Method for improving CD7 blocking lentivirus titer and application thereof

Abstract

The invention discloses a method for improving CD7 blocking lentivirus titer and application of the method. The method comprises the following steps: reducing serum in three stages, carrying out serum-free domestication to obtain suspension cells, carrying out mixed incubation on a transfection reagent and CD7 blocking plasmid DNA, and carrying out mixed culture on the obtained cell suspension after incubation. According to the method disclosed by the invention, efficient large-scale production can be realized through cell domestication and optimization of a harvest liquid development process strategy, the operation is simple, conditions are clear, parameters are clear, harmful ingredients are avoided, the CD7 blocking lentivirus titer can be remarkably improved, and the uniformity among batches is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a method for improving the titer of CD7-blocking lentivirus and its application. Background Art

[0002] T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematological malignancy. The 5-year survival rate of pediatric T-ALL patients is 80%-90%, and that of adult patients is 30%-40%. However, 20% of T-ALL patients still die from relapse or refractory diseases in the end. Based on the significant progress made by CAR-T therapy in the field of tumor treatment, more and more studies are currently using CAR-T to treat T-ALL / LBL. CD7 is a transmembrane molecule. Since CD7 is highly expressed in T-ALL / LBL cells, it is an important target for targeted killing of T-ALL / LBL. Since normal human T cells also express the CD7 molecule, preparing CD7-CAR-T cells using normal T cells will cause CAR-T to attack itself. To effectively avoid the self-killing of CD7-CAR-T cells, a CD7 nanobody and an endoplasmic reticulum retention sequence are introduced in the design of CD7-CAR-T cells to block its expression on the cell membrane, thereby preventing normal T cells from being recognized and killed by the CD7-CAR molecule.

[0003] Lentivirus is a powerful tool for preparing CAR-T cells. Many factors such as its yield, purity, and infection efficiency significantly affect the preparation efficiency of CAR-T, hindering the large-scale production of CAR-T and the process of clinical treatment. Currently, the production of lentiviral preparations faces problems such as low titer in the harvest fluid and unstable titer between different batches. To accelerate the clinical application of lentivirus, it is extremely urgent to improve the production efficiency of lentivirus.

[0004] Calcium phosphate, liposome, and PEI are several transfection reagents commonly used in the production of lentivirus. Calcium phosphate is an economical transfection reagent, but this method requires strict control of component concentration, temperature, aggregation time, and pH. In addition, if there is no serum or albumin in the cell culture medium, calcium phosphate transfection may cause cytotoxicity, so it is difficult to be scaled up for use. Liposomes are expensive and have too high production costs, making it difficult to be used in the large-scale production of lentivirus. PEI is an economical cationic polymer transfection reagent that binds to DNA and condenses into cationic nanoparticles (Polyplex). Polyplex binds to the phosphate groups on the host cell membrane and is endocytosed into the cell. After entering the cell, it dissociates due to the change in osmotic pressure and is free in the cytoplasm. Research shows that the size of Polyplex is a key factor in transfection efficiency. The size of Polyplex is closely related to the total amount of DNA packaging, incubation time, and incubation volume.

[0005] Therefore, there is an urgent need to provide a method for improving the titer of CD7-blocking lentivirus with simple operation, good packaging effect and high virus titer. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art and the actual needs, the present invention provides a method for improving the titer of CD7-blocking lentivirus, which solves the problems of low titer of lentivirus harvest fluid, high impurity content, large difference between different batches, and difficulty in clinical application. The lentivirus packaging method of the present invention has simple operation, clear conditions, clear parameters and no harmful components.

[0007] To achieve the object of the present invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides a method for improving the titer of CD7-blocking lentivirus, the method comprising: performing serum-free acclimation by reducing serum in three stages to obtain suspension cells, mixing a transfection reagent with CD7-blocking plasmid DNA for incubation, and after incubation, mixing with the obtained cell suspension for culture.

[0009] The method of the present invention can achieve efficient large-scale production through cell acclimation and optimization of the harvest fluid development process strategy, has simple operation, clear conditions, clear parameters and no harmful components, can significantly improve the titer of CD7-blocking lentivirus, and has good uniformity between batches.

[0010] Preferably, the first stage of performing serum-free acclimation by reducing serum in three stages to obtain suspension cells includes: transferring ATCC cells from DMEM medium containing 10% NBS to medium containing 5% NBS, thawing the cryopreserved cell line, mixing the cell suspension with DMEM medium containing 10% FBS, centrifuging to remove the supernatant, resuspending with DMEM medium containing 10% FBS, and adding medium for culture and subculture after supplementation.

[0011] Preferably, the number of passages is 4-5 times, and the number of cells for each passage is 1.5×10 6 -2×10 6 cells.

[0012] The specific point value among the above 4-5 times can be selected as 4 times or 5 times.

[0013] The specific point value among the above 1.5×10 6 -2×10 6 can be selected as 1.5×10 6 , 1.6×10 6 , 1.7×10 6 , 1.8×10 6 , 1.9×10 6 , 2×10 6 etc.

[0014] Preferably, the second stage of obtaining suspension cells by reducing serum in three stages for serum-free acclimation includes: transferring the cells obtained in the first stage to a medium containing 1% FBS for culture and subculture, and the number of subculture generations is 7-9 times, and the number of cells for each subculture is 1.5×10 6 -2×10 6 cells.

[0015] Specific point values among the above 7-9 times can be selected as 7 times, 8 times, 9 times, etc.

[0016] Specific point values among the above 1.5×10 6 -2×10 6 can be selected as 1.5×10 6 、1.6×10 6 、1.7×10 6 、1.8×10 6 、1.9×10 6 、2×10 6 etc.

[0017] Preferably, the third stage of obtaining suspension cells by reducing serum in three stages for serum-free acclimation includes: after digesting and centrifuging the cells obtained in the second stage, transferring them to a serum-free medium for culturing for 2-3 days, then transferring the cells to a six-well plate without adsorption for culturing for 6-8 days, and then culturing in an incubator for 2-3 days, standing to obtain a cell suspension supernatant, adding medium to continue subculture for 6-8 days, repeating the standing operation to obtain a cell suspension supernatant, and adding medium to continue subculture for 11 days.

[0018] Specific point values among the above 2-3 days can be selected as 2 days, 3 days, etc.

[0019] Specific point values among the above 6-8 days can be selected as 6 days, 7 days, 8 days, etc.

[0020] Preferably, the temperature for culturing in the incubator is 35℃-37℃, the rotation speed is 110-150 rpm, and the CO2 concentration is 5%-6%.

[0021] Specific point values among the above 35℃-37℃ can be selected as 35℃, 36℃, 37℃, etc.

[0022] Specific point values among the above 110-150 rpm can be selected as 110 rpm, 115 rpm, 120 rpm, 130 rpm, 140 rpm, 145 rpm, 150 rpm, etc.

[0023] Specific point values among the above 5%-6% can be selected as 5%, 5.5%, 6%, etc.

[0024] Preferably, the packaging amount of the CD7-blocking plasmid DNA is 0.5 μg / mL - 1 μg / mL.

[0025] Specific point values within the range of 0.5 μg / mL - 1 μg / mL can be selected as 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, etc.

[0026] Preferably, the volume of the incubation is 4.4% - 22% of the total cell culture volume.

[0027] Specific point values within the range of 4.4% - 22% can be selected as 4.4%, 5%, 6%, 8%, 10%, 15%, 16%, 18%, 20%, 22%, etc.

[0028] Preferably, the incubation time is 5 - 20 min.

[0029] Specific point values within the range of 5 - 20 min can be selected as 5 min, 6 min, 8 min, 10 min, 15 min, 16 min, 18 min, 20 min, etc.

[0030] In a second aspect, the present invention provides the use of the method described in the first aspect for enhancing the lentivirus titer.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Lentivirus packaging mainly relies on adherent cells, with limited production scale and containing serum, presenting safety risks and being difficult to be widely applied clinically. The method of the present invention can achieve efficient large-scale production through cell domestication and optimization of the harvest fluid development process strategy. It is simple to operate, with clear conditions, clear parameters, and no harmful components, can significantly improve the CD7-blocking lentivirus titer, and has good uniformity among batches. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a graph showing the corresponding results of the culture days at the time of virus harvest;

[0034] Figure 2 It is a statistical graph of the optimal total DNA packaging amount of the CD7-blocking lentivirus;

[0035] Figure 3 It is a statistical graph of the optimal incubation volume of the CD7-blocking lentivirus;

[0036] Figure 4 It is a statistical graph of the optimal incubation time of the CD7-blocking lentivirus;

[0037] Figure 5 It is a graph showing the lentivirus titers of different batches of CD7-blocking lentivirus;

[0038] Figure 6 It is the result diagram before and after the optimization of the CD7-blocking virus packaging process;

[0039] Figure 7 It is the result diagram of the impurity content before and after the optimization of the CD7-blocking virus packaging process. Specific Embodiments

[0040] To further elaborate on the technical means and their effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention.

[0041] For those not specifying specific techniques or conditions in the embodiments, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0042] Example 1

[0043] Domestication of 293T adherent cells and testing of the optimal packaging culture days.

[0044] In this example, adherent cells were serially acclimated to serum-free conditions with the aim of converting adherent cells cultured in serum to suspension cells cultured without serum.

[0045] (1) In the first stage, ATCC cells were transferred from DMEM medium containing 10% NBS to medium containing 5% NBS (prepared by mixing DMEM containing 10% FBS and serum-free medium OPM-293CD05 at a ratio of 1:1).

[0046] Cell resuscitation: Rapidly thaw the cryopreserved cell line at 37°C. Add the cell suspension to DMEM medium containing 5 mL of 10% FBS. After centrifuging at 1000 rpm for 3 min, discard the supernatant, resuspend with 5 mL of DMEM medium containing 10% FBS, transfer to a T175 flask, supplement with 15 mL of DMEM medium containing 10% FBS, and culture in a carbon dioxide incubator at 37°C and 5% carbon dioxide.

[0047] Cell passage: Use a T25 flask for passage, and control the number of cells per passage to be 1.5×10 6 cells; passage 4 times, and from the second passage, set up a control group by sub-bottling, and compare the cell morphology, viability and viable cell density between the experimental group and the control group;

[0048] (2) In the second stage, transfer the cells to a medium containing 1% FBS (prepared by mixing DMEM containing 10% FBS and serum-free medium OPM-293CD05 at a ratio of 1:9), and passage them using a T25 flask. Control the number of cells for each passage to be 1.5×10 6 cells, and passage for 7 generations;

[0049] (3) In the third stage, after digesting and centrifuging the cells, transfer them to serum-free medium OPM-293CD05 and culture for 2 days; then transfer the cells to a non-adsorbent six-well plate for culture (seed 0.5×10 6 cells per well), select a well with better viability and cell number for continuous culture. After a total of 6 days of passage culture, transfer to a 125 mL flask and culture at 37 °C, 5% CO2, and 110 rpm for 2 days. Then let the cells stand in a biosafety cabinet for 20 min to obtain the cell suspension supernatant. Add an appropriate amount of medium and continue passage culture for 6 days. Repeat the standing operation to obtain the cell suspension supernatant, add an appropriate amount of medium and continue passage culture for 11 days. After the acclimation process, the cell viability is maintained above 90%;

[0050] (4) Passage the suspension cells cultured in serum-free medium obtained after acclimation, with a passage density of 1×10 6 cells / mL, and passage after culturing for 48 h when the culture volume is 30 mL; with a passage density of 0.5×10 6 cells / mL, and passage after culturing for 72 h when the culture volume is 30 mL;

[0051] (5) Lentivirus packaging and harvesting

[0052] Cell seeding: Use the suspension cells cultured in serum-free medium obtained after acclimation for seeding, with a seeding density of 2×10 6 cells / mL and a culture volume of 30 mL;

[0053] Lentivirus packaging: In a biosafety cabinet, take two 1.8 mL cryotubes, add 666 μL of serum-free medium OPM-293CD05 Medium to each, and label them Vec1-2 and TR1-2 respectively. In the Vec tubes, package according to the mass ratio of CD7 blocking plasmid: pMDLg-pRRE: pRSV-Rev: pMD2.G of 6:1.5:2.5:2. The dosage of a single plasmid for packaging = (DNA packaging amount × cell density at packaging × packaging volume × the proportion of this plasmid in the four ratios) / sum of the four plasmid ratios / concentration of this plasmid; the dosage of PEIpro = total DNA packaging amount × 3, mix well and let stand; add the corresponding volume of PEI to the TR tubes, mix well and let stand for 2 min; then add the plasmid mixture in the Vec tubes to the TR tubes, mix well and let stand for 8 min, and then add to the cell suspension. After packaging is completed, continue culturing in a large-capacity stacked thermostatic culture oscillator at 37 °C, 5% CO2, and 135 rpm.

[0054] Harvest: Virus harvesting was performed after 48 hours of cultivation.

[0055] The results were as Figure 1 shown. After continuous passage for 190 days, the titer of the cells packaged gradually decreased. The titer results were relatively stable between 190 and 220 days. Therefore, the number of days of cultivation for the domesticated cells to obtain stable titer results was before 220 days. In addition, after the cells were passaged to 220 days, the titer decreased to zero. Therefore, the maximum number of days for the domesticated cells to be continuously cultivated and packaged with lentivirus was 240 days.

[0056] Example 2

[0057] Explore the total amount of plasmid DNA packaging.

[0058] In this example, by exploring the total amount of plasmid DNA packaging, the aim was to determine the optimal packaging amount of CD7-blocking lentivirus.

[0059] The steps for packaging CD7-blocking virus are as follows:

[0060] (1) Inoculate the domesticated 293 suspension cells in Example 1 at a cell density of 2.0×10 6 cells / mL.

[0061] (2) After culturing for 24 hours, when the cell density is 4.0×10 6 cells / mL, perform the lentivirus packaging experiment.

[0062] (3) Package according to the mass ratio of CD7-blocking plasmid: pMDLg-pRRE: pRSV-Rev: pMD2.G of 6:1.5:2.5:2. The dosage of a single plasmid for packaging = (DNA packaging amount × cell density at packaging × packaging volume × the proportion of this plasmid in the four ratios) / sum of the four plasmid ratios / concentration of this plasmid. The dosage of PEIpro = DNA total packaging amount × 3.

[0063] (4) Add DNA to the container with culture medium, pipette and mix well. Add PEIpro to the container with culture medium, pipette and mix well.

[0064] (5) Add the PEI mixture to the DNA mixture, pipette and mix well, and incubate in the dark at room temperature.

[0065] (6) After the incubation time ends, add the mixture to the cell suspension and place it in the incubator for cultivation.

[0066] The CD7-blocking lentivirus was packaged at a mass ratio of CD7-blocking plasmid:pMDLg-pRRE:pRSV-Rev:pMD2.G of 6:1.5:2.5:2, and the DNA packaging amounts were set at 0.5 μg / mL, 0.75 μg / mL, and 1 μg / mL. The results were as Figure 2 shown that when the DNA packaging amount was 0.5 μg / mL, the titer of the CD7-blocking lentivirus was significantly higher than those with the total DNA packaging amounts of 0.75 μg / mL and 1 μg / mL, and the titer of the CD7-blocking lentivirus decreased successively with the increase of the total DNA packaging amount.

[0067] Example 3

[0068] Optimal incubation volume of the CD7-blocking lentivirus.

[0069] After the transfection reagent was mixed with the plasmid, a transfection complex was formed after a short incubation. The incubation volume affected the formation of the transfection complex. The incubation volumes were set at 4.4%, 13.2%, and 22% of the cell culture volume. As Figure 3 shown, where V1 was the incubation volume of 4.4% of the packaging volume, V2 was the incubation volume of 13.2% of the packaging volume, and V3 was the incubation volume of 22% of the packaging volume. When the incubation volume was 4.4% of the packaging volume, the titer of the CD7-blocking lentivirus was as high as 1.0×10 7 IU / mL to 1.5×10 7 IU / mL, and the lentivirus titer decreased successively with the increase of the incubation volume.

[0070] Example 4

[0071] Optimal incubation time of the CD7-blocking lentivirus.

[0072] Finally, the effect of the incubation time on the formation of the transfection complex was investigated, and the incubation times were set at 5 min, 10 min, 15 min, and 20 min. As Figure 4 shown, when the DNA packaging amount was 0.5 μg / mL, the incubation volume was 4.4% of the cell culture volume, and the incubation time was 5 min, the transfection effect of the CD7-blocking transfection complex formed under these conditions was the best and the virus titer was the highest.

[0073] Example 5

[0074] Determination of the CD7-blocking virus packaging process.

[0075] Taking the mass ratio of CD7-blocking plasmid:pMDLg-pRRE:pRSV-Rev:pMD2.G of 6:1.5:2.5:2, the DNA packaging amount of 0.5 μg / mL, the incubation volume of 4.4% of the cell culture volume, and the incubation time of 5 min as the packaging conditions, 8 batches of CD7-blocking lentivirus were produced. AsFigure 5 As shown, the differences in CD7-blocking viruses between different batches are small, and the titer values are relatively uniform, indicating that the lentivirus production process of this method is stable.

[0076] Example 6

[0077] Results before and after optimization of the CD7-blocking virus packaging process.

[0078] Before the optimization of the CD7-blocking virus packaging process, the packaging amount of CD7-blocking plasmid DNA was 0.5 μg / mL, the volume of PEIpro incubated with DNA was 4.4% of the total cell culture volume, and the incubation time was 15 min. After the optimization of the CD7-blocking virus packaging process, the packaging amount of CD7-blocking plasmid DNA was 0.5 μg / mL, the volume of PEIpro incubated with DNA was 4.4% of the total cell culture volume, and the incubation time was 5 min. As Figure 6 shown, the harvested titer of the optimized CD7-blocking virus was 2.0×10 7 IU / mL - 4.0×10 7 IU / mL, significantly increasing the virus titer, which was 10 - 26 times that of the original process.

[0079] Example 7

[0080] Results of the impurity content harvested before and after optimization of the CD7-blocking virus packaging process.

[0081] Host protein (Human cell Protein, HCP) and host DNA (Human cell DNA, HCD) can affect the biological activity of protein products, cause immunogenicity to the receptor, and then produce adverse reactions. Moreover, the residual viral DNA may transfer the DNA to the receptor, posing potential safety risks to the host in terms of viral infectivity, tumorigenicity, immunogenicity, and mutagenicity. Therefore, to meet regulatory standards, the generation of impurities should be reduced during the lentivirus production process. Before the optimization of the CD7-blocking virus packaging process, the packaging amount of CD7-blocking plasmid DNA was 0.5 μg / mL, the incubation volume of DNA and PEIpro was 4.4% of the total cell culture volume, and the incubation time was 15 min. After the optimization of the CD7-blocking virus packaging process, the packaging amount of CD7-blocking plasmid DNA was 0.5 μg / mL, the incubation volume of DNA and PEIpro was 4.4% of the cell culture packaging volume, and the incubation time was 5 min. As Figure 7 shown, the contents of HCP and HCD in the harvested CD7-blocking virus after optimization were significantly lower than those before optimization.

[0082] Example 8

[0083] Verification of CD7-blocking virus transfection of T cells.

[0084] To verify the cell preparation effect, CD7-blocking lentivirus was transfected into T cells, and the cells were given sufficient nutrients and space to recover. Before the optimization of the CD7-blocking virus packaging process, the packaging amount of CD7-blocking plasmid DNA was 0.5 μg / mL, the incubation volume of DNA and PEIpro was 4.4% of the total cell culture volume, and the incubation time was 15 min. After the optimization of the CD7-blocking virus packaging process, the packaging amount of CD7-blocking plasmid DNA was 0.5 μg / mL, the incubation volume of DNA and PEIpro was 4.4% of the total cell culture volume, and the incubation time was 15 min. As shown in Table 1, the CD7-blocking virus before optimization needed to transfect T cells at an MOI of 0.2, and the CD7 molecules could be blocked 14 days later (the expression level of CD7 molecules was lower than 5%, indicating that the CD7 molecules had been blocked). However, the CD7-blocking virus after process optimization only needed to transfect T cells at an MOI of 0.05, and the CD7 molecules were completely blocked 14 days later. It shows that after the optimization of the lentivirus packaging process, the activity of the CD7-blocking lentivirus is higher than that before optimization, and the transfection efficiency is better than that before optimization.

[0085] Table 1

[0086]

[0087]

[0088] In summary, the method of the present invention can achieve efficient large-scale production through cell domestication and optimization of the harvest solution development process strategy. It is simple to operate, with clear conditions, clear parameters, and no harmful components. It can significantly improve the titer of the CD7-blocking lentivirus, and the uniformity between batches is good.

[0089] The applicant declares that the present invention uses the above embodiments to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for improving the titer of CD7-blocking lentivirus, characterized in that, The method includes: obtaining suspension cells through serum-free acclimation by reducing serum in three stages, mixing a transfection reagent with CD7-blocking plasmid DNA for incubation, and culturing the mixture with the obtained cell suspension after incubation.

2. The method according to claim 1, wherein The first stage of obtaining suspension cells through serum-free acclimation by reducing serum in three stages includes: transferring ATCC cells from DMEM medium containing 10% NBS to medium containing 5% NBS, thawing the cryopreserved cell line, mixing the cell suspension with DMEM medium containing 10% FBS, centrifuging to remove the supernatant, resuspending with DMEM medium containing 10% FBS, supplementing the medium, and then culturing and subculturing.

3. The method according to claim 2, wherein The number of passages is 4 - 5 times, and the number of cells for each passage is 1.5×10 6 - 2×10 6 cells.

4. The method according to any one of claims 1 to 3, characterized in that, The second stage of obtaining suspension cells by serum-free acclimation with serum reduction in three stages includes: transferring the cells obtained in the first stage to a medium containing 1% FBS for culture and subculture, the number of subculture generations being 7-9 times, and the number of cells for each subculture being 1.5×10 6 -2×10 6 cells.

5. The method according to any one of claims 1-4, characterized in that, The third stage of obtaining suspension cells through serum-free acclimation by reducing serum in three stages includes: digesting and centrifuging the cells obtained in the second stage, transferring them to serum-free medium for culture for 2 - 3 days, transferring the cells to a non-adsorbent six-well plate for culture for 6 - 8 days, then culturing in an incubator for 2 - 3 days, standing to obtain the cell suspension supernatant, supplementing the medium and continuing subculture for 6 - 8 days, repeating the standing operation to obtain the cell suspension supernatant, and supplementing the medium and continuing subculture for 11 days.

6. The method according to claim 5, characterized in that The temperature for culturing in the incubator is 35°C - 37°C, the rotation speed is 110 - 150 rpm, and the CO2 concentration is 5% - 6%.

7. The method according to any one of claims 1-6, characterized in that, The packaging amount of the CD7-blocking plasmid DNA is 0.5 μg / mL - 1 μg / mL.

8. The method according to any one of claims 1-7, characterized in that, The volume of the incubation is 4.4% - 22% of the total cell culture volume.

9. The method according to any one of claims 1-8, characterized in that, The time of the incubation is 5 - 20 min.

10. Application of the method according to any one of claims 1 - 9 in enhancing the lentivirus titer.

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