Composition for expanding natural killer cells containing feeder cells

Abstract

The present invention relates to a composition for expanding natural killer cells, which comprises feeder cells, and a method for expanding natural killer cells. Specifically, the present invention relates to a method for expanding natural killer cells using feeder cells transformed to express at least one gene selected from the group consisting of B7H6, CD137L, IL-15, and IL-15Rα.

Description

Technical Field

[0001] The present invention relates to a composition for natural killer cell proliferation containing feeder cells and a method for natural killer cell proliferation. Specifically, the present invention relates to a method for proliferating natural killer cells using feeder cells transformed to express at least one gene selected from the group consisting of B7H6, CD137L, IL-15, and IL-15Rα.

Background Art

[0002] Natural killer cells (NK cells) are representative cells responsible for innate immunity. They are a type of lymphocyte with large granules, showing cytotoxicity against various types of tumor cells and virus-infected cells. NK cells account for 5 - 10% of human peripheral blood lymphocytes and, unlike T cells, mature in the liver and bone marrow. Generally, immune cells detect infected cells via major histocompatibility complex (MHC) proteins presented on their surfaces and remove the infected cells by secreting various cytokines and chemicals that induce apoptosis. However, NK cells can recognize and remove abnormal cells even without the major histocompatibility complex bound to an antigen, thus inducing an immediate immune response.

[0003] Defects in the function of NK cells have been reported in many diseases. In particular, in cancer patients, it is known that most NK cells are inactivated. Some researchers have advanced research on methods for proliferating and activating NK cells in vitro. When NK cells amplified in vitro were reacted against various carcinomas, it was proven that they showed excellent cytotoxicity.

[0004] Therefore, methods for proliferating NK cells have been studied, and as one of the efficient methods for the selective amplification of NK cells, research using feeder cells has been conducted. However, the exact mechanism of the method for amplifying NK cells is not known.

[0005] As feeder cells for the amplification of NK cells, allogenic or autologous peripheral blood mononuclear cells (PBMC) and cancer cell lines are used. As cancer cell lines, HFWT, a Wilms tumor-derived cell line, EBV-LCL (EBV transformed lymphoblastoid cells), etc. are known.

[0006] The present inventors have discovered that feeder cells expressing B7H6 have an excellent NK cell proliferation effect, and have also found that in addition to the previously known feeder cells, ARH77 cells are also excellent feeder cells and have an NK cell proliferation effect, thus completing the present invention.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

[0008] An object of the present invention is to provide a composition for natural killer cell proliferation containing feeder cells transformed to express at least one gene selected from the group consisting of B7H6, CD137L, IL-15, and IL-15Rα.

[0009] Another object of the present invention is to provide a feeder cell line transformed to express at least one gene selected from the group consisting of B7H6, CD137L, IL-15, and IL-15Rα, and a method for natural killer cell proliferation using the same. Means for Solving the Problems

[0010] The present invention provides a composition for natural killer cell proliferation containing feeder cells transformed to express the B7H6 gene.

[0011] The present invention provides a composition for natural killer cell proliferation containing feeder cells transformed to express at least one gene selected from the group consisting of B7H6, CD137L, IL-15, and IL-15Rα.

[0012] The composition of the present invention may contain feeder cells expressing B7H6, and the feeder cells may further express at least one selected from the group consisting of CD137L, IL-15, and IL-15Rα.

[0013] Preferably, the composition of the present invention may contain feeder cells expressing B7H6, CD137L, and IL-15.

[0014] More preferably, the composition of the present invention may contain feeder cells expressing B7H6, CD137L, IL-15, and IL-15Rα.

[0015] The feeder cells of the present invention may be ARH77 or K562 cells, and preferably may be ARH77 cells.

[0016] The present invention provides a feeder cell line expressing B7H6.

[0017] The feeder cell line of the present invention may further express at least one selected from the group consisting of CD137L, IL-15, and IL-15Rα.

[0018] The feeder cell line of the present invention may be an ARH77 or K562 cell line.

[0019] Furthermore, the present invention (1) a step of transforming the B7H6 gene with a viral expression vector to produce a recombinant virus; (2) a step of adding the recombinant virus to feeder cells and culturing them; and provides a method for producing feeder cells expressing B7H6.

[0020] The transformed gene of the present invention may further contain at least one gene selected from the group consisting of CD137L, IL-15, and IL-15Rα.

[0021] The virus of the present invention may be a lentivirus or a retrovirus.

[0022] The feeder cells of the present invention may be ARH77 or K562 cells.

[0023] Furthermore, the present invention provides a method for natural killer cell proliferation, which includes the step of culturing feeder cells expressing B7H6 and peripheral blood mononuclear cells.

[0024] The feeder cells of the present invention may further express at least one selected from the group consisting of CD137L, IL-15, and IL-15Rα.

[0025] The feeder cells of the present invention may be ARH77 or K562 cells.

[0026] The culture medium of the present invention may contain at least one selected from the group consisting of penicillin, streptomycin, glutamine, gentamycin, fetal bovine serum, human serum, mercaptoethanol, ethanolamine, ascorbic acid, and sodium selenite. Preferably, it may contain at least one selected from the group consisting of glutamine, gentamycin, human serum, mercaptoethanol, ethanolamine, ascorbic acid, and sodium selenite.

[0027] The above-mentioned proliferation method of the present invention may further include the step of adding at least one selected from the group consisting of recombinant human Interleukin-2, recombinant human Interleukin-21, and recombinant human Interleukin-15.

[0028] Preferably, it may further include the steps of (a) adding recombinant human Interleukin-2 and recombinant human Interleukin-21, and (b) adding recombinant human Interleukin-2 and recombinant human Interleukin-15.

[0029] In addition, a natural killer cell line produced by the proliferation method of the present invention is provided.

Effect of the Invention

[0030] When natural killer cells (NK cells) are proliferated using feeder cells in which at least one gene selected from the group consisting of B7H6, CD137L, IL-15, and IL-15Rα according to the present invention is expressed, compared with the method using well-known feeder cells, there is an effect that NK cells having a higher amplification rate, purity, and cytotoxicity can be obtained, and thus it can be very usefully used in various immunocyte therapies using NK cells.

Brief Description of the Drawings

[0031]

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Modes for Carrying Out the Invention

[0032] Hereinafter, with reference to the accompanying drawings, the embodiments and examples of the present application will be described in detail so that those having ordinary knowledge in the technical field to which the present invention pertains can easily implement it. However, since the present application can be implemented in various forms, it is not limited to the embodiments and examples described below.

[0033] Throughout this specification, when a part includes a certain component, unless otherwise specified, it does not exclude other components, but means that other components may be further included.

[0034] The present invention relates to a composition for natural killer cell proliferation and a proliferation method, which include feeder cells transformed to express the B7H6 gene.

[0035] The present invention relates to a composition for natural killer cell proliferation and a proliferation method, which include feeder cells transformed to express at least one gene selected from the group consisting of B7H6, CD137L, IL-15, and IL-15Rα.

[0036] In the present invention, the term "B7H6" means a ligand of the natural killer cell receptor NKp30.

[0037] The B7H6 gene according to the present invention may consist of the nucleotide sequence of SEQ ID NO: 1, and homologs of the nucleotide sequence are included within the scope of the present invention. Specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence of SEQ ID NO: 1.

[0038] In the present invention, the term "CD137L" means a ligand of CD137.

[0039] The CD137L gene according to the present invention may consist of the nucleotide sequence of SEQ ID NO: 2, and homologs of the nucleotide sequence are included within the scope of the present invention. Specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence of SEQ ID NO: 1.

[0040] In the present invention, the term "IL-15" refers to interleukin-15, which means a cytokine produced by epithelial cells.

[0041] The IL-15 gene according to the present invention may consist of the nucleotide sequence of SEQ ID NO: 3, and homologs of the said nucleotide sequence are included within the scope of the present invention. Specifically, the said gene may include a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence of SEQ ID NO: 1.

[0042] In the present invention, the term "IL-15Rα" refers to the interleukin 15 receptor α-chain.

[0043] The IL-15Rα gene according to the present invention may consist of the nucleotide sequence of SEQ ID NO: 4, and homologs of the said nucleotide sequence are included within the scope of the present invention. Specifically, the said gene may include a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence of SEQ ID NO: 1.

[0044] The term "feeder cells (support cells)" used in the present invention means cells that do not have the ability to divide and proliferate but have metabolic activity, and thus produce various metabolites to assist the proliferation of target NK cells. Feeder cells that may be used in the present invention include animal cell lines into which genes have been introduced, peripheral blood leukocyte cells (PBL) treated with various cytokines or compounds, autologous or allogeneic peripheral blood leukocyte cells, T cells, B cells, or monocytes, etc. Preferably, an animal cell line into which a gene has been introduced may be used, more preferably, it may be ARH77 cells, but it is not limited thereto. Other feeder cells that are known to be normally usable in the technical field to which the present invention pertains can be used without limitation as long as they meet the object of the present invention.

[0045] As used herein, the term "expression vector" refers to a vector capable of expressing a target protein or target RNA in a suitable host cell, and refers to a gene construct containing essential regulatory elements operably linked such that the gene insert is expressed.

[0046] In the present invention, the term "operably linked" means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked so as to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA are functionally linked and can affect the expression of the encoding nucleic acid sequence. The operative linkage with a recombinant expression vector can be achieved using gene recombination techniques well known in the art, and site-specific DNA cleavage and ligation use enzymes generally known in the art.

[0047] Examples of expression vectors that can be used in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. Preferably, a viral vector may be used, and more preferably, a lentivirus or retrovirus vector may be used, but it is not limited thereto. Further, as an appropriate expression vector, in addition to expression control sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion may be variously produced according to the purpose. The promoter of the expression vector may be constitutive or inducible. In addition, the expression vector contains a selection marker for selecting a host cell containing the vector, and when it is an expression vector capable of replication, it contains an origin of replication.

[0048] In the present invention, the term "peripheral blood mononuclear cell (PBMC)" means a cell having a spherical nucleus present in the blood, and such peripheral blood mononuclear cells include immune cells such as B cells, T cells, macrophages, dendritic cells, and natural killer cells (NK cells).

[0049] The "IL-2 (Interleukin-2)" of the present invention has been reported to have a function of promoting the proliferation and activation of mature NK cells. In humans and mice lacking IL-2, there are reports that the number of NK cells significantly decreases. On the other hand, there are also research results indicating that the deficiency of IL-2 and IL-2Ra indirectly affects the number and activation of NK cells. The IL-2R chain is known to be involved in the formation of the IL-15 receptor.

[0050] Regarding the "IL-15 (Interleukin-15)" of the present invention, in mice lacking IL-15 or IL-15Rα, the fact that no NK cells are observed, and in mice lacking the transcription factor interferon (IFN)-regulatory factor 1 required for IL-15 production, it has been found that NK cells are deficient. Therefore, it is known that IL-15 is involved in NK cell differentiation and that IL-15 directly promotes the proliferation and differentiation of NK cells via the IL-15 receptor expressed on NK cells.

[0051] The "IL-21 (Interleukin-21)" of the present invention is known to increase the telomere length of NK cells by stimulating transcription-3 (STAT-3) and play an important role in the activity, growth, and amplification of NK cells. In particular, in human CD34+ hematopoietic stem cells, it has been reported to assist in the growth of NK cells in combination with IL-15. When amplifying NK cells via membrane-bound (mb) IL-21, an increase in the expression of interferon gamma (IFN-γ) and cytotoxicity has been confirmed. Furthermore, it has been reported that injecting IL-21 only once is more effective for amplifying NK cells than continuously injecting it into the culture medium.

[0052] Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0053] [Production Example] Production of B7H6, CD137L, IL-15 and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells Genes of B7H6, CD137L, IL-15 and IL-15Rα were cloned, and then cDNA of each gene was transferred into the plasmid pLVX-EF1α-IRES-Puro (Takara, Japan), a lentiviral expression vector, to produce lentivirus.

[0054] As shown in Fig. 1, the CD137L gene, the B7H6 and EGFP genes, the IL-15 and RFP-647 genes, and the IL-15Rα and BFP genes were inserted into the multiple cloning sites of their respective pLVX-EF1α-IRES-Puro plasmid. The lentiviral plasmid (10 μg) into which each gene was introduced was transfected into HEK 293T cells together with Lenti-X TM Packaging Single shots (Takara), and cultured in DMEM culture medium containing 10% FBS for 48 to 72 hours to produce recombinant lentivirus.

[0055] To transduce each gene into feeder cells, ARH77 cells and K562 cells were dispensed into 24-well plates at 2×10 5 cells per well, and then recombinant lentivirus was added to each well together with polybrene (8 μg / mL). Centrifugation was performed at 25°C and 1,500 g for 2 hours, and then the cells were cultured in an incubator at 37°C and 5% CO 2 for 48 hours. After removing the culture medium, the culture was continued while replacing it with fresh culture medium (2 mL / well). One week after transduction, all the cells were collected respectively, and only the fluorescent positive cells were separated using a cell sorter BD FACSAria (registered trademark) III, and these cells were continuously cultured in RPMI1640 culture medium containing 10% FBS.

[0056] In the same method as described above, each gene was introduced into ARH77 cells and K562 cells according to the procedure shown in Figure 2, and after the selection process, ARH77-B7H6-CD137L-IL15-IL15Rα and K562-B7H6-CD137L-IL15-IL15Rα cells were produced.

[0057] The nucleotide sequences of B7H6, CD137L, IL-15 and IL-15Rα are shown in Table 1 below.

[0058]

Table 1

Example

[0059] Effect of NK cell activity by B7H6 or CD137L-expressing feeder cells 1-1. Evaluation of NK cell expansion rate To confirm the effect of NK cell activity by the expression of B7H6 or CD137L, the expansion rate of NK cells was evaluated using K562 cells, B7H6-expressing K562 (K562-B7H6) cells and CD137L-expressing K562 (K562-CD137L) cells.

[0060] Using the density gradient centrifugation method, PBMCs were separated from the peripheral blood of healthy blood donors and washed twice with PBS. Then, 3×10 6 PBMCs were irradiated with gamma rays at 100 Gy, and together with 5×10 5 cells of each feeder cell (K562 cells, K562-B7H6 cells or K562-CD137L cells), they were co-cultured in a 24-well plate with a culture medium of 2 mL / well in an incubator under the conditions of 37°C and 5% CO 2 2.

[0061] The culture medium was used by adding 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin, and 4 mmol / L L-glutamine to RPMI 1640 culture medium. For the first week from the start of culture, 10 U / mL recombinant human interleukin (rhIL)-2 and 5 ng / mL rhIL-21 were added to the culture medium. After 1 week of culture, 100 U / mL rhIL-2 and 5 ng / mL rhIL-15 were added to the culture medium and cultured for 28 days. The culture medium was changed to a new one every 1 - 2 days.

[0062] Cells cultured on days 14, 21, and 28 were collected, and the proportion (purity) of NK cells (CD3−, CD56+ cells) in the collected cells was confirmed by flow cytometry. The proportion of NK cells was multiplied by the total number of living cells among the collected cells to calculate the total number of NK cells. In the same way, the total number of NK cells in PBMC before culture was calculated, and the amplification rate of NK cells was calculated by dividing the total number of NK cells on days 14, 21, and 28 of culture by the number of NK cells before culture.

[0063] The experimental results are shown in Tables 2 and 3 and Figures 3 and 4 below.

[0064]

Table 2

[0065]

Table 3

[0066] As shown in Table 2 and Figure 3, the NK cells obtained via K562 cells showed an amplification rate of 291-fold after 4-week culture, while the NK cells obtained via B7H6-expressing K562 (K562-B7H6) cells showed an amplification rate of 1,383-fold after 4-week culture.

[0067] In addition, as shown in Table 3 and FIG. 4, the NK cells obtained from CD137L-expressing K562 (K562-CD137L) cells showed an amplification rate of 3,344-fold after 4 weeks of culture.

[0068] Therefore, B7H6-expressing K562 (K562-B7H6) cells showed an amplification rate more than 4 times that of K562 cells, and CD137L-expressing K562 (K562-CD137L) cells showed an amplification rate more than 11 times that of K562 cells, indicating that they have a better NK cell amplification effect.

[0069] 1-2. Evaluation of the purity of NK cells To confirm the effect of NK cell activity by the expression of B7H6 or CD137L, the purity of NK cells was evaluated using K562 cells, B7H6-expressing K562 (K562-B7H6) cells, and CD137L-expressing K562 (K562-CD137L) cells.

[0070] PBMC was separated in the same manner as in Example 1-1 and co-cultured with each irradiated feeder cell. Each cell collected before culture and 14 days, 21 days, and 28 days after culture was stained with fluorescently conjugated human CD3 and CD56 monoclonal antibodies, and then the purity of NK cells was evaluated by flow cytometry. The ratio of CD3 - CD56+ NK cells in each cell was evaluated as the purity of NK cells.

[0071] The experimental results are shown in Tables 4 and 5 below, and FIGS. 5 and 6.

[0072]

Table 4

[0073]

Table 5

[0074] As shown in Tables 4 and 5, and Figures 5 and 6, NK cells obtained via K562 cells, B7H6-expressing K562 (K562-B7H6) cells, and CD137L-expressing K562 (K562-CD137L) cells all showed a purity of 90% or higher.

[0075] Therefore, it can be seen that both B7H6-expressing K562 (K562-B7H6) cells and CD137L-expressing K562 (K562-CD137L) cells can proliferate highly pure NK cells.

[0076] 1-3. Evaluation of cytotoxicity of NK cells To confirm the effect of B7H6 or CD137L expression on NK cell activity, the cytotoxic performance of the proliferated NK cells against cancer cells was evaluated using K562 cells, B7H6-expressing K562 (K562-B7H6) cells, and CD137L-expressing K562 (K562-CD137L) cells.

[0077] The killing ability of NK cells proliferated by the same method as in Example 1-1 against K562 cells was measured by a sensitive colorimetric assay using WST-8 (Biotool, USA). K562 cells (1×10 5 cells / 100 μL culture medium), which are the target cancer cells of NK cells, were dispensed into a 96-well plate. Then, 2.5×10 4 proliferated NK cells were added and mixed (effector:target ratio = 0.25:1), followed by centrifugation at 1,500 rpm for 3 minutes, and then cultured in an incubator at 37°C and 5% CO 2 conditions. After 3 hours of culture, 10 μL of WST-8 was added to each well, and after further culturing for 1 hour, the absorbance (A450) was measured at a wavelength of 450 nm using SpectraMax (Molecular Devices, USA). The percentage of cancer cells killed by NK cells was calculated using the following formula.

[0078] Cytotoxicity (%) = 100% - 100 × [A450 of effector cell treated target cells - A450 of effector cells] / [A450 of target cells - A450 of target cells without WST-8]

[0079] The above experimental results are shown in Tables 6 and 7 below, and Figures 7 and 8.

[0080]

Table 6

[0081]

Table 7

[0082] As shown in Tables 6 and 7, and Figures 7 and 8, NK cells obtained through B7H6-expressing K562 (K562-B7H6) cells and CD137L-expressing K562 (K562-CD137L) cells showed higher cytotoxicity than NK cells obtained through K562 cells.

[0083] Therefore, it can be seen that B7H6-expressing K562 (K562-B7H6) cells and CD137L-expressing K562 (K562-CD137L) cells can proliferate NK cells showing higher levels of cytotoxicity.

Example

[0084] Effect of IL15Rα-expressing feeder cells on NK cell activity 2-1. Evaluation of NK cell expansion rate To confirm the effect of IL15Rα expression on NK cell activity, the expansion rate of NK cells was evaluated using ARH77 cells and ARH77 cells expressing IL15Rα (ARH77-IL15Rα).

[0085] The experiment was conducted in the same manner as in Example 1-1. Only IL-2 and IL-15 were added to the culture medium for culturing, and the evaluation was carried out for 3 weeks. The experimental results are shown in Table 8 and Figure 9 below.

[0086]

Table 8

[0087] As shown in Table 8 and Figure 9, the NK cells obtained via ARH77 cells showed an amplification rate of 222-fold after 3 weeks of culture, while the NK cells obtained via IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells showed an amplification rate of 388-fold after 3 weeks of culture.

[0088] Therefore, it can be seen that IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells showed an amplification rate of more than 1.5 times that of ARH77 cells and had a better NK cell amplification effect.

[0089] 2-2. Evaluation of the purity of NK cells To confirm the effect of IL15Rα expression on NK cell activity, the purity of NK cells was evaluated using ARH77 cells and IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells.

[0090] The experiment was conducted in the same manner as in Example 1-2 and evaluated for 3 weeks. The experimental results are shown in Table 9 and Figure 10 below.

[0091]

Table 9

[0092] As shown in Table 9 and Figure 10, the NK cells obtained via ARH77 cells showed a purity of 59-70%, while the NK cells obtained via IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells showed a higher purity of 63-79%.

[0093] Therefore, it can be seen that NK cells obtained through IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells have higher purity, and IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells can proliferate NK cells with higher purity.

[0094] 2-3. Evaluation of cytotoxicity of NK cells To confirm the effect of IL15Rα expression on NK cell activity, the cytotoxic performance of the proliferated NK cells against cancer cells was evaluated using ARH77 cells and IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells.

[0095] Experiments were conducted in the same manner as in Examples 1-3 and evaluated for 3 weeks. The experimental results are shown in Table 10 and Figure 11 below.

[0096]

Table 10

[0097] As shown in Table 10 and Figure 11, NK cells obtained through IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells showed higher cytotoxicity than NK cells obtained through ARH77 cells.

[0098] Therefore, it can be seen that IL15Rα-expressing ARH77 (ARH77-IL15Rα) cells can proliferate NK cells that show a higher level of cytotoxicity.

Example

[0099] Effect of ARH77 cells on NK cell stimulation 3-1. Evaluation of NK cell expansion rate To confirm the effect of ARH77 cells on NK cell stimulation, the expansion rate of NK cells was evaluated using ARH77 cells and K562 cells.

[0100] PBMCs were isolated in the same manner as in Example 1-1 and co-cultured with irradiated ARH77 cells or K562 cells. Each cell collected before culture and 14 days, 21 days, and 28 days after culture was stained with fluorescently conjugated human CD3 and CD56 monoclonal antibodies, and then the purity of NK cells (CD3−CD56+ cells) was evaluated by flow cytometry. The total number of NK cells was calculated by multiplying the proportion of NK cells by the total number of viable cells among the collected cells. In the same manner, the total number of NK cells in PBMCs before culture was calculated, and the amplification rate of NK cells was calculated by dividing the total number of NK cells on days 14, 21, and 28 of culture by the number of NK cells before culture.

[0101] The experimental results are shown in Table 11 and FIG. 12 below.

[0102]

Table 11

[0103] As shown in Table 11 and FIG. 12, the NK cells obtained via K562 cells showed an amplification rate of 627-fold after 4 weeks of culture, whereas the NK cells obtained via ARH77 cells showed an amplification rate of 1376-fold after 4 weeks of culture.

[0104] Therefore, it can be seen that ARH77 cells have a more than two-fold superior NK cell amplification effect.

[0105] 3-2. Evaluation of the purity of NK cells To confirm the effect of NK cell stimulation by ARH77 cells, the purity of the obtained NK cells was evaluated using ARH77 cells and K562 cells.

[0106] In the same manner as in Example 1-2, the proportion of intracellular CD3−CD56+ NK cells was measured by flow cytometry to evaluate the purity. The experimental results are shown in Table 12 below, as well as FIGS. 13 and 14.

[0107]

Table 12

[0108] As shown in Table 12 and Figures 13 and 14, the NK cells obtained via K562 cells showed a purity of 46.1 - 68.7%, while the NK cells obtained via ARH77 cells showed a higher purity of 69.0 - 83.1%.

[0109] Therefore, it can be seen that the NK cells obtained via ARH77 cells have a higher purity.

[0110] 3 - 3. Evaluation of cytotoxicity of NK cells To confirm the effect of NK cell stimulation by ARH77 cells, the cytotoxicity of the obtained NK cells was evaluated using ARH77 cells and K562 cells.

[0111] In the same manner as in Example 1 - 3, the cytotoxic performance of the NK cells obtained using ARH77 cells or K562 cells against cancer cells was measured. The experimental results are shown in Table 13 and Figure 15 below.

[0112] [Table 13]

[0113] As shown in Table 13 and Figure 15, the cytotoxicity of the NK cells obtained via ARH77 cells and K562 cells was confirmed to be significant.

[0114] Therefore, it can be seen that ARH77 cells can proliferate NK cells showing a cytotoxicity level similar to that of K562 cells.

[0115] 3 - 4. Evaluation of NK cell phenotype To confirm the NK cell stimulation effect of ARH77 cells, the receptor expression of the NK cells obtained using ARH77 cells and K562 cells was evaluated. The results are shown in Tables 14 - 20 and Figures 16 - 22 below.

[0116]

Table 14

[0117]

Table 15

[0118]

Table 16

[0119]

Table 17

[0120]

Table 18

[0121]

Table 19

[0122]

Table 20

[0123] As shown in Tables 14 to 20 and Figures 16 to 22, ARH77 cells showed a higher mean fluorescence intensity (MFI) than K562 cells in all measured values except for CD158b, and showed a lower mean fluorescence intensity in CD158b, which suppresses the function of NK cells. Therefore, it was confirmed that ARH77 cells express better receptors important for NK cell activity than K562 cells.

[0124] From this, it can be seen that ARH77 cells have a better NK cell stimulating effect as feeder cells than K562 cells.

Example

[0125] Effect of B7H6, CD137L, IL15 and IL15Rα-expressing feeder cells on NK cell stimulation To confirm the effect of B7H6, CD137L, IL15- and IL15Rα-expressing feeder cells on NK cell stimulation, ARH77 cells and K562 cells were used to express each gene, and the following experiments were conducted.

[0126] 4-1. Evaluation of NK cell expansion rate To confirm the effect of B7H6, CD137L, IL15 and IL15Rα-expressing feeder cells on NK cell stimulation, the expansion rate of NK cells was evaluated using ARH77 cells and K562 cells that expressed the above genes.

[0127] PBMC was isolated in the same manner as in Example 1-1 and co-cultured with each irradiated feeder cell. The purity of NK cells (CD3-CD56+ cells) was evaluated by flow cytometry in the cells collected before culture and 14 days, 21 days and 28 days after culture, and then the expansion rate of NK cells was calculated in the same manner as above.

[0128] The above experimental results are shown in Table 21 below.

[0129]

Table 21

[0130] As shown in Table 21, it was confirmed that B7H6, CD137L, and IL-15-expressing ARH-77 (ARH77-B7H6-CD137L-IL15) cells, B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells, and B7H6, CD137L, IL-15, and IL-15Rα-expressing K562 (K562-B7H6-CD137L-IL15-IL15Rα) cells showed the best NK cell amplification rate at the third week.

[0131] 4-2. Evaluation of the purity of NK cells To confirm the effect of NK cell stimulation by B7H6, CD137L, IL15, and IL15Rα-expressing feeder cells, the purity of NK cells was evaluated using ARH77 cells and K562 cells expressing the said genes.

[0132] In the same manner as in Example 1-2, the proportion of intracellular CD3-CD56+ NK cells was measured by flow cytometry to evaluate the purity. The experimental results are shown in Table 22 below.

[0133]

Table 22

[0134] As shown in Table 22, it was confirmed that NK cells obtained through B7H6, CD137L, and IL-15-expressing ARH-77 (ARH77-B7H6-CD137L-IL15) cells, B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells, B7H6, CD137L, and IL-15-expressing K562 (K562-B7H6-CD137L-IL15) cells, and B7H6, CD137L, IL-15, and IL-15Rα-expressing K562 (K562-B7H6-CD137L-IL15-IL15Rα) cells showed a purity of about 90% or more by the fourth week.

[0135] 4-3. Evaluation of the cytotoxicity of NK cells To confirm the effect of NK cell stimulation by B7H6, CD137L, IL15, and IL15Rα-expressing feeder cells, the cytotoxicity of the proliferated NK cells against cancer cells was evaluated using ARH77 cells and K562 cells expressing the said genes.

[0136] In the same manner as in Examples 1-3, the cytotoxic performance of NK cells proliferated using each gene-expressing feeder cell against cancer cells was measured. The experimental results are shown in Table 23 below.

[0137]

Table 23

[0138] As shown in Table 23, the NK cells obtained via B7H6, CD137L, and IL-15-expressing ARH-77 (ARH77-B7H6-CD137L-IL15) cells, B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells, and B7H6, CD137L, IL-15, and IL-15Rα-expressing K562 (K562-B7H6-CD137L-IL15-IL15Rα) cells maintained toxicity until the 4th week, while the NK cells using the remaining cells were confirmed to have reduced toxicity.

[0139] Therefore, it can be seen that B7H6, CD137L, and IL-15-expressing ARH-77 (ARH77-B7H6-CD137L-IL15) cells, B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells, and B7H6, CD137L, IL-15, and IL-15Rα-expressing K562 (K562-B7H6-CD137L-IL15-IL15Rα) cells have an excellent effect of stimulating NK cells as feeder cells.

Example

[0140] Effect of Feeder Cells on NK Cell Stimulation in NK Cell Optimization Medium In Example 3, the following experiments were conducted using B7H6, CD137L, and IL-15-expressing ARH-77 (ARH77-B7H6-CD137L-IL15) cells, B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells, and B7H6, CD137L, IL-15, and IL-15Rα-expressing K562 (K562-B7H6-CD137L-IL15-IL15Rα) cells, which have excellent effects on NK cell stimulation as feeder cells.

[0141] 5-1. Evaluation of NK Cell Expansion Rate To confirm the effect of NK cell stimulation using the three genetically modified feeder cells, the expansion rate of NK cells was evaluated using a medium optimized for NK cells.

[0142] PBMCs were isolated in the same manner as in Example 1-1 and co-cultured with each irradiated feeder cell. The culture medium was used by adding 30% HAM’S F12, 10% human serum (HS), 10 μg / mL gentamicin, 1x Glutamax, 10 μM β-mercaptoethanol, 50 μM ethanolamine, 20 μg / mL ascorbic acid, and 5 ng / mL sodium selenite to DMEM medium. From the start day of culture, 10 U / mL recombinant human interleukin (rhIL)-2 and 5 ng / mL rhIL-21 were added to the culture medium. After 1 week of culture, 100 U / mL rhIL-2 and 5 ng / mL rhIL-15 were added to the culture medium and cultured for 28 days.

[0143] The culture medium was changed to a new one every 1 to 2 days. Cells cultured for 14 days, 21 days, and 28 days were collected, and the proportion (purity) of NK cells (CD3−, CD56+ cells) in the collected cells was confirmed by flow cytometry. The proportion of NK cells was multiplied by the total number of living cells among the collected cells to calculate the total number of NK cells.

[0144] In the same manner, the total number of NK cells in PBMC before culture was calculated, and the amplification rate of NK cells was calculated by dividing the total number of NK cells on each of the 14th, 21st, and 28th days of culture by the number of NK cells before culture.

[0145] The experimental results are shown in Table 24 and FIG. 23 below.

[0146]

Table 24

[0147] As shown in Table 24 and FIG. 23, it was confirmed that B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells had the most excellent effect on NK cell amplification.

[0148] Specifically, it was confirmed that NK cells using B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells had an amplification rate approximately 3.5 times higher than that of NK cells using B7H6, CD137L, IL-15, and IL-15Rα-expressing K562 (K562-B7H6-CD137L-IL15-IL15Rα) cells after 4 weeks of culture.

[0149] 5-2. Evaluation of purity of NK cells To confirm the effect of NK cell stimulation by the three gene-recombinant feeder cells in Example 4-1, the purity of NK cells was evaluated using a medium optimized for NK cells.

[0150] In the same manner as in Example 1-2, the proportion of intracellular CD3−CD56+ NK cells was measured by flow cytometry to evaluate the purity. The experimental results are shown in Table 25 and FIG. 24 below.

[0151]

Table 25

[0152] As shown in Table 25 and FIG. 24, NK cells obtained via B7H6, CD137L, and IL-15-expressing ARH-77 (ARH77-B7H6-CD137L-IL15) cells, B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells, and B7H6, CD137L, IL-15, and IL-15Rα-expressing K562 (K562-B7H6-CD137L-IL15-IL15Rα) cells were all confirmed to maintain a purity of about 85% or more until 4 weeks. In particular, it was confirmed that the purity of NK cells using ARH77 cells was more excellent.

[0153] 5-3. Evaluation of cytotoxicity of NK cells To confirm the effect of NK cell stimulation by the three gene recombinant feeder cells, the cytotoxicity of NK cells was evaluated using a medium optimized for NK cells.

[0154] In the same manner as in Example 1-3, the cytotoxicity of NK cells grown using each gene-expressing feeder cell against cancer cells was measured. The experimental results are shown in Table 26 and FIG. 25 below.

[0155]

Table 26

[0156] As shown in Table 26 and FIG. 25, NK cells obtained through B7H6, CD137L, and IL-15-expressing ARH-77 (ARH77-B7H6-CD137L-IL15) cells, B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells, and B7H6, CD137L, IL-15, and IL-15Rα-expressing K562 (K562-B7H6-CD137L-IL15-IL15Rα) cells were all confirmed to exhibit excellent cytotoxicity.

[0157] Ultimately, from the above experimental results, it can be seen that B7H6, CD137L, IL-15, and IL-15Rα-expressing ARH77 (ARH77-B7H6-CD137L-IL15-IL15Rα) cells have the most excellent effect of stimulating NK cells as feeder cells.

Claims

1. A composition for expanding natural killer cells, comprising ARH77 cells as feeder cells expressing B7H6, CD137L and IL-15.

2. The composition for expanding natural killer cells according to claim 1, wherein the ARH77 cells further express IL-15Rα. Composition.

3. A feeder cell line, ARH77 cells, expressing B7H6, CD137L and IL-15.

4. The feeder cell line according to claim 3, wherein the feeder cell line is an ARH77 cell, and further expresses IL-15Rα.

5. (1) producing a recombinant virus by transforming a B7H6 gene, a CD137L gene and an IL-15 gene with a viral expression vector; (2) A method for producing feeder cells which are ARH77 cells and express B7H6, CD137L and IL-15, comprising the step of adding the recombinant virus to feeder cells which are ARH77 cells and culturing the cells.

6. The method for producing feeder cells which are ARH77 cells according to claim 5, further comprising transfecting the cells with an IL-15Rα gene in the step (1).

7. The method for producing feeder cells which are ARH77 cells according to claim 5, wherein the virus is a lentivirus or a retrovirus.

8. A method for expanding natural killer cells, comprising the step of culturing peripheral blood mononuclear cells with feeder cells, which are ARH77 cells expressing B7H6, CD137L and IL-15.

9. The method for expanding natural killer cells according to claim 8, wherein the feeder cells which are ARH77 cells further express IL-15Rα.

10. 9. The method of claim 8, wherein the culture medium contains at least one selected from the group consisting of penicillin, streptomycin, glutamine, gentamycin, fetal bovine serum, human serum, mercaptoethanol, ethanolamine, ascorbic acid, and sodium selenite.

11. 9. The method for expanding natural killer cells according to claim 8, wherein the culture medium contains at least one selected from the group consisting of glutamine, gentamycin, human serum, mercaptoethanol, ethanolamine, ascorbic acid, and sodium selenite.

12. 9. The method for expanding natural killer cells according to claim 8, further comprising the step of adding at least one selected from the group consisting of recombinant human interleukin-2, recombinant human interleukin-21, and recombinant human interleukin-15.

13. 9. The method for expanding natural killer cells according to claim 8, further comprising the steps of: (a) adding recombinant human interleukin-2 and recombinant human interleukin-21; and (b) adding recombinant human interleukin-2 and recombinant human interleukin-15.

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