Method for introducing target genes into immune cells

Abstract

The present invention relates to a method for efficiently expressing a target gene in immune cells, specifically, natural killer cells, using a viral vector. The present invention can dramatically improve the transduction efficiency of a viral vector without the addition of enzymes or low molecular weight compounds by adding a simple step of freezing and thawing natural killer cells before introducing a target gene. In particular, the present invention can be usefully used to produce natural killer cells into which a gene of a desired trait has been introduced and in which the function, activity, and productivity as cells for immunotherapy have been maximized by significantly improving the efficiency of lentiviral vectors, which have low gene transfer efficiency into natural killer cells.

Description

[Technical field]

[0001] The present invention relates to a method for improving the transduction efficiency of a target gene via a viral vector by subjecting immune cells, specifically natural killer cells, to a process of freezing and thawing. [Background technology]

[0002]

[0003] Natural killer cells are lymphocytes that account for about 15% of peripheral blood lymphocytes and play an important role in innate immune defense against malignant cells. Specifically, natural killer cells activate dendritic cells and induce cytotoxic T lymphocytes (CTLs) to react specifically to tumors to eliminate tumor cells, and in particular directly kill malignant tumors such as sarcoma, myeloma, carcinoma, lymphoma, and leukemia. However, immune cell therapy using natural killer cells has been limited due to the difficulty of ex vivo cell expansion and the difference in natural killer cell activity between patients.

[0004] Meanwhile, in immune cell therapy using T cells, a method has been developed to reprogram autologous immune cells to activate them to increase the therapeutic effect against a specific cancer, or to express chimeric antigen receptors that enable cancer cells to overcome resistance to treatment, and such chimeric antigen receptors have recently been applied to natural killer cells. In addition, in order to overcome the fact that transplanted NK cells cannot be sustained for a long time in the recipient in treatment using allogeneic natural killer cells, it is necessary to introduce foreign genes or suppress or overexpress endogenously expressed genes to maximize the function and activity of immune therapy cells, such as expressing cytokine IL-2 that can induce NK cell proliferation, or suppressing NK cell inhibitory receptors (KIR, etc.) to block the recipient's immune response. However, natural killer cells have a disadvantage in that the transduction efficiency is low, especially when using viral vectors such as lentivirus, and there is a continuous demand for the development of more efficient gene transfer methods.

[0005]

[0006] Throughout this specification, a number of papers and patent documents are referenced and are indicated by incorporation, the disclosures of which are incorporated by reference in their entirety into this specification in order to more clearly describe the state of the art to which the present invention pertains and the contents of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007]

[0008] The present inventors have made extensive research efforts to develop a method for efficiently expressing various target genes in immune cells in order to maximize the function, activity and productivity of immune cells as a cell resource for therapeutic use. As a result, the present inventors have found that when immune cells, specifically natural killer cells, are subjected to a freezing and thawing process before introducing a target gene into the cells using a viral vector, the gene transfer efficiency is significantly improved, and natural killer cells with an optimized phenotype for immunotherapy can be efficiently obtained, thereby completing the present invention.

[0009] It is therefore an object of the present invention to provide a method for expressing a target gene in an immune cell.

[0010] Another object of the present invention is to provide natural killer cells in which a target gene is expressed by the method of the present invention, and a pharmaceutical composition containing the natural killer cells for preventing or treating tumors or infectious diseases.

[0011]

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0013]

[0014] According to one aspect of the invention, the invention provides a method for expressing a target gene in an immune cell comprising the steps of:

[0015] (a) freezing immune cells;

[0016] (b) thawing the frozen immune cells; and

[0017] (c) introducing into the thawed immune cells a gene carrier into which the target gene is inserted.

[0018] The present inventors have made extensive research efforts to develop a method for efficiently expressing various target genes in immune cells in order to maximize the function, activity, and productivity of immune cells as a cell resource for therapeutic use. As a result, they have found that when immune cells, specifically natural killer cells, are subjected to a freezing and thawing process before introducing a gene of interest into the cells using a viral vector, the gene transfer efficiency is significantly improved, and natural killer cells with an optimized phenotype for immunotherapy can be efficiently obtained.

[0019] As used herein, the term "target gene" refers to a nucleic acid molecule that is artificially expressed in immune cells so that the target immune cells have a desired trait, or that is inserted into a gene carrier to artificially increase the natural expression level. As used herein, the term "nucleic acid molecule" is intended to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogs in which the sugar or base moiety is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0020] As used herein, the term "express" refers to making a target cell express an exogenous gene or to artificially introduce an endogenous gene using a gene carrier to increase the natural expression level of the gene, thereby making the gene replicable in the target cell as an extrachromosomal element or by chromosomal integration. Thus, the term "express" has the same meaning as "transformation," "transfection," or "transduction."

[0021] As used herein, the term "gene carrier" or "gene delivery system" refers to any means for carrying a gene into a cell, and gene delivery has the same meaning as intracellular transduction of a gene. At the tissue level, the term gene delivery has the same meaning as gene spread. Thus, the gene delivery system of the present invention may be described as a gene transduction system and a gene spread system.

[0022] To prepare the gene delivery vehicle of the invention, the nucleotide sequence of the invention is preferably present in a suitable expression construct. In said expression construct, the nucleotide sequence of the invention is preferably operably linked to a promoter. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or reading of the other nucleic acid sequence. The promoter linked to the target gene of the present invention is specifically one that can operate in an animal cell, more specifically a mammalian cell, most specifically an immune cell to regulate the transcription of the target gene, and includes a promoter derived from a mammalian virus and a promoter derived from the genome of a mammalian cell, such as, but not limited to, a CMV (mammalian cytomegalovirus) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of HSV, a RSV promoter, an EF1 alpha promoter, a metallothionine promoter, a beta-actin promoter, a promoter of the human IL-2 gene, a promoter of the human IFN gene, a promoter of the human IL-4 gene, a promoter of the human lymphotoxin gene, and a promoter of the human GM-CSF gene.

[0023] The nucleotide sequence of the target gene can be applied to all gene transfer systems used in conventional gene transfer, and preferably includes plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 (1997)), adeno-associated viruses (AAV, Lashford LS., et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentiviruses (Wang G. et al., J. Clin. Invest. 104(11): R55-62 (1999)), herpes simplex viruses (Chamber R., et al., Proc. Natl. Act. Sci. 1999). USA 92:1411-1415 (1995)), vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10:649-657 (1999)), liposomes (Methods in Molecular Biology, 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes.

[0024] According to a specific embodiment of the present invention, the gene delivery vehicle used in the present invention is a viral vector.

[0025]

[0026] More particularly, said virus is selected from the group consisting of lentiviruses, adenoviruses, adeno-associated viruses (AAV), retroviruses, herpes simplex viruses and vaccinia viruses, most particularly a lentivirus.

[0027] In the present invention, when the gene transfer construct is based on a viral vector, the contacting step is carried out by a viral infection method known in the art, and infection of host cells with a viral vector is described in the above cited references.

[0028] As used herein, the term "immune cell" refers to any cell involved in the initiation or promotion of an immune response, and more specifically, refers to an immune effector cell. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, and mast cells. More specifically, the immune cell is a natural killer cell.

[0029]

[0030] In the present specification, the term "freezing" or "cryopreservation" means to stably maintain cells for a short or long period of time at a low temperature, specifically, at an ultra-low temperature of -70°C to -200°C. In general, mutations occur in cells at a rate of about 1 in 10,000 during the culture process, and if cells are subcultured for a long period of time, they may degenerate into a cell population different from the original cell population, or the cell's inherent biological activity and function may be lost. In addition, the risk of infection by mycoplasma increases in proportion to the period, so a method of freezing cells is widely used to preserve the inherent characteristics of cells. The present inventors first found that when such a freezing process is performed in immune cells, particularly natural killer cells, which have a very low gene transfer efficiency using a lentiviral vector, the gene transfer efficiency is greatly improved. Therefore, the freezing step performed in the present invention may be performed only for a short period of time, unlike cryopreservation, which is performed for the purpose of long-term conservation of cells.

[0031] Freezing of cells can be achieved by treating with a cryoprotectant that can minimize cell damage due to ice crystal formation and ionic and osmotic imbalances that inevitably accompany the freezing and thawing process. The freezing medium may contain, for example, dextran, albumin, and / or DMSO to improve safety and stability while the cells are frozen.

[0032] As used herein, the term "thawing" refers to the process of increasing the temperature of frozen or cryopreserved cells until they become soft, viable cells in order to resume their vital activities. Thawing can be carried out quickly by removing cells that have been cryopreserved at ultra-low temperatures from a liquid nitrogen tank or the like and placing them in a thermostatic water bath at 37°C.

[0033] Since the freezing and thawing process of the present invention is intended to improve the efficiency of gene transfer into cells, not to store the cells for a long period of time, the freezing (step (a) of the present invention) and thawing (step (b) of the present invention) may be performed without a time interval, and the thawing process may be started immediately after the freezing is completed, or, if necessary, there may be an appropriate time lag between the freezing process and the thawing process.

[0034]

[0035] According to a specific embodiment of the present invention, the method of the present invention may further include, prior to step (a), a step of in vitro culturing the immune cells isolated from a subject for 6 to 11 days, more specifically for 6 to 9 days, even more specifically for 6 to 8 days, and most specifically for about 7 days.

[0036] According to a specific embodiment of the present invention, the method of the present invention may additionally comprise, after step (b), a step of culturing the thawed immune cells for 2 to 4 days, more specifically for 3 to 4 days, even more specifically for 2 to 3 days, and most specifically for about 3 days.

[0037] According to a specific embodiment of the present invention, step (c) is performed by adding a TAK1 MAPKKK (transforming growth factor-β-activated kinase 1 mitogen-activated protein kinase kinase kinase) inhibitor, a TBK1 (TANK Binding Kinase1) inhibitor, or a combination thereof to the culture medium of the thawed immune cells.

[0038] Specifically, the TAK1 MAPKKK inhibitor may be a compound represented by the following formula 1 or a pharma- ceutically acceptable salt thereof:

[0039]

[0040] [ka]

[0041] Specifically, the TBK1 inhibitor may be a compound represented by the following formula 2 or a pharma- ceutically acceptable salt thereof:

[0042]

[0043] [ka]

[0044] As used herein, the term "pharmaceutical acceptable salt" includes salts derived from pharmaceutical acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases can include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium, and the like.

[0045]

[0046] In another aspect, the present invention provides a natural killer cell in which a target gene is expressed by the method of the present invention described above.

[0047] The method for introducing a target gene of the present invention and immune cells (e.g., natural killer cells) into which the target gene is introduced by the method have already been described in detail, so a detailed description thereof will be omitted to avoid excessive duplication.

[0048] In yet another aspect, the present invention provides a pharmaceutical composition for preventing or treating tumors or infectious diseases, comprising the natural killer cells of the present invention as an active ingredient.

[0049] According to yet another aspect of the present invention, there is provided a method for preventing or treating a tumor or an infectious disease, comprising the step of administering the natural killer cells of the present invention to a subject.

[0050] When the present invention is applied to immune cells, it can be applied to the treatment of tumors and infectious diseases. The NK cells prepared by the method according to the present invention can be applied to all kinds of tumors including solid cancer and blood cancer. Unlike blood cancer, solid cancer means cancer formed in a mass in an organ, and corresponds to cancer occurring in most organs. There is no particular limitation on tumors that can be treated using the NK cells according to the present invention, and they may be, but are not limited to, gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, etc. Infectious diseases that can be treated using the immune cells of the present invention are diseases caused by infection with viruses or pathogenic bacteria, and include all diseases that can be transmitted and infected through the respiratory tract, blood, skin contact, etc. Such infectious diseases include, but are not limited to, hepatitis B and C, human papilloma virus (HPV) infection, cytomegalovirus infection, viral respiratory diseases, and influenza.

[0051] As used herein, the term "treatment" means (a) suppression of the development of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. When immune cells into which the target gene of the present invention (e.g., a nucleic acid molecule encoding a chimeric antigen receptor that specifically recognizes a surface antigen of a pathogenic cell) has been introduced are administered to a subject, they also serve to suppress the development of, eliminate, or alleviate symptoms caused by tumors or infectious diseases by inducing the death of cancer cells or infected cells. Thus, the composition of the present invention may be a cell therapy composition for a disease by itself, or may be administered together with other pharmacological ingredients and applied as a therapeutic adjuvant for the disease. For this reason, as used herein, the term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

[0052] As used herein, the term "administration" or "administering" refers to administering a therapeutically effective amount of a composition of the present invention directly to a subject, thereby causing the same amount to be formed in the subject's body.

[0053] In the present invention, the term "therapeutically effective amount" means the content of the composition contained in an amount sufficient to provide a therapeutic or prophylactic effect to an individual to whom the composition of the present invention is to be administered, and thus includes a "prophylactically effective amount."

[0054] As used herein, the term "subject" includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, the subject of the present invention is a human.

[0055]

[0056] According to another aspect of the present invention, there is provided a method for enhancing the efficiency of target gene expression in immune cells, comprising the steps of freezing immune cells; and thawing the frozen immune cells.

[0057] The immune cells used in the present invention, the freezing and thawing process thereof, and the method for transducing the target gene have already been described in detail, so the description thereof will be omitted to avoid excessive duplication. Effect of the Invention

[0058]

[0059] The features and advantages of the present invention can be summarized as follows:

[0060] (a) The present invention provides a method for efficiently expressing a target gene in immune cells, specifically, natural killer cells, using a viral vector.

[0061] (b) The present invention enables a dramatic improvement in the transduction efficiency of viral vectors by adding a simple step of freezing and thawing natural killer cells before introducing a target gene, without the addition of enzymes or low molecular weight compounds.

[0062] (c) The present invention is particularly useful for producing natural killer cells into which genes of desired traits have been introduced and whose function, activity, and productivity as immunotherapeutic cells have been maximized, by significantly improving the efficiency of lentiviral vectors, which have a low efficiency of gene transfer into natural killer cells. [Brief description of the drawings]

[0063]

[0064] [Figure 1] This figure shows the expression efficiency of HER2 CAR introduced into a lentiviral vector after freezing the cells at various time points during NK cell culture and culturing for 3 days from the time of freezing.

[0065] [Diagram 2] This figure shows the gene transfer efficiency of HER2 CAR using a lentiviral vector when NK cells were frozen at various time points and then cultured for 3 days, and when they were cultured without freezing.

[0066] [Diagram 3] FIG. 13 shows a comparison of lentiviral vector transduction efficiency depending on the culture period after thawing frozen NK cells.

[0067] [Figure 4] FIG. 4A shows the transduction efficiency of lentiviral vectors depending on the culture period, and the results of comparing the efficiency between freezing and not freezing NK cells on day 10 of culture, confirmed by FACS analysis (FIG. 4B).

[0068] [Diagram 5]This figure shows the transduction efficiency of lentiviral vectors into NK cells using the enhancers BX795 (FIG. 5A) and (5Z)-7-oxozeaenol (FIG. 5B).

[0069] [Figure 6] FIG. 6A shows the results of confirming the transduction efficiency of lentiviral vectors expressing various CAR scFvs based on the CAR expression rate (FIG. 6A) and VCN (Vector Copy Number) (FIG. 5B) values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070]

[0071] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely intended to more specifically illustrate the present invention, and that the scope of the present invention is not limited to these examples according to the gist of the present invention. EXAMPLES

[0072]

[0073]

[0074] Example 1: Comparison of lentiviral vector transduction efficiency of NK cells by freezing time point

[0075] 1-1) Cultivation and freezing of NK cells

[0076] NK (cord blood natural killer, CBNK) cells derived from umbilical cord blood donated by donors were isolated and cultured for use. On the day of initiation of culture, the cells were stimulated and cultured using support cells, which were CD4+ T cells transformed with 4-1BBL, mbIL-21 (membrane bound IL-21), and mTNF-a (membrane TNF-α) genes. The NK cell culture medium was CellGro® serum-free medium (Cellgenix) containing 1% (v / v) human plasma, 1333 IU hIL-2 (Proleukin strain, Norvatis Korea), and 4 mM glutamine (Hyclone), and the cell concentration during NK cell culture was 1 × 10 6 / mL and cultured in a culture vessel with an appropriate volume.

[0077] The freezing medium used was PBS (Irvine Scientific) containing 25% dextran-40 (Hospira), 20% albumin (Akron Biotech), and 5% DMSO (Avantor). NK cells were frozen using a freezing container or an automatic cell freezer, and the frozen NK cells were stored in a cryogenic refrigerator at -70°C or a cryogenic liquid nitrogen tank at -130°C or lower until use.

[0078]

[0079] 1-2) Transduction of NK cells using lentiviral vectors

[0080] The conditions for cell freezing, transduction (TD), and expression confirmation at each time point of NK culture are as shown in Table 1 below. NK cells were cultured and frozen for 0 days (immediately after isolation), 3 days, 5 days, 7 days, 9 days, 11 days, and 13 days, respectively, and then thawed at the same time after being stored at ultra-low temperatures. To prevent cell damage that may occur during the freezing and thawing process, the cells were statically cultured in NK culture medium for 3 days, and then transduced with the anti-HER2 scFv CAR (HER2 CAR) gene. The NK cells cultured for 3 days after thawing were counted using a cell counter and found to be 3 x 10 5NK cells were placed in each well of a 48-well plate. The lentiviral vector for expressing the HER2 CAR gene was prepared by ICGM (Daejeon, Korea). To transduce HER2 CAR into NK cells, a complex of 20 MOI (multiplicity of infection) HER2 CAR lentiviral vector and 10 μg / mL Protransduzin (JPT, TE-PTDB-2) was prepared and left at room temperature for more than 5 minutes, and then applied to NK cells in a 48-well plate. IL-21 (Biolegend / 571204) 20 ng / mL was added to the culture medium to promote intracellular introduction and expression of the lentiviral vector. The day after transduction, the same amount of fresh NK cell culture medium containing 20 ng / mL IL-21 was additionally added to each well of the plate.

[0081] [Table 1]

[0082]

[0083] 1-3) Confirmation of expression of genes transduced into NK cells

[0084] The expression level of HER2 CAR in NK cells cultured for 6 days after transduction was confirmed using a flow cytometer, FACS. NK cells cultured for 9 days after thawing without transduction of HER2 CAR were used as a HER2 CAR negative control group. 5NK cells were collected and centrifuged at 1,200 rpm for 5 minutes, the supernatant was removed, and the cells were suspended in 2 mL of FACS buffer supplemented with 2% FBS. The cells were centrifuged at 2,000 rpm for 3 minutes, the supernatant was removed, and 100 uL of FACS buffer and 1 uL of 50 ng / uL of recombinant human HER2 protein with 6X histidine at the C-terminus (R&D systems) were added again and reacted at 4°C for 30 minutes. For subtype staining, 100 uL of FACS buffer was added without recombinant human HER2 protein and reacted under the same conditions. Then, 2 mL of FACS buffer was added, centrifuged at 2,000 rpm for 3 minutes, and the supernatant was removed. For secondary staining, 100 μL of FACS buffer was added to the washed NK cells, and the antibodies to be analyzed as shown in Table 2 were added and reacted at 4°C for 30 minutes. Thereafter, after washing with 2 mL of FACS buffer, the cells were fixed with 200 μL of fixation solution (BD, 554655), and the expression of NK cell surface was analyzed using a flow cytometer, FACS (LSRFortessa, BD Bioscience).

[0085] [Table 2]

[0086] The gene transduction efficiency of anti-HER2 scFv CAR was compared by calculating the percentage of HER2 CAR expression positive compared to cells stained for the subtype among NK cells with 7-AAD negative, CD3 negative, and CD56 positive phenotypes. NK cells frozen on days 0, 3, and 5 of culture were thawed and cultured for 3 days, and on days 3 (D0F-TD3), 6 (D3F-TD6), and 8 (D5F-TD8) of culture, HER2 CAR expression showed low efficiency of between 8-9% when transduced with lentiviral vectors. However, in NK cells transduced on the 10th day of culture (D7F-TD10), after thawing NK cells frozen on the 7th day of culture and culturing for 3 days, the expression of HER2 CAR rapidly increased to 29.3%, and in NK cells transduced on the 11th day of culture (D9F-TD11), the 13th day of culture (D11F-TD13), and the 15th day of culture (D13F-TD15), after thawing NK cells frozen on the 9th, 11th, and 13th days of culture in the same manner, the expression of HER2 CAR decreased over time to 26.4%, 20.5%, and 13.7%, respectively (Figure 1). The detailed values ​​of the graph in Figure 1 are as shown in Table 3. As shown in Figure 1, when NK cell culture was performed by freezing the cells at various times during the culture period and then thawing them again to transfer genes to lentiviral vectors, the gene expression efficiency was higher when the cells were frozen and transduced on the 7th day of culture than when they were initially cultured. In particular, the highest expression was observed under the D7F-TD10 condition, where the cells were frozen on the 7th day of culture, thawed, cultured for an additional 3 days, and then transduced with the gene on the 10th day of culture.

[0087] [Table 3]

[0088]

[0089] Example 2: Comparison of gene transfer efficiency with and without freezing of NK cells

[0090] Table 4 shows the detailed culture schedule and experimental conditions for comparing the efficiency of gene transfer with or without freezing NK cells. Frozen NK cells were thawed and cultured for three days, and the results of HER2 CAR expression were compared under conditions in which gene transfer was performed (freeze-thaw) and conditions in which gene transfer was performed after the same culture period without freezing (fresh).

[0091] [Table 4]

[0092]

[0093] In the experimental group that underwent freezing and thawing, NK cell culture and lentiviral vector introduction were performed in the same manner as in Example 1. In the non-frozen group, NK cells on day 0 of culture were cultured without a freezing process after stimulation with supporting cells, and were transduced with lentiviral vectors on days 6, 8, 10, 12, 14, and 16 of culture, respectively. Gene introduction was performed in the same manner as in Examples 1-2. All experimental groups were cultured for 6 days after gene introduction, and then the expression of the transgene was analyzed using a FACS device in the same manner as in Examples 1-3. As a result, the highest expression efficiency of HER2 CAR was confirmed in D7F-TD10, which was thawed on day 7 of culture and transduced on day 10 of culture (Figure 2). The detailed values ​​of the graph in Figure 2 are as shown in Table 5. In addition, when comparing the expression of HER2 CAR between days 10 and 14 of culture, the expression efficiency of HER2 CAR was higher in NK cells transduced after freezing than in NK cells transduced without a freezing process. From the above results, it was confirmed that after the same culture period, NK cells that underwent the freeze-thaw process during transduction with lentiviral vectors had a higher gene transfer efficiency, and that the freezing on day 7 of culture / transduction on day 10 of culture (D7F-TD10) conditions were optimal for transferring foreign genes into NK cells (Figure 2, Table 5).

[0094] [Table 5]

[0095]

[0096] Example 3: Comparison of transduction efficiency of lentiviral vectors depending on the days of culture after thawing of NK cells

[0097] From the results of Examples 1 and 2, it was confirmed that freezing NK cells on the 7th day of culture resulted in the highest gene transfer efficiency. Based on this, the transduction efficiency of lentiviral vectors according to the culture period for cell recovery after thawing NK cells frozen on the 7th day of culture was compared under the experimental conditions in Table 6 below.

[0098] [Table 6]

[0099] Immediately after thawing (D7F-TD7), on the first day of culture after thawing (D7F-TD8), on the second day of culture after thawing (D7F-TD9), on the third day of culture after thawing (D7F-TD10), and on the fourth day of culture after thawing (D7F-TD11), transduction of lentiviral vectors was performed using the same experimental method as in Example 1-2. Four types of lentiviral vectors (Lentigen, MD, USA) expressing anti-HER2 scFv CAR (HER2 CAR), anti-CD5 scFv CAR (CD5 CAR), anti-CD19 scFv CAR (CD19 CAR) and GFP genes were used. The expression of HER2 CAR, CD5 CAR, and CD19 CAR in NK cells cultured for 6 days after transduction was measured using the same method as in Example 1-3. The recombinant proteins used for expression analysis of each gene and the antibodies used for secondary staining are summarized in Tables 7 and 8, respectively. After secondary staining, NK cells were fixed with 200μL of fixative solution, and the gene expression efficiency of CAR was compared by calculating the percentage of cells positive for HER2 CAR, CD5 CAR, or CD19 CAR expression compared to the subtype-stained cells among 7-AAD-negative, CD3-negative, and CD56-positive cells. The gene expression efficiency of GFP was compared by calculating the mean fluorescent intensity (MFI) value compared to the negative control NK among 7-AAD-negative, CD3-negative, and CD56-positive cells.

[0100] [Table 7]

[0101] [Table 8]

[0102] NK cells frozen on day 7 of culture showed gradually higher transgene expression on days 1, 2, and 3 of culture after thawing, starting immediately after thawing, and showed the highest expression efficiency on day 3 of culture after thawing, i.e., day 10 of NK culture, after which the expression efficiency decreased from day 4 of culture. All four types of transgenes showed similar gene expression trends, confirming once again that the D7F-TD10 condition, in which NK cells frozen on day 7 of culture were thawed and then cultured for 3 days to introduce genes, was the most efficient lentiviral vector transduction condition for NK cells (Figure 3). Detailed values ​​of the graph in Figure 3 are shown in Table 9.

[0103] [Table 9]

[0104]

[0105] Example 4: Transduction of HER2 CAR into NK cells using the D7F-TD10 method

[0106] NK cells were cultured for 10 days without freezing, and the transduction efficiency at various time points was compared by HER2 CAR expression. In the case of the 10-day culture condition, the transduction efficiency was compared with that of the D7F-TD10 condition in which frozen NK cells were thawed on the 7th day of culture and cultured for 3 days. Detailed comparison conditions are shown in Table 10.

[0107] [Table 10]

[0108] Cord blood-derived NK cells were cultured using support cells in the same manner as in Example 1-1, and HER2 CAR gene transfer was carried out using a lentiviral vector on the 3rd, 7th, and 10th days of culture, respectively. NK cells under D7F-TD10 conditions were frozen on the 7th day of culture, and then thawed and cultured for 3 days to allow HER2 CAR gene transfer to proceed. As for the detailed gene transfer method, NK cells were treated with a lentiviral vector expressing HER2 CAR at 20 MOI, 10 μg / mL protransduzin, and 20 ng / mL human IL-21 recombinant protein in the same manner as in Example 1-2. In addition, (5Z)-7-oxozeaenol (TOCRIS, 3604), a TAK1 (transforming growth factor-β-activated kinase 1) inhibitor known as an enhancer that enhances the transduction efficiency of lentivirus, was treated at a concentration of 6 μM. The day after transduction, fresh NK cell culture medium containing 20ng / mL IL-21 and 6μM (5Z)-7-oxozeaenol was added to each well of the plate in the same amount. The expression of HER2 CAR in NK cells cultured for 6 days after transduction was confirmed by FACS analysis in the same manner as in Examples 1-3. In the case of NK cells without a freezing process, the expression of HER2 CAR increased with increasing culture period, and the expression of HER2 CAR was highest in TD10. However, compared with the D7F-TD10 condition, it was confirmed that the gene transduction efficiency was significantly higher in the D7F-TD10 condition despite the same period of NK cell culture (Figure 4A). In addition, when comparing the expression of NK cells transfected with HER2 CAR under TD10 or D7F-TD10 conditions in the FACS counter plot analysis, the expression of HER2 CAR was more than twice as high in D7F-TD10 as in TD10, and the MFI was also more than 10 times higher (Figure 4B).

[0109]

[0110] Example 5: Comparison of gene transfer efficiency by use of enhancers

[0111] The gene transfer efficiency under D7F-TD10 conditions was evaluated using an enhancer that increases the transduction efficiency of lentivirus. The detailed experimental conditions are as shown in Table 11.

[0112] [Table 11]

[0113] 5-1) Transduction of lentiviral vectors using enhancers and FACS expression analysis

[0114] Cord blood-derived NK cells were cultured using support cells in the same manner as in Example 1-1, and were transduced with lentiviral vectors expressing HER2 CAR and IL-15 genes simultaneously under D7F-TD10 conditions on the 3rd and 10th days of culture. As for the detailed gene transduction method, NK cells were treated with 20 MOI lentiviral vectors expressing HER2 CAR and IL-15, 10 μg / mL Protransduzin, and 20 ng / mL human IL-21 recombinant protein in the same manner as in Example 1-2. Additionally, (5Z)-7-oxozeaenol was treated at a concentration of 6 μM as an enhancer to increase the transduction efficiency of the lentivirus, or BX795 (TOCRIS, 4318), a TBK1 inhibitor, was treated at a concentration of 1.5 μM. The day after transduction, fresh NK cell culture medium containing 20 ng / mL IL-21 and 6 μM (5Z)-7-oxozeoenol or 1.5 μM BX795 was added to each well of the plate in the same amount and cultured. NK cells cultured for 6 days after transduction were analyzed for HER2 CAR expression as a percentage value in the same manner as in Examples 1-3.

[0115] Regardless of the type of enhancer that increases gene transfer efficiency, the results using both enhancers showed the highest expression of HER2 CAR in NK cells transduced on day 10 of culture after freezing on day 7 of culture (D7F-TD10) compared to TD3 and TD10 (Figure 5A).

[0116]

[0117] 5-2) Cell culture to confirm the IL-15 secretion ability of gene-transfected cells

[0118] NK cells were cultured for 6 days after transduction, and HER2 CAR-positive cells were isolated to confirm the expression level of IL-15 as follows: NK cells were centrifuged at 1,200 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended in MACS buffer (PBS containing 0.5% FBS and 2 mM EDTA) until the cell concentration reached 1 × 10 7 The NK cells were resuspended at 1 × 10 NK cells / mL. Biotin-tagged recombinant human HER2 protein (Acrobiosystems, HE2-H822R) at a concentration of 50 ng / mL was added to 1 × 10 NK cells. 6 1.5 μL per well was added and left at 4°C for 30 minutes. MACS buffer equivalent to 10 times the volume of NK cells was added and centrifuged at 340 g, 4°C for 10 minutes, after which the supernatant was removed. The cell concentration was adjusted to 1 × 10 8 / mL, and then anti-biotin beads (Miltenyi Biotec, 130-092-357) were added at 4 × 10 7 37.5 μL per 1 mL of NK cells was added and left at 4° C. for 30 minutes. MACS buffer equivalent to 10 times the volume of the NK cells reacted with the beads was added, and the mixture was centrifuged at 340 g at 4° C. for 10 minutes, after which the supernatant was removed.

[0119] QuadroMACS TM An LS column (Miltenyi Biotec) was attached to a Separator (Miltenyi Biotec) device, and 3 mL of MACS buffer was poured through the column to activate it. The NK cells that had reacted with the beads were thoroughly dissolved by pipetting, and 500 μL was poured into each column, after which 3 mL of MACS buffer was poured through the column to wash it three times. The washed columns were then placed on a QuadroMACS TM After separation with a separator, 5 mL of MACS buffer was added and the CAR-positive cells in the column were separated using a plunger. After centrifugation, the separated cells were diluted to 1 × 10 6 The NK cells were suspended in NK cell culture medium at a concentration of 100 / mL, placed in a well plate of an appropriate size, and restimulated with feeder cells in the same manner as in Example 1-1, and then further cultured for 13 days. The NK cells cultured for a total of 29 days were stored in an ultra-low temperature liquid nitrogen tank until use after freezing.

[0120]

[0121] 5-3) Confirmation of IL-15 secretion ability

[0122] To confirm the expression of IL-15 introduced simultaneously with HER2 CAR, NK cells frozen on day 29 of culture were thawed and co-cultured with the target cancer cell line, and the amount of IL-15 secreted was measured by ELISA. HER2-positive human breast cancer cells, HCC1954 (American Type Culture Collection, Manassas, VA, USA), were used as the target cancer cell line. The target cells were placed in assay medium (RPMI1640 containing 10% FBS, Gibco, 11875093) and centrifuged at 1,200 rpm for 5 minutes, after which the supernatant was removed and the cell pellet obtained after washing was placed in assay medium at 6 × 10 5 After thawing, the frozen NK cells were resuspended at a concentration of 2 × 10 6The cells were prepared at a concentration of 100 μL / mL. A 96-well round-bottom plate was prepared, and 100 μL of the prepared target cells and NK cells were placed in each well to be co-cultured. For the NK cell only control group, 100 μL of assay medium was placed in place of target cells and 100 μL of NK cells. After 24 hours of co-culture, the plate was removed and centrifuged at 2000 rpm and 4°C for 3 minutes, and 170 μL of the supernatant was collected and the expression level of IL-15 was measured using an IL-15 ELISA kit (R&D systems, S1500). In summary, the highest standard was prepared at 250 pg / mL, and then diluted 1 / 2 to prepare a total of seven standards (250, 125, 62.5, 31.25, 15.63, 7.81, and 3.91 pg / mL), including the lowest standard value of 3.9 pg / mL. 50μL of samples and standards were placed in a 96-well plate pre-coated with the antibody provided in the ELISA kit, and 50μL of dilution buffer was placed in the standard blank well. After leaving it at room temperature for 3 hours, each well was washed 4 times with 300μL of washing buffer. 200μL of human IL-15 conjugate was placed in each well and left at room temperature for 45 minutes. Each well was washed 4 times with 300μL of washing buffer. 200μL of substrate solution made by mixing equal amounts of solution A and solution B was placed in each well and left at room temperature for 30 minutes, and 50μL of stop solution was placed in each well and mixed slowly so that the solutions were mixed. The absorbance was measured at a wavelength of 450nM using a Spectra max190 device to calculate the expression level of IL-15 in the sample.

[0123] When co-cultured with HER2 CAR-positive target cells HCC1954, the D7F-TD10 condition, which was frozen and thawed on day 7 and transfected on day 10, showed the highest IL-15 expression. In contrast, the control group, which contained only NK cells without target cells, showed low IL-15 expression of less than 5 pg / mL with no significant difference depending on the time of transfection (Figure 5B). From the above results, it was confirmed that the D7F-TD10 method showed the highest transfection efficiency even when various enhancers that increase the transfection efficiency of lentiviral vectors were used (Figure 5).

[0124]

[0125] Example 6: Comparison of transduction efficiency and vector copy number (VCN) of lentiviral vectors

[0126] The gene transfer efficiency of D7F-TD10 was evaluated according to the type of carrier used for lentivirus transduction. The detailed experimental conditions are shown in Table 12.

[0127] [Table 12]

[0128] For NK cells under the TD3 and D7F-TD10 conditions, transduction of lentiviral vectors expressing HER2 CAR, CD5 CAR, and CD19 CAR was performed in NK cells in the same manner as in Example 3. For the D7F-TD10(L) condition, the NK cell concentration was 1 × 10 6After suspending the cells in NK cell culture medium to a concentration of 1 / mL, the lentiviral vector was added at 20 MOI in the presence of Lentiboost-P (sirion biotec, P-grade) equivalent to 1 / 100 of the total volume and human IL-21 recombinant protein (Biolegned) at a concentration of 20 ng / mL. NK cells without lentiviral vector were used as a negative control group. Three types of lentiviral vectors expressing anti-HER2 scFv CAR (HER2 CAR), anti-CD5 scFv CAR (CD5 CAR), and anti-CD19 scFv CAR (CD19 CAR) genes were used, all of which were produced by Lentigen (MD, USA). The expression of HER2 CAR, CD5 CAR, and CD19 CAR in NK cells cultured for 6 days after transduction was confirmed by the same method as in Examples 1-3. The recombinant proteins and antibodies used for staining NK cells for flow cytometry of each gene are summarized in Tables 7 and 8, respectively. After secondary staining, NK cells were fixed with 200 μL of fixative solution, and the expression of CAR protein on the surface of NK cells was analyzed as a percentage value using a FACS device, with the lowest expression value of TD3 being converted to 1. The expression values ​​of D7F-TD10 or D7F-TD10(L) using Protransduzen or Lentiboost-P as a delivery reagent were calculated as a ratio to the TD3 cell expression level, which was converted to 1, and the differences in gene expression were compared. As a result, it was confirmed that various scFv CARs showed high gene expression efficiency under D7F-TD10 conditions regardless of the type of delivery reagent (Figure 6A).

[0129] In addition, to measure VCN, which can confirm the degree of integration of the lentiviral vector into NK cells, genomic DNA was extracted from the same NK cells used in the above experiment and real-time PCR was performed. Genomic DNA was extracted using NucleoSpin (登録商標)The final extracted genomic DNA concentration was measured using a Nanodrop device with a Tissue (MachereyNagel, 740952.250) kit, and then diluted to between 10-50ng / uL using nuclease-free water (Ambion, AM9937). The standard DNA, primers, and probe information used for real-time PCR are summarized in Tables 13 and 14 below.

[0130] [Table 13]

[0131] [Table 14]

[0132] To measure the VCN of anti-HER2 scFv CAR, standard DNA stocks (4 × 10 7 After mixing equal amounts of the same antibody, the mixture was serially diluted with nuclease-free water to give 2 x 10 (STD1), 2 x 10 (STD2), 2 x 10 (STD3), and 2 x 10 (STD4). 2 (STD2), 2×10 3 (STD3), 2×104 4 (STD4), 2×10 5 (STD5) copies / uL were prepared. The spiking control group was prepared by mixing equal amounts of STD3 diluted 1 / 5 with wild-type NK cell genomic DNA (25 ng / uL). The PCR control group for the anti-HER2 scFv CAR target was prepared with wild-type NK cell genomic DNA at 10 ng / uL, and the PCR blank control group was prepared with nuclease-free water. The NK cell genomic DNA to be measured for VCN was prepared by diluting it with nuclease-free water at 10 ng / uL. PCR premix was prepared at the doses shown in Table 15 per well for the target gene.

[0133] [Table 15]

[0134] A MicroAmp® Optical 96-well reaction plate (Applied biosystems, N8010560) was prepared, and 5 μL of PCR template and 20 μL of premix were dispensed into each well, followed by PCR with the cycles shown in Table 17. The copy numbers of the CAR and RPPH1 genes in each sample were calculated based on the standard curve, the cell number was estimated using the calculated RPPH1 copy number, and the VCN of the CAR gene per NK cell was calculated by dividing the CAR copy number by the estimated cell number. The VCN was finally calculated by correcting for the expression rate (%) of the CAR in each NK cell.

[0135] The method for measuring the VCN of anti-CD5 and CD19 scFv CAR was the same as the method for measuring anti-HER2 scFv VCN, and the standard plasmid in Table 13, the primers and probes in Table 14, and the PCR premix in the doses shown in Table 16 were prepared in a manner similar to that of the method for measuring anti-HER2 scFv VCN. A MicroAmp (registered trademark) Optical 96-well reaction plate (Applied biosystems, N8010560) was prepared, and 5 μL of PCR template and 20 μL of premix were dispensed into each well, followed by PCR at the cycles shown in Table 17.

[0136] [Table 16]

[0137] [Table 17]

[0138] VCN is a value calculated by the number of lentiviral vectors inserted into one cell, and is a method for confirming gene transduction. The VCN of the condition (TD3) in which genes were transduced on the third day of culture was converted to 1, and the relative VCN of each condition to the TD3 VCN was calculated as a ratio and the difference was compared. For various scFv CARs, regardless of the type of delivery reagent, a higher VCN was shown when genes were transduced using the D7F-TD10 method rather than TD3 (Figure 6B). From the above results, high gene transduction and expression efficiency was confirmed when NK cells were frozen on the 7th day of culture, thawed, and cultured for 3 days, and transduced using a lentiviral vector on the 10th day of culture.

[0139]

[0140] Although certain parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and therefore do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for expressing a target gene in an immune cell, comprising: (a) freezing the immune cells; (b) thawing the frozen immune cells; and (c) introducing into the thawed immune cells a gene carrier into which the target gene is inserted.

2. The method of claim 1, further comprising the step of in vitro culturing the immune cells isolated from the subject for 6 to 11 days prior to step (a).

3. The method of claim 1, further comprising the step of culturing the thawed immune cells for 2 to 4 days after step (b).

4. The method of claim 1, wherein the immune cells are selected from the group consisting of T cells, natural killer (NK) cells and natural killer T (NKT) cells.

5. The method of claim 1 , wherein the gene delivery vehicle is a viral vector.

6. 6. The method of claim 5, wherein the virus is selected from the group consisting of lentivirus, adenovirus, adeno-associated viruses (AAV), retrovirus, herpes simplex virus and vaccinia virus.

7. 7. The method of claim 6, wherein the virus is a lentivirus.

8. The method according to claim 1, wherein the step (c) is carried out by adding one or more compounds selected from the group consisting of a compound represented by the following formula 1, a compound represented by the following formula 2, and pharma- ceutically acceptable salts thereof: 【Chemistry 1】 【Chemistry 2】

9. A natural killer cell in which a target gene is expressed by the method according to any one of claims 1 to 8.

10. A pharmaceutical composition for preventing or treating tumors or infectious diseases, comprising the natural killer cell according to claim 9 as an active ingredient.

11. A method for enhancing the efficiency of target gene expression in immune cells, comprising the steps of freezing immune cells; and thawing the frozen immune cells.

12. The method of claim 11, wherein the target gene is inserted into a viral vector and introduced into the immune cells.

13. 13. The method of claim 12, wherein the virus is a lentivirus.

14. The method of claim 11, wherein the immune cells are selected from the group consisting of T cells, natural killer (NK) cells and natural killer T (NKT) cells.

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