Method for culturing natural killer cells derived from umbilical cord blood using transformed T cells

Abstract

The present invention relates to a method for culturing natural killer cells derived from umbilical cord blood using transformed T cells. The method for culturing natural killer cells using transformed T cells according to the present invention can efficiently propagate and produce natural killer cells from a small amount of original cells. In addition, the method can also improve the cell killing ability of natural killer cells. Therefore, the method for culturing natural killer cells using transformed T cells according to the present invention can be usefully used to commercialize cell therapeutic agents. In addition, the natural killer cells produced by the culturing method of the present invention can be usefully used as cell therapeutic agents.

Description

Technical Field

[0001] The present invention relates to a method for culturing natural killer cells derived from umbilical cord blood using transformed T cells. Background Art

[0002] Immunotherapies have been developed that aim to use the patient's own immunity to treat cancer patients and prevent the patient's cancer from recurring. Specifically, immunotherapies using natural killer cells, which can be produced in large quantities and frozen, have also been studied. Natural killer cells are lymphoid cells that make up approximately 15% of all peripheral blood lymphocytes and play a key role in the innate immune response.

[0003] Specifically, natural killer cells activate dendritic cells and induce cytotoxic T lymphocytes (CTLs) to specifically respond to tumors and eliminate tumor cells. Natural killer cells directly kill malignant tumors such as sarcomas, myelomas, epithelial cancers, lymphomas, and leukemias. However, most natural killer cells in healthy subjects exist in an inactive state and need to be activated to eliminate tumors. The immune escape mechanisms of cancer cells lead to functional defects of natural killer cells in cancer patients.

[0004] Therefore, activation of natural killer cells is very important for their use in therapeutic applications.Since the number of natural killer cells in the body is limited, it is necessary to develop a technology for large-scale expansion and freezing of natural killer cells from the blood of healthy subjects or patients.

[0005] Large-scale proliferation of natural killer cells using ex vivo expansion methods In addition, methods for mass-culturing natural killer cells using peripheral blood lymphocytes (PBMCs), cord blood (CB), or human induced pluripotent stem cells as raw materials have been studied.

[0006] Specifically, unlike bone marrow, umbilical cord blood can be obtained from the umbilical cord discarded after childbirth by simple processing. Due to the revitalization of the umbilical cord blood banking industry and the ease of donor acquisition, methods of culturing natural killer cells from umbilical cord blood have been actively studied.

[0007] Specifically, the ex vivo expansion of natural killer cells derived from umbilical cord blood is based on the proliferation of natural killer cells from mononuclear cells (MNCs) as seed cells or the proliferation of natural killer cells from hematopoietic progenitor cells (CD34+ cells) as seed cells. The method using mononuclear cells as seed cells involves the use of interleukin-2 (IL-2), interleukin-15 (IL-15) and FLT-3L alone or in combination to help proliferate natural killer cells. However, this method has the problem of low proliferation rate and purity (Biossel L. et al., Biology of Blood and Marrow Transplantation, 14, 1031-1038, 2008). On the other hand, the method using hematopoietic progenitor cells as seed cells is advantageous in terms of proliferation rate and purity, and requires a long culture period and the use of a mixture of multiple cytokines and growth factors, making it difficult to commercialize natural killer cells in terms of cost (Fias AM et al., Experimental Hematology 36 (1): 61-68, 2008).

[0008] Seed cells (such as PBMC, CD3- cells, CD3-CD56+ cells and CD56+ cells) are used for ex vivo expansion culture of natural killer cells. Cytokines (such as IL-2, IL-12, IL-15 and IL-21) and OKT-3 antibodies that stimulate LPS (Goodier et al., J. Immunol. 165 (1): 139-147, 2000) and CD3 (Condiotti et al., Experimental Hematol. 29 (1): 104-113, 2001) are also used as proliferation factors for natural killer cells. The proliferation factors mentioned above allow natural killer cells to proliferate by about 3-10 times. However, the proliferation rate alone is not enough to commercialize natural killer cells for therapeutic applications.

[0009] Recent research efforts have focused on methods for using various types of feeder cells to proliferate natural killer cells in large quantities. Peripheral blood mononuclear, EBV-LCL and K562 cell lines are used as representative feeder cell lines. K562 cell line is a cell line lacking HLA derived from leukemia. K562 cell line is a typical target that can be easily attacked by natural killer cells. Most feeder cells used to culture natural killer cells are propagated by known methods, for example, by expressing 4-1BBL and membrane-bound IL-15 in K562 cell line (Fujisaki et al., Cancer Res.69(9):4010-4017,2009), expressing MICA, 4-1BBL and IL-15 (Gong et al., Tissue Antigens, 76(6):467-475,2010) and expressing 4-1BBL and membrane-bound IL-21 (Cecele JD et al., PloSONE, 7(1):e30264,2012).

[0010] Detailed description of the invention

[0011] Problems to be solved by the present invention

[0012] In some cases, the inventors of the present invention have successfully developed an effective method for the ex vivo proliferation of natural killer cells from umbilical cord blood by co-culturing the natural killer cells from umbilical cord blood with CD4+ T cells expressing co-stimulatory factors and growth factors that can increase the proliferation of natural killer cells.

[0013] Specifically, the inventors of the present invention have produced and used transformed CD4(+)T cells as feeder cells to effectively culture natural killer cells, and have found that co-culture of transformed CD4(+)T cells with cord blood-derived mononuclear cells results in increased proliferation rate and cytotoxicity of natural killer cells. Based on this finding, the present invention has been completed.

[0014] Means of solving the problem

[0015] One aspect of the present invention provides a method for culturing natural killer cells, which comprises co-culturing transformed CD4+T cells and seed cells.

[0016] Another aspect of the present invention provides natural killer cells produced by the method.

[0017] Effects of the Invention

[0018] The method of the present invention uses transformed T cells to culture natural killer cells and enables efficient proliferation and production of natural killer cells from a small amount of umbilical cord blood-derived seed cells. In addition, the natural killer cells produced by the method of the present invention have improved cytotoxicity. Therefore, the method of the present invention can be used to commercialize natural killer cells for cell therapy. In addition, the natural killer cells produced by the method of the present invention can be used for cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1a Shown is the expression of different genes in the Hut78 cell line, as determined by FACS.

[0020] Figure 1b Shown is the expression of single genes transduced into the Hut78 cell line, as determined by FACS.

[0021] Figure 1c Shown is the expression of dual genes mTNF-α / OX40L and mTNF-α / 4-1BBL transduced into the Hut78 cell line, as determined by FACS.

[0022] Figure 1d Shown is the expression of dual gene mbIL-21 / OX40L and mbIL-21 / 4-1BBL transduced into the Hut78 cell line, as determined by FACS.

[0023] Figure 1e Shown is the expression of three genes transduced into the Hut78 cell line, as determined by FACS.

[0024] Figure 1f Shown is the expression of four genes transduced into the Hut78 cell line, as determined by FACS.

[0025] Figure 2a Shown are the proliferation rates of natural killer cells generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0026] Figure 2b Shown are the proliferation rates of natural killer cells generated by co-culturing H9 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0027] Figure 2c Shown are the proliferation rates of natural killer cells generated by co-culturing Jurkat cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0028] Figure 2dShown are the proliferation rates of natural killer cells generated by co-culturing Peer cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0029] Figure 2e Shown are the proliferation rates of natural killer cells generated by restimulation at intervals of 14 or 16 days during co-culture of Hut78 cell line transduced with triple genes and cord blood-derived CD3(-) monocytes.

[0030] Figure 3a The activity of natural killer cells generated by co-culturing Hut78 cell lines transduced with different genes with umbilical cord blood-derived CD3(-) monocytes is shown.

[0031] Figure 3b The activity of natural killer cells generated by co-culturing H9 cell lines transduced with different genes with umbilical cord blood-derived CD3(-) monocytes is shown.

[0032] Figure 3c The activity of natural killer cells generated by co-culturing Jurkat cell lines transduced with different genes with umbilical cord blood-derived CD3(-) monocytes is shown.

[0033] Figure 3d The activity of natural killer cells generated by co-culturing Peer cell lines transduced with different genes with umbilical cord blood-derived CD3(-) monocytes is shown.

[0034] Figure 3e The viability of natural killer cells generated by restimulation during co-culture of Hut78 cell line transduced with triple genes with cord blood-derived CD3(-) monocytes at intervals of 14 or 16 days is shown.

[0035] Figure 4a Shown is the purity of natural killer cells (CD3-CD56+) generated by co-culturing Hut78 cell lines transduced with different genes with cord blood-derived CD3(-) monocytes.

[0036] Figure 4b The purity of natural killer cells (CD3-CD56+) generated by co-culturing H9 cell lines transduced with different genes with cord blood-derived CD3(-) monocytes is shown.

[0037] Figure 4c Shown is the purity of natural killer cells (CD3-CD56+) generated by co-culturing Jurkat cell lines transduced with different genes with cord blood-derived CD3(-) monocytes.

[0038] Figure 4dShown is the purity of natural killer cells (CD3-CD56+) generated by co-culturing Peer cell lines transduced with different genes with cord blood-derived CD3(-) monocytes.

[0039] Figure 4e The purity of natural killer cells (CD3-CD56+) generated by restimulation at intervals of 14 or 16 days during co-culture of Hut78 cell line transduced with triple genes with cord blood-derived CD3(-) monocytes is shown.

[0040] Figure 5a The activity of natural killer cells (CD16+CD56+) generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes is shown.

[0041] Figure 5b Shown is the expression level of NKG2D as a phenotypic marker of natural killer cells generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0042] Figure 5c Shown is the expression level of NKp30 as a phenotypic marker of natural killer cells generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0043] Figure 5d Shown is the expression level of NKp44 as a phenotypic marker of natural killer cells generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0044] Figure 5e Shown is the expression level of NKp46 as a phenotypic marker of natural killer cells generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0045] Figure 5f Shown is the expression level of DNAM-1 as a phenotypic marker of natural killer cells generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0046] Figure 5g Shown is the expression level of CXCR3 as a phenotypic marker of natural killer cells generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0047] Figure 6aThe activity of natural killer cells (CD16+CD56+) generated by co-culturing Hut78 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes is shown.

[0048] Figure 6b Shown is the expression level of NKG2D as a phenotypic marker of natural killer cells generated by co-culturing H9 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0049] Figure 6c Shown is the expression level of NKp30 as a phenotypic marker of natural killer cells generated by co-culturing H9 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0050] Figure 6d Shown is the expression level of NKp44 as a phenotypic marker of natural killer cells generated by co-culturing H9 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0051] Figure 6e Shown is the expression level of NKp46 as a phenotypic marker of natural killer cells generated by co-culturing H9 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0052] Figure 6f Shown is the expression level of DNAM-1 as a phenotypic marker of natural killer cells generated by co-culturing H9 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0053] Figure 6g Shown is the expression level of CXCR3 as a phenotypic marker of natural killer cells generated by co-culturing H9 cell lines transduced with various genes with cord blood-derived CD3(-) monocytes.

[0054] Figure 7a Shown are the activity of natural killer cells (CD16+CD56+) generated by restimulation at intervals of 14 or 16 days during co-culture of Hut78 cell line transduced with triple genes with cord blood-derived CD3(-) monocytes and the expression level of NKG2D as a phenotypic marker of natural killer cells.

[0055] Figure 7b Shown are the expression levels of NKp30, NKp44, NKp46, DNAM-1, and CXCR3 as phenotypic markers of natural killer cells generated by restimulation at intervals of 14 or 16 days during co-culture of Hut78 cell line transduced with the triple genes with cord blood-derived CD3(-) monocytes.

[0056] Figure 8a The cytotoxicity of natural killer cells against tumor cells generated by co-culturing Hut78 cell lines transduced with different genes with umbilical cord blood-derived CD3(-) monocytes was shown.

[0057] Figure 8b The cytotoxicity of natural killer cells against tumor cells was shown by co-culturing H9 cell lines transduced with different genes with umbilical cord blood-derived CD3(-) monocytes.

[0058] Figure 8c The cytotoxicity of natural killer cells against tumor cells was shown by co-culturing Jurkat cell lines transduced with different genes with umbilical cord blood-derived CD3(-) monocytes.

[0059] Figure 8d The cytotoxicity of natural killer cells against tumor cells was shown by co-culturing Peer cell lines transduced with different genes with umbilical cord blood-derived CD3(-) monocytes.

[0060] Figure 8e The cytotoxicity of natural killer cells against tumor cells generated by restimulation during co-culture of Hut78 cell line transduced with triple genes with umbilical cord blood-derived CD3(-) monocytes at intervals of 14 or 16 days was shown.

[0061] Figure 9a is a schematic diagram showing the administration schedule for evaluating the efficacy of natural killer cells using the Raji mouse model.

[0062] Figure 9b Shown is the survival rate of the Raji animal model, which was measured to confirm the efficacy of NK cells, RTX, and their co-administration.

[0063] Fig.10a is a schematic diagram showing the administration schedule for evaluating the efficacy of natural killer cells using the Ramos mouse model.

[0064] Fig.10b Shown is the survival rate of the Ramos animal model, which was measured to confirm the efficacy of NK cells, RTX, and their co-administration. DETAILED DESCRIPTION

[0065] The present invention will now be described in detail.

[0066] One aspect of the present invention provides a method for culturing natural killer cells, which comprises co-culturing transformed CD4+T cells and seed cells.

[0067] The transformed CD4+T cells may express at least one gene selected from the group consisting of 4-1BBL, mbIL-21, OX40L and mTNF-α genes.

[0068] Specifically, a single gene can be expressed in the CD4+T cells of conversion. In this case, the gene can be 4-1BBL, mbIL-21, OX40L or mTNF-α. Alternatively, two genes can be expressed in the CD4+T cells of conversion. In this case, gene combination can be mbIL-21 / 4-1BBL, 4-1BBL / OX40L, mTNF-α / 4-1BBL, mbIL-21 / OX40L, mbIL-21 / mTNF-α or mTNF-α / OX40L. In the embodiment sections hereinafter, mbIL-21 / 4-1BBL, mTNF-α / OX40L, mTNF-α / 4-1BBL and mbIL-21 / OX40L as gene combination are transduced into T cells.

[0069] Alternatively, three genes can be expressed in transformed CD4+T cells. In this case, the gene combination can be 4-1BBL / mbIL-21 / OX40L, mbIL-21 / OX40L / mTNF-α, mTNF-α / mbIL-21 / 4-1BBL or 4-1BBL / OX40L / mTNF-α. In the examples below, mTNF-α / mbIL-21 / 4-1BBL is transduced into T cells.

[0070] Alternatively, four genes can be expressed in transformed CD4+T cells. In this case, the gene combination can be mTNF-α / mbIL-21 / OX40L / 4-1BBL. In the Examples section below, mTNF-α / mbIL-21 / OX40L / 4-1BBL is transduced into T cells.

[0071] As used herein, the term "4-1BBL" refers to a ligand that forms a trimer and binds to 4-1BB as a receptor. 4-1BBL is a member of the TNF superfamily (TNFSF) and is also known as CD137L. The 4-1BBL gene may be derived from humans.

[0072] Specifically, the 4-1BBL gene may be the NCBI reference sequence: NM_003811, but is not limited thereto. The 4-1BBL gene may have a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1. The encoding nucleotide sequence may be shown in SEQ ID NO: 2.

[0073] As used herein, the term "mbIL-21" refers to IL-21 designed to bind to a cell membrane. mbIL-21 may be a functional protein in which IL-21 and a transmembrane protein are fused together. The transmembrane protein may be CD8α, specifically the transmembrane domain of CD8α.

[0074] Specifically, the IL-21 gene can be the NCBI reference sequence: NM_021803.3, but not limited thereto. The CD8α gene can be the NCBI reference sequence: NM_001768, but not limited thereto. mbIL-21 is expressed in the form of IL-21 bound to the cell membrane. The mbIL-21 gene can have a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:3. The encoding nucleotide sequence can be shown in SEQ ID NO:4.

[0075] As used herein, the term "OX40L" refers to a ligand that binds to OX40. OX40L is also known as TNFSF4, gp34, TXGP1, CD252, or CD134L. Specifically, the OX40L gene may be the NCBI reference sequence: NM_003326, but is not limited thereto. The OX40L gene may have a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:5. The encoding nucleotide sequence may be shown in SEQ ID NO:6.

[0076] As used herein, the term "mTNF-α" refers to a gene in which alanine-valine (a tumor necrosis factor-α converting enzyme (TACE) recognition site in the amino acid sequence of tumor necrosis factor-α) is point-mutated to proline-valine on DNA. The alanine mutated to proline is randomly selected.

[0077] Specifically, the mTNF-α gene may have a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 8. The encoding nucleotide sequence may be shown in SEQ ID NO:9.

[0078] The 4-1BBL, mbIL-21, OX40L or mTNF-α gene can be transduced via a recombinant lentivirus. The gene transduction vector is not limited to a recombinant lentivirus.

[0079] One or more genes can be transfected into cells by biochemical, physical or virus-mediated transfection. FuGene6 (Roche, USA), Lipofectamine TM2000 (Invitrogen, USA) or ExGen 500 (MBI Fermentas International Inc., Canada) can be used for biochemical transfection. Lipid-mediated transfection using lipofectamine can also be used.

[0080] As used herein, the term "vector" refers to a gene construct containing necessary regulatory elements, which are operably linked so that the gene insert introduced into the vector is expressed. The vector may be an expression vector that can express a target gene in a cell into which the vector has been introduced.

[0081] The expression vector containing the gene can be any expression vector that can express the gene in a CD4+ cell line. In the Examples section below, pCDH-CMV-MCS-EF1-Puro (SBI, CD510B-1) or pCDH-CMV-MCS-EF1-Neo (SBI, CD514B-1) lentiviral vectors are used as expression vectors.

[0082] Lentivirus refers to a virus belonging to the retrovirus family, which is characterized by a long incubation period. Lentivirus can transfer genetic information to the DNA of host cells. The use of lentivirus is one of the most effective methods of using gene delivery vectors that can replicate in non-dividing cells.

[0083] CD4+T cells can be CD4+T cells isolated in vitro, CD4+T cells cultured in vitro amplified, or CD4+ cell lines (T lymphoma cell lines). CD4+T cells can be helper T cells, and can be hybridomas obtained by fusion with cancer cells. Specifically, CD4+T cells can be selected from Hut78, H9, Jurkat, Loucy, Molt-3, Molt-13, Peer, RPMI8402, and TALL-01 cells. Preferably, CD4+T cells are Hut78, H9, Jurkat, or Peer cells.

[0084] As used herein, the term "feeder cell" refers to a cell that does not proliferate but can produce various metabolites to help target cells proliferate due to its metabolic activity. Feeder cells are also referred to as culture helper cells and can be transformed CD4+T cells that express at least one gene selected from 4-1BBL, mbIL-21, OX40L and mTNF-α genes.

[0085] The T cells used as feeder cells may be inactivated cells whose division / proliferation is inhibited, or may be non-inactivated cells. Preferably, the T cells are inactivated to ensure safety. The inactivation may be accomplished by any suitable method known in the art, for example, by irradiation with gamma rays. Most non-inactivated T cells are tumor cells, which may be killed by activated natural killer cells during culture.

[0086] As used herein, the term "seed cells" refers to cells that can be proliferated into natural killer cells by appropriate culture. Specifically, the seed cells can be cord blood-derived mononuclear cells or cord blood-derived natural killer cells, but are not limited thereto. The seed cells are preferably CD3(-) cells in which CD3(+) cells are eliminated.

[0087] According to the method of the present invention, natural killer cells can be cultured by mixing feeder cells with seed cells at a ratio of ≥0.1:1. The ratio of feeder cells to seed cells can be specifically 0.1:1 to 50:1, more specifically 0.5:1 to 40:1, even more specifically 1:1 to 30:1, most specifically 2:1 to 20:1. In one embodiment, the ratio of feeder cells to seed cells can be 2.5:1, but is not particularly limited thereto. The "ratio" is based on the number of feeder cells and seed cells.

[0088] According to the method of the present invention, natural killer cells can be cultured by mixing seed cells with feeder cells only once for 5 to 60 days or by mixing seed cells with feeder cells twice or more for at least 60 days. Preferably, the culture is performed by mixing seed cells with feeder cells only once for 14 to 21 days, but is not limited to these conditions.

[0089] According to the method of the present invention, natural killer cells can be cultured together with T lymphoma cell lines in a general animal cell culture medium, such as AIM-V culture medium, RPMI1640 culture medium, CellGro SCGM, X-VIVO20 culture medium, IMDM or DMEM. Suitable substances can be added to the culture medium. The suitable substances can be antibodies and interleukins that have low affinity for T cells and stimulate T cells, but are not limited thereto.

[0090] As used herein, the term "antibody having low affinity for T cells and stimulating T cells" refers to a protein that specifically reacts to the CD3 antigen, which belongs to a group of molecules that associate with the T cell receptor (TCR) to form an antigen recognition complex. The intracellular region of the CD3 molecule is longer than that of the TCR, and plays a role in transmitting antigen recognition signals to cells.

[0091] The antibody having low affinity for T cells and stimulating T cells is preferably an anti-CD3 antibody. Specifically, the anti-CD3 antibody may be OKT-3, UCHT1 or HIT3a.

[0092] As used herein, the term "interleukin (IL)" refers to a biologically active proteinaceous substance produced by immunocompetent cells such as lymphocytes, monocytes and macrophages. Interleukins belong to the cytokine group and can be IL-2, IL-15, IL-12, IL-18 or IL-21.

[0093] In the Examples section below, OKT-3 and IL-2 are added for culture. The antibody may be added at a concentration of 0.1 ng / ml to 1,000 ng / ml, preferably 10 ng / μl. The interleukin may be added at a concentration of 10 U / ml to 2,000 U / ml, preferably 1,000 U / ml. The culture medium may be supplemented with serum or plasma and additional growth factors that support lymphocyte proliferation. The serum or plasma is not particularly limited to a specific type and is selected from commercially available serum and plasma products of animal origin. The serum or plasma is preferably serum or plasma of human origin.

[0094] As used herein, the term "culture" refers to the growth of cells under appropriate artificially controlled environmental conditions. The transformed CD4+T cells can be cultured by suitable methods well known in the art. Specifically, the culture can be carried out in batches or fed-batch processes, or can be carried out continuously in repeated fed-batch processes.

[0095] Progenitor cells suitable for culture medium can be used. The above raw materials can be added to the culture in batches, fed-batch or continuous mode by appropriate methods during the culture period, but the addition method is not particularly limited. Basic compounds (such as sodium hydroxide, potassium hydroxide or ammonia) or acidic compounds (such as phosphoric acid or sulfuric acid) can be used to adjust the pH of the culture in an appropriate manner.

[0096] The use of T cells as feeder cells selectively induces the culture of natural killer cells from seed cells. When compared with the use of PBMC feeder cells from a donor, the use of T cells as feeder cells enables more stable culture of natural killer cells without any difference depending on who is the donor during proliferation. In addition, the use of MNCs from a donor as feeder cells makes it difficult to perform in vitro culture of umbilical cord blood seed cells. Therefore, the use of T cells as feeder cells in the method of the present invention can ensure a large amount of therapeutic natural killer cells in an efficient and stable manner.

[0097] Another aspect of the present invention provides natural killer cells produced by the method.

[0098] The natural killer cells can be frozen and do not lose their functions even when thawed. In addition, the natural killer cells have increased cytotoxicity against tumor cell lines and secrete a large amount of cytokines due to their ability to express high levels of activation receptors (such as NKp46), and therefore, their outstanding anti-cancer activity can be expected. Therefore, when activated, clinically applicable natural killer cells can be used in large quantities to produce effective cell therapeutic agents for tumors.

[0099] The natural killer cells can be used as an active ingredient of a pharmaceutical composition for preventing or treating infectious diseases. In this case, the natural killer cells are present in an amount of 10% to 95% by weight based on the total weight of the composition. The composition may also include one or more active ingredients having the same or similar functions.

[0100] The pharmaceutical compositions may be formulated for administration with one or more pharmaceutically acceptable carriers.

[0101] The dosage of the pharmaceutical composition can be adjusted according to various factors, including the type and severity of the disease, the type and amount of the active ingredient and other ingredients, the type of formulation, the patient's age, weight, general health, sex and diet, the time and route of administration, the secretion rate of the composition, the treatment period and the presence of concomitant medications. For the desired effect, the natural killer cells are 0.01×10 7 cells / kg to 1.0×10 9 cells / kg or 0.5×10 7 cells / kg to 1.0×10 8 The composition can be administered in a single or divided dose per day.

[0102] The pharmaceutical composition can be administered to a subject by various methods known in the art. A person skilled in the art can appropriately select an administration route after considering the administration method, body fluid volume, composition viscosity and other factors.

[0103] Another aspect of the present invention provides a composition for culturing natural killer cells, comprising transformed CD4+T cells as an active ingredient. Since CD4+T cells and one or more genes transduced into the cells have been described above, their description is omitted to avoid excessive repetition.

[0104] Mode for Carrying Out the Invention

[0105] The present invention will be explained in detail with reference to the following examples. However, these examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0106] Example 1. Production of recombinant lentivirus

[0107] Example 1.1. Construction of recombinant lentiviral vector

[0108] pCDH-CMV-MCS-EF1-Puro (SBI, CD510B-1) or pCDH-CMV-MCS-EF1-Neo (SBI, CD514B-1) was used as a lentiviral vector. 4-1BBL (TNF superfamily member 9, TNFSF9), mbIL-21 (membrane-bound IL-21), OX40L (TNF superfamily member 4 (TNFSF4) transcript variant 1) and mTNF-α (membrane-bound TNFα) were used as genes for transduction.

[0109] Specifically, the 4-1BBL gene expression vector (Origene, RC211160) was used for the 4-1BBL gene (SEQ ID NO: 2), and the pcDNA3.1 vector (Genscript, USA) carrying the codon-optimized mbIL-21 gene sequence was used for the mbIL-21 gene (SEQ ID NO: 4). The synthesis of the OX40L gene (SEQ ID NO: 6) was requested from Bioneer.

[0110] For the mTNF-α gene (SEQ ID NO: 9), RNA was extracted from peripheral blood mononuclear cells (PBMCs) and CDS was obtained by reverse transcriptase (RT)-PCR. TNF-α was cut with tumor necrosis factor-α converting enzyme (TACE) for secretion. For this cut, alanine-valine (TACE recognition site in the amino acid sequence of TNF-α) was point mutated to proline-valine on DNA so that TNF-α remained attached to the cell membrane. The point mutation was performed by replacing the guanine and adenine residues at positions 226 and 228 of the human mTNF-α gene (SEQ ID NO: 7) with cytosine and guanine, respectively.

[0111] The coding sequence (CDS) of each gene was amplified by PCR using primers appropriate for the gene (Table 1).

[0112] [Table 1]

[0113]

[0114] The gene and the lentiviral vector are treated with EcoRI and BamHI as restriction enzymes, and then connected with In-Fusion HD cloning kit (Clontech, 639649). The connected lentiviral vector is transformed into DH5α competent cells and then cultured. Plasmid DNA is isolated from the transformed DH5α competent cells using a plasmid miniprep kit (MACHEREY-NAGEL / 740422.50). All plasmid DNA samples are outsourced for sequencing to confirm the matching of DNA sequences. The desired gene is transduced into cLV-CMV-MCS-IRES-Puro (puromycin), cLV-CMV-MCS-IRES-Neo (neomycin) and cLV-CMV-MCS-IRES-Bsd (blasticidin) in the same manner as above. This gene transduction is outsourced to an external manufacturer.

[0115] Example 1.2. Production of concentrated lentivirus

[0116] In this example, recombinant lentivirus was produced. First, 1.5×10 cells of 293T cell line were cultured 2 days before transfection. 6 -2×10 6 The cells were inoculated into 75T flasks (Nunc, 156499). The cells were cultured in an incubator at 5% CO2 and 37°C. When the 293T cells were cultured to about 80-90% confluence, the culture medium was replaced with 6ml of OPTI-MEM (Gibco, 31985-088). The cells were cultured at 37°C and 5% CO2 for 30min. DNA mixtures and lipofectamine mixtures (lipofectamine 2000, Life technologies, 11668500) were prepared as shown in Table 2.

[0117] [Table 2]

[0118]

[0119] The components of each mixture are fully mixed in a vortexer and allowed to stand at room temperature for 3min. Afterwards, the two mixtures are mixed together and allowed to stand at room temperature for at least 20min. The 293T cells cultured in 6ml OPTI-MEM culture medium are treated with 2ml of DNA / lipofectamine mixture. After 4h, the culture medium is replaced with DMEM (Gibco, 11995073) supplemented with 10% (v / v) FBS. The cells are cultured for 48h at 37°C and 5% CO2. The 8ml culture solution of 293T cells is collected and passed through a 0.45μm filter (Millipore, SLHP033RS). The filtrate is concentrated to ≤250μl with an Amicon Ultra-15 centrifugal filter device (Merckmillipore, UFC910096) with an Ultracel-100 membrane. The concentrated virus is aliquoted in appropriate amounts and stored at -80°C.

[0120] Example 2. Generation of transgenic T cells

[0121] Example 2.1. Lentivirus infection

[0122] 0.5×10 6 Cells, 1 ml OPTI-MEM, 50 μl thawed lentiviral solution and 10 μg / ml polybrene (Santa Cruz, C2013) were mixed and plated in a 6-well plate (Nunc, 140675), followed by spinning at 1800 × g and 32° C. for 90 min. The cells were then cultured in an incubator at 5% CO2 and 37° C. for 2 h. The culture medium was replaced with new culture medium, followed by culture for 48 h.

[0123] Hut78 cell line (ATCC, TIB-161) was cultured in IMDM (ATCC, 30-2005) supplemented with 20% (v / v) FBS. TM During the subculture period, the cell density was maintained at 1.5 × 10 5 -2.0×10 5 cells / ml. H9 cell line (ATCC, HTB-176 TM ) and Jurkat cell lines (ATCC, TIB-152 TM ) were cultured in RPMI1640 (ATCC, 30-2001) medium supplemented with 10% (v / v) FBS, and the cell density was maintained at 1.0×10 5 -1.5×10 5 cells / ml and 0.5×10 5 -1.0×10 5Peer cell lines were cultured in RPMI1640 medium supplemented with 20% (v / v) FBS. During subculture, the cell density was maintained at 3.0×10 5 -5.0×10 5 Cells / ml. All cell lines were subcultured at intervals of 2-3 days. 75T flasks were used as culture vessels, and the volume of the culture medium was maintained at 15-20 ml.

[0124] Antibiotics were used to select cell lines infected with the recombinant lentivirus (Table 3).

[0125] [Table 3]

[0126]

[0127] Antibiotics used for transgenic cell lines are shown in Table 3.

[0128] Example 2.2. Confirmation of expression of transduced gene

[0129] In this example, the expression of the transduced gene was confirmed by flow cytometry. Each cell line subcultured in Example 2.1 was collected and centrifuged at 1,200 rpm for 5 min. Then, the culture solution was removed by aspiration. 2% (v / v) FBS was added to PBS to prepare FACS buffer. The cells were counted after dilution with 1 ml of FACS buffer. The cells were diluted to 5×10 6 The density of individual cells / ml. The diluted cell solution is added to 5ml FACS tube (Falcon, 352052) (100 μl / tube). Anti-human TNF-α (membrane)-PE (R&D systems, FAB210P), anti-human OX40L-PE (BD, 558184), anti-human 4-1BBL-PE (BD, 559446), anti-human IL-21-PE (eBioscience, 12-7219-42), 7-AAD (Beckmancoulter, IM3630c), PE mouse IgG1κ isotype control (BD Pharmingen, 555749) and PerCP-Cy5.5 mouse IgG1κ isotype control (BD, 550795) antibody are used to stain the cells. The expression level of the gene is analyzed by FACS ( Figures 1a to 1f ).

[0130] The expression of the transduced gene was confirmed by real-time (RT)-qPCR. To this end, each cell line subcultured in Example 2.1 was collected and centrifuged at 1,200 rpm for 5 min. Then, the culture solution was removed by aspiration. The cells were counted after dilution with PBS. RNA was prepared from 1×10 6RNA was extracted from each cell and quantified. cDNA was synthesized using a cDNA synthesis kit. RT-qPCR was performed using the synthesized cDNA. The primers used for RT-qPCR are shown in Table 4.

[0131] [Table 4]

[0132]

[0133] The expression levels of the transduced genes in the cell lines are shown in Table 5.

[0134] [Table 5]

[0135]

[0136]

[0137] As can be seen from the results in Table 5, high expression levels of the genes were induced when transduced into the cell lines.

[0138] Example 3. Co-culture of CD3(-) PBMC and transgenic T cells

[0139] Example 3.1. Preparation of CD3(-) PBMCs from umbilical cord blood as seed cells

[0140] Umbilical cord blood donated for research purposes was placed in a 50 ml tube and centrifuged at 1,500 rpm for 10 min. After removing the supernatant plasma, phosphate buffered saline (PBS, LONZA, 17-516Q) was added at a 1:1 ratio. Afterwards, cord blood mononuclear cells (MNCs) were separated by ficoll density gradient centrifugation (Ficoll-Paque Plus, GE Healthcare, 17-1440-03) and the cell number was measured using an ADAM cell counter system (Nano Entec).

[0141] Seed cells from which CD3(+) cells were eliminated were obtained by the following procedure. First, 5×10 7 The cord blood mononuclear cells were transferred to a new 50 ml tube and centrifuged at 1,200 rpm and 4 ° C for 5 min. 2% (v / v) FBS and 2mM EDTA were mixed in PBS to prepare MACS running buffer. After centrifugation, 400 μl MACS running buffer and 100 μl CD3 magnetic beads (Miltenyi biotech, 130-050-101) were added to the precipitation, and incubation was allowed to continue at 4 ° C for 20 min. The culture was washed with 10 ml MACS running buffer, centrifuged at 13,500 rpm and 4 ° C for 8 min, and suspended in 0.5 ml MACS running buffer.

[0142] Cells were separated on a VarioMACS (Miltenyi Biotech) equipped with a CS column (column, Miltenyi Biotech, 130-041-305). Cells were recovered by washing the column until the final volume reached 20 ml. The recovered cells were placed in a new 50 ml tube, centrifuged at 1,200 rpm and 4 °C for 5 min, and suspended in freezing medium. Cells were counted using an ADAM cell counter system. 5 × 10 6 Cells / vial were frozen in liquid nitrogen.

[0143] The frozen CD3(-) cord blood mononuclear cells in each vial were thawed in a thermostatic bath at 37°C, transferred to a 50 ml tube, suspended in PBS containing 0.6% (v / v) citrate-dextrose solution (ACD, Sigma-Aldrich, C3821), 0.2% (v / v) fetal bovine serum (FBS) and 2mM EDTA, and centrifuged at 1,500 rpm and 4°C for 10 min. CD3(-) cord blood mononuclear cells were suspended in CellGro medium (Cellgenix, 20802-0500) and the cell number was measured using the ADAM cell counter system. CD3(-) cord blood mononuclear cells were suspended to 1×10 in CellGro medium. 6 The density of cells / ml.

[0144] Example 3.2. Co-culture of CD3(-) umbilical cord blood mononuclear cells and transgenic T cells

[0145] The transgenic T cells produced in Example 2 were recovered from the culture flask and centrifuged at 1,200 rpm and 4°C for 5 min. Thereafter, the cells were suspended in CellGro medium and counted using the ADAM cell counter system. The transgenic T cells were suspended in CellGro medium to 2.5×10 6 The cells were then inactivated by irradiation with gamma rays at 20,000 cGy.

[0146] Natural killer cells were cultured by the following procedure. First, 1,000 IU IL-2 (Proleukin injection, Novartis Korea) and 10 ng / ml OKT-3 (eBioscience, 16-0037-85) were plated in a plastic culture plate. On day 0 of culture, CD3(-) cord blood mononuclear cells and transgenic T cells (0.25 ml each) were added at a ratio of 1:2.5, and 0.25 ml of CellGro medium supplemented with 2% (v / v) human plasma was added. The cells were statically cultured in an incubator at 37°C for 4 days.

[0147] On the 4th day of culture, an equal amount of CellGro medium supplemented with 1% (v / v) human plasma and 1,000 IU / ml IL-2 was added, followed by static culture. Thereafter, cells were counted at intervals of 2-3 days. CellGro medium supplemented with 1% (v / v) human plasma and 1,000 IU / ml IL-2 was added and suspension culture was performed until the 21st day, so that the cell density was 1×10 6 When using cells of the Jurkat cell line or Peer cell line as feeder cells, suspension culture was continued until day 11. When using cells of the transgenic H9 and Hut78 cell lines as feeder cells, suspension culture was continued until day 21.

[0148] The proliferation rates of the cultured natural killer cells were compared based on total nucleated cells (TNC). As a result, the proliferation rate of natural killer cells increased by up to 93 times when co-cultured with the non-transgenic Hut78 cell line. In contrast, the proliferation rate of natural killer cells increased significantly when co-cultured with the Hut78 cell line transduced with one or more genes (mTNF-α, mbIL-21, 4-1BBL). Specifically, the proliferation rate of natural killer cells increased by up to 957 times when co-cultured with the Hut78 cell line transduced with the genes mbIL-21 / 4-1BBL, and increased by up to 1,138 times when co-cultured with the Hut78 cell line transduced with the genes mTNF-α / mbIL-21 / 4-1BBL (Table 6, Figure 2a ).

[0149] [Table 6]

[0150]

[0151]

[0152] When co-cultured with the non-transgenic H9 cell line, the proliferation rate increased 13-fold. In contrast, when co-cultured with the H9 cell lines transduced with mbIL-21 / 4-1BBL and mTNF-α / mbIL-21 / 4-1BBL, the proliferation rate increased up to 367-fold and 979-fold, respectively (Tables 7 and Figure 2b ).

[0153] [Table 7]

[0154] One or more H9+ transduction genes average value STDEV H9 parent 12.6 4.3 mbIL21+4.1BBL 367.4 80.1 mTNF-α+mbIL21+4.1BBL 978.8 287.7

[0155] When co-cultured with other cell lines (Jurkat cell line and Peer cell line), the culture could be continued until culture day 11. Relatively high proliferation rates were observed in cell lines transduced with the gene mbIL-21 / 4.1BBL or the gene mTNF-α / mbIL-21 / 4-1BBL (Table 8 and Figure 9, Figure 2c and Figure 2b ).

[0156] [Table 8]

[0157] One or more Jurkat+ transducing genes Average value (after 11 days of cultivation) STDEV Jurkat parent 0.9 0.7 mbIL21+4.1BBL 36.3 4.8 mTNF-α+mbIL21+4.1BBL 43.6 6.6

[0158] [Table 9]

[0159] One or more Peer+transduced genes Average value (after 11 days of cultivation) STDEV Peer Parent 1.6 0.7 mbIL21+4.1BBL 14.3 4.1

[0160] The above results revealed that natural killer cells can be cultured at a higher proliferation rate for 21 days from CD3(-) cells isolated from umbilical cord blood mononuclear cells when transgenic feeder cells are used than when non-transgenic feeder cells are used.

[0161] Example 3.3. Re-stimulation of natural killer cell cultures using Hut78 cells transduced with the genes mTNF-α / mbIL-21 / 4-1BBL

[0162] The transgenic T cells prepared in Example 2 were recovered from the culture flask and centrifuged at 1,200 rpm and 4°C for 5 min. Thereafter, the cells were suspended in CellGro medium and counted using an ADAM cell counter system. The transgenic T cells were suspended in CellGro medium to 2.5×10 6 The cells were then inactivated by irradiation with gamma rays at 20,000 cGy.

[0163] Natural killer cells were cultured by the following procedure. First, 1,000 IU IL-2 and 10 ng / ml OKT-3 were plated in a plastic culture plate. On culture day 0, CD3(-) cord blood mononuclear cells and transgenic T cells (0.25-1 ml each) were added at a ratio of 1:2.5, and 0.25-1 ml of CellGro medium supplemented with 2% (v / v) human plasma was added. The cells were statically cultured in an incubator at 37°C for 4 days.

[0164] On the fourth day of culture, an equal amount of CellGro medium supplemented with 1% (v / v) human plasma and 1,000 IU / ml IL-2 was added, followed by static culture. Thereafter, cells were counted at intervals of 2-3 days. CellGro medium supplemented with 1% (v / v) human plasma and 1,000 IU / ml IL-2 was added and culture was continued until the cell density was 1×10 6 cells / ml.

[0165] For restimulation, Hut78 cells transduced with mTNF-α / mbIL-21 / 4-1BBL were used at the same ratio on culture day 0. On culture day 16, the first restimulation was performed. First, the cultured natural killer cells were counted using the ADAM cell counter system and diluted to 1.5 × 10 6 0.25 ml of the dilution was plated on a plastic culture plate. Hut78 cells transduced with mTNF-α / mbIL-21 / 4-1BBL were suspended in CellGro medium to 2.5×10 6 cells / ml and then inactivated by irradiation with gamma rays at 10,000 cGy.

[0166] 0.25 ml of Hut78 cells transduced with the inactivated gene mTNF-α / mbIL-21 / 4-1BBL were plated in a plastic culture plate. 1000 IU / ml IL-2, 10 ng / ml OKT-3 and 1% (v / v) human plasma were added to the plastic culture plate, followed by static culture at 37°C in an incubator for 3 days. Afterwards, the cell number was measured at intervals of 2-3 days. Culture was continued in CellGro medium supplemented with 1% (v / v) human plasma and 1000 IU / ml IL-2 until the cell number reached 1×10 6 After the first restimulation, restimulation was performed with feeder cells in the same manner as above at 14-day intervals (on days 32, 46, and 60). Culture was continued until day 70.

[0167] As a result, the proliferation rate of natural killer cells increased by 6.9 × 10 on day 32 of culture after the first restimulation. 4 times, and increased by 3.7×10 on the 46th day of culture after the second restimulation. 6 times, and increased by 2.3×10 on the 60th day of culture after the third restimulation. 8 times, and increased to 5.9×10 on the 70th day of culture after the fourth restimulation. 9 That is, natural killer cells continued to proliferate at a high rate (Table 10, Figure 2e ).

[0168] [Table 10]

[0169] Cultivation days average value STDEV 32 <![CDATA[6.9×10 4 ]]> <![CDATA[3.2×10 3 ]]> 46 <![CDATA[3.7×10 6 ]]> <![CDATA[3.1×10 5 ]]> 60 <![CDATA[2.3×10 8 ]]> <![CDATA[1.4×10 8 ]]> 70 <![CDATA[5.9×10 9 ]]> <![CDATA[1.1×10 8 ]]>

[0170] The results in Table 10 reveal that periodic restimulation using the Hut78 cell line transduced with the genes mTNF-α / mbIL-21 / 4-1BBL leads to a continuous increase in proliferation factors, indicating that cells of the Hut78 cell line are very suitable as feeder cells.

[0171] Experimental Example 1. Confirmation of cell viability of natural killer cells based on transduced genes

[0172] ADAM cell counter system is used to compare and evaluate cell viability in vitro. ADAM cell counter system is a cell counter using a PI staining solution that can be combined with the nucleus. The number of living cells is calculated by deducting the number of dead cells from the total number of counted cells. Cell viability is calculated using the following equation 1.

[0173] [Equation I]

[0174] Cell viability (%) = (number of live cells / total number of counted cells) × 100

[0175] When co-cultured with the transgenic Hut78 cell line, the viability of the natural killer cells was about 90%, regardless of whether the cell line was transduced with the gene or not (Table 11, Figure 3a ).

[0176] [Table 11]

[0177]

[0178]

[0179] When cultured in H9, Jurkat, and Peer cell lines transduced with the gene mbIL-21 / 4-1BBL and the gene mTNF-α / mbIL-21 / 4-1BBL, the cell viability of natural killer cells was >90% on culture day 21 (H9) and culture day 11 (Jurkat, Peer) (Tables 12 to 14, Figure 3b to Figure 3d ).

[0180] [Table 12]

[0181] One or more H9+ transduction genes average value STDEV Parent 86 6.1 mbIL21+4.1BBL 91 3.1 mTNF-α+mbIL21+4.1BBL 94 0.6

[0182] [Table 13]

[0183] One or more Jurkat+ transducing genes average value STDEV Parent 80 6.1 mbIL21+4.1BBL 91 0.6 mTNF-α+mbIL21+4.1BBL 92 2.0

[0184] [Table 14]

[0185] One or more Peer+transduced genes average value STDEV Parent 83.5 6.1 mbIL21+4.1BBL 91 0.6

[0186] Natural killer cells were cultured in the Hut78 cell line transduced with the gene mTNF-α / mbIL-21 / 4-1BBL, and the number of restimulations was increased. As a result, despite the increase in the number of restimulations, the viability of natural killer cells was > about 90% (Table 15, Figure 3e ).

[0187] [Table 15]

[0188] Cultivation days 32 42 60 70 average value 96.0 93.5 97.5 91.5 STDEV 1.4 0.7 0.7 4.9

[0189] These results revealed that the viability of natural killer cells was maintained at a high level even during continuous culture for a long period of time, thus providing the possibility of long-term expansion culture of natural killer cells.

[0190] Experimental Example 2. Confirmation of the purity of natural killer cells

[0191] Natural killer cells cultured for 21 days or cultured by repeated restimulation were collected and centrifuged at 1,200 rpm for 5 min. The culture solution was removed by aspiration. The cells were counted after dilution with 1 ml of FACS buffer. Cell lines were referred to as 5 × 10 6 Cells / ml. Add the diluted cell solution to a 5 ml FACS tube (Falcon, 352052) (100 μl / tube). Analyze the phenotype of the cells with the following antibodies:

[0192] Tube 1: anti-human CD3-FITC (BD Pharmingen, 555332), anti-human CD16-PE (BD Pharmingen, 555407), anti-human CD56-BV421 (BD Pharmingen, 562751)

[0193] Tube 2: anti-human CD14-FITC (BD Pharmingen, 555397), anti-human CD19-PE (BD Pharmingen, 555413), anti-human CD3-BV421 (BD Pharmingen, 562438)

[0194] Tube 3: anti-human CD3-FITC, anti-human NKG2D-PE (R&D system, FAB139P), anti-human CD56-BV421

[0195] Tube 4: anti-human CD3-FITC, anti-human NKp30-PE (BD Pharmingen, 558407), anti-human CD56-BV421

[0196] Tube 5: anti-human CD3-FITC, anti-human NKp44-PE (BD Pharmingen, 558563), anti-human CD56-BV421

[0197] Tube 6: anti-human CD3-FITC, anti-human NKp46-PE (BD Pharmingen, 557991), anti-human CD56-BV421

[0198] Tube 7: anti-human CD3-FITC, anti-human DNAM-1-PE (BD Pharmingen, 559789), anti-human CD56-BV421

[0199] Tube 8: anti-human CD3-FITC, anti-human CXCR3-PE (BD Pharmingen, 557185), anti-human CD56-BV421

[0200] Tube 9: anti-human CD3-FITC, PE mouse IgG1κ isotype control (BD Pharmingen, 555749), anti-human CD56-BV421

[0201] Tube 10: FITC mouse IgG1κ isotype control (BD Pharmingen, 555748), PE mouse IgG1κ isotype control, BV421 mouse IgG1κ isotype control (BD Pharmingen, 562438)

[0202] The cells in each tube were stained with one of three fluorescent antibodies. Specifically, the cells in tube 1 were stained with anti-human CD56 antibody, the cells in tube 2 were stained with anti-human CD3 antibody, the cells in tubes 3-9 were stained with anti-human CD56 antibody, and the cells in tube 10 were stained with isotype control antibody.

[0203] The cells in the tube were dyed at freezing temperature for 30 min. Afterwards, 2 ml FACS buffer was added to the dyed cells, followed by centrifugation at 1,500 rpm for 3 min. The supernatant was discarded, 2 ml FACS buffer was added, followed by centrifugation at 2,000 rpm for 3 min. After removing the supernatant, the cells were suspended in 200 μl cytofix buffer (fixation buffer, BD, 554655), then FACS LSRII Fortessa (BD Biosciences) was used to identify and check purity and phenotype.

[0204] After co-culturing CD3(-) cells isolated from cord blood mononuclear cells with the transgenic Hut78 cell line for 21 days, natural killer cells were identified and analyzed for purity. As a result, it was found that the content of natural killer cells (CD3-CD56+) was >90% under all conditions, regardless of whether the cell line was transduced with the gene (Table 16, Figure 4a ).

[0205] [Table 16]

[0206] One or more Hut78+ transduced genes average value STDEV Parent 92.4 9.6 mTNF-α 96.8 2.5 mbIL-21 98.6 0.9 OX40L 95.7 3.6 4.1BBL 98 1.8 mTNF-α+OX40L 97.2 2.5 mTNF-α+4.1BBL 98.6 1 mbIL-21+OX40L 98.4 1.3 mbIL-21+4.1BBL 98.5 0.9 mTNF-α+mbIL-21+4.1BBL 98.7 0.9 QD 99.3 0.5

[0207] After co-culture with H9, Jurkat or Peer cell lines transduced with the gene mbIL-21 / 4-1BBL or mTNF-α / mbIL-21 / 4-1BBL, the cultured natural killer cells were identified and the purity was measured. As a result, the purity of the natural killer cells produced by co-culture with the transgenic cell lines was maintained at a higher level than those produced by co-culture with the non-transgenic cell lines (Tables 17 to 19, Figure 4b to Figure 4d ).

[0208] [Table 17]

[0209]

[0210]

[0211] [Table 18]

[0212] One or more Jurkat+ transducing genes average value STDEV Parent 88.6 6.9 mbIL21+4.1BBL 97.6 1.3 mTNF-α+mbIL21+4.1BBL 97.5 0.8

[0213] [Table 19]

[0214] One or more Peer+transduced genes average value STDEV Parent 79.2 14.6 mbIL21+4.1BBL 94.9 2.1

[0215] Natural killer cells were cultured while increasing the number of restimulations with the cell line transduced with the three genes mTNF-α / mbIL-21 / 4-1BBL. It was found that the content of natural killer cells (CD3-CD56+) was high (>90%) until the 60th day of culture (Table 20, Figure 4e ).

[0216] [Table 20]

[0217] Cultivation days 32 60 average value 99.7 97.8 STDEV 0.1 0.8

[0218] Experimental Example 3. Analysis of activation markers of natural killer cells

[0219] After co-culturing CD3(-) cells isolated from cord blood mononuclear cells with transgenic feeder cells for 21 days, the expression of representative receptors for natural killer cells was analyzed.

[0220] When co-cultured with the Hut78 cell line, all CD16 cells expressed at high levels. When feeder cells were transduced with dual genes, the activation markers NKG2D, NKp30, NKp44, NKp46, and DNAM-1 were expressed at higher levels with no differences between donors compared to when feeder cells were not transduced or transduced with a single gene ( Figure 5a to Figure 5g ).

[0221] Higher expression levels of CD16, NKG2D, DNAM-1, and CXCR3 were found when co-cultured with feeder cells of the non-transgenic H9 cell line compared to when co-cultured with feeder cells transduced with the genes mbIL-21 / 4-1BBL or the triple genes mTNF-α / mbIL-21 / 4-1BBL. The expression levels of other activation markers NKp30, NKp44, and NKp46 were found to be high and did not differ between donors. In conclusion, feeder cells transduced with dual and triple genes can be used to increase the activity and tumor targeting ability of NK cells ( Figure 6a to Figure 6g ).

[0222] Natural killer cells were generated by restimulating the co-culture using the Hut78 cell line transduced with the triple gene mTNF-α / mbIL-21 / 4-1BBL, and their phenotypes were identified. As a result, when restimulation was performed 4 times during the culture period, the expression of activation markers (including NKG2D, NKp44, NKp46, DNAM-1, and CXCR3) showed a tendency to decrease compared to when restimulation was performed only once. These results reveal that increasing the number of restimulations leads to a long culture period, which affects the expression levels of some activation markers ( Figure 7a and Figure 7b ).

[0223] Experimental Example 4. Confirmation of cytotoxicity to natural killer cells based on genes transduced into T cells and co-culture with T cells

[0224] 1×10 K562 cancer cells 6 The cells were placed in a 15 ml tube and centrifuged. The cell pellet was suspended in RPMI1640 medium supplemented with 1 ml 10% (v / v) FBS. After adding 30 μl 1 mM calcein-AM (Molecularprobe, C34852), the suspension was protected from light with aluminum foil and then stained in an incubator at 37° C. for 1 h.

[0225] The tumor cell lines stained with Calcein-AM were washed in RPMI1640 medium supplemented with 10-15 ml 10% (v / v) FBS and centrifuged. The pellet was suspended to 1×10 in RPMI1640 medium supplemented with 10 ml 10% (v / v) FBS. 5 The density of 1×10 cells / ml. 6 NK cells were placed in a 15 ml tube and centrifuged. The pellet was suspended in RPMI1640 medium supplemented with 10% (v / v) FBS at the desired ratio (1:1) with K562 cancer cell line. The mixture of K562 cancer cell line and NK cell line (100 μl each) was plated in a 96-well U-bottom plate (Nunc, 163320). Fluorescence was measured in triplicate for each well, and the measured values ​​were averaged.

[0226] The stained K562 cancer cell line was added to the spontaneous release well (100 μl / well), and RPMI1640 medium supplemented with 10% (v / v) FBS was added thereto (100 μl / well). The stained K562 cancer cell line was added to the maximum release well (100 μl / well), and three times distilled water containing 2% (v / v) Triton-X 100 (100 μl / well) was added thereto.

[0227] The autofluorescence values ​​of RPMI1640 medium supplemented with 10% (v / v) FBS and RPMI1640 medium supplemented with 2% (v / v) Triton-X 100 were corrected in the following manner: the fluorescence value of a mixture of 100 μl of RPMI1640 medium supplemented with 10% (v / v) FBS and 100 μl of RPMI1640 medium supplemented with 2% (v / v) Triton-X 100 was subtracted from the fluorescence value of 200 μl of RPMI1640 medium supplemented with 10% (v / v) FBS, and the difference A was added to the maximum release value.

[0228] The incubation was allowed to continue for 4 h in an incubator at 37 °C in the dark, and the plate was then centrifuged at 2,000 rpm for 3 min. The supernatant was plated in a 96-well black plate (Nunc, 237108) (100 μl / well). Fluorescence values ​​(OD 480 / 535 The cytotoxicity (killing %) of natural killer cells against tumor cells was calculated using the following equation II:

[0229] [Equation II]

[0230] Killing % = (average fluorescence value of sample wells - average fluorescence value of spontaneous wells) / [(average fluorescence value of maximum wells + A) - average fluorescence value of spontaneous wells] × 100

[0231] The direct cytotoxicity of natural killer cells cultured using various types of feeder cells to the K562 cancer cell line was measured. As a result, the cytotoxicity of natural killer cells cultured using feeder cells transduced with the gene mbIL-21 / 4-1BBL and feeder cells transduced with the gene mTNF-α / mbIL-21 / 4-1BBL was higher than those of natural killer cells cultured using non-transgenic feeder cells ( Figures 8a to 8d ).

[0232] Despite increasing the number of restimulations with the Hut78 cell line transduced with the genes mTNF-α / mbIL-21 / 4-1BBL, the cytotoxicity of natural killer cells remained high and did not differ significantly until day 60 of culture ( Figure 8e ).

[0233] These results suggest that feeder cells transduced with the gene mbIL-21 / 4-1BBL or the gene mTNF-α / mbIL-21 / 4-1BBL can be used for the ex vivo expansion of highly purified natural killer cells with high cytotoxicity and activity compared to non-transgenic feeder cells.

[0234] Example 4. Animal Experiment

[0235] Example 4.1. Cultivation of natural killer cells using transgenic T feeder cells

[0236] 500 or 1,000 IU IL-2 (Proleukin injection, Novartis Korea) and 10 ng / ml OKT-3 (eBioscience, 16-0037-85) were plated in plastic culture plates. On culture day 0, CD3(-) cord blood mononuclear cells or peripheral blood mononuclear cells and transgenic T cells were added at a ratio of 1:2.5, and 0.25 ml of CellGro medium supplemented with 2% (v / v) human plasma was added. The cells were statically cultured at 37°C in an incubator for 4 days. Cord blood mononuclear cells were used at a concentration of 1000 IU / mL, and peripheral blood mononuclear cells were used at a concentration of 500 IU / mL.

[0237] Umbilical cord blood-derived natural killer cells were cultured by the following procedure. On the 4th day of culture, an equal amount of CellGro medium supplemented with 1% (v / v) human plasma and 1,000 IU / ml IL-2 was added, followed by static culture. Thereafter, cells were counted at intervals of 2-3 days. CellGro medium supplemented with 1% (v / v) human plasma and 1,000 IU / ml IL-2 was added and culture was continued until the 14th day, until the cell density reached 1×10 6 On day 14, the culture of natural killer cells was restimulated with transgenic T feeder cells at a ratio of 1:2.5. Natural killer cells were cultured in CellGro medium supplemented with 1% (v / v) human plasma, OKT3, and IL-2. Thereafter, cells were counted at 2-3 day intervals and CellGro medium supplemented with 1% (v / v) human plasma and 1000 IU / mL IL-2 was added. Culture was continued for another 14 days (total 28 days) to a cell number of 1×10 6 cells / mL.

[0238] Umbilical cord blood-derived natural killer cells were cultured by the following procedure. On the 4th day of culture, an equal amount of CellGro medium supplemented with 1% (v / v) human plasma and 500 IU / ml IL-2 was added, followed by static culture. Thereafter, cells were counted at intervals of 2-3 days. CellGro medium supplemented with 1% (v / v) human plasma and 500 IU / ml IL-2 was added and culture was continued until the 11th day, until the cell density reached 1×10 6On day 11, the culture of natural killer cells was restimulated with transgenic T feeder cells at a ratio of 1:2.5. Natural killer cells were cultured in CellGro medium supplemented with 1% (v / v) human plasma, OKT3 and IL-2. Thereafter, cells were counted at 2-3 day intervals and CellGro medium supplemented with 1% (v / v) human plasma and 1000 IU / mL IL-2 was added. Culture was continued for another 8-10 days (total 19-21 days) to a cell number of 1×10 6 cells / mL.

[0239] The cultured cells were suspended in freezing medium to a volume of 1 × 10 6 cells / mL, frozen in a temperature-controlled freezer, and stored in liquid nitrogen.

[0240] The proliferation rates of the cultured natural killer cells were compared. As a result, the natural killer cells derived from umbilical cord blood (CB-enFeeder) proliferated approximately 80,000 times, and the natural killer cells derived from peripheral blood (PBMC-enFeeder) proliferated 55,000 times. There was no significant difference in the proliferation rate between CB-enFeeder and PBMC-enFeeder (Table 21).

[0241] [Table 21]

[0242] average value STDEV CB-enFeeder 79288.3 37381.0 PBMC-enFeeder 53649.3 16827.9

[0243] Example 4.2. Efficacy evaluation using the Raji animal model

[0244] On the last day of culture, the cancer cells of the Raji-luc cell line were collected and the cell density was adjusted to 5 × 10 5 The cells were injected into mice via the tail vein (0.2 mL (1×10 5 cells / mouse). 2×10 7 Natural killer cells were injected via the tail vein at a density of 10 cells / 200 μL. Rituxan (RTX) (Mabthera injection, Roche Korea) was diluted to 0.01 μg / 100 μL with PBS. 100 μL of the dilution was injected subcutaneously into the weakened area between the shoulder blade and the chest wall of each mouse. On the second day after cancer cell transplantation, NK cells were administered via the tail vein using a fixator for a total of 6 times. RTX was subcutaneously administered once (Table 22, Figure 9a ).

[0245] [Table 22]

[0246]

[0247] All animals (including systemic symptoms and dead animals) were observed twice a day. After observing the death or survival of the animals, the median survival time of the freezing medium control group and the group treated with natural killer cells and RTX was calculated to evaluate the effect of natural killer cells in prolonging the survival rate. Dead animals were observed in the group where natural killer cells and RTX were administered alone or in combination 26-122 days after transplantation of the Raji-luc cell line. Until the last day (day 122), the median survival time of the freezing medium control group was 30 days. In contrast, the median survival time of the group administered RTX, the group administered PBMC-enFeeder, and the group administered CB-enFeeder were 48.5 days, 43 days, and 47 days, respectively. The median survival time of the group administered PBMC-enFeeder and RTX and the group administered CB-enFeeder and RTX were 55 days and 75.5 days, respectively (Table 23, Figure 9b ).

[0248] [Table 23]

[0249]

[0250] Example 4.3. Efficacy evaluation using the Ramos animal model

[0251] On the last day of culture, the cancer cells of the Ramos cell line were collected and the cell density was adjusted to 5 × 10 6 The cells were injected into mice via the tail vein (0.2 mL (1×10 6 cells / mouse). 2×10 7 Natural killer cells were injected via the tail vein at a density of 10 cells / 200 μL. RTX was diluted to 0.3 μg / 100 μL with PBS. 100 μL of the dilution was injected subcutaneously into the weakened area between the shoulder blade and the chest wall of each mouse. Natural killer cells were administered via the tail vein using a fixator for a total of 6 times from the fourth day after cancer cell transplantation, and RTX was administered via the tail vein for a total of 6 times from the third day after cancer cell transplantation (Table 24, Fig.10a ).

[0252] [Table 24]

[0253]

[0254] All animals (including systemic symptoms and dead animals) were observed twice a day. After observing the death or survival of the animals, the median survival time of the cryomedia control group and the group treated with natural killer cells and RTX was calculated to evaluate the effect of natural killer cells in prolonging the survival rate. Dead animals were observed in the groups where natural killer cells and RTX were administered alone or in combination 34-110 days after transplantation of the Ramos cell line. Until the last day (day 124), the median survival time of the cryomedia control group was 31 days. In contrast, the median survival time of the group administered RTX, the group administered PBMC-enFeeder, and the group administered CB-enFeeder were 49.5 days, 42 days, and 42.5 days, respectively. The median survival time of the group administered PBMC-enFeeder and RTX and the group administered CB-enFeeder and RTX were 63.5 days and 87.5 days, respectively (Table 25, Fig.10b ).

[0255] [Table 25]

[0256]

[0257] Although the details of the present invention have been described in detail, it will be apparent to those skilled in the art that such details are only preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents. <110> GC Cell Therapy <120> Method for culturing natural killer cells derived from umbilical cord blood using transformed T cells <130> FPD / 201810-0021 <160> 25 <170> KoPatentIn 3.0 <210> 1 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of 4-1BBL <400> 1 Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro 1 5 10 15 Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val 20 25 30 Ala Gly Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe 35 40 45 Leo Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser 50 55 60 Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp 65 70 75 80 Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val 85 90 95 Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp 100 105 110 Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu 115 120 125 Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe 130 135 140 Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser 145 150 155 160 Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala 165 170 175 Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala 180 185 190 Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 195 200 205 Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His 210 215 220 Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val 225 230 235 240 Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 245 250 <210> 2 <211> 765 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide Sequence of 4-1BBL <400> 2 atggaatacg cctctgacgc ttcactggac cccgaagccc cgtggcctcc cgcgccccgc 60 gctcgcgcct gccgcgtact gccttgggcc ctggtcgcgg ggctgctgct gctgctgctg 120 ctcgctgccg cctgcgccgt cttcctcgcc tgcccctggg ccgtgtccgg ggctcgcgcc 180 tcgcccggct ccgcggccag cccgagactc cgcgagggtc ccgagctttc gcccgacgat 240 cccgccggcc tcttggacct gcggcagggc atgtttgcgc agctggtggc ccaaaatgtt 300 ctgctgatcg atgggcccct gagctggtac agtgacccag gcctggcagg cgtgtccctg 360 acggggggcc tgagctacaa agaggacacg aaggagctgg tggtggccaa ggctggagtc 420 tactatgtct tctttcaact agagctgcgg cgcgtggtgg ccggcgaggg ctcaggctcc 480 gtttcacttg cgctgcacct gcagccactg cgctctgctg ctggggccgc cgccctggct 540 ttgaccgtgg acctgccacc cgcctcctcc gaggctcgga actcggcctt cggtttccag 600 ggccgcttgc tgcacctgag tgccggccag cgcctgggcg tccatcttca cactgaggcc 660 agggcacgcc atgcctggca gcttacccag ggcgccacag tcttgggact cttccgggtg 720 acccccgaaa tcccagccgg actcccttca ccgaggtcgg aataa 765 <210> 3 <211> 223 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of membrane-bound IL-21 (mbIL-21) <400> 3 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Asp Arg His Met Ile Arg Met Arg Gln Leu 20 25 30 Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro 35 40 45 Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser 50 55 60 Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly 65 70 75 80 Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys 85 90 95 Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys 100 105 110 Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu 115 120 125 Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser 130 135 140 Arg Thr His Gly Ser Glu Asp Ser Ala Lys Pro Thr Thr Thr Pro Ala 145 150 155 160 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 165 170 175 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 180 185 190 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 195 200 205 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 210 215 220 <210> 4 <211> 672 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of membrane-bound IL-21 (mbIL-21) <400> 4 atggctctgc ccgtcaccgc tctgctgctg cctctggccc tgctgctgca cgccgcaaga 60 ccacaggacc gccacatgat tcggatgagg cagctgatcg acattgtgga tcagctgaag 120 aactacgtga atgacctggt ccccgagttc ctgcccgctc ctgaggatgt cgaaacaaac 180 tgcgaatggt ctgcattcag ttgttttcag aaggctcagc tgaaatctgc aaacactgga 240 aacaatgagc gaatcattaa tgtgagcatc aagaaactga agcggaaacc cccttccact 300 aatgcaggcc ggagacagaa gcaccgactg acctgcccct catgtgacag ctatgaaaag 360 aaaccaccca aagagttcct ggagcggttc aagagcctgc tccagaaaat gatccaccag 420 cacctgagca gccggaccca cggcagcgag gattctgcca agcctaccac aactccagct 480 ccccgccctc caacccctgc accaacaatt gccagtcagc ccctgtcact gaggcctgaa 540 gcatgccgcc cagccgctgg cggagcagtc cacacacgag gcctggactt tgcttgtgat 600 atctacattt gggcacctct ggctggaact tgcggcgtcc tgctgctgtc cctggtcatc 660 accctgtatt ga 672 <210> 5 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of OX40L <400> 5 Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg 1 5 10 15 Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gln 20 25 30 Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser 35 40 45 Ala Leu Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val 50 55 60 Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln 65 70 75 80 Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn 85 90 95 Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu 100 105 110 Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln 115 120 125 Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr 130 135 140 Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu 145 150 155 160 Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn 165 170 175 Pro Gly Glu Phe Cys Val Leu 180 <210> 6 <211> 552 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of OX40L <400> 6 atggaacggg tgcaacctct ggaggagaat gtgggcaatg cagcccggcc aagatttgag 60 aggaacaagc tactgctggt ggcctctgtg atacagggac tggggctcct cctgtgcttc 120 acctacatct gcctgcactt cagcgctctt caggtgtcac accggtatcc ccgcatacaa 180 agtatcaagg tccagtttac cgagtataag aaagagaagg gtttcatcct cactagtcag 240 aaggaggatg aaatcatgaa ggtgcagaac aactcagtta ttattaactg tgacggcttt 300 tatctgatca gcctgaaggg ctacttctcc caggaagtaa acatcagcct gcattaccag 360 aaggatgagg agcccctgtt tcagctgaag aaggtccggt ccgtgaactc ccttatggtc 420 gcctctctga cgtacaagga caaggtgtac ctcaatgtga ccacagacaa tacatccctg 480 gatgacttcc atgtgaatgg cggagaactg attctgattc accagaaccc tggggagttc 540 tgtgtcttgt ga 552 <210> 7 <211> 705 <212> DNA <213> Homo sapiens <400> 7 atgagcactg aaagcatgat ccgggacgtg gagctggccg aggaggcgct ccccaagaag 60 acaggggggc cccagggctc caggcggtgc ttgttcctca gcctcttctc cttcctgatc 120 gtggcaggcg ccaccacgct cttctgcctg ctgcactttg gagtgatcgg cccccagagg 180 gaagagttcc ccagggacct ctctctaatc agccctctgg cccaggcagt cagatcatct 240 tctcgaaccc cgagtgacaa gcctgtagcc catgttgtag caaaccctca agctgagggg 300 cagctccagt ggctgaaccg ccgggccaat gccctcctgg ccaatggcgt ggagctgaga 360 gataaccagc tggtggtgcc atcagagggc ctgtacctca tctactccca ggtcctcttc 420 aagggccaag gctgcccctc cacccatgtg ctcctcaccc acaccatcag ccgcatcgcc 480 gtctcctacc agaccaaggt caacctcctc tctgccatca agagcccctg ccagagggag 540 accccagagg gggctgaggc caagccctgg tatgagccca tctatctggg aggggtcttc 600 cagctggaga agggtgaccg actcagcgct gagatcaatc ggcccgacta tctcgacttt 660 gccgagtctg ggcaggtcta ctttgggatc attgccctgt gagga 705 <210> 8 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of mutant TNF alpha (mTNF-a) <400> 8 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Pro Val Arg Ser Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln Val Tyr Phe Gly Free Mp3 Download 225 230 <210> 9 <211> 702 <212> DNA <213> The snowstorm <220> <223> The anti-TNF alpha (mTNF-a) anti-inflammatory activity <400> 9 atgagcactg aaagcatgat ccgggacgtg gagctggccg aggaggcgct ccccaagaag acaggggggc cccagggctc caggcggtc ttgttcctca gcctcttc cttcctgatc 120 gtggcaggcg ccaccacgct cttctgcctg ctgcactttg gagtgatcgg cccccagagg 180 gaagagttcc ccagggacct ctctctaatc agccctctgg cccagccggt cagatcatct 240 300. tctcgaaccc cgagtgacaa gcctgtagcc catgttgtag caaaccctca agctgagggg cagctccagt ggctgaaccg ccgggccaat gccctcctgg ccaatggcgt ggagctgaga 360 gataaccagc tggtggtgcc atcagagggc ctgtacctca tctactccca ggtcctcttc 420 aagggccaag gctgcccctc cacccatgtg ctcctcaccc acaccatcag ccgcatcgcc 480 gtctcctacc agaccaaggt caacctcctc tctgccatca agagcccctg ccagagggag 540 accccagagg gggctgaggc caagccctgg tatgagccca tctatctggg aggggtcttc 600 cagctggaga agggtgaccg actcagcgct gagatcaatc ggcccgacta tctcgacttt 660 gccgagtctg ggcaggtcta ctttgggatc attgccctgt ga 702 <210> 10 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Forward primer for 4-1BBL (PCR) <400> 10 tctagagcta gcgaattcgc caccatggaa tacgcctctg acgctt 46 <210> 11 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for 4-1BBL (PCR) <400> 11 ttcgcggccg cggatcctta ttccgacctc ggtgaagg 38 <210> 12 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Forward primer for mbIL-21 (PCR) <400> 12 tagagctagc gaattcgcca ccgccaccat ggctctgccc 40 <210> 13 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for mbIL-21 (PCR) <400> 13 tcgcggccgc ggatcctcaa tacagggtga tgacc 35 <210> 14 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Forward primer for OX40L (PCR) <400> 14 tagagctagc gaattcgcca ccatggaacg ggtgcaac 38 <210> 15 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for OX40L (PCR) <400> 15 tcgcggccgc ggatcctcac aagacacaga actcccc 37 <210> 16 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Forward primer for mTNF-a (PCR) <400> 16 tagagctagc gaattcgcca ccgccaccat ggctctgccc 40 <210> 17 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for mTNF-a (PCR) <400> 17 tcgcggccgc ggatcctcac agggcaatga tccc 34 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer for 4-1BBL (RT-qPCR) <400> 18 tctgagacag ggcatgtttg 20 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for 4-1BBL (RT-qPCR) <400> 19 ccaccagttc tttggtgtcc 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer for mTNF-a (RT-qPCR) <400> 20 aacctcctct ctgccatcaa 20 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for mTNF-a (RT-qPCR) <400> twenty one atagtcgggc cgattgatct 20 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer for mbIL-21 (RT-qPCR) <400> twenty two tggaaacaat gagcgaatca 20 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for mbIL-21 (RT-qPCR) <400> twenty three aaccgctcca ggaactcttt 20 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer for hTOP1 (RT-qPCR) <400> twenty four ccagacggaa gctcggaaac 20 <210> 25 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer for hTOP1 (RT-qPCR) <400> 25 gtccaggagg ctctatcttg aa 22

Claims

1. A method for culturing natural killer cells, comprising co-culturing transformed CD4+T cells and seed cells, wherein the transformed CD4+T cells express the 4-1BBL gene. 2 . The method according to claim 1 , wherein the transformed CD4+ T cells further express at least one gene selected from the group consisting of mbIL-21, OX40L and mTNF-α genes. 3 . The method according to claim 1 , wherein the 4-1BBL gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

1. 4 . The method according to claim 3 , wherein the encoding nucleotide sequence is shown in SEQ ID NO:

2. 5 . The method according to claim 2 , wherein the mbIL-21 gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

3. The method according to claim 5 , wherein the encoding nucleotide sequence is shown in SEQ ID NO:

4.

7. The method according to claim 2, wherein the OX40L gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

5.

8. The method of claim 7, wherein the encoding nucleotide sequence is shown in SEQ ID NO:

6.

9. The method according to claim 2, wherein the mTNF-α gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

8.

10. The method according to claim 9, wherein the encoding nucleotide sequence is shown in SEQ ID NO:

9. The method according to claim 1 , wherein the gene is transduced by a recombinant lentivirus.

12. The method according to claim 2, wherein the CD4+ T cells express the 4-1BBL and mbIL-21 genes.

13. The method according to claim 2, wherein the CD4+ T cells express the 4-1BBL, mbIL-21 and mTNF-α genes.

14. The method according to claim 2, wherein the CD4+ T cells express the 4-1BBL, mbIL-21, OX40L and mTNF-α genes.

15. The method according to claim 1, wherein the CD4+ T cells are selected from Hut78, H9, Jurkat, Loucy, Molt-3, Molt-13, Peer, RPMI8402 and TALL-01 cells. The method according to claim 1 , wherein the seed cells are mononuclear cells derived from umbilical cord blood. The method according to claim 1 , wherein the seed cells are cells in which CD3(+) cells are depleted. The method according to claim 1 , wherein the transformed CD4+ T cells and the seed cells are mixed at a ratio of 0.1:1 to 50:

1.

19. The method of claim 1, wherein the seed cells are mixed with the feeder cells only once for 5 to 60 days, or are mixed two or more times for at least 60 days.

20. The method of claim 1, wherein the natural killer cells are cultured in a medium supplemented with anti-CD3 antibodies and interleukin proteins.

21. The method of claim 20, wherein the anti-CD3 antibody is selected from the group consisting of OKT-3, UCHT1 and HIT3a.

22. The method of claim 20, wherein the interleukin protein is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, and IL-21.

23. A composition for culturing natural killer cells, comprising transformed CD4+T cells as an active ingredient, wherein the transformed CD4+T cells express 4-1BBL gene and mbIL-21 gene.

24. The composition according to claim 23, wherein the transformed CD4+ T cells further express at least one gene selected from the group consisting of OX40L and mTNF-α genes.

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