A composition, method and use for increasing the titre of mouse leukemia virus

Abstract

The application provides a composition, a method and an application for improving the titer of mouse leukemia virus, and belongs to the technical field of biotechnology.The method comprises the following steps: before a host cell is infected with xenotropic mouse leukemia virus, the host cell is pretreated by using SC79; during virus infection of the pretreated host cell and during cell passage, mifepristone is added to a culture medium; and during continuous virus harvesting, LR3 IGF-1 is added to the culture medium.Through adding additives with different functional mechanisms at different stages of virus production, the application realizes dynamic optimization of the whole process of virus production.The experimental results show that the combination of SC79, mifepristone and LR3 IGF-1 can increase the titer of xenotropic mouse leukemia virus by 14.32 times, which is significantly better than the effect of single additive or two additives in combination.The application solves the problems of low titer of virus stock solution and low production efficiency in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to virus culture and preparation technology, and more specifically to a composition, method, and application for increasing the titer of mouse leukemia virus. Background Technology

[0002] Xenotropic murine leukemia virus (X-MuLv) is an important indicator virus widely used in the virus clearance and inactivation validation processes of biotechnological therapeutic products (such as monoclonal antibodies, recombinant proteins, vaccines, and gene therapy viral vectors) produced from Chinese hamster ovary cells (CHO). To ensure the safety of viruses derived from human or animal cell lines in biotechnological products, relevant national and international regulations explicitly require virus clearance or inactivation validation for these products. In these process validation experiments, the indicator virus is typically added artificially to assess the effectiveness of the process. This involves incorporating the indicator virus into the sample during the virus clearance and inactivation steps for inactivation, followed by detection of changes in infectious virus levels using relevant indicator cells. To avoid dilution effects or alterations to product properties caused by virus addition, regulations require the indicator virus titer to be as high as possible. However, the low titer of X-MuLv stock solution remains a critical technical challenge in this field. In the process of virus production through cell culture, increasing the titer of the virus stock solution can reduce the pressure on subsequent virus concentration and purification, reduce resource waste, and improve the economic efficiency of enterprise production.

[0003] Currently, strategies to increase viral titers can be approached from multiple angles, including improving the efficiency of viral infection of host cells, increasing the density of live host cells, enhancing host cell metabolism or altering the physiological state of host cells, and promoting viral synthesis and release. Existing research indicates that adding the glucocorticoid receptor antagonist mifepristone during retroviral infection of target cells can increase the sensitivity of target cells to retroviral infection, which works by promoting viral integration into the host genome. However, most existing technologies focus on the application of a single additive at a specific stage of viral production, lacking a systematic, multi-stage, dynamic optimization solution for the entire viral production process. For example, Chinese patent CN119709641A discloses the application of mifepristone in improving the titer and stability of heterophilic mouse leukemia virus, but only adds mifepristone during the viral infection and passage amplification stages. Therefore, there is still a need in the field to develop a multi-stage, dynamically optimized production process that can significantly improve X-MuLv viral titers to address the technical problems of low viral stock titers and low production efficiency in existing technologies. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for increasing the titer of mouse leukemia virus, which significantly increases the titer of heterophilic mouse leukemia virus and solves the technical problems of low titer of virus stock solution and low production efficiency in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for increasing the titer of mouse leukemia virus, comprising the following steps: Before infecting host cells with mouse leukemia virus, host cells were pretreated with SC79. Mifepristone was added to the culture medium during infection of pretreated host cells with mouse leukemia virus and during cell passage. During continuous virus harvesting, LR3 IGF-1 was added to the culture medium.

[0006] Preferably, the concentration of SC79 is 5-20 μM; the concentration of mifepristone is 0.1-2 μM; and the concentration of LR3 IGF-1 is 5-100 ng / mL.

[0007] Preferably, the pretreatment time is 12 to 48 hours.

[0008] Preferably, the cells are passaged and expanded at least three times.

[0009] Preferably, the culture medium used during the continuous harvesting of the virus is a culture medium containing a low concentration of fetal bovine serum, wherein the volume fraction of the low concentration of fetal bovine serum is 0.5% to 8%.

[0010] Preferably, the host cell is a mouse skin fibroblast.

[0011] Preferably, the mouse leukemia virus is a heterophilic mouse leukemia virus.

[0012] The present invention also provides a composition for increasing the titer of mouse leukemia virus, the composition comprising a signaling pathway activator, a glucocorticoid receptor antagonist, and an insulin-like growth factor-1 analog.

[0013] Preferably, the signaling pathway activator is SC79; the glucocorticoid receptor antagonist is mifepristone; and the insulin-like growth factor-1 analog is LR3 IGF-1.

[0014] This invention provides a method or composition thereof for the preparation of retroviruses and / or for increasing retrovirus titers.

[0015] Compared with the prior art, the present invention has the following advantages: This invention achieves dynamic optimization of the entire virus production process by adding additives SC79, mifepristone, and LR3 IGF-1 with different functional mechanisms during the cell pretreatment, virus infection and amplification, and continuous virus harvesting stages. In the cell pretreatment stage, SC79, as an Akt signaling pathway activator, significantly increases the viable cell density of host cells. In the virus infection and amplification stage, mifepristone, as a glucocorticoid receptor antagonist, promotes the integration of the viral genome into the host cell genome, improving virus infection efficiency. In the continuous virus harvesting stage, LR3 IGF-1 maintains host cell viability under low-concentration fetal bovine serum culture conditions, overcoming the bottleneck of decreased yield in the later stages of the SC79 and mifepristone combination. Experimental results show that pretreatment of host cells with 10 μM SC79 alone can increase the virus titer by 2.05 times (0.31 log...). 10 TCID 50 / mL); adding 0.5 μM mifepristone alone during inoculation and passage increased the viral titer by 2.51 times (0.40 log) 10 TCID 50 / mL); while the combination of SC79 and mifepristone (combination 1) increased the viral titer by 7.24 times (0.86 log 1 / mL). 10 TCID 50 The viral titer was significantly higher than the combined effect of the two drugs used alone, demonstrating an excellent synergistic effect. Furthermore, adding 20 ng / mL LR3 IGF-1 during continuous virus harvesting resulted in a 14.32-fold (1.16 log) increase in viral titer. 10 TCID 50 The titer of X-MuLv virus stock solution was significantly higher than that of combination 1 (7.66 times), further demonstrating the synergistic effect of the three combinations. This remarkable technical effect indicates that the method provided by this invention can effectively overcome the problem of low X-MuLv virus stock solution titer in the prior art. Attached Figure Description

[0016] Figure 1 This is a flowchart of the X-MuLv manufacturing process.

[0017] Figure 2 The effects of different signaling pathway activators on the viable cell density of M. dunnni.

[0018] Figure 3 The effect of different additives on X-MuLv virus titers.

[0019] Figure 4 The effect of the combination of SC79 and mifepristone on X-MuLv viral amplification.

[0020] Figure 5 The effect of different concentrations of LR3 IGF-1 on the viable cell density of M. dunnni.

[0021] Figure 6 The effect of different concentrations of LR3 IGF-1 on X-MuLv viral titers.

[0022] Figure 7 The effect of the combination of SC79 and mifepristone on X-MuLv virus amplification under different conditions.

[0023] Figure 8 The fold change in X-MuLv viral titer under different conditions. Detailed Implementation

[0024] This invention provides a method for increasing the titer of mouse leukemia virus, comprising the following steps: Before infecting host cells with mouse leukemia virus, host cells were pretreated with SC79. Mifepristone was added to the culture medium during infection of pretreated host cells with mouse leukemia virus and during cell passage. During continuous virus harvesting, LR3 IGF-1 was added to the culture medium.

[0025] In this invention, to increase the titer of heterophilic murine leukemia virus, the host cells used for virus amplification are first pretreated. This pretreatment step is performed before viral infection. As a preferred embodiment, mouse skin fibroblasts (M. dunnni cells) are used as the host cells, which have good sensitivity to heterophilic murine leukemia virus. The pretreatment procedure is preferably as follows: After M. dunnni cells are cultured to a suitable density, SC79 is added to the cell culture medium. SC79 is an Akt signaling pathway activator that can promote cell proliferation, increase cell metabolic activity, and inhibit apoptosis by activating the Akt signaling pathway, thereby significantly increasing the viable cell density. Experiments have shown that the concentration of SC79 added in the range of 5–20 μM can effectively increase the viable cell density, with 10 μM showing the most significant effect. The pretreatment time is 12–48 hours, preferably 24 hours. In practice, SC79 can be pre-prepared as a stock solution. For example, SC79 powder can be dissolved in DMSO to prepare a 10mM stock solution. After sterilization by filtration through a 0.22μm filter, it can be added to MC-5A medium containing 10% fetal bovine serum at the desired concentration, mixed well, and then used for the culture of *M. dunni* cells. After culturing under these pretreatment conditions for 24 hours, the viable cell density of *M. dunni* cells significantly increased. Cell counting verification showed that the viable cell density of the 10μM SC79 treatment group reached 1.82 × 10⁻⁶ cells on day 5.6 The concentration of cells / mL was 1.55 × 10⁻⁶, while the untreated control group had only 1.55 × 10⁻⁶. 6 The number of host cells / mL indicates that SC79 pretreatment can effectively increase the number of host cells.

[0026] In this invention, after the host cell pretreatment is completed, the virus infection and amplification stage begins. A key operation in this stage is the addition of mifepristone to the culture medium during the infection of pretreated host cells with heterophilic mouse leukemia virus and during subsequent cell passage. Mifepristone is a glucocorticoid receptor antagonist whose mechanism of action is to promote the integration of the retroviral genome into the host cell genome, thereby significantly improving viral infection efficiency and stable amplification. As a preferred embodiment, during infection, *M. dunnni* cells pretreated with SC79 are first seeded into culture containers, and viral infection is performed when the cell confluence reaches 70%–80%. The heterophilic mouse leukemia virus strain is mixed with an inoculation medium containing mifepristone and diluted to an MOI of 0.01–0.5, preferably 0.25. The inoculation medium is preferably MC-5A basal medium with added polygel to enhance viral infection efficiency. Simultaneously, mifepristone is added to achieve a final concentration of 0.1–2 μM, preferably 0.5 μM. After inoculating the above-mentioned virus dilution into *M. dunnni* cells, incubate them at 37°C in a 5% CO2 incubator for 2–5 hours, preferably 3 hours, to allow the virus to fully contact and enter the host cells. Then, add complete culture medium containing the same concentration of mifepristone and continue culturing. Three days after infection, perform cell passage amplification using a passage medium containing mifepristone. The passage medium is MC-5A basal medium supplemented with 10% fetal bovine serum and mifepristone. The mifepristone concentration is maintained at the same level as at the infection stage, 0.1–2 μM, preferably 0.5 μM. Passage culture is performed at least three times to ensure that the viral genome can stably integrate into the host cell genome and complete viral amplification.

[0027] In this invention, after viral infection and passage amplification are completed, the continuous virus harvesting stage begins. A key operation in this stage is the addition of LR3 IGF-1 to the culture medium during continuous virus harvesting. LR3 IGF-1 is an analog of insulin-like growth factor-1, and its mechanism of action is to maintain host cell viability and inhibit apoptosis under low-serum or serum-free culture conditions, thereby solving the yield bottleneck caused by decreased cell viability in the later stages of virus production. In the continuous harvesting operation, after three passage amplifications, *M. dunnni* cells have stably carried the viral genome, and continuous harvesting of the viral supernatant begins. At each harvest, after collecting the cell culture supernatant, fresh virus harvesting medium is immediately added to the culture system. This virus harvesting medium is MC-5A basal medium, to which low-concentration fetal bovine serum (FBS) is added, with a volume fraction of 0.5%–8%, preferably 0.5%–5%, more preferably 2%; and LR3 IGF-1, with a concentration of 5–100 ng / mL, preferably 20 ng / mL. LR3 IGF-1 can be pre-prepared as a stock solution. For example, LR3IGF-1 lyophilized powder can be dissolved in sterile water containing 0.1% BSA to prepare a 5 μg / mL stock solution. After sterilization by filtration through a 0.22 μm filter, it can be added to the virus harvesting medium at the required concentration. Continuous harvesting is usually carried out in 6 batches, with a certain time interval between each batch. The virus solution harvested each time is centrifuged at 2000-2500 rpm for 15-20 minutes, and the supernatant is collected as the virus stock solution. TCID45 is used for further analysis. 50 Virus titer was detected by a method. Experimental results showed that adding 20 ng / mL LR3 IGF-1 alone during virus harvesting significantly increased viable cell density. When LR3 IGF-1 was used in combination with SC79 and mifepristone, the virus titer increased by 14.32 times compared to the original process, significantly higher than the 7.66-fold increase achieved by combining SC79 and mifepristone alone. This demonstrates that LR3 IGF-1 not only maintains cell viability on its own but also has a synergistic effect with SC79 and mifepristone, effectively overcoming the technical bottleneck of yield decline in the later stages of continuous virus harvesting.

[0028] In this invention, the host cell is mouse skin fibroblasts, specifically M. dunnni cells. This cell line is derived from the skin tissue of Dunni mice and has been established as a stable cell line through long-term passage culture. It exhibits high sensitivity to heterophilic mouse leukemia virus and is an ideal cell matrix for retroviral amplification. Those skilled in the art may also use other cell lines sensitive to heterophilic mouse leukemia virus as host cells.

[0029] In this invention, the mouse leukemia virus is preferably heterophilic mouse leukemia virus, which is an important member of the γ retrovirus genus of the retroviridae family and is widely used as an indicator virus in the virus clearance verification process of biotechnology therapeutic products.

[0030] This invention also provides a composition for increasing the titer of mouse leukemia virus, comprising a signaling pathway activator, a glucocorticoid receptor antagonist, and an insulin-like growth factor-1 analog. The composition of this invention is not a simple mixture of multiple additives, but rather a scientific combination of three active ingredients with different functional mechanisms, targeting the specific needs of different stages of virus production. These components correspond to applications in the cell pretreatment stage, the virus infection and amplification stage, and the continuous virus harvesting stage. Specifically, the signaling pathway activator is used to pretreat host cells before virus infection, promoting cell proliferation, increasing cellular metabolic activity, and inhibiting apoptosis by activating intracellular signal transduction pathways, thereby increasing viable cell density and providing sufficient host cells for subsequent virus infection. The glucocorticoid receptor antagonist is used in the virus infection and passage amplification stage; its mechanism of action is to antagonize glucocorticoid receptors, thereby promoting the integration of the retroviral genome into the host cell genome, significantly improving virus infection efficiency and stable amplification capacity. The insulin-like growth factor-1 analog is used in the continuous virus harvesting stage, maintaining host cell viability and inhibiting apoptosis under low serum or serum-free culture conditions, overcoming the yield bottleneck caused by decreased cell viability in the later stages of virus production. In a preferred embodiment, the signaling pathway activator is SC79, a specific Akt signaling pathway activator that effectively activates the Akt signaling pathway, promoting cell proliferation and increasing cell density; the glucocorticoid receptor antagonist is mifepristone, a classic glucocorticoid receptor antagonist that efficiently promotes retroviral genome integration; and the insulin-like growth factor-1 analog is LR3 IGF-1, a long-acting analog of insulin-like growth factor-1 that exhibits excellent effects in maintaining cell viability under low serum conditions. The compositions of this invention can be provided in various forms, for example, as three individually packaged liquid components, each containing SC79, mifepristone, and LR3 IGF-1, prepared as appropriate concentrations of stock solutions or working solutions, which users can add according to different stages of the virus production process; alternatively, they can be provided as kits, with SC79, mifepristone, and LR3 IGF-1 dispensed as lyophilized powders in individual containers, along with sterile solvents and culture medium powders, which can be prepared according to the instructions.

[0031] This invention provides a method or composition thereof for the preparation of retroviruses and / or for increasing retrovirus titers.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The test materials used in the following examples include SB216763 powder, purchased from Beyotime (product number SC0384-5mg). The 10mM SB216763 stock solution was prepared as follows: 5mg of SB216763 powder was added to 1.35 mL of DMSO, and after complete dissolution, it was filtered through a 0.22μm filter for sterilization. SC79 powder was purchased from Beyotime (product number SF2730-5mg). The 10mM SC79 stock solution was prepared as follows: 5mg of SC79 powder was added to 1.37 mL of DMSO, and after complete dissolution, it was filtered through a 0.22μm filter for sterilization. Mifepristone powder was purchased from MERCK, catalog number M8046. A 100mM mifepristone stock solution was prepared as follows: 100mg of mifepristone powder was dissolved in 2.33mL of anhydrous ethanol. After complete dissolution, the solution was filtered through a 0.22μm syringe filter for sterilization and stored at 2–8℃ for later use. Inoculation medium (MC-5A medium + mifepristone + polybrene) and passage medium (MC-5A medium + 10% FBS + mifepristone) were prepared using MC-5A medium and stored at 2–8℃ for later use. MC-5A medium was McCoy's 5A medium, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. Cholesterol powder was purchased from Sigma-Aldrich, catalog number C3045. A 10mM cholesterol stock solution was prepared as follows: 30mg of cholesterol was dissolved in 7.76mL of sterile water and filtered through a 0.22μm filter. Sodium butyrate powder was purchased from Aladdin, catalog number S102956-100g. The 0.5M sodium butyrate stock solution was prepared as follows: 5.5g of sodium butyrate was dissolved in 99.9mL of sterile water and filtered through a 0.22μm filter. Dexamethasone powder was purchased from Beyotime, catalog number ST1258-50mg. The 10mM dexamethasone stock solution was prepared as follows: 50mg of dexamethasone was dissolved in 12.74mL of anhydrous ethanol and filtered through a 0.22μm filter. LR3 IGF-1 was purchased from YEASEN, catalog number 92210ES08. The 5μg / mL LR3 IGF-1 stock solution was prepared as follows: 100μg of LR3 IGF-1 was dissolved in 20mL of sterile water containing 0.1% BSA and filtered through a 0.22μm filter.

[0034] Example 1 A method for increasing the titer of mouse leukemia virus, comprising the following steps: (1) M. dunni cells were pretreated with MC-5A + 10% FBS + 10 μM SC79 cell passage medium for 24 h.

[0035] (2) The pretreated M. dunnni cells were seeded into T75 cells. On the second day, when the cell confluence was about 80%, the X-MuLv virus (MOI: 0.01~0.5) was diluted with inoculation medium (MC-5A + 21.4 μM Polybrene + 0.5 μM mifepristone) and then seeded into M. dunnni cell culture flasks. The flasks were incubated in a CO2 incubator. After 2~5 hours, MC-5A medium containing 10% FBS, 21.4 μM Polybrene and 0.5 μM mifepristone was added to the culture flasks.

[0036] (3) Three days after inoculation, the cells were passaged three times using passage medium to ensure that the X-MuLv virus genome was stably integrated into the M. dunnni cell genome. The passage medium was MC-5A + 10% FBS + 0.5 μM mifepristone.

[0037] (4) After three passages, the virus culture supernatant was harvested in six batches and fresh virus harvesting medium was added. The virus harvesting medium was MC-5A + 2% FBS + 20 ng / mL LR3 IGF-1. The virus solution was centrifuged at 2000~2500 rpm / min for 15~20 min, the supernatant was collected, and the supernatant was purified by TCID45. 50 Virus titer is detected by a specific method. See the process flow diagram below. Figure 1 .

[0038] Example 2: Screening of the optimal signaling pathway activators and their concentrations for increasing live cell density This embodiment aims to screen signaling pathway activators that can increase the viable cell density of host cells and determine their optimal concentration.

[0039] 1. Experimental Methods (1) M. dunni cells were pretreated with MC-5A + 10% FBS medium containing 1 μM SB216763, 5 μM SB216763, 10 μM MSC79 and 20 μM SC79 for 24 h. The control group was MC-5A + 10% FBS.

[0040] (2) Pretreated M. dunnni cells were subjected to a 2×10⁻⁶ thiocyanate infusion. 5 Cells were seeded at a density of / wells into six-well plates and cultured in MC-5A medium containing the corresponding components in Table 1 and 10% FBS.

[0041] (3) After inoculation, cells were digested daily for cell counting, and the effects of different concentrations of activators SB216763 and SC79 on the density of viable cells were analyzed.

[0042] Table 1. Pretreatment conditions for M. dunni cells

[0043] 2. Experimental Results According to Table 2 and Figure 2 The results showed that, compared with the control group, the viable cell density of the 10 μM SC79 treatment group was significantly higher from day 1 to day 5 of culture (P<0.05), reaching 1.82 × 10⁻⁶ cells / day on day 5. 6 The concentration was 1.55 × 10⁻⁶ / mL, while the control group had only 1.55 × 10⁻⁶ / mL. 6 Cells / mL. The 20 μM SC79 treatment group did not significantly increase viable cell density and showed a certain inhibitory trend in cell growth at high concentrations. The SB216763 treatment group had no significant effect on viable cell density at any concentration. Therefore, 10 μM SC79 was determined to be the optimal cell pretreatment condition.

[0044] Table 2 Viable cell density of different signaling pathway activator treatment groups (10⁻⁶) 5 / mL)

[0045] Note: In the table, " / " indicates that no significant difference analysis was performed, and ns indicates no significant difference. P <0.05, P <0.01.

[0046] Example 3: Screening of Additives to Improve X-MuLv Virus Titer This embodiment aims to screen functional additives that can effectively increase X-MuLv virus titers during the virus infection and amplification phases.

[0047] 1. Experimental Methods (1) M.dunni cells were seeded into T75 cells and groups were set according to the functional mechanisms of different additives, as shown in Table 3.

[0048] (2) When the cell confluence is about 80% on the second day, dilute the X-MuLv virus with medium containing or without 0.5 / 1μM mifepristone and inoculate it into M. dunnni cell culture flasks. Incubate in a CO2 incubator. After 2-5 hours, add MC-5A medium containing 10% FBS, Polybrene and 0.5 / 1μM mifepristone to the culture flasks.

[0049] (3) After inoculation, the cells were passaged three times using passage medium. The passage medium used was: MC-5A + 10% FBS + (0.5 μM / 1 μM) mifepristone / (0.01 mM / 0.03 mM) cholesterol / (0.5 mM / 1 mM) sodium butyrate / (100 nM / 1 μM) dexamethasone.

[0050] (4) After three passages, the virus culture supernatant was harvested in six batches, and fresh virus harvesting medium was added. The virus solution was centrifuged at 2000-2500 rpm / min for 15-20 min, the centrifuged supernatant was collected, and the virus titer was detected by TCID50 method. The virus harvesting medium was: MC-5A + 5% FBS + (0.01 / 0.03mM) cholesterol / (0.5 / 1mM) sodium butyrate / (100nm / 1μM) dexamethasone.

[0051] Table 3. Different virus amplification conditions settings

[0052] 2. Experimental Results According to Table 4 and Figure 3 The results showed that the treatment group receiving 0.5 μM mifepristone during inoculation and three passages achieved an average viral titer of 5.85 log. 10 TCID 50 / mL, compared to 5.40 log in the control group. 10 TCID 50 / mL increased by 0.45log 10 TCID 50 / mL, equivalent to 2.84 times. The viral titer in the 1 μM mifepristone treatment group increased by 0.33 log compared to the control group. 10 TCID 50 / mL, equivalent to 2.13 times. The cholesterol and dexamethasone treatments had no significant effect on increasing viral titer, while the sodium butyrate treatment group showed a decrease in viral titer due to inhibition of cell growth. Therefore, 0.5 μM mifepristone was determined to be the optimal additive for viral infection and amplification.

[0053] Table 4 Virus titers (log) of different additive treatment groups 10 TCID 50 / mL)

[0054] Note: " / " indicates that no significant difference analysis was performed, and ns indicates no significant difference. P <0.05.

[0055] Example 4: Effect of SC79 combined with mifepristone on X-MuLv viral titer This embodiment aims to verify the synergistic effect of the combined use of SC79 and mifepristone.

[0056] 1. Experimental Methods (1) Pre-treat M. dunni cells for 24 h with passage medium containing 10 μM SC79 MC-5A + 10% FBS or without treatment.

[0057] (2) The pretreated / untreated M. dunnni cells from step (1) were seeded into T75 cells. On the second day, when the cell confluence was about 80%, the X-MuLv virus was diluted with medium containing or without 0.5 μM mifepristone and then seeded into M. dunnni cell culture flasks. The flasks were incubated in a CO2 incubator. After 2-5 hours, MC-5A medium containing 10% FBS, Polybrene and 0.5 / 1 μM mifepristone was added to the culture flasks. The group settings are shown in Table 5.

[0058] (3) After inoculation, the cells were passaged three times using passage medium. The passage medium used was MC-5A + 10% FBS or MC-5A + 10% FBS + 0.5 μM mifepristone to ensure that the X-MuLv virus genome was stably integrated into the M.dunni cell genome.

[0059] (4) After three passages, the virus culture supernatant was harvested in six batches. The virus harvesting medium was MC-5A + low concentration FBS. The virus solution was centrifuged at 2000~2500 rpm / min for 15~20 min, and the supernatant was collected and purified by TCID. 50 Methods for detecting viral titers.

[0060] Table 5. Different virus amplification conditions settings

[0061] Note: The original process steps are as follows: ① M. dunni cell culture: M. dunni cells are cultured for X-MuLv amplification; ② Virus infection (inoculation): M. dunni cells are inoculated in culture flasks (such as T75). When the cell confluence reaches 70-80% on the second day, the X-MuLv virus is infecting the M. dunni cells with an MOI of 0.01-0.5; ③ 3 or more passages: After 3 days of X-MuLv infection, the M. dunni cells are passaged 3 or more times to promote the integration of the X-MuLv virus genome into the M. dunni cell genome and complete the amplification of the X-MuLv virus; ④ Continuous virus harvest: After 3 passages, 6 batches of X-MuLv virus stock solution are harvested continuously.

[0062] 2. Experimental Results According to Table 6 and Figure 4 The results showed that pretreatment of *M. dunnni* cells with 10 μM SC79 alone for 24 h significantly increased the X-MuLv viral titer by 0.31 log. 10 TCID 50 Adding 0.5 μM mifepristone alone, at a concentration of 2.05 times the normal concentration, during inoculation and three passages significantly increased the X-MuLv viral titer by 0.40 log₂ / mL. 10 TCID 50 / mL (2.51 times), while the combined use of the two significantly increased the X-MuLv viral titer by 0.85 log. 10 TCID 50 / mL (7.24 times). It has a synergistic effect of significantly increasing the X-MuLv viral titer, and this combination is named Combination 1.

[0063] Table 6. X-MuLv viral titers (log) in different treatment groups of SC79 and mifepristone 10 TCID 50 / mL)

[0064] Note: In the table, " / " indicates that no significant difference analysis was performed, and ns indicates no significant difference. P <0.05, P <0.01, P <0.01.

[0065] Example 5: Screening for the optimal concentration of LR3 IGF-1 to maintain viable cell density This embodiment aims to screen the optimal concentration of LR3 IGF-1 that can maintain host cell viability during the continuous virus harvesting phase. To focus on the role of LR3 IGF-1 during continuous virus harvesting, this experiment was conducted under conditions simulating the virus harvesting period. Specifically, M. dunnni cells infected with X-MuLv and passaged twice were used, and the medium was replaced with MC-5A+ low-concentration FBS virus harvesting medium supplemented with 0 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL LR3 IGF-1.

[0066] 1. Experimental Methods (1) M. dunnni cells that had been inoculated with X-MuLv and passaged twice were digested and counted, and seeded into 6-well plates at a cell density of 3.5E5 / well. Four wells were seeded for each treatment group, with virus supernatant harvested from two wells and cell counting used in the other two wells. The group settings are shown in Table 7 below.

[0067] (2) The virus culture supernatant was harvested in six batches and fresh virus harvesting medium was added. The virus harvesting medium was MC-5A + low concentration FBS + (0 / 5 / 10 / 20 / 50 / 100 ng / mL) LR3 IGF-1. The virus solution was centrifuged at 2000~2500 rpm / min for 15~20 min, the centrifuged supernatant was collected, and the virus titer was detected by TCID50 method.

[0068] Table 7 LR3 IGF-1 Treatment Group Settings

[0069] 2. Experimental Results Cell counting results are shown in Table 8 and Figure 5 As shown, the viable cell density in the 20 ng / mL LR3 IGF-1 treatment group was significantly higher than that in the control group at all time points, reaching 2.04 × 10⁻⁶ on day 6. 6 The concentration of cells / mL was only 1.65 × 10⁻⁶ in the control group. 6 Virus titer / mL. Results are shown in Table 8. Figure 6 As shown, the viral titer in the 20 ng / mL LR3 IGF-1 treatment group was 0.26 log higher than that in the control group. 10 TCID 50 / mL, equivalent to 1.82 times, while the improvement effects of the 50ng / mL and 100ng / mL treatment groups were not significantly different from those of the 20ng / mL group. Considering both efficacy and cost, 20ng / mL was determined to be the optimal concentration of LR3 IGF-1.

[0070] Table 8 Viral titers (log) of different concentrations of LR3 IGF-1 treatment groups 10 TCID 50 / mL)

[0071] Note: In the table, " / " indicates that no significant difference analysis was performed, and ns indicates no significant difference. P <0.05, P <0.01.

[0072] Example 6: Efficacy verification of the combined use of SC79, mifepristone, and LR3 IGF-1 This embodiment aims to verify the synergistic effect of the combined use of SC79, mifepristone, and LR3 IGF-1, and to determine the final optimal production process.

[0073] In the continuous harvesting of X-MuLv virus using SC79 and mifepristone (combination 1) in Example 4, the virus harvesting medium used was MC-5A medium with a low concentration of FBS. This was to reduce the pressure on downstream virus purification, as it was anticipated that virus yield would be limited later due to decreased cell viability. Therefore, during continuous virus harvesting, LR3IGF-1 was added to the MC-5A medium to maintain M. dunnni cell viability and maximize X-MuLv virus yield.

[0074] 1. Experimental Methods Following the steps in Example 4, the group settings are shown in Table 9.

[0075] (1) Pretreat M. dunnni cells for 24 h with passage medium containing 10 μM SC79 in MC-5A + 10% FBS or without treatment. Add or not add 0.5 μM mifepristone during inoculation and 3 cell passages.

[0076] (2) After three passages, the virus culture supernatant was harvested in six batches and fresh virus harvesting medium was added, as shown in Table 7. The virus harvesting medium for groups ① and ② was MC-5A + 2% FBS medium, and the virus harvesting medium for groups ③ and ④ was MC-5A + 2% FBS + 20 ng / mL LR3 IGF-1. The virus solution was centrifuged at 2000~2500 rpm / min for 15~20 min, and the supernatant was collected and purified by TCID45. 50 Methods for detecting viral titers.

[0077] Table 9. Combination 1 + Different Virus Amplification Conditions Settings

[0078] Note: The original process steps are as follows: ① M. dunni cell culture: M. dunni cells are cultured for X-MuLv amplification; ② Virus infection (inoculation): M. dunni cells are inoculated in culture flasks (such as T75). When the cell confluence reaches 70-80% on the second day, the X-MuLv virus is infecting the M. dunni cells with an MOI of 0.01-0.5; ③ 3 or more passages: After 3 days of X-MuLv infection, the M. dunni cells are passaged 3 or more times to promote the integration of the X-MuLv virus genome into the M. dunni cell genome and complete the amplification of the X-MuLv virus; ④ Continuous virus harvest: After 3 passages, 6 batches of X-MuLv virus stock solution are harvested continuously.

[0079] 2. Experimental Results According to Table 10 and Figure 7-8 The results showed that pretreatment of *M. dunnni* cells with 10 μM SC79 for 24 h, followed by the addition of 0.5 μM mifepristone during inoculation and three passages, and the addition of 20 ng / mL LR3IGF-1 during continuous virus harvest, significantly increased the X-MuLv virus titer by 1.16 log. 10 TCID 50 / mL, which is approximately 14.32 times higher than the original process (control group), and significantly higher than combination 1 (combination 1 only increased the viral titer by 0.88 log / mL compared to the control group). 10 TCID 50 The problem of titer reduction during continuous virus harvesting was greatly alleviated (approximately 7.66 times the normal value). However, using LR3 IGF-1 alone did not significantly improve X-MuLv virus titer, increasing it by only 1.62 times. These results indicate that although using 20 ng / mL LR3 IGF-1 alone during virus harvesting has little effect on X-MuLv virus titer, during virus harvesting, due to the maintenance of viable cell density under low-concentration FBS culture conditions, LR3 IGF-1 can specifically overcome the late-stage bottleneck of the combination 1 (SC-79 + mifepristone) process, producing an unpredictable synergistic effect, thereby maximizing X-MuLv virus yield.

[0080] Table 10. X-MuLv viral titers amplified under different conditions in combination 1 (log 10 TCID 50 / mL)

[0081] Note: In the table, " / " indicates that no significant difference analysis was performed, and ns indicates no significant difference. P <0.05, P <0.01, P <0.01.

[0082] Example 7 A composition for increasing the titer of mouse leukemia virus, said composition comprising three separately packaged liquid components: Component A (cell pretreatment agent): Cell maintenance medium containing SC79; Component B (viral infection promoter): inoculation medium and passage medium containing mifepristone; Component C (Virus Harvest Maintenance Agent): Virus harvesting medium containing LR3 IGF-1. The composition comprises the following components: SC79, mifepristone and LR3 IGF-1.

[0083] The preparation method of the above composition is as follows: (1) Preparation of component A Dissolve 10 mg of SC79 powder in 2.74 mL of DMSO, shake thoroughly until completely dissolved, and filter sterilize using a 0.22 μm filter to obtain a 10 mM SC79 stock solution. Add the SC79 stock solution to MC-5A cell maintenance medium (containing 10% fetal bovine serum) at a volume ratio of 1:1000, and mix thoroughly to obtain component A. The final concentration of SC79 in component A is 10 μM. This component should be stored at 2–8 °C protected from light and has a shelf life of 6 months.

[0084] (2) Preparation of component B Dissolve 100 mg of mifepristone powder in 2.33 mL of anhydrous ethanol, shake thoroughly until completely dissolved, and filter sterilize using a 0.22 μm filter to obtain a 100 mM mifepristone stock solution. Add the mifepristone stock solution to MC-5A inoculation medium (containing 21.4 μM Polybrene) at a volume ratio of 1:200, and mix thoroughly to obtain inoculation component B1. Simultaneously, add the mifepristone stock solution to MC-5A passage medium (containing 10% fetal bovine serum) at a volume ratio of 1:200, and mix thoroughly to obtain passage component B2. The final concentration of mifepristone in both components B1 and B2 is 0.5 μM. These components should be stored at 2–8 °C protected from light and have a shelf life of 6 months.

[0085] (3) Preparation of component C Take 100 μg of LR3 IGF-1 lyophilized powder and dissolve it in 20 mL of sterile water containing 0.1% BSA. Filter the solution through a 0.22 μm filter to obtain a 5 μg / mL LR3 IGF-1 stock solution. Add the LR3 IGF-1 stock solution to MC-5A virus harvesting medium (containing low-concentration fetal bovine serum, volume fraction 0.5%–5%) at a volume ratio of 1:250 and mix thoroughly to obtain component C. The final concentration of LR3 IGF-1 in component C is 20 ng / mL. This component should be stored at -20℃ protected from light and has a shelf life of 12 months. After thawing, it can be stored at 2–8℃ for 7 days.

[0086] This composition is used in the production of heterophilic mouse leukemia virus according to the following steps: Cell pretreatment stage: Take component A and add it to the host cell culture system 24 hours before virus infection for cell pretreatment; Viral infection and amplification phase: Component B1 was added during viral infection, and component B2 was added during the subsequent three cell passages; Continuous virus harvesting phase: Component C was added to fresh culture medium after each virus harvest.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for increasing the titer of mouse leukemia virus, characterized in that, Includes the following steps: Before infecting host cells with mouse leukemia virus, host cells were pretreated with SC79. Mifepristone was added to the culture medium during infection of pretreated host cells with mouse leukemia virus and during cell passage. During continuous virus harvesting, LR3 IGF-1 was added to the culture medium.

2. The method according to claim 1, characterized in that, The concentration of SC79 added is 5~20μM; the concentration of mifepristone added is 0.1~2μM; and the concentration of LR3 IGF-1 added is 5~100ng / mL.

3. The method according to claim 1, characterized in that, The pretreatment time is 12 to 48 hours.

4. The method according to claim 1, characterized in that, The cells are passaged and expanded at least three times.

5. The method according to claim 1, characterized in that, The culture medium used during the continuous harvesting of the virus is a culture medium containing a low concentration of fetal bovine serum, wherein the volume fraction of the low concentration of fetal bovine serum is 0.5% to 8%.

6. The method according to claim 1, characterized in that, The host cells are mouse skin fibroblasts.

7. The method according to claim 1, characterized in that, The mouse leukemia virus mentioned is a heterophilic mouse leukemia virus.

8. A composition for increasing the titer of mouse leukemia virus, characterized in that, The composition comprises a signaling pathway activator, a glucocorticoid receptor antagonist, and an insulin-like growth factor-1 analog.

9. The composition according to claim 8, characterized in that, The signaling pathway activator is SC79; the glucocorticoid receptor antagonist is mifepristone; and the insulin-like growth factor-1 analog is LR3 IGF-1.

10. The method according to any one of claims 1-7, or the composition according to claim 8 or 9, in the preparation of retroviruses and / or the enhancement of retrovirus titers.

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