Transformant and method for producing target protein

Abstract

Provided is a transformant used for producing a target protein in vitro, in which a gene that encodes the target protein and a suicide gene have been introduced into a host cell. Also provided is a method for producing a target protein, the method comprising: a culture step in which the transformant is cultured in the presence of a cell death inducer that cooperates with a protein encoded by the suicide gene of the transformant to induce the cell death of the transformant, thereby producing a target protein; and a recovery step in which the target protein is recovered from the culture after the culture step.

Description

Transformant and method for producing target protein

[0001] The present invention relates to a transformant and a method for producing a target protein using the transformant.

[0002] Genetic recombination methods are widely used for the production of proteins used for medical, diagnostic, industrial, etc. Various cells, such as mammalian cells, insect cells, plant cells, yeast, and Escherichia coli, are used as host cells for obtaining transformants. In particular, Chinese hamster ovary-derived CHO cells are widely used for the production of recombinant proteins used as active ingredients in biopharmaceuticals because they allow for advanced post-translational modification of recombinant proteins and enable serum-free suspension culture.

[0003] When producing a recombinant protein, it is important to improve the productivity of the recombinant protein in order to reduce production costs. To date, various techniques for genetically modifying host cells have been proposed to improve the productivity of recombinant proteins (see, for example, Patent Documents 1 and 2). In addition, Chinese hamster lung-derived CHL-YN cells have recently been established, which have the excellent characteristics of CHO cells but grow at approximately twice the rate of CHO cells (see Non-Patent Document 1).

[0004] International Publication No. 2020 / 158877 Japanese Patent Application Laid-Open No. 2021-101733

[0005] Yamano-Adachi, N. et al., Sci. Rep., 10, 17612 (2020)

[0006] When producing a recombinant protein, not only the productivity of the recombinant protein but also the quality of the resulting recombinant protein is important.

[0007] In conventional production methods, it is known that as the culture period of a transformant increases, the structure of the produced recombinant protein changes (in other words, the purity of the produced recombinant protein decreases). In this regard, it is thought that the purity of the produced recombinant protein can be maintained by shortening the culture period, but a decrease in productivity is unavoidable.

[0008] Furthermore, it is generally known that when the number of cells increases during culture and the cells become overcrowded, the properties of the cells change, which also causes a decrease in the purity of the recombinant protein produced.

[0009] Therefore, an objective of the present invention is to provide a technique that enables efficient production of highly pure recombinant proteins.

[0010] Specific means for solving the above problems include the following embodiments: <1> A transformant used for producing a target protein in vitro, wherein a gene encoding the target protein and a suicide gene are introduced into a host cell.

[0011] <2> The transformant according to <1>, wherein the target protein is an active ingredient of a biopharmaceutical.

[0012] <3> The transformant according to <1> or <2>, wherein the suicide gene is a herpes simplex virus thymidine kinase gene, a cytosine deaminase gene, a uracil phosphoribosyltransferase gene, a guanine phosphoribosyltransferase gene, a nitroreductase gene, or an inducible caspase 9 gene.

[0013] <4> The transformant according to any one of <1> to <3>, wherein the host cell is a mammalian cell, an insect cell, a plant cell, a yeast cell, or an Escherichia coli cell.

[0014] <5> The transformant according to any one of <1> to <4>, wherein the host cell is a cell capable of being cultured in suspension.

[0015] <6> The transformant according to any one of <1> to <5>, wherein the host cell is a Chinese hamster ovary-derived CHO cell, a Chinese hamster lung-derived CHL-YN cell, a Syrian hamster kidney-derived BHK cell, a human embryonic kidney cell line HEK293 cell, a mouse myeloma cell line NS0 cell, or a mouse myeloma cell line SP2 / 0 cell.

[0016] <7> The transformant according to any one of <1> to <6>, wherein the target protein is an antibody and the suicide gene is a herpes simplex virus thymidine kinase gene.

[0017] <8> A method for producing a target protein in vitro, comprising: a culturing step of culturing the transformant according to any one of <1> to <7> in the presence of a cell death inducer that cooperates with a protein encoded by a suicide gene carried by the transformant to induce cell death in the transformant, thereby producing the target protein; and a recovery step of recovering the target protein from the culture resulting from the culturing step.

[0018] <9> The method for producing a target protein according to <8>, wherein the culturing step is performed so as to maintain the logarithmic growth phase.

[0019] <10> The method for producing a target protein according to <8> or <9>, wherein the combination of the suicide gene and the cell death inducer is any one of the following (a) to (f): (a) the suicide gene is a herpes simplex virus thymidine kinase gene, and the cell death inducer is ganciclovir, acyclovir, penciclovir, adefovir, or tenofovir; (b) the suicide gene is a cytosine deaminase gene, and the cell death inducer is 5-fluorocytosine; (c) the suicide gene is a uracil phosphoribosyltransferase gene, and the cell death inducer is 5-fluorouracil; (d) the suicide gene is a guanine phosphoribosyltransferase gene, and the cell death inducer is 6-thioxanthine or 6-thioguanine; or (e) the suicide gene is a nitroreductase gene, and the cell death inducer is CB1954. (f) the suicide gene is an inducible caspase-9 gene, and the cell death inducer is AP1903 or AP20187.

[0020] According to the present invention, a technique can be provided that enables efficient production of highly pure recombinant proteins.

[0021] 1 is a graph showing the daily change in viable cell count when CHL-YN-IgG cells are cultured. FIG. 2 is a graph showing the daily change in viable cell count when CHL-YN-IgG-HSVtk cells are cultured using media containing various concentrations of ganciclovir. FIG. 3 is a graph showing the daily change in viable cell count when CHL-YN-IgG cells are cultured using media containing 5 μg / mL of ganciclovir. FIG. 4 is a graph showing the daily change in viable cell count when CHL-YN-IgG-HSVtk cells are cultured using media containing various concentrations of ganciclovir. FIG. 5 is a graph showing the daily change in viable cell count when CHL-YN-IgG-HSVtk cells are cultured using media containing various concentrations of ganciclovir. FIG. 6 is a graph showing the daily change in viable cell count when CHL-YN-IgG-HSVtk cells are cultured using media containing various concentrations of ganciclovir. FIG. 7 is a graph showing the daily change in viable cell count when CHL-YN-IgG-HSVtk cells are cultured using media containing 0.625 μg / mL of ganciclovir. 1 is a diagram showing the amount of protein in the culture supernatant when CHL-YN-IgG-HSVtk cells are cultured using a medium containing 0.625 μg / mL of ganciclovir, and when CHL-YN-IgG-HSVtk cells or CHL-YN-IgG cells are cultured using a medium containing no ganciclovir. 2 is a diagram showing the amount of IgG in the culture supernatant when CHL-YN-IgG-HSVtk cells are cultured using a medium containing 0.625 μg / mL of ganciclovir, and when CHL-YN-IgG-HSVtk cells or CHL-YN-IgG cells are cultured using a medium containing no ganciclovir. 3 is a diagram showing microscopic images of C3H10T1 / 2 / HSVtk / IFN-γ cells on days 3 and 7 of culture when cells are cultured using media containing various concentrations of ganciclovir.

[0023] Figure 1 shows the amount of IFN-γ in the culture supernatant when C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured for 7 days using a medium containing 0.125 μg / mL of ganciclovir. Figure 2 shows the amount of IFN-γ in the culture supernatant when C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured for a total of 7 days using a medium containing 0.125 μg / mL of ganciclovir, with passage every 3 days. Figure 3 shows the amount of IFN-γ in the culture supernatant when C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured for 7 days using a medium not containing ganciclovir.

[0022] Hereinafter, specific embodiments of the present invention will be described. In this specification, a numerical range indicated using "to" indicates a range that includes the numerical values ​​written before and after "to."

[0023] <Method for Producing a Target Protein> The method for producing a target protein according to this embodiment (hereinafter also referred to as "the production method according to this embodiment") is an in vitro method for producing a target protein, and includes a culturing step of culturing a transformant, in which a gene encoding the target protein and a suicide gene have been introduced into a host cell, in the presence of a cell death inducer that cooperates with the protein encoded by the suicide gene carried by the transformant to induce cell death of the transformant, thereby producing the target protein, and a recovery step of recovering the target protein from the culture after the culturing step.

[0024] As described above, when producing a recombinant protein, it is known that the structure and other properties of the produced recombinant protein change as the culture period of a transformant increases (in other words, the purity of the produced recombinant protein decreases). In addition, it is generally known that when the number of cells increases during culture and the cells become overcrowded, the properties of the cells change, which also causes a decrease in the purity of the produced recombinant protein.

[0025] In this regard, in the production method according to the present embodiment, a transformant into which a suicide gene has been introduced along with a gene encoding a target protein is cultured in the presence of a specific cell death inducer, and while the transformant grows, some of it dies during the culture process. As a result, viable cells that have been cultured for a longer period are gradually replaced by cells that have been cultured for a shorter period. Furthermore, in the production method according to the present embodiment, since the transformant grows while some of it dies during the culture process, the cells are less likely to become overcrowded. As a result, the production method according to the present embodiment makes it possible to produce a highly pure target protein over a long period of time.

[0026] Each element of the production method according to this embodiment will be described in detail below.

[0027] [Transformant] The transformant used in the production method according to this embodiment (hereinafter also referred to as "the transformant according to this embodiment") is a host cell into which a gene encoding a target protein and a suicide gene have been introduced. The transformant according to this embodiment expresses the target protein and the protein encoded by the suicide gene.

[0028] Examples of target proteins include recombinant proteins used for medical, diagnostic, industrial, etc., and particularly preferred are recombinant proteins used as active ingredients in biopharmaceuticals. Target proteins are, for example, secretory proteins that are secreted outside of cells. Specific examples of recombinant proteins used as active ingredients in biopharmaceuticals include antibodies (Fab, Fab', F(ab') ２ erythropoietin, polyethylene glycol (PEG)-modified erythropoietin, and other erythropoietic proteins; cytokines such as granulocyte colony-stimulating factor (G-CSF) and basic fibroblast growth factor (bFGF); fusion proteins such as tumor necrosis factor (TNF) receptor-Fc fusion protein and vascular endothelial growth factor (VEGF) receptor-Fc fusion protein; and the like.

[0029] The suicide gene is not particularly limited as long as it can induce cell death in the transformant in cooperation with the cell death inducer described below. Specific examples of suicide genes include the herpes simplex virus thymidine kinase (HSVtk) gene, cytosine deaminase gene, uracil phosphoribosyltransferase gene, guanine phosphoribosyltransferase gene, nitroreductase gene, and inducible caspase 9 (iCasp9) gene. The inducible caspase 9 gene is a combination of a sequence encoding the catalytic domain of caspase 9 and a sequence encoding an FK506-binding protein having the F36V mutation.

[0030] The suicide gene may be a modified suicide gene in which one or more bases have been substituted, deleted, or added in the base sequence of the wild-type suicide gene, as long as it can induce cell death in the transformant in cooperation with the cell death-inducing agent described below. The sequence identity between the base sequence of the wild-type suicide gene and the base sequence of the modified suicide gene is usually 90% or more, or alternatively 92% or more, 94% or more, 96% or more, or even 98% or more. Note that "sequence identity" refers to the match between the two base sequences when they are aligned, and can be calculated, for example, using the BLAST program (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0031] The transformant according to this embodiment can be obtained by introducing a gene encoding a target protein and a suicide gene into a host cell via a vector.

[0032] Examples of host cells include mammalian cells, insect cells, plant cells, yeast, Escherichia coli, etc. The host cells may be suspension cells or adherent cells, but from the viewpoint of production efficiency of the target protein, cells that can be cultured in suspension are preferred.

[0033] Preferred mammalian cells that can be cultured in suspension include Chinese hamster ovary-derived CHO cells, Chinese hamster lung-derived CHL-YN cells, Syrian hamster kidney-derived BHK cells, human embryonic kidney cell line HEK293 cells, mouse myeloma cell line NS0 cells, and mouse myeloma cell line SP2 / 0 cells, with CHO cells and CHL-YN cells being more preferred. Each of these cells encompasses subspecies. For example, CHO cells encompass subspecies such as CHO-K1 cells, CHO-S cells, CHO-MK cells, CHO-DG44 cells, and CHO-DXB11 cells.

[0034] Preferred mammalian cells for adhesion culture include mouse mesenchymal stem cell line C3H10T1 / 2 cells, human mesenchymal stem cell line UE7T-13 cells, mouse fibroblast cell line NIH3T3 cells, mouse insulinoma cell line MIN6 cells, mouse hepatoma-derived cell line HEPA1-6 cells, human hepatoma-derived cell line HepG2 cells, human lung cancer-derived cell line A549 cells, human epidermal keratinocyte cell line HaCaT cells, etc. Each of these cells includes subspecies.

[0035] Vectors include plasmid vectors, virus vectors, cosmid vectors, fosmid vectors, and artificial chromosome vectors, and can be selected depending on the type of host cell.

[0036] The method for introducing a vector into a host cell is not particularly limited, and lipofection, calcium phosphate method, microinjection method, etc. can be appropriately used.

[0037] [Culturing step] In the culturing step, the transformant according to the present embodiment is cultured in the presence of a specific cell death inducer to produce the target protein. In the culturing step, suspension culture or adhesion culture is performed depending on the properties of the transformant.

[0038] (Cell Death Inducer) The cell death inducer is not particularly limited as long as it can cooperate with the protein encoded by the suicide gene contained in the transformant of this embodiment to induce cell death of the transformant.

[0039] When the suicide gene is the herpes simplex virus thymidine kinase gene, cytosine deaminase gene, uracil phosphoribosyltransferase gene, guanine phosphoribosyltransferase gene, or nitroreductase gene, the cell death inducer can be a prodrug that is converted into a cytotoxic drug by the enzyme encoded by these suicide genes. When the suicide gene is the inducible caspase-9 gene, the cell death inducer can be a chemical inducer that causes dimerization of the catalytic domain of caspase-9.

[0040] Preferred combinations of suicide genes and cell death inducers include, for example, the following combinations (a) to (f): (a) the suicide gene is the herpes simplex virus thymidine kinase gene, and the cell death inducer is ganciclovir, acyclovir, penciclovir, adefovir, or tenofovir; (b) the suicide gene is the cytosine deaminase gene, and the cell death inducer is 5-fluorocytosine; (c) the suicide gene is the uracil phosphoribosyltransferase gene, and the cell death inducer is 5-fluorouracil; (d) the suicide gene is the guanine phosphoribosyltransferase gene, and the cell death inducer is 6-thioxanthine or 6-thioguanine; (e) the suicide gene is the nitroreductase gene, and the cell death inducer is CB1954; (f) the suicide gene is the inducible caspase 9 gene, and the cell death inducer is AP1903 or AP20187.

[0041] The mechanism by which cell death in a transformant is induced by a suicide gene and a cell death inducer will be explained using an example in which the suicide gene is the herpes simplex virus thymidine kinase (HSVtk) gene and the cell death inducer is ganciclovir. When a transformant expressing the HSVtk gene is cultured in the presence of ganciclovir, the ganciclovir taken up into the cell is phosphorylated by the action of HSVtk to become ganciclovir 5'-triphosphate. This ganciclovir 5'-triphosphate inhibits the DNA polymerase of the transformant, thereby inducing cell death in the transformant.

[0042] (Suspension culture) When suspension culture is performed in the culture step, a liquid medium containing a cell death inducer is used in the culture step. As the medium composition other than the cell death inducer, a known medium composition depending on the type of host cell used to obtain the transformant can be used.

[0043] In the culture step, it is preferable to culture the cells at a viable cell density that maintains the logarithmic growth phase before reaching confluence. The viable cell density can be controlled by the concentration of the cell death inducer in the liquid medium. The concentration of the cell death inducer in the liquid medium may be constant throughout the culture step or may be increased or decreased during the culture step.

[0044] Specifically, when the viable cell density at the time of maximum viable cell density when cultured in the same liquid medium except for not containing the cell death inducer is taken as 100%, the viable cell density during the culture step excluding the initial stage of culture (days 1 to 5 of culture) is preferably maintained at 5 to 50%, more preferably at 10 to 25%. For example, the viable cell density during the culture step excluding the initial stage of culture is preferably maintained at 5 x 10 ７ ~5 x 10 ８ It is preferable to maintain the concentration at 1 x 10 cells / mL. ８ ~2.5 x 10 ８ It is more preferable to maintain the concentration at 1000kJ / mL. The day on which the transformant according to this embodiment is seeded in a liquid medium containing a cell death inducer and culture is started is defined as the first day of culture.

[0045] In order to stably produce the target protein, it is preferable to minimize the rate of change in viable cell density during the culture process, except for the initial stage of culture. １ The viable cell density at the time point is D １ Let t ２ (=t １ +24 hours) was used as the viable cell density. ２ Then, {(D ２ -D １ ) / D １ The absolute value of the rate of change (%) defined by {circle around (3)}×100 is preferably 25% or less, and more preferably 10% or less.

[0046] The concentration of the cell death inducer in the liquid medium is set appropriately depending on the type of host cells used to obtain the transformant, the type of cell death inducer, etc. For example, when the host cells are CHL-YN cells, the suicide gene is the HSVtk gene, and the cell death inducer is ganciclovir, the concentration of ganciclovir in the liquid medium is preferably 0.32 to 1.25 μg / mL, and more preferably 0.5 to 0.9 μg / mL.

[0047] The culture in the culture step can be carried out aerobically, for example, by aeration culture or shaking culture. The culture may be any of batch culture, fed-batch culture (semi-batch culture), and continuous culture (perfusion culture). The culture temperature can be any known condition depending on the type of transformant.

[0048] The culture period in the culture step is not particularly limited and may be 1 to 60 days, or 1 to 14 days. During the culture step, dead cells may be removed, and the entire medium may be replaced.

[0049] (Adhesion culture) When adhesion culture is performed in the culture step, a liquid medium containing a cell death inducer is used in the culture step. As the medium composition other than the cell death inducer, a known medium composition depending on the type of host cell used to obtain the transformant can be used.

[0050] In the culturing step, the cells are preferably cultured at a cell density that maintains the logarithmic growth phase before reaching confluence. The cell density can be controlled by the concentration of the cell death inducer in the liquid medium. The concentration of the cell death inducer in the liquid medium may be constant throughout the culturing step or may be increased or decreased during the culturing step.

[0051] Specifically, when a state in which the entire culture surface of a culture vessel is covered with cells is defined as 100% confluence, the cell density during the culture process, excluding the initial stage of culture (days 1 to 4 of culture), is preferably maintained at 60 to 90% confluence, and more preferably at 70 to 80% confluence. Note that the day on which the transformant according to this embodiment is seeded and culture is initiated in a liquid medium containing a cell death inducer is defined as day 1 of culture.

[0052] The concentration of the cell death inducer in the liquid medium is set appropriately depending on the type of host cells used to obtain the transformant, the type of cell death inducer, etc. For example, when the host cells are C3H10T1 / 2 cells, the suicide gene is the HSVtk gene, and the cell death inducer is ganciclovir, the concentration of ganciclovir in the liquid medium is preferably 0.05 to 0.24 μg / mL, and more preferably 0.1 to 0.15 μg / mL.

[0053] The culture period in the culture step is not particularly limited, and may be 1 to 60 days, or 1 to 7 days. During the culture step, dead cells may be removed, and the entire medium may be replaced.

[0054] [Pre-culture step] The production method according to this embodiment may include a pre-culture step for increasing the cell number of the transformant according to this embodiment before the culture step. In the pre-culture step, a liquid medium containing no cell death inducer can be used.

[0055] [Recovery Step] In the recovery step, the target protein is recovered from the culture after the culturing step. Examples of the culture include culture supernatant, cultured cells, and disrupted cells thereof.

[0056] The method for recovering the target protein from the culture is not particularly limited, and any known method can be used depending on the type of target protein. For example, when the target protein is a secretory protein, the target protein can be recovered by removing cells contained in the culture supernatant by centrifugation or filtration, followed by a combination of dialysis, chromatography, and other means.

[0057] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0058] <Reference Example 1: Growth of CHL-YN-IgG cells> CHL-YN-IgG cells are cells obtained by transforming CHL-YN cells by introducing a gene encoding a human IgG1 antibody (see Non-Patent Document 1), and were provided by the Next Generation Biopharmaceutical Manufacturing Technology Research Association.

[0059] CHL-YN-IgG cells were suspended in 20 mL of CHL culture medium (EX-CELL CD CHO Fusion medium (Merck) containing 0.05% puromycin (InvivoGen) and 3% L-glutamine solution (Fujifilm Wako Pure Chemical Industries, Ltd.)) at a concentration of 5 × 10 ４ The cells were adjusted to 0.15 cells / mL and seeded into 125 mL flasks (Corning) (n=3). Culture was continued for 10 days with daily medium changes, and the viable cell count was measured over time. A shaker MMS-320 (Tokyo Rikakikai Co., Ltd.) was used for the culture. The viable cell count was measured after staining the cells with 0.4 w / v% trypan blue solution (Fujifilm Wako Pure Chemical Industries, Ltd.) using a Countess II FL Automated Cell Counter (Invitrogen) and Countess Cell Counting Chamber Slides (Invitrogen).

[0060] The daily change in viable cell count is shown in Figure 1. As shown in Figure 1, the viable cell count peaked on day 6 of culture and then gradually decreased. This result confirmed that CHL-YN-IgG cells exhibit a confluency-like growth profile in long-term culture.

[0061] Test Example 1: Preparation of CHL-YN-IgG-HSVtk cells Preparation of a plasmid vector encoding the HSVtk gene A cDNA fragment of the HSVtk gene was amplified by PCR using the following primers: Forward primer: 5'-CGCGTCGACATGGCTTCTTACCCT-3' (SEQ ID NO: 1) Reverse primer: 5'-CCGGATATCTTAGTTGGCCTCTCCCAT-3' (SEQ ID NO: 2)

[0062] The resulting PCR product and pEBMulti-Hyg vector (Fujifilm Wako Pure Chemical Industries, Ltd.) were treated overnight at 37°C with the restriction enzymes SalI and EcoRV, and the reaction products were separated by agarose gel electrophoresis to obtain specific sequences. Subsequently, ligation and cloning using E. coli were performed according to a previous report (J. Control. Release 275, 78-84 (2018)), yielding the pEBMulti-HSVtk vector encoding the HSVtk gene.

[0063] [Preparation of CHL-YN-IgG-HSVtk cells] CHL-YN-IgG cells were suspended in 1.8 mL of CHL culture medium to give a total of 1 x 10 ６ The culture medium was adjusted to 100 μg / mL and seeded into a 6-well plate (WATSON) for suspension cells. Separately, 1.6 μg (DNA amount) of pEBMulti-HSVtk vector was added to 200 μL of CHL culture medium, and 3.2 μL of FectoPRO (Polyplus) was added, mixed, and incubated at room temperature for 10 minutes. The incubated solution was added to CHL-YN-IgG cells cultured in a 6-well plate for transfection. The next day, the cells were collected as a pellet, suspended in 2 mL of CHL culture medium containing 250 μg / mL Hygromycin B Gold (InvivoGen), and cultured for 7 days. The medium was changed daily during culture. After 7 days, the medium was replaced with regular CHL culture medium and expanded to obtain CHL-YN-IgG-HSVtk cells.

[0064] Test Example 2: Ganciclovir sensitivity of CHL-YN-IgG-HSVtk cells CHL-YN-IgG-HSVtk cells or CHL-YN-IgG cells were suspended in 2 mL of CHL culture medium and diluted to 1 x 10 ４ The cells were adjusted to 0.5 μg / mL and seeded onto a 6-well plate (WATSON) for suspension cells (n=3). Ganciclovir (Tokyo Chemical Industry Co., Ltd.) was added to each cell culture medium to achieve various final concentrations (0, 2.5, 5 μg / mL), and the cells were cultured for 4 days with daily medium changes. The number of viable cells was then counted daily as in Reference Example 1.

[0065] The daily change in the number of viable CHL-YN-IgG-HSVtk cells is shown in Figure 2A, and the daily change in the number of viable CHL-YN-IgG cells is shown in Figure 2B. The results in Figures 2A and 2B confirmed that CHL-YN-IgG-HSVtk cells died in response to ganciclovir.

[0066] Test Example 3: Growth control of CHL-YN-IgG-HSVtk cells (1) CHL-YN-IgG-HSVtk cells were suspended in 2 mL of CHL culture medium and incubated at 5 × 10 ４ The cells were adjusted to 100 μg / mL and seeded onto a 6-well plate (WATSON) for suspension cells (n=3). Ganciclovir (Tokyo Chemical Industry Co., Ltd.) was added to the cell culture medium at various concentrations (0, 0.03125, 0.625, 1.25, 2.5, and 5 μg / mL), and the cells were cultured for 6 days with daily medium changes. The number of viable cells was then counted daily as in Reference Example 1.

[0067] The change in viable cell count over time is shown in Figure 3A, and the viable cell count after 72 hours of culture is shown in Figure 3B. As shown in Figures 3A and 3B, CHL-YN-IgG-HSVtk cells died in a ganciclovir concentration-dependent manner, and particularly when the ganciclovir concentration was 1.25 μg / mL or higher, most of the cells died after 72 hours of culture.

[0068] Test Example 4: Growth control of CHL-YN-IgG-HSVtk cells (2) CHL-YN-IgG-HSVtk cells were suspended in 2 mL of CHL culture medium and incubated at 5 × 10 ４ The cells were adjusted to a final concentration of 0.625 μg / mL and seeded onto a 6-well plate (WATSON) for suspension cells (n=3). Ganciclovir (Tokyo Chemical Industry Co., Ltd.) was added to the cell culture medium to a final concentration of 0.625 μg / mL, and the cells were cultured for 14 days with daily medium changes. The number of viable cells was then counted daily as in Reference Example 1.

[0069] The change in viable cell count over time is shown in Figure 4. As shown in Figure 4, when CHL-YN-IgG-HSVtk cells were cultured in a medium containing 0.625 μg / mL of ganciclovir, the viable cell count remained approximately constant. This is thought to be due to a balance between the cell proliferation rate and the cell death (apoptosis) rate.

[0070] Test Example 5: Protein amount in culture supernatant of CHL-YN-IgG-HSVtk cells CHL-YN-IgG-HSVtk cells were suspended in 2 mL of CHL culture medium and diluted to 5 × 10 ４ The cells were adjusted to a final concentration of 0.625 μg / mL and seeded onto a 6-well plate (WATSON) for suspension cells (n=3). Ganciclovir (Tokyo Chemical Industry Co., Ltd.) was added to the cell culture medium to a final concentration of 0.625 μg / mL, and the cells were cultured for 14 days with daily medium changes. The viable cell counts on days 7 and 14 of culture were 2-3 × 10 ６ For comparison, CHL-YN-IgG-HSVtk cells or CHL-YN-IgG cells were cultured for 14 days in the same manner as above, except that a medium containing no ganciclovir was used. The viable cell counts on days 7 and 14 of culture were 3-4 × 10 ６ cells / mL.

[0071] The cells were collected on days 1, 7, and 14 of culture and suspended in 2 mL of Opti-MEM I Reduced Serum Medium (Gibco) at a concentration of 1 × 10 ６ The cell concentration was adjusted to 1000 cells / mL and seeded into a 6-well suspension cell plate (WATSON) for 24 hours. The cell suspension was then centrifuged at 17,000 × g for 2 minutes to collect the culture supernatant. The protein content in the culture supernatant was then measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). Measurements were performed by measuring absorbance at 555 nm using an ARVO X4 Multimode Plate Reader (Perkin Elmer).

[0072] The amount of protein in the culture supernatant is shown in Figure 5. As shown in Figure 5, when CHL-YN-IgG-HSVtk cells were cultured in a medium containing 0.625 μg / mL of ganciclovir, the amount of protein produced remained almost unchanged even after 14 days of culture. On the other hand, when CHL-YN-IgG-HSVtk cells or CHL-YN-IgG cells were cultured in a medium not containing ganciclovir, the amount of protein produced significantly decreased after 14 days of culture.

[0073] Test Example 6: Amount of IgG in the culture supernatant of CHL-YN-IgG-HSVtk cells CHL-YN-IgG-HSVtk cells were suspended in 2 mL of CHL culture medium and diluted to 5 × 10 ４ The cells were adjusted to a final concentration of 0.625 μg / mL and seeded onto a 6-well plate (WATSON) for suspension cells (n=3). Ganciclovir (Tokyo Chemical Industry Co., Ltd.) was added to the cell culture medium to a final concentration of 0.625 μg / mL, and the cells were cultured for 14 days with daily medium changes. The viable cell counts on days 7 and 14 of culture were 2-3 × 10 ６ For comparison, CHL-YN-IgG-HSVtk cells or CHL-YN-IgG cells were cultured for 14 days in the same manner as above, except that a medium containing no ganciclovir was used. The viable cell counts on days 7 and 14 of culture were 3-4 × 10 ６ cells / mL.

[0074] The cells were collected on days 1, 7, and 14 of culture and suspended in 2 mL of Opti-MEM I Reduced Serum Medium (Gibco) at a concentration of 1 × 10 ６ The cells were adjusted to 0.1 cells / mL and seeded into a 6-well suspension cell plate (WATSON) and cultured for 24 hours. The cell suspension was then centrifuged at 17,000 × g for 2 minutes, and the culture supernatant was collected. The amount of IgG in the culture supernatant was then measured using an IgG, Human, ELISA Quantification Kit, FastELISA (RD-Biotech). Measurements were performed by measuring absorbance at 450 nm using an ARVO X4 Multimode Plate Reader (Perkin Elmer).

[0075] The amount of IgG in the culture supernatant is shown in Figure 6. As shown in Figure 6, when CHL-YN-IgG-HSVtk cells were cultured using a medium containing 0.625 μg / mL of ganciclovir, the amount of IgG produced remained almost unchanged even after 14 days of culture. On the other hand, when CHL-YN-IgG-HSVtk cells or CHL-YN-IgG cells were cultured using a medium not containing ganciclovir, the amount of IgG produced significantly decreased after 14 days of culture.

[0076] When CHL-YN-IgG-HSVtk cells or CHL-YN-IgG cells were cultured in a medium containing no ganciclovir, the change in the amount of IgG shown in Figure 6 was greater than the change in the amount of protein shown in Figure 5. This is presumably because the produced proteins contained mutant IgGs with altered structures, etc., and these mutant IgGs were not detected by ELISA.

[0077] Test Example 7: Ganciclovir sensitivity of C3H10T1 / 2 / HSVtk / IFN-γ cells In Test Example 7, C3H10T1 / 2 / HSVtk / IFN-γ cells were used, which were C3H10T1 / 2 cells transfected with the HSVtk gene and the IFN-γ gene.

[0078] 1 × 10 C3H10T1 / 2 / HSVtk / IFN-γ cells ５ The cells were adjusted to 0.125, 0.25, or 0.5 μg / mL and seeded onto 6-well plates. Ganciclovir (Tokyo Chemical Industry Co., Ltd.) was added to each cell culture medium to achieve various final concentrations (0, 0.125, 0.25, or 0.5 μg / mL), and the cells were cultured for 7 days. During culture, the medium was replaced daily with 15% FBS-containing DMEM medium containing various concentrations of ganciclovir (0, 0.125, 0.25, or 0.5 μg / mL). The cells were then observed using an APEXVIEW APX100 (Evident Co., Ltd.).

[0079] Microscopic images of the cells on days 3 and 7 of culture are shown in Figure 7. The results in Figure 7 confirmed that C3H10T1 / 2 / HSVtk / IFN-γ cells died in response to ganciclovir.

[0080] Test Example 8: Amount of IFN-γ in the culture supernatant of C3H10T1 / 2 / HSVtk / IFN-γ cells C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured at 1×10 ５ The cells were adjusted to a final concentration of 1 × 10 cells / mL and seeded onto a 6-well plate (n = 3). Ganciclovir (Tokyo Chemical Industry Co., Ltd.) was added to each cell culture solution to a final concentration of 0.125 μg / mL, and the cells were cultured for 7 days. During the culture, the medium was changed daily with 15% FBS-containing DMEM medium containing 0.125 μg / mL of ganciclovir. For comparison, 1 × 10 cells were cultured every 3 days. ５ A group of cells was cultured for a total of 7 days in the same manner as above except that they were passaged at a cell concentration of 1000 cells / mL, and a group of cells was cultured for 7 days in the same manner as above except that a medium containing no ganciclovir was used.

[0081] The cells were collected on the first and seventh days of culture and suspended in 2 mL of Opti-MEM I Reduced Serum Medium (Gibco) at a concentration of 1 × 10 ６ The cells were adjusted to 100 cells / mL, seeded onto a 6-well plate, and cultured for 24 hours. The culture supernatant was then collected, and the amount of IFN-γ in the culture supernatant was measured using ELISA MAX Deluxe Set Mouse IFN-γ (BioLegend). Measurements were performed by measuring absorbance at a wavelength of 450 nm using a GloMax Discover Microplate Reader (Promega).

[0082] Figure 8A shows the amount of IFN-γ in the culture supernatant when C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured for 7 days using a medium containing 0.125 μg / mL of ganciclovir. Figure 8B shows the amount of IFN-γ in the culture supernatant when C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured for a total of 7 days using a medium containing 0.125 μg / mL of ganciclovir, with passage every 3 days. Figure 8C shows the amount of IFN-γ in the culture supernatant when C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured for 7 days using a medium not containing ganciclovir. As shown in Figures 8A and 8B, when C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured in medium containing 0.125 μg / mL of ganciclovir, the amount of IFN-γ produced remained almost unchanged even after 7 days of culture, and was comparable to that produced when the cells were passaged every 3 days (ns: not significant). On the other hand, as shown in Figure 8C, when C3H10T1 / 2 / HSVtk / IFN-γ cells were cultured in medium without ganciclovir, the amount of IFN-γ produced decreased after 7 days of culture (*p<0.05 vs. untreated group).

Claims

1. A transformant used for producing a target protein in vitro, in which a gene encoding the target protein and a suicide gene are introduced into a host cell.

2. The transformant according to claim 1, wherein the target protein is an active ingredient of a biopharmaceutical.

3. The transformant according to claim 1, wherein the suicide gene is a herpes simplex virus thymidine kinase gene, a cytosine deaminase gene, a uracil phosphoribosyltransferase gene, a guanine phosphoribosyltransferase gene, a nitroreductase gene, or an inducible caspase 9 gene.

4. The transformant according to claim 1, wherein the host cell is a mammalian cell, an insect cell, a plant cell, a yeast cell, or an E. coli cell.

5. The transformant according to claim 1, wherein the host cell is a cell capable of being cultured in suspension.

6. The transformant according to claim 1, wherein the host cell is a Chinese hamster ovary-derived CHO cell, a Chinese hamster lung-derived CHL-YN cell, a Syrian hamster kidney-derived BHK cell, a human embryonic kidney cell line HEK293 cell, a mouse myeloma cell line NS0 cell, or a mouse myeloma cell line SP2 / 0 cell.

7. The transformant according to claim 1, wherein the target protein is an antibody and the suicide gene is a herpes simplex virus thymidine kinase gene.

8. A method for producing a target protein in vitro, comprising: a culturing step of culturing the transformant according to any one of claims 1 to 7 in the presence of a cell death inducer that cooperates with a protein encoded by a suicide gene possessed by the transformant to induce cell death of the transformant, thereby producing the target protein; and a recovery step of recovering the target protein from the culture after the culturing step.

9. The method for producing a target protein according to claim 8, wherein the culturing step is carried out so as to maintain the logarithmic growth phase.

10. The method for producing a target protein according to claim 8, wherein the combination of the suicide gene and the cell death inducer is any one of the following (a) to (f): (a) the suicide gene is a herpes simplex virus thymidine kinase gene and the cell death inducer is ganciclovir, acyclovir, penciclovir, adefovir, or tenofovir; (b) the suicide gene is a cytosine deaminase gene and the cell death inducer is 5-fluorocytosine; (c) the suicide gene is a uracil phosphoribosyltransferase gene and the cell death inducer is 5-fluorouracil; (d) the suicide gene is a guanine phosphoribosyltransferase gene and the cell death inducer is 6-thioxanthine or 6-thioguanine; (e) the suicide gene is a nitroreductase gene and the cell death inducer is CB1954; (f) the suicide gene is an inducible caspase 9 gene and the cell death inducer is AP1903 or AP20187.

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