellular signaling domain may be mediated by two different classes of intracellular signaling domains. For example, immune cell activation may be mediated by a primary signaling domain that initiates antigen-dependent primary activation and a co-stimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal. Accordingly, the intracellular signaling domain may include a primary signaling domain and a co-stimulatory signaling domain.

[0060] The above primary signaling domain refers to a signaling domain that regulates immune cell activation in a stimulating or inhibitory manner. A primary signaling domain acting in a stimulating manner may contain an immune receptor tyrosine-based activation motif or a signaling motif known as an ITAM. An ITAM containing a primary signaling domain may be TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD66d, etc., but is not limited thereto. More specifically, the above primary signaling domain may be CD3ζ(zeta), but is not limited thereto.

[0061] The above co-stimulatory signaling domain refers to an intracellular signaling domain of a co-stimulatory molecule. The above co-stimulatory signaling domain may include, but is not limited to, a co-stimulatory signaling domain selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a / CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, CD5, CD83, etc.

[0062] The chimeric antigen receptor may include two or more intracellular signaling domains, and if it includes two or more intracellular signaling domains, the intracellular signaling domains may be connected in series with each other. Alternatively, they may be connected via a polypeptide linker consisting of 2 to 10 amino acids, and the linker sequence may be, for example, a glycine-serine continuous sequence. The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (where n and m are each 1 to 10), for example, (GnS)m (where n and m are each 1 to 10), but is not limited thereto.

[0063] The chimeric antigen receptor may additionally include an immune function-promoting factor of an immune cell, for example, the immune function-promoting factor of the immune cell may be an interleukin signal sequence. The interleukin signal sequence may be characterized by inducing the expression of IL-12, IL-8, IL-2, etc., but is not limited thereto. Additionally, if the immune cell is a T cell, the immune function-promoting factor may be IL-7, CCL19, etc., but is not limited thereto.

[0064] Meanwhile, the chimeric antigen receptor may further include a signal peptide for domain exposure. The signal peptide may be any type of secreted or transmembrane protein that directs the chimeric antigen receptor to be transported to the cell membrane or cell surface and can provide precise localization. Specific types may be CD8 alpha or mouse light kappa signal peptide, but are not limited thereto.

[0065] In one embodiment of the present invention, a CRISPR / Cas system was used to insert a nucleotide sequence (Sequence No. 11) encoding a CD5-CAR protein into the FTO gene location of human pluripotent stem cells, thereby producing stem cells capable of deleting the FTO gene while simultaneously expressing the CD5-CAR protein. The CD5-CAR gene (Sequence No. 11) was composed of a CD8aSP sequence (Sequence No. 17), a CD5 scFV sequence (Sequence No. 18), a Myc tag sequence (Sequence No. 19), a hinge sequence (Sequence No. 20), a transmembrane domain sequence (Sequence No. 21), a DAP10 sequence (Sequence No. 22), and a CD3ζ sequence (Sequence No. 23).

[0067] 2. Immune cells with suppressed FTO gene expression

[0068] In addition, another aspect of the present invention provides an immune cell in which the expression of the FTO (fat mass and obesity-associated protein) gene is suppressed.

[0069] Duplicate entries are ' 1. Stem cells with suppressed FTO gene expression As it is identical to what was stated above, the description is omitted.

[0070] The above "immune cell" may be used without restriction as long as it is a cell capable of inducing immunity to produce a desired therapeutic effect, and for example, it may be any one selected from the group consisting of macrophages, natural killer cells (NK cells), T cells, natural killer T cells (NKT cells), cytokine-induced killer cells (CIKs), and dendritic cells; specifically, it may be a macrophage, a natural killer cell, or a T cell, but is not limited thereto. The above T cell may be cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), T cells isolated from peripheral blood mononuclear cells (PBMCs), etc., but is not limited thereto.

[0071] Among the aforementioned immune cells, "natural killer cells" are key innate immune cells that immediately recognize and eliminate infections caused by viruses, bacteria, fungi, and parasites, as well as abnormal self cells (especially cancer cells). Unlike T cells, which recognize target cells by expressing antigen-specific receptors, natural killer cells recognize abnormal changes in target cells (especially cancer cells and infected cells), such as the balance of inhibitory or activating receptors like Killer Immunoglobulin Receptors (KIR), Natural Cytotoxicity Receptors (NCR), DNAM-1 (DNAX accessory molecule-1), and NKG2D (NK group 2 member D), and the loss of surface MHC (Major Histocompatibility Complex) Class I antigens, without specificity to antigens or human leukocyte antigen (HLA) matching, and exhibit contact-dependent cytotoxicity through various mechanisms. Unlike T cells, which can cause graft-versus-host disease (GVHD) in non-self allogeneic cells that do not match human leukocyte antigens (HLA), allogeneic NK cells have almost no side effects of graft-versus-host disease and may instead show strong anticancer effects.

[0072] In one embodiment of the present invention, it was confirmed that when a tumor is formed in mice after deleting the FTO gene using a CRISPR / Cas9 system, metastatic nodules are hardly formed in the lungs compared to normal mice, the tumor size is reduced, and the survival rate is also increased. Additionally, when NK cells were extracted from FTO-deleted mice, activated, and then treated with tumor cell lines, it was confirmed that the cytolytic activity against tumor cells was significantly higher than that of NK cells extracted from normal mice.

[0073] In addition, in another embodiment of the present invention, it was confirmed that when FTO gene expression in human NK cell lines is suppressed using siRNA and then treated with tumor cell lines, the cytolytic activity and cytokine secretion amount against tumor cell lines are significantly increased compared to normal NK cell lines.

[0074] In other words, the present invention has confirmed that the activity of NK cells can be significantly increased by deleting the FTO gene in NK cells, and thereby effectively suppress the survival, growth, and metastasis of cancer cells. Therefore, immune cells with suppressed expression of the FTO gene of the present invention can be effectively utilized for the prevention or treatment of cancer.

[0075] Meanwhile, in the present invention, the immune cells may be differentiated from the stem cells of the present invention described above. That is, the immune cells of the present invention may have the expression of the FTO gene suppressed by differentiating from stem cells in which the expression of the FTO gene is suppressed.

[0076] Immunotherapy drugs have limitations such as immune mismatch and a limited number of cells, but the immune cells of the present invention can overcome such limitations by differentiating from stem cells.

[0077] In one embodiment of the present invention, a stem cell with suppressed expression of the FTO gene was produced by deleting the FTO gene by inserting a nucleotide sequence encoding mCherry fluorescent protein at the FTO gene site of a human pluripotent stem cell using a CRISPR / Cas system, and the stem cell was differentiated into an NK cell with suppressed expression of the FTO gene by treating it under differentiation-specific conditions, and it was confirmed that the NK cell had excellent anti-tumor activity.

[0078] Meanwhile, if the above immune cell is differentiated from a stem cell among the stem cells of the present invention described above in which a gene encoding a chimeric antigen receptor is inserted at the location of the FTO gene, the immune cell of the present invention may further express a chimeric antigen receptor on its cell surface.

[0079] When immune cells express chimeric antigen receptors on their cell surface, they express antigen-binding sites capable of specifically recognizing and binding to tumor antigens that may be specifically expressed in cancer cells. Consequently, the immune cells can specifically bind to cancer cells expressing the tumor antigens, and signal transduction may occur via the chimeric antigen receptors. This leads to further enhancement of the cytotoxic or cytolytic activity of the immune cells and an increase in the secretion of cytokines. Therefore, among the immune cells of the present invention, immune cells that express chimeric antigen receptors may exhibit enhanced cytolytic activity against cancer cells.

[0080] In one embodiment of the present invention, a CRISPR / Cas system was used to insert a nucleotide sequence encoding mCherry fluorescent protein into the FTO gene location of human pluripotent stem cells, thereby producing stem cells capable of deleting the FTO gene while simultaneously expressing CD5-CAR protein. Then, by treating the stem cells with differentiation-specific conditions to differentiate them into NK cells, NK cells were produced in which the expression of the FTO gene was suppressed while simultaneously expressing CD5-CAR protein, and it was confirmed that these NK cells possessed significantly high anti-tumor activity.

[0082] 3. The therapeutic use of immune cells of the present invention against cancer

[0083] Another aspect of the present invention provides a cell therapeutic agent comprising the immune cells as an active ingredient.

[0084] In addition, another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the immune cells as an active ingredient.

[0085] Duplicate entries are ' 1. Stem cells with suppressed FTO gene expression ' and ' 2. Immune cells with suppressed FTO gene expression As it is identical to what was stated above, the description is omitted.

[0086] In the cell therapeutic agent of the present invention, the immune cells may further express a chimeric antigen receptor (CAR) on the cell surface.

[0087] In addition, in the pharmaceutical composition of the present invention, the immune cells may further express a chimeric antigen receptor (CAR) on the cell surface.

[0088] The cell therapeutic agent or pharmaceutical composition of the present invention may have a preventive or therapeutic effect against cancer by including the immune cell of the present invention, which has excellent anti-tumor activity, as an active ingredient.

[0089] In this invention, the terms "cancer" and "tumor" are used interchangeably and refer to any condition in which a problem occurs in the regulatory functions of normal cell division, differentiation, or apoptosis, resulting in abnormal overproliferation and the infiltration of surrounding tissues or organs to form a mass, thereby destroying or altering existing structures.

[0090] Cancers that can be treated with the cell therapy or pharmaceutical composition of the present invention may include all types of cancer, neoplasms, or malignant tumors found in mammals, including leukemia, lymphoma, carcinoma, and sarcoma. For example, the above cancers include liver cancer, thyroid cancer, papillary thyroid carcinoma, medullary thyroid carcinoma, pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, bronchogenic carcinoma, basal cell carcinoma, testicular cancer, bone marrow cancer, multiple myeloma, myelodysplasia, osteosarcoma, osteogenic sarcoma, colon cancer, glioblastoma, oral cancer, lip cancer, mycosis fungoides, ovarian cancer, ovarian germ cell tumor, male breast cancer, cystic carcinoma, endothelial sarcoma, brain cancer, meningioma, pituitary adenoma, biliary duct carcinoma, colorectal cancer, head and neck cancer, craniopharyngioma, biliary tract cancer, gallbladder cancer, multiple myeloma, lymphoangioendothelial sarcoma, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, lymphangioma, membranous carcinoma, retinoblastoma, choroidal melanoma, diffuse large B-cell lymphoma, bladder cancer, preleukemia, acute myeloid Leukemia, acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia (BALL), acute leukemia, acute lymphoblastic leukemia (ALL), chronic leukemia, chronic myeloid leukemia (CML), chronic lymphoblastic leukemia (CLL), T-cell acute lymphoblastic leukemia (TALL), small lymphoblastic leukemia (SLL), ampullary carcinoma, non-melanoma skin cancer, bladder cancer, peritoneal cancer, parathyroid carcinoma, adrenal cancer, paranasal sinus cancer, squamous cell carcinoma, non-small cell lung cancer, Wilms' tumor, epithelial carcinoma, ependymoma, germ cell cancer, kidney cancer, renal cell carcinoma, glioma, neuroblastoma, renal pelvis cancer, neuroblastoma, neuroendocrine tumor, duodenal cancer, tongue cancer, fibrosarcoma, adenocarcinoma, astrocytoma, small cell pineal cell tumor, pediatric brain tumor, pediatric lymphoma, small intestine cancer, meningioma, medulloblastoma, renal cell carcinoma, Esophageal cancer, ocular cancer, malignant soft tissue tumor, malignant endoplasmic reticulum, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye tumor, vulvar cancer, Ewing's chondrosarcoma, ureteral cancer, urethral cancer, cancer of unknown primary site, sarcoma, carcinoid tumor, breast cancer, choriocarcinoma, gastric cancer, gastric lymphoma, gastric carcinoid tumor, gastrointestinal stromal tumor, Wilms tumor, penile cancer, pharyngeal cancer, gestational trophoblastic disease, uterine cancer, cervical cancer, endometrial cancer,Uterine sarcoma, prostate cancer, myxosarcoma, seminoma, liposarcoma, mesothelioma, rectal cancer, vaginal cancer, metastatic bone tumor, metastatic brain tumor, mediastinal cancer, spinal cord cancer, chordoma, acoustic neuroma, acoustic neuroma, salivary gland cancer, pancreatobiliary cancer, pancreaticobiliary cancer, gingival cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, sebaceous gland carcinoma, oligodendroglioma retinoblastoma, leiomyosarcoma, squamous cell carcinoma, squamous cell carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, skin cancer, anal cancer, laryngeal cancer, pleural cancer, thymoma, hemangioma, hematological cancer, angiosarcoma, synovialoma, rhabdomyosarcoma, melanoma, rare cancer, embryonic carcinoma, sweat gland carcinoma, sarcoma, and combinations thereof, but not limited thereto.

[0091] In the present invention, "prevention" refers to any act of suppressing cancer or delaying its onset by administering a cell therapeutic agent or pharmaceutical composition according to the present invention, and "treatment" refers to any act of improving or beneficially altering the symptoms of cancer by administering a cell therapeutic agent or pharmaceutical composition according to the present invention.

[0092] The above cell therapy contains 1.0 x 10 to 1.0 x 10 of the immune cells relative to the total weight of the cell therapy. 10 Dog cells / ml, specifically 1.0 x 10⁶ 6 Up to 1.0X10 9 It may contain cells / ml, but is not limited thereto.

[0093] The cell therapy product described above may be formulated into a unit-dose pharmaceutical preparation suitable for administration into a patient's body according to conventional methods in the pharmaceutical field, and the preparation contains an effective dosage amount for one or several administrations. Suitable formulations for this purpose may include parenteral preparations such as injectable ampoules, injectables such as infusion bags, and sprays such as aerosol preparations. The injectable ampoule may be prepared by mixing with an injectable solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, etc., may be used as the injectable solution. Additionally, the infusion bag may be made of polyvinyl chloride or polyethylene, and examples include infusion bags from Baxter, Becton Dickinson, Medcep, National Hospital Products, or Terumo.

[0094] In addition to the active ingredient, the above pharmaceutical formulation may further include one or more pharmaceutically acceptable conventional inert carriers, for example, in the case of an injectable formulation, a preservative, an analgesic, a solubilizing agent, or a stabilizer, and in the case of a formulation for topical administration, a base, an excipient, a lubricant, or a preservative.

[0095] The cell therapy agent or pharmaceutical preparation thereof prepared according to the present invention may be administered in the form of a mixture with other cells used for cancer treatment or together with such cells using administration methods commonly used in the art. Specifically, it may be directly engrafted or implanted into the disease site of a patient requiring treatment, or directly implanted or injected into the abdominal cavity, but is not limited thereto. Furthermore, the administration may be performed using both non-surgical methods such as administration via a catheter and surgical methods such as injection or implantation after incision of the disease site. In addition, in addition to parenteral administration according to conventional methods, for example, direct administration to the lesion, implantation via intravascular injection is also possible.

[0096] The cell therapeutic agent may be administered at a dose of 0.0001 to 1,000 mg / kg per day, specifically 0.01 to 100 mg / kg, and the administration may be performed once a day or divided into several doses. However, it should be understood that the actual dosage of the active ingredient must be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and gender; therefore, the above dosage does not limit the scope of the present invention in any way.

[0097] The pharmaceutical composition of the present invention comprises 1.0 x 10 to 1.0 x 10 of the immune cells based on the total weight of the composition. 10 Dog cells / ml, specifically 1.0 x 10⁶ 6 Up to 1.0X10 9 It may contain cells / ml, but is not limited thereto.

[0098] The above pharmaceutical composition may be in the form of a quasi-drug composition, a health food composition, etc., in addition to a pharmaceutical composition.

[0099] The above pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0100] In addition, the above pharmaceutical composition may be formulated and used in the form of tablets, pills, powders, granules, capsules, suspensions, liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, transdermal absorption agents, gels, lotions, ointments, creams, patches, cataplasmas, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages, or suppositories, respectively, according to conventional methods.

[0101] Specifically, when formulating, the formulation may be prepared using diluents or excipients such as fillers, weights, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid dosage forms for oral administration include, but are not limited to, tablets, pills, powders, granules, and capsules. These solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. In addition to liquids and liquid paraffin for oral administration, various excipients, for example, wetting agents, sweeteners, flavoring agents, and preservatives, may be added. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc. may be used as bases for suppositories.

[0102] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including individual type and severity, age, gender, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. For example, the pharmaceutical composition may be administered at a dose of 0.0001 to 1,000 mg / kg per day, specifically 0.01 to 100 mg / kg, and the administration may be divided into one dose or several doses per day.

[0103] The above pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0104] The term "administration" above means introducing the composition of the present invention into an individual by any appropriate method, and the route of administration of the composition may be any general route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, or orally, topically, or intranasally, but is not limited thereto.

[0105] The term "individual" above refers to any animal, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, that has developed or may develop cancer. The types of individuals may be included without limitation as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the individual.

[0106] Another aspect of the present invention provides a method for preventing or treating cancer, comprising the step of administering the cell therapeutic agent or pharmaceutical composition to an individual.

[0108] The present invention will be explained in detail below through examples.

[0109] However, the following examples are intended to specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0111] [Example 1]

[0112] Analysis of the effects of the FTO (Fat mass and obesity-associated protein) gene on NK cell activity

[0113] FTO (Fat mass and obesity-associated protein) is a protein that removes m6A and is known to be able to regulate gene expression by controlling the characteristics of specific RNAs containing m6A. Accordingly, experiments were conducted to determine how the expression of the FTO gene affects NK cell activity.

[0115] 1-1. Mouse Experiment

[0116] First, FTO-deleted mice were prepared by deleting 76 nucleotides, including the start codon, from the first exon of the FTO gene using the CRISPR / Cas9 system (pT7 plasmid) (Fig. 1). Subsequently, splenocytes and bone marrow cells were extracted from normal mice and FTO-deleted mice, and total RNA was obtained from the cells to measure the m6A content. The results showed that the m6A content was significantly increased in the splenocytes and bone marrow cells of FTO-deleted mice, confirming that the FTO gene was deleted normally (Fig. 2).

[0117] Subsequently, B16F10 mouse melanoma cell lines were intravenously injected into the tails of normal mice and FTO-deficient mice to measure the extent of cancer cell metastasis to the lungs, tumor size, and mouse survival rate. As a result, in normal mice, extensive metastatic nodules formed in the lungs, tumor size increased, and survival rate decreased significantly; however, in FTO-deficient mice, almost no metastatic nodules formed in the lungs, tumor size decreased compared to normal mice, and survival rate was higher compared to normal mice (Fig. 3).

[0118] Next, to confirm whether the above-mentioned activity in FTO-deficient mice was due to NK cell activity, normal mice and FTO-deficient mice were treated with an anti-NK1.1 antibody to deplete NK cells, and it was found that B16F10 metastatic nodules formed in FTO-deficient mice as in normal mice (Fig. 4). Through this, it was confirmed that the non-formation of B16F10 metastatic nodules in FTO-deficient mice was due to NK cell activity.

[0119] In addition, NK cells were extracted from normal mice and FTO-deficient mice, activated by treating them with IL-2 and IL-15, and then treated with EL4 lymphoma cell lines and YAC1 lymphoma cell lines. As a result, the cytotoxicity of the activated NK cells extracted from FTO-deficient mice was found to be significantly higher than that of NK cells derived from normal mice (Fig. 5). Furthermore, the secretion and expression levels of IFNγ in NK cells derived from FTO-deficient mice were measured to be significantly higher than those in NK cells derived from normal mice (Fig. 6).

[0120] Through the above experiment, it was confirmed that deleting the FTO gene can increase the activity of NK cells, and thus effectively inhibit the survival, growth, and metastasis of cancer cells.

[0122] 1-2. Human NK Cell Experiment

[0123] Next, experiments were conducted to confirm the role of the FTO gene in human NK cells. First, to suppress the expression of the FTO gene in NK92 cells, NK92 cell lines were treated with two types of siRNA (si-hFTO#1: SEQ ID NO. 1, si-hFTO#2: SEQ ID NO. 2) (Fig. 7). Subsequently, the expression of the FTO gene and the m6A content in NK92 cells were measured. The results showed that in the NK92 cell lines treated with the two types of siRNA (NK92-siFTO), FTO gene expression was suppressed in a concentration-dependent manner, and the m6A content increased in a concentration-dependent manner, confirming that FTO gene expression was normally suppressed (Fig. 8).

[0124] In addition, when NK92 cell lines were co-cultured with K562 lymphoma cell lines, the cytotoxicity and degranulation activity of NK92-siFTO cell lines against K562 lymphoma cell lines were found to be significantly higher than that of the control NK92 cell lines (Fig. 9).

[0125] In addition, after treating NK92 cell lines with IL-2, the expression of granzyme B protein and the release of cytokines (TNFα, IFNγ) were measured, and it was found that the expression and release of the said proteins in the NK92-siFTO cell line were significantly increased compared to the control NK92 cell line (Fig. 10).

[0126] Conversely, when the FTO gene was introduced into the NK92 cell line to overexpress the FTO gene (NK92+FTO), cytotoxicity against the K562 lymphoma cell line was reduced, and IFNγ expression was also reduced (Fig. 11).

[0127] In addition, when comparing the expression levels of activating receptors (NKG2D, NKp30, NKp44, NKp46, CD16), IL-2 receptor (CD25), TNF receptor (CD27), and TGFβ receptor (CD105) in NK92 cell lines, it was confirmed that the expression level of the NKp30 receptor in the NK92-siFTO cell line was significantly higher than that of the control NK92 cell line (Fig. 12).

[0128] Next, the expression profiles of the JAK / STAT signaling pathway, known to activate cytokine signaling pathways, were compared in the NK92-siFTO cell line and the control NK92 cell line. As a result, total JAK1 and JAK3 levels, as well as phosphorylated JAK1 and phosphorylated JAK3 levels, were all found to be higher in the NK92-siFTO cell line compared to the control NK92 cell line, and phosphorylated STAT3 and phosphorylated STAT5 levels were also found to be significantly higher (Fig. 13). Conversely, when the FTO gene was introduced into the NK92 cell line to overexpress FTO gene expression (NK92+FTO), phosphorylated JAK1 and phosphorylated JAK3 levels were found to decrease (Fig. 14).

[0129] In addition, the expression of SOCS family genes, known to inhibit cytokine signaling pathways, was compared in the NK92-siFTO cell line and the control NK92 cell line. As a result, the expression of SOCS family genes and proteins in the NK92-siFTO cell line was significantly reduced, and in particular, the expression of SOCS3 gene and protein was found to be significantly reduced (Fig. 15).

[0130] Furthermore, when m6A methylation mRNA (MeRIP) levels were measured, the mRNA levels of SOCS1 and SOCS3 decreased in the NK92-siFTO cell line, while MeRIP levels increased, whereas the opposite result was observed in the NK92 cell line (NK92+FTO) with overexpression of the FTO gene (Fig. 16).

[0131] Meanwhile, 3'UTR reporter system analysis confirmed that m6A is present in the 3'UTR of SOCS3 and CISH and that said m6A is regulated by FTO (Fig. 17). These results indicate that FTO regulates the JAK / STAT signaling pathway by regulating the stability of SOCS3 mRNA through demethylation.

[0132] Next, human umbilical cord blood-derived CD3 - CD56 + Experiments similar to the above were performed using NK cell lines. First, CD3 using siRNA - CD56 + After reducing the expression of the FTO gene in NK cell lines (CD56 + -siFTO), as a result of treatment on K562 lymphoma cell lines, control CD56 + Compared to NK cell lines, cytotoxicity was significantly increased (Fig. 18), and mRNA expression levels of cytokines and effector molecules were found to be significantly increased (Fig. 19).

[0133] Also, CD56 +Phosphorylation levels of JAK1 and STAT3 in -siFTO cell lines compared to control CD56 + Compared to NK cell lines, it was found to be significantly increased (Fig. 20), while the protein expression levels of CISH, SOCS1, and SOCS3 were found to be decreased (Fig. 21).

[0134] Through the above experiment, when FTO gene expression is inhibited, human NK92 cell lines and CD3 - CD56 + It was found that lysis activity and cytokine secretion increase in NK cell lines, and that this activity is due to the FTO gene regulating SOCS family gene expression to inhibit the JAK / STAT signaling pathway.

[0136] [Example 2]

[0137] Analysis of antitumor activity of FTO gene-suppressed NK92 cells

[0138] Experiments were performed to confirm the antitumor activity of NK92 cells in which the expression of the FTO gene was suppressed. To this end, shRNA was first produced by cloning si-hFTO #2 (sequence number 2) into the pLKO.1 TRC plasmid (Addgene, #10878), and an NK92 cell line (NK92-shFTO) in which the expression of the FTO gene was always suppressed was produced by infecting NK92 cells with this via a lentivirus (Fig. 22).

[0139] When the above NK92-shFTO cell line was treated to the K562 lymphoma cell line, it was found that cytotoxicity increased significantly compared to the control cell line (NK92-TRC) and degranulation activity also increased (Fig. 23).

[0140] Subsequently, a luciferase-expressing K562 lymphoma cell line (K562-Luc) was injected into the tail veins of NSG mice, and after injecting either the NK92-shFTO cell line or the control NK92 cell line three times, the luminescence signal and survival rate were compared (Fig. 24). As a result, mice injected with the NK92-shFTO cell line showed a significantly lower luminescence signal and a significantly higher survival rate compared to mice injected with the control NK92 cell line (Fig. 25).

[0141] Through the above experiment, NK92 cell lines in which FTO gene expression was suppressed in vivo It was confirmed that it has excellent antitumor activity, and the specific mechanism of action of the FTO gene is as shown in Fig. 26.

[0143] [Example 3]

[0144] Production of FTO gene-deleted stem cell lines and analysis of the activity of NK cells differentiated therefrom

[0145] NK cells have problems such as immune mismatch and a limited number of cells. Accordingly, to solve the above problems, an experiment was conducted to obtain FTO gene-deleted NK cells by deleting the FTO gene in stem cells and differentiating NK cells from them.

[0147] 3-1. Production of FTO gene-deleted stem cell lines and verification of stability

[0148] First, the FTO gene was deleted in human pluripotent stem cells (hPSCs) by inserting a sequence encoding mCherry fluorescent protein (Sequence No. 3) into the FTO gene site using the CRISPR-Cas9 system (Fig. 27). Specifically, based on the pGEM-T Easy Vector, the FTO-KO plasmid and the FTO gRNA-CAS9 plasmid were prepared. The FTO 5'-arm and 3'-arm sequences of the plasmid were composed of the sequences of Sequence No. 4 and Sequence No. 6, respectively, and the EF1 promoter (Sequence No. 6) and IRES sequence (Sequence No. 7) were introduced. The FTO-gRNA was composed of the sequence of Sequence No. 8, and a puromycin resistance gene sequence (Sequence No. 9) was introduced to select the cells into which the plasmid was introduced. The plasmid (Sequence No. 10) constructed as described above was transformed into hPSCs using the P3 Primary Cell 4D-Nucleofector® X Kit (Lonza) and the 4D-Nucleofector™ System (Lonza), and screening was performed using puromycin. Subsequently, FTO gene expression was measured via PCR in the stem cell line, confirming that the FTO gene was not expressed (Fig. 28), and the mCherry gene was confirmed to be normally expressed by analyzing the fluorescence of the mCherry protein (Fig. 29).

[0149] Subsequently, the stability of the FTO-deleted stem cell line (FTO-KO hPSCs) prepared as described above was verified. First, the expression levels of pluripotency markers were measured by immunocytochemistry and flow cytometry, and it was confirmed that the expression levels of pluripotency markers specific to pluripotency stem cell lines (Oct4, Sox2, Nanog, SSEA4, and TRA-1-60) were stably maintained in the FTO-KO hPSCs cell line (Fig. 30).

[0150] In addition, karyotyping analysis was performed, and no chromosomal abnormalities that can occur due to the CRISPR-Cas9 system were observed, confirming that chromosomal stability was maintained (Fig. 31). To determine whether germ layers could differentiate normally, the expression levels of endoderm, mesoderm, and ectoderm markers were measured. As a result, mesoderm markers (αSMA, TBXT), ectoderm markers (Nestin, PAX6), and endoderm markers (Sox17, FOXA2) were all found to be stably expressed, confirming that the trigerm differentiation ability of the FTO-KO hPSCs cell line was also stable (Fig. 32).

[0152] 3-2. Analysis of NK cell differentiation and activity in FTO gene-deleted stem cell lines

[0153] Next, differentiation-specific conditions were applied to differentiate NK cells from FTO-KO hPSCs cell lines. Specifically, APELII, 0.5% P / S, 40 ng / ml BMP4, 50 ng / ml VEGF, 40 ng / ml SCF, and 5 ng / ml bFGF were applied for mesoderm induction, followed by APELII, 0.5% P / S, 40 ng / ml BMP4, 50 ng / ml VEGF, 40 ng / ml SCF, 5 ng / ml bFGF, 30 ng / ml TPO, and 50 ng / ml Flt3L for hemogenic endothelium induction. Next, APELII, 0.5% P / S, 5 ng / ml IL-3, 20 ng / ml IL-7, 10 ng / ml IL-15, 20 ng / ml SCF, 10 ng / ml Flt3L, and 30 ng / ml TPO were treated for differentiation into NK cells, and APELII, 0.5% P / S, 20 ng / ml IL-7, 10 ng / ml IL-15, 20 ng / ml SCF, 10 ng / ml Flt3L, and 30 ng / ml TPO were treated for maturation of NK cells.

[0154] Subsequently, analysis of cells expressing CD34, a marker for hematopoietic stem cells; CD45, a marker for differentiated blood cells; and CD56, a marker for NK cells, revealed that the FTO-KO hPSCs cell line expressed CD34 in larger quantities compared to the control NK cell line. + CD45 + Hematopoietic stem cells and CD45 + CD56 + It was confirmed that it differentiates into NK cells (Fig. 33).

[0155] To analyze the activity of NK cells (FTO-KO) differentiated from the FTO-KO hPSCs cell line as described above, gene expression related to NK cell activity was compared. As a result, compared to the control NK cell line, SOCS family gene expression in the FTO-KO NK cell line was significantly reduced, while the expression of TNFα, Granzyme B, MYC, and Prf1 genes, which promote NK cell activity, was significantly increased (Fig. 34).

[0156] In addition, to confirm the cancer cell killing ability of the FTO-KO NK cell line, MOLT-4 lymphoma cell line was treated, and the cytolytic activity was found to be significantly increased compared to the control NK cell line (Fig. 35).

[0157] Through the above experiment, it was confirmed that an NK cell line differentiated from stem cells with the FTO gene deleted has excellent cytolytic activity, similar to an NK cell line with the FTO gene directly deleted. Therefore, by differentiating NK cells from the above stem cell line, the limitations of NK cell therapeutics can be overcome.

[0159] [Example 4]

[0160] Production of stem cell lines with CD5-CAR gene introduced into the FTO gene locus and analysis of the activity of NK cells differentiated therefrom

[0161] In Example 3 above, the mCherry gene was introduced into the FTO gene location of human pluripotent stem cells to produce a stem cell line with a simple deletion of the FTO gene and NK cells differentiated therefrom. In the following experiment, the CD5-CAR gene, instead of the mCherry gene, was introduced into the FTO gene location of the human pluripotent stem cell line so that not only the FTO gene could be deleted but also the chimeric antigen receptor (CAR) could be additionally expressed.

[0163] 4-1. Production of stem cell lines with CD5-CAR gene introduced into the FTO gene locus and verification of stability

[0164] First, using the CRISPR-Cas9 system, the CD5-CAR gene (SEQ No. 11) was introduced into the FTO gene locus in human pluripotent stem cell lines to construct stem cell lines (FTO-CD5 hPSCs) that express the CD5-CAR gene while deleting the FTO gene (Fig. 36). Specifically, based on the pGEM-T Easy Vector, the FTO-KO plasmid and the FTO gRNA-CAS9 plasmid were prepared. The AAVS1-HA-R and AAVS1-HA-L sequences of the plasmids were composed of the nucleotide sequences of SEQ No. 12 and SEQ No. 13, respectively, and the EF1α promoter (SEQ No. 14) and IRES sequence were introduced. The AAVS1-gRNA was composed of the nucleotide sequence of SEQ No. 15, and a neomycin resistance gene sequence (SEQ No. 16) was introduced to select cells into which the plasmids were introduced. The plasmid (sequence number 24) configured as described above was transformed into hPSCs using the P3 Primary Cell 4D-Nucleofector® X kit (Lonza) and the 4D-Nucleofector™ system (Lonza), and screening was performed using neomycin.

[0165] Meanwhile, the CD5-CAR gene (sequence number 11) was composed of a CD8aSP sequence (sequence number 17), a CD5 scFV sequence (sequence number 18), a Myc tag sequence (sequence number 19), a hinge sequence (sequence number 20), a transmembrane domain sequence (sequence number 21), a DAP10 sequence (sequence number 22), and a CD3ζ sequence (sequence number 23).

[0166] Subsequently, FTO gene expression was measured in the above stem cell line via PCR, and it was confirmed that the CD5-CAR gene was normally expressed (Fig. 37). Using Myc, a marker of CD5-CAR, it was confirmed that the CD5-CAR protein was normally expressed on the cell membrane of the FTO-CD5 hPSCs cell line (Fig. 38).

[0167] In addition, to verify the stability of the FTO-CD5 hPSCs cell line, the expression levels of pluripotency markers were measured, and it was confirmed that the expression levels of pluripotency markers specific to pluripotency stem cell lines (Oct4, Sox2, Nanog, SSEA4, and TRA-1-60) were maintained stably (Fig. 39). Furthermore, karyotype analysis was performed, and it was confirmed that chromosomal stability was also maintained, as no chromosomal abnormalities that can occur due to the CRISPR-Cas9 system were observed (Fig. 40).

[0169] 4-2. Analysis of NK cell differentiation and activity in stem cell lines in which the CD5-CAR gene was introduced to the FTO gene locus

[0170] Next, differentiation-specific conditions were applied to differentiate NK cells from FTO-CD5 hPSCs cell lines. Specifically, APELII, 0.5% P / S, 40 ng / ml BMP4, 50 ng / ml VEGF, 40 ng / ml SCF, and 5 ng / ml bFGF were applied for mesoderm induction, followed by APELII, 0.5% P / S, 40 ng / ml BMP4, 50 ng / ml VEGF, 40 ng / ml SCF, 5 ng / ml bFGF, 30 ng / ml TPO, and 50 ng / ml Flt3L for hemogenic endothelium induction. Next, APELII, 0.5% P / S, 5 ng / ml IL-3, 20 ng / ml IL-7, 10 ng / ml IL-15, 20 ng / ml SCF, 10 ng / ml Flt3L, and 30 ng / ml TPO were treated for differentiation into NK cells, and APELII, 0.5% P / S, 20 ng / ml IL-7, 10 ng / ml IL-15, 20 ng / ml SCF, 10 ng / ml Flt3L, and 30 ng / ml TPO were treated for maturation of NK cells.

[0171] Subsequently, analysis of cells expressing NK cell markers revealed that the FTO-CD5 hPSCs cell line showed a higher amount of CD45 compared to the control NK cell line.+ CD56 + It was confirmed that it differentiates into NK cells (Fig. 41).

[0172] As described above, in order to analyze the activity of NK cells (FTO-CD5) differentiated from the FTO-CD5 hPSCs cell line and to confirm the cancer cell killing ability of NK cells, MOLT-4 lymphoma cell line was treated, and the cytolytic activity of FTO-CD5 NK cells against cancer cells was found to be significantly higher than that of NK cells (WT-CD5) containing the CD5-CAR gene as a control (Fig. 42).

[0174] Through the above experiment, it was confirmed that an NK cell line differentiated from a stem cell configured to express the CD5-CAR gene while deleting the FTO gene by introducing the CD5-CAR gene to the FTO gene site has significantly superior cytolytic activity compared to NK cells expressing the CD5-CAR gene. Thus, the above stem cell and NK cell can overcome the limitations of existing NK cell therapies and are therefore expected to be useful as effective anticancer immunotherapy.

[0176] Although representative embodiments of the present invention have been described above by way of example, the scope of the present invention is not limited to such specific embodiments, and those skilled in the art will be able to make appropriate modifications within the scope described in the claims of this application.

Claims

Claim 1 A stem cell in which a gene encoding a chimeric antigen receptor (CAR) is inserted into the location of the fat mass and obesity-associated protein (FTO) gene within a chromosome, thereby suppressing the expression of the FTO gene and expressing the CAR on the cell surface. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 A stem cell according to claim 1, wherein the chimeric antigen receptor specifically binds to a tumor antigen. Claim 7 The stem cell of claim 1, wherein the stem cell is a pluripotent stem cell (PSC), an embryonic stem cell (ESC), or an induced pluripotent stem cell (iPSC). Claim 8 An immune cell in which a gene encoding a Chimeric Antigen Receptor (CAR) is inserted into the location of the FTO (fat mass and obesity-associated protein) gene within a chromosome, thereby suppressing the expression of the FTO gene and expressing the CAR on the cell surface. Claim 9 In claim 8, the immune cell is an immune cell differentiated from a stem cell of any one of claims 1, 6 and 7. Claim 10 In claim 8, the immune cell is a macrophage, a natural killer cell, or a T cell. Claim 11 delete Claim 12 In claim 8, the chimeric antigen receptor is an immune cell that specifically binds to a tumor antigen. Claim 13 A cell therapeutic agent comprising immune cells that express a CAR on their surface and have a gene encoding a chimeric antigen receptor inserted into the location of the fat mass and obesity-associated protein (FTO) gene within a chromosome, thereby suppressing the expression of the FTO gene. Claim 14 delete Claim 15 A pharmaceutical composition for the prevention or treatment of cancer comprising immune cells that express a CAR on their surface and have a gene encoding a chimeric antigen receptor inserted into the location of a fat mass and obesity-associated protein (FTO) gene within a chromosome, thereby suppressing the expression of the FTO gene. Claim 16 delete

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