Packaging method of CAR virus

Abstract

The invention relates to the technical field of virus packaging. The invention provides a packaging method of a CAR (chimeric antigen receptor) virus. The packaging method comprises the following steps: mixing a plasmid pCMX-dR8.91, a plasmid pCMV-BaEV, a plasmid pAdVAnti and an opti-MEM culture medium, standing, mixing with a PEI transfection reagent, standing, mixing with a 293T cell, culturing, replacing the culture medium, continuously culturing, and harvesting the virus. A second-generation lentivirus packaging system is adopted, transfer plasmids are second-generation transfer plasmids needing to be activated by a Tat element, packaging plasmids are second-generation packaging plasmids pCMV-dR8.91 containing ag, pol, rev and tat, enveloping plasmids are second-generation enveloping plasmids pCMV-BaEV, plasmids pAdVAnti beneficial to improvement of transfection efficiency are added, and the packaging efficiency of the CAR virus is improved through the system.

Description

Technical Field

[0001] The present invention relates to the technical field of virus packaging, and in particular to a packaging method for a CAR virus. Background Art

[0002] The CAR molecule is a tumor-coupled chimeric antigen receptor composed of antibody-derived fragments that can recognize and bind to tumors and promote T cell expansion. It contains an extracellular ligand binding region (scFV), a transmembrane region, and an intracellular signaling region.

[0003] The CAR structure has gone through five generations of development. The first generation of CAR targets the CD19 or CD20 antigens of the tumor, and the signal transduction region is composed of the intracellular CD3ζ chain. The second generation CAR signal transduction region adds a co-stimulatory signal molecule CD28 or 4-1BB. The third generation CAR signal transduction region adds two co-stimulatory signal molecules CD28, 4-1BB or CD134, which improves the proliferation and killing ability of CAR-T. The fourth generation CAR adds cytokines or co-stimulatory ligands (such as IL-12 / IL15 / anti-PD-1, etc.) on the basis of the third generation, which can regulate the tumor microenvironment and recruit effector cells to kill tumors by locally releasing cytokines. The fifth generation CAR is a universal CAR, which uses gene editing tools to knock out TCR, MHC and related signal pathway genes on allogeneic T cells to prevent host rejection of allogeneic CAR-T. In addition, knocking out T cell inhibitory signal molecules such as PD-1 and CTLA4 can further enhance the function of CAR-T cells.

[0004] CAR virus packaging is usually lentivirus packaging, and the lentivirus genome is double-stranded RNA, which has the ability to infect both dividing and non-dividing cells. Recombinant lentiviral vectors are tool vectors developed based on HIV-1 (human immunodeficiency virus type 1). Its toxic genes have been removed and replaced by exogenous target genes. Lentivirus can integrate the target gene into the host genome and can stably express it in cell lines to screen stable cell lines.

[0005] Common lentiviral packaging systems include the second-generation lentiviral system (i.e., the three-plasmid system) and the third-generation lentiviral system (i.e., the four-plasmid system). The second-generation lentiviral system contains packaging plasmids, envelope plasmids, and transfer plasmids. The packaging plasmid encodes the ag, pol, rev, and tat genes. The four virulence genes vif, vpr, vpu, and nef have been deleted, eliminating the pathogenicity of the virus but not affecting the titer and infectivity of the virus. The envelope plasmid contains the VSV-G sequence (to replace the env sequence) and encodes the envelope protein. The transfer plasmid contains the LTRs and Ψ packaging signal of the virus. Unless there is an internal promoter, the target sequence of this transfer plasmid is driven by the 5'LTR, which is a weak promoter that requires Tat element activation. All second-generation lentiviral transfer plasmids must use the second-generation lentiviral packaging system because its LTR is Tat-dependent. The third-generation lentiviral packaging system cannot package the second-generation transfer plasmid because Tat is not expressed. The third-generation lentiviral packaging system divides the packaging plasmid into two plasmids, one encoding rev and the other encoding gag and pol, which improves the safety of the packaging system but reduces the efficiency of virus packaging. The third-generation lentiviral packaging system can only package the third-generation lentiviral transfer plasmid, but the second-generation lentiviral packaging system can package both the second-generation and the third-generation lentiviral transfer plasmids.

[0006] The current CAR virus packaging method has low efficiency. In order to improve the transfection efficiency of the CAR virus, the present invention is carried out. In addition, there are few reports on the four-plasmid packaging system based on the second-generation lentiviral packaging system. Summary of the invention

[0007] The object of the present invention is to provide a packaging method for CAR virus, using a second-generation lentiviral packaging system and further improving the virus transfection efficiency. The transfer plasmid is a second-generation transfer plasmid that requires Tat element activation, the packaging plasmid is a second-generation packaging plasmid pCMV-dR8.91 containing ag, pol, rev and tat, the envelope plasmid is a second-generation envelope plasmid pCMV-BaEV, and the plasmid pAdVAntage that is beneficial to improving the transfection efficiency is added, and the packaging efficiency of the CAR virus is improved through this system.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for packaging a CAR virus, comprising the following steps:

[0010] (1) Plasmid pCMX-dR8.91, plasmid pCMV-BaEV, plasmid pAdVAntage and opti-MEM medium were mixed and allowed to stand to obtain material 1;

[0011] (2) mixing material 1 and PEI transfection reagent, and allowing to stand to obtain material 2;

[0012] (3) Material 2 is mixed with 293T cells, the culture medium is replaced after cultivation, and the virus is harvested after continued cultivation.

[0013] Preferably, the method further comprises the step of concentrating the virus after harvesting the virus;

[0014] The virus concentration method comprises: mixing the harvested virus with 4-6*PEG 8000, reacting at 2-6°C for 10-14h, centrifuging at 3500-4500g and 2-6°C for 35-45min, removing the supernatant, mixing the precipitate with DMEM serum-free culture medium, and obtaining concentrated virus;

[0015] The volume ratio of the precipitate to the DMEM serum-free culture medium is 1:90-110.

[0016] Preferably, the dosage ratio of the plasmid pCMX-dR8.91, plasmid pCMV-BaEV, plasmid pAdVAntage and opti-MEM culture medium in step (1) is: 2.5-3.5 μg: 0.5-1.5 μg: 0.4-0.6 μg: 0.9-1.1 mL.

[0017] Preferably, the standing time in step (1) is 4 to 6 minutes.

[0018] Preferably, the concentration of the PEI transfection reagent in step (2) is 0.8-1.2 μg / mL, and the volume ratio of the PEI transfection reagent to the opti-MEM culture medium is 28-29:1000.

[0019] Preferably, the standing time in step (2) is 13 to 17 minutes.

[0020] Preferably, the amount of material 2 in step (3) is: per 1×10 8 0.8-1.2 mL of material 2 was used for each 293T cell. The 293T cells were cultured for 12-24 h before mixing, and a new DMEM serum-free medium was replaced before mixing.

[0021] Preferably, the culture conditions in step (3) are 35-39° C. and 4-6% CO 2 , and the culture time is 6-8 h.

[0022] Preferably, DMEM serum-containing medium is used when replacing the culture medium in step (3).

[0023] Preferably, the virus is harvested in step (3) at 46 to 50 hours and 70 to 74 hours after material 2 is mixed with 293T cells.

[0024] The virus was harvested by filtration using a 0.45 μm filter membrane.

[0025] The present invention provides a packaging method for CAR virus, comprising the following steps: (1) mixing plasmid pCMX-dR8.91, plasmid pCMV-BaEV, plasmid pAdVAntage and opti-MEM culture medium, and obtaining material 1 after standing; (2) mixing material 1 and PEI transfection reagent, and obtaining material 2 after standing; (3) mixing material 2 with 293T cells, replacing the culture medium after culturing, and harvesting the virus after continuing to culture. The present invention adopts a second-generation lentivirus packaging system, the transfer plasmid is a second-generation transfer plasmid that requires Tat element activation, the packaging plasmid is a second-generation packaging plasmid pCMV-dR8.91 containing ag, pol, rev and tat, the envelope plasmid is a second-generation envelope plasmid pCMV-BaEV, and the plasmid pAdVAntage that is beneficial to improving the transfection efficiency is added, and the packaging efficiency of the CAR virus is improved by this system. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram of the virus packaging efficiency of the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a method for packaging a CAR virus, comprising the following steps:

[0028] (1) Plasmid pCMX-dR8.91, plasmid pCMV-BaEV, plasmid pAdVAntage and opti-MEM medium were mixed and allowed to stand to obtain material 1;

[0029] (2) mixing material 1 and PEI transfection reagent, and allowing to stand to obtain material 2;

[0030] (3) Material 2 is mixed with 293T cells, the culture medium is replaced after cultivation, and the virus is harvested after continued cultivation.

[0031] In the present invention, the step of harvesting the virus preferably further includes a step of concentrating the virus;

[0032] The method for concentrating the virus is preferably: mixing the harvested virus with 4-6*PEG 8000, reacting at 2-6°C for 10-14h, centrifuging at 3500-4500g and 2-6°C for 35-45min, removing the supernatant, mixing the precipitate with DMEM serum-free medium to obtain concentrated virus, and further preferably: mixing the harvested virus with 5*PEG 8000, reacting at 4°C for 12h, centrifuging at 4000g and 4°C for 40min;

[0033] In the present invention, the volume ratio of the precipitate to the DMEM serum-free medium is preferably 1:90-110, and more preferably 1:100.

[0034] In the present invention, the dosage ratio of the plasmid pCMX-dR8.91, plasmid pCMV-BaEV, plasmid pAdVAntage and opti-MEM culture medium in step (1) is preferably: 2.5-3.5 μg: 0.5-1.5 μg: 0.4-0.6 μg:: 0.9-1.1 mL, and further preferably: 3 μg: 1 μg: 0.5 μg: 1 mL.

[0035] In the present invention, the standing time in step (1) is preferably 4 to 6 minutes, more preferably 5 minutes.

[0036] In the present invention, the concentration of the PEI transfection reagent in step (2) is preferably 0.8-1.2 μg / mL, and more preferably, the volume ratio of the PEI transfection reagent is preferably 28-29:1000, and more preferably 28.5:1000.

[0037] In the present invention, the standing time in step (2) is preferably 13 to 17 minutes, more preferably 15 minutes.

[0038] In the present invention, the amount of material 2 in step (3) is preferably: 8 0.8-1.2 mL of material 2 is used for each 293T cell, and more preferably, 0.8-1.2 mL of material 2 is used for each 1×10 8 1 mL of material 2 is used for each 293T cell. The 293T cells are preferably cultured for 12 to 24 hours, more preferably for 18 hours before mixing. A new DMEM serum-free medium is replaced before mixing.

[0039] In the present invention, the culture conditions in step (3) are preferably 35-39° C., 4-6% CO 2 , more preferably 37° C., 5% CO 2 , and the culture time is preferably 6-8 h, more preferably 7 h.

[0040] In the present invention, when replacing the culture medium in step (3), preferably DMEM serum-containing culture medium is used.

[0041] In the present invention, the time for harvesting the virus in step (3) is preferably 46 to 50 hours and 70 to 74 hours after material 2 is mixed with 293T cells, and more preferably 48 hours and 72 hours.

[0042] When harvesting the virus, preferably a 0.45 μm filter membrane is used for filtration.

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] The raw materials and reagents used in the following examples:

[0045] pCMX-dR8.91, pAdVAntage, pCMV-BaEV are universal plasmids, and pHIV-EGFP is the control plasmid in the target plasmid, which were purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0046] The brand of opti-MEM medium is Gibco, catalog number 31985070;

[0047] PEI is brand MCE, item number HY-K2014;

[0048] The brand of DMEM serum-free culture medium is cytiva, product number SH30243.FS.

[0049] Example 1 Preparation before virus packaging

[0050] 1. Prepare the target plasmid, packaging plasmid pCMX-dR8.91, pAdVAntage, pCMV-BaEV, control plasmid pHIV-EGFP, culture medium opti-MEM, and cationic polymer transfection reagent PEI.

[0051] Among them: pCMX-dR8.91, pAdVAntage, pCMV-BaEV are universal plasmids, pHIV-EGFP is the control plasmid in the target plasmid, purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0052] The culture medium is opti-MEM, brand Gibco, item number 31985070; PEI, brand MCE, item number HY-K2014.

[0053] 2. After 293T cells are subcultured for 24 hours, the cell density reaches 90% of the culture dish. Replace with fresh DMEM serum-free medium before virus packaging. DMEM serum-free medium brand cytiva, catalog number SH30243.FS.

[0054] Example 2 Virus packaging and harvesting

[0055] 1. Virus packaging

[0056] (1) Select a 10 cm culture dish to culture 293T cells (the number of 293T cells in each culture dish is 1×10 8 The plasmids were mixed with 1 ml of the culture medium opti-MEM according to the following proportions.

[0057] Proportion1:pHIV-EGFP:pCMX-dR8.1:pCMV-BaEV:pAdVAntage=3μg:3μg:1.5μg:0.35μg;

[0058] Proportion2:pHIV-EGFP:pCMX-dR8.91:pCMV-BaEV:pAdVAntage=2μg:2μg:1μg:0.25μg;

[0059] Proportion3: pHIV-EGFP: pCMX-dR8.91: pCMV-BaEV: pAdVAntage=4μg: 4μg: 2μg: 0.5μg;

[0060] Proportion4: pHIV-EGFP: pCMX-dR8.91: pCMV-BaEV: pAdVAntage=2.4μg: 3.6μg: 1.2μg: 0.3μg;

[0061] Proportion5: pHIV-EGFP: pCMX-dR8.91: pCMV-BaEV: pAdVAntage=3.6μg: 2.4μg: 1.2μg: 0.8μg;

[0062] Proportion6: pHIV-EGFP: pCMX-dR8.91: pCMV-BaEV: pAdVAntage=5μg: 3μg: 1μg: 0.5μg.

[0063] After mixing, blow and beat evenly and let stand for 5 minutes;

[0064] (2) After standing, add 28.5 μl of cationic polymer transfection reagent PEI (concentration 1 μg / ml), shake well and let stand for 15 min;

[0065] (3) Add the above mixed reagent to be transfected to a 293T culture dish that has been replaced with fresh DMEM medium (serum-free), shake well and place in a 37°C 5% CO2 incubator for culture. After 8 hours, replace with DMEM medium with serum;

[0066] (4) Collect the virus for the first time 48 hours after packaging, filter it through a 0.45 μm filter membrane, and then put it into a new sterile centrifuge tube;

[0067] (5) Collect the virus for the second time 72 hours after packaging.

[0068] 2. Virus Concentration

[0069] (1) Add 5*PEG 8000 (Sigma-Aldrich, Catalog No. 25322-68-3) to the collected virus solution for concentration and invert evenly;

[0070] (2) Centrifuge at 4000 g, 4°C for 40 min, remove the supernatant, collect the precipitate, mix the precipitate with 100 times the volume of DMEM (serum-free) culture medium, and store at -80°C.

[0071] Results

[0072] The packaging efficiency results of different groups in Example 2 are as follows Figure 1 As shown. Figure 1 As shown, according to the cell fluorescence ratio, the transfection efficiency of Proportion 1 is about 5%; the transfection efficiency of Proportion 2 is about 5%; the transfection efficiency of Proportion 3 is about 30%; the transfection efficiency of Proportion 4 is about 20%; the transfection efficiency of Proportion 5 is about 40%; and the transfection efficiency of Proportion 6 is above 80%.

[0073] As can be seen from the above embodiments, the present invention provides a packaging method for CAR virus, comprising the following steps: (1) mixing plasmid pCMX-dR8.91, plasmid pCMV-BaEV, plasmid pAdVAntage and opti-MEM culture medium, and obtaining material 1 after standing; (2) mixing material 1 and PEI transfection reagent, and obtaining material 2 after standing; (3) mixing material 2 with 293T cells, replacing the culture medium after culturing, and harvesting the virus after continuing to culture. The present invention adopts a second-generation lentiviral packaging system, the transfer plasmid is a second-generation transfer plasmid that requires Tat element activation, the packaging plasmid is a second-generation packaging plasmid pCMV-dR8.91 containing ag, pol, rev and tat, the envelope plasmid is a second-generation envelope plasmid pCMV-BaEV, and the plasmid pAdVAntage that is beneficial to improving the transfection efficiency is added, and the packaging efficiency of the CAR virus is improved by this system.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for packaging a CAR virus, characterized in that: The steps include: (1) Plasmid pCMX-dR8.91, plasmid pCMV-BaEV, plasmid pAdVAntage and opti-MEM medium were mixed and allowed to stand to obtain material 1; (2) mixing material 1 and PEI transfection reagent, and allowing to stand to obtain material 2; (3) Material 2 is mixed with 293T cells, the culture medium is replaced after cultivation, and the virus is harvested after continued cultivation.

2. The packaging method according to claim 1, characterized in that: The step of harvesting the virus also includes concentrating the virus; The virus concentration method comprises: mixing the harvested virus with 4-6*PEG 8000, reacting at 2-6°C for 10-14h, centrifuging at 3500-4500g and 2-6°C for 35-45min, removing the supernatant, mixing the precipitate with DMEM serum-free culture medium, and obtaining concentrated virus; The volume ratio of the precipitate to the DMEM serum-free culture medium is 1:90-110.

3. The packaging method according to claim 1 or 2, characterized in that: The dosage ratio of the plasmid pCMX-dR8.91, plasmid pCMV-BaEV, plasmid pAdVAntage and opti-MEM culture medium in step (1) is: 2.5-3.5 μg: 0.5-1.5 μg: 0.4-0.6 μg: 0.9-1.1 mL.

4. The packaging method according to claim 3, characterized in that: The standing time in step (1) is 4 to 6 minutes.

5. The packaging method according to claim 4, characterized in that: The concentration of the PEI transfection reagent in step (2) is 0.8-1.2 μg / mL, and the volume ratio of the PEI transfection reagent to the opti-MEM culture medium is 28-29:1000.

6. The packaging method according to claim 5, characterized in that: The standing time in step (2) is 13 to 17 minutes.

7. The packaging method according to claim 6, characterized in that: The amount of material 2 in step (3) is: per 1×10 8 0.8-1.2 mL of material 2 was used for each 293T cell. The 293T cells were cultured for 12-24 h before mixing, and a new DMEM serum-free medium was replaced before mixing.

8. The packaging method according to claim 7, characterized in that: The culture conditions in step (3) are 35-39° C. and 4-6% CO 2 , and the culture time is 6-8 hours.

9. The packaging method according to claim 8, characterized in that: When replacing the culture medium in step (3), DMEM serum-containing culture medium is used.

10. The packaging method according to claim 9, characterized in that: The virus is harvested at 46-50h and 70-74h after material 2 is mixed with 293T cells in step (3). The virus was harvested by filtration using a 0.45 μm filter membrane.

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