Reagent composition for improving lentivirus infection NK cell efficiency and application thereof

Abstract

The invention discloses a reagent composition for improving lentivirus infection NK cell efficiency and application thereof. The reagent combination comprises a suspension culture type 293T cell, a lentivirus transfer plasmid, a lentivirus packaging plasmid and a lentivirus packaging helper plasmid. The lentivirus prepared by using the suspension culture type 293T cell package can significantly improve the NK cell infection efficiency of the lentivirus, and compared with the lentivirus prepared by using adherent cells, the virus titer is higher, the culture medium does not need to be added with serum, and the use amount of the lentivirus in a subsequent infection experiment can be reduced, so that the cost is saved, and the cost is reduced. In addition, operation is easy and convenient, time is saved, and large-scale production is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a reagent combination for improving the efficiency of lentivirus infection of NK cells and its application. Background Art

[0002] With the continuous progress of technology, tumor treatment methods have experienced a development process from surgery, radiotherapy, chemotherapy to targeted drugs and immunotherapy. In immunotherapy, CAR-T immunotherapy has achieved amazing efficacy in hematological malignancies. However, CAR-T therapy is time-consuming and expensive in the large-scale production process, and there are still problems such as insufficient efficacy and inability to effectively target the target in the treatment of solid tumors with a higher incidence. Research shows that CAR-NK therapy has potential advantages in the treatment of solid tumors, is expected to reduce side effects such as cytokine release syndrome (CRS) and neurotoxicity that occur in CAR-T therapy, and has unique advantages such as higher safety, multiple target cell killing pathways, and easy preparation of universal cell products. Therefore, CAR-NK therapy has a broader clinical application prospect.

[0003] Although CAR-NK therapy has a broad application prospect, it also faces various challenges. Among them, when preparing CAR-NK cells, how to effectively deliver the CAR gene into NK cells is a major problem. Currently, CAR gene delivery is divided into two major systems: virus-free delivery systems and virus delivery systems. Virus-free delivery systems mainly use electroporation technology, which can effectively deliver the CAR gene into cells. However, for the CAR-NK cells prepared by this method, the expression of the CAR gene is transient, and the treatment effect is limited in clinical applications. In addition, the electroporation method can cause a relatively high cell mortality rate, which is another major drawback. Virus delivery systems mainly use retroviruses and lentiviruses. Currently, lentiviruses are mainly used to prepare CAR-T and CAR-NK cells. However, lentiviruses have a low infection efficiency in immune cells, and in the process of preparing CAR-NK cells, the infection efficiency cannot be effectively improved by increasing the virus dosage, and too high a virus dosage will cause slow cell proliferation, and may even affect the killing effect of NK cells on tumor cells and reduce the clinical efficacy. Therefore, how to effectively improve the infection efficiency of lentiviruses on NK cells is the key issue for the effective clinical translational application of CAR-NK cells.

[0004] The prior art mainly improves the infection efficiency of lentivirus in NK cells through two methods: replacing the viral envelope protein and adding co-infection reagents. Lentivirus enters cells by recognizing the corresponding receptors on the cell surface through the envelope protein on its surface and entering the cells by means of membrane fusion or endocytosis. Both RD114 and BaEV can recognize the sodium-dependent neutral amino acid transporter ASCT-2. Therefore, the envelope proteins of RD114 and BaEV can be used to improve the lentivirus infection efficiency of the above cells. Compared with RD114, BaEV can also recognize ASCT-1, which makes its application more extensive. However, this method is relatively complex and has low repeatability, making it difficult to promote and apply. When lentivirus infects NK cells, adding co-infection reagents can effectively improve the infection efficiency. For example, CN108893493A discloses a method for improving the transfection efficiency of NK cells. When NK cells are infected with lentivirus, co-infection agent 1 is added at the first infection while adding a lentiviral vector containing CAR, and co-infection agents 2, 3, and 4 are added at the second infection while adding a lentiviral vector containing CAR; the co-infection agent 1 is a solution containing polybrene; the co-infection agent 2 is a solution containing IL2; the co-infection agent 3 is a solution containing IL12; the co-infection agent 4 is a solution containing PHA, but the co-infection reagents used have a high cost and complex operations.

[0005] In summary, how to develop a method for lentivirus to infect NK cells with high efficiency, low cost, and easy to promote and apply is one of the urgent problems to be solved in the field of CAR-NK cell application. Summary of the Invention

[0006] In view of the deficiencies of the prior art and the actual needs, the present invention provides a reagent combination for improving the efficiency of lentivirus infecting NK cells and its application, aiming to improve the efficiency and safety of lentivirus infecting NK cells, while reducing costs and simplifying operations.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a reagent combination for improving the efficiency of lentivirus infecting NK cells, characterized in that the reagent combination includes suspension-cultured 293T cells, a lentiviral transfer plasmid, a lentiviral packaging plasmid, and a lentiviral packaging auxiliary plasmid.

[0009] The present invention discovers that using suspension-cultured 293T cells to package and prepare lentivirus can significantly improve the efficiency of lentivirus infecting NK cells, and the virus titer of the lentivirus prepared using adherent cells is higher. The amount of lentivirus used in subsequent infection experiments can be reduced, thus saving costs, and then designing a reagent combination for improving the efficiency of lentivirus infecting NK cells, providing new ideas and means for the genetic modification of NK cells.

[0010] It is understood that the lentivirus preparation plasmids commonly used in the art are all applicable to the present invention. In addition, the suspension-cultured 293T cells described in the present invention refer to 293T cells that can grow in suspension, and the suspension-cultured 293T cells prepared by the commonly used preparation methods in the art are all applicable to the present invention and should be within the protection scope of the present invention.

[0011] Preferably, the lentiviral packaging plasmid includes the pMD2.G plasmid containing the VSVG sequence.

[0012] Preferably, the lentiviral packaging auxiliary plasmid includes the psPAX2 plasmid.

[0013] Preferably, the lentiviral transfer plasmid further contains the CAR structural sequence.

[0014] In the present invention, the CAR structural sequence can be inserted into the lentiviral transfer plasmid as the target gene, and then CAR-NK cells can be prepared. The CAR structure refers to the chimeric antigen receptor structure well known in the art, which usually consists of 4 main parts: an extracellular target antigen-binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains.

[0015] Preferably, the lentiviral packaging plasmid further contains the coding gene of the BaEV envelope protein and / or the RD114 envelope protein.

[0016] In the present invention, the lentivirus using the BaEV envelope protein or the RD114 envelope protein can further improve the infection efficiency.

[0017] In the present invention, using suspension-cultured 293T cells to package lentivirus can significantly improve the infection efficiency, and further improving the infection efficiency can be achieved by cooperating with specific co-infection agents.

[0018] Preferably, the reagent further includes a co-infection agent.

[0019] Preferably, the co-infection agent includes any one or a combination of at least two of recombinant human fibrin fragment (RetroNectin), polybrene, or Vectofusin-1 (a histidine-rich cationic amphiphilic short peptide).

[0020] In the second aspect, the present invention provides the application of the reagent combination for improving the lentivirus infection efficiency of NK cells described in the first aspect in the lentivirus infection of NK cells.

[0021] In the third aspect, the present invention provides a method for improving the lentivirus infection efficiency of NK cells. The method for improving the lentivirus infection efficiency of NK cells includes preparing lentivirus using the reagent combination for improving the lentivirus infection efficiency of NK cells described in the first aspect and infecting NK cells.

[0022] In the present invention, lentivirus is packaged and prepared using suspension-cultured 293T cells, which can significantly improve the efficiency of lentivirus infection of NK cells, and has a higher virus titer compared to the lentivirus prepared using adherent cells, reducing the amount of lentivirus used in subsequent infection experiments, thereby saving costs.

[0023] Preferably, the method for improving the efficiency of lentivirus infection of NK cells includes:

[0024] Insert the target gene into the lentivirus transfer plasmid, co-transfect the suspension-cultured 293T cells with the lentivirus packaging plasmid and the lentivirus packaging helper plasmid for lentivirus packaging, collect the lentivirus; use the lentivirus to infect NK cells.

[0025] Preferably, the preparation method of the suspension-cultured 293T cells includes the following steps:

[0026] (1’) Resuscitate 293T cells in a medium containing 85 - 95% DMEM and 5 - 15% FBS (the % refers to volume percentage, and the total is 100%, and the same applies hereinafter), and culture until 80% - 90% confluence;

[0027] (2’) Discard the medium, add trypsin for digestion, centrifuge the digested cells, and collect the cell pellet;

[0028] (3’) Resuspend the cell pellet with a medium containing 75 - 85% DMEM + 5 - 15% OPM + 5 - 15% FBS, culture until 80% - 90% confluence, and collect the cell pellet using the method of step (2’);

[0029] (4’) Resuspend the cell pellet with a medium containing 45 - 55% DMEM + 35 - 45% OPM + 5 - 15% FBS, and culture until 80% - 90% confluence, and collect the cell pellet using the method of step (2’);

[0030] (5’) Resuspend the cell pellet with a medium containing 5 - 15% DMEM + 75 - 85% OPM + 5 - 15% FBS, and culture until 80% - 90% confluence, and collect the cell pellet using the method of step (2’);

[0031] (6’) Resuspend the cell pellet with a medium containing 85 - 95% OPM + 5 - 15% FBS, and culture until 80% - 90% confluence, and collect the cell pellet using the method of step (2’);

[0032] (7’) Resuspend the cell pellet with a medium containing 95 - 99% OPM + 1 - 5% FBS, and culture until 80% - 90% confluence, and collect the cell pellet using the method of step (2’);

[0033] (8’) Resuspend the cell pellet completely with OPM medium and culture for 48 - 72 h, at which time the cells float in sheets;

[0034] (9’) After pipetting the above cells evenly, continue to culture until the cells are completely transformed into a suspended state, expand the culture, and restore the cell viability to over 95%.

[0035] The present invention designs a method for preparing suspension - cultured 293T cells, with a shorter domestication period than traditional methods, being simple and easy to operate, and having a higher virus titer of the lentivirus prepared compared to that using adherent cells, and the culture medium does not require the addition of serum.

[0036] Preferably, the culture medium of the suspension - cultured 293T cells does not contain animal serum.

[0037] In the present invention, when preparing lentivirus using suspension - cultured 293T cells, compared with using adherent 293T cells, the culture medium does not require the addition of animal serum (such as 10% FBS). Therefore, in subsequent clinical applications, the lentivirus packaged by suspension cells can effectively avoid potential safety risks such as immune reactions that may be caused by animal - derived serum, significantly improving safety.

[0038] Preferably, the method for constructing the lentiviral packaging plasmid includes replacing the VSVG sequence in pMD2.G with the coding sequence of BaEV envelope protein and / or RD114 envelope protein.

[0039] Preferably, the target gene includes the coding gene of the CAR structure.

[0040] Preferably, the step of infecting NK cells further includes adding a co - infecting agent.

[0041] Preferably, the co - infecting agent includes any one or a combination of at least two of RetroNectin, Polybrene, or Vectofusin - 1.

[0042] [[ID=((27))]]As a preferred technical solution, the method for improving the efficiency of lentivirus infecting NK cells includes the following steps: As a preferred technical solution, the method for improving the efficiency of lentivirus infecting NK cells includes the following steps:

[0043] (1) Insert the target gene into the lentiviral transfer plasmid, co - transfect the suspension - cultured 293T cells with the lentiviral packaging plasmid containing the coding gene of BaEV envelope protein and / or RD114 envelope protein and the lentiviral packaging helper plasmid for lentiviral packaging, and collect the lentivirus;

[0044] (2) Mix the lentivirus with the co - infecting agent and NK cells for infection.

[0045] Compared with the prior art, the present invention adopts the following technical solutions:

[0046] The present invention discovers that preparing lentivirus by packaging with suspension culture type 293T cells can significantly improve the efficiency of lentivirus infecting NK cells, achieve efficient infection without the use of co-infection agents, and has a higher virus titer compared to lentivirus prepared with adherent cells and does not require adding serum to the culture medium, which can reduce the usage amount of lentivirus in subsequent infection experiments, thereby saving costs and improving safety. In addition, the operation is simple, time-saving, and is beneficial to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a microscopic examination result diagram of domesticating adherent cell 293T into suspension cell AZ293TS for Example 1. Among them, Figure A is 293T cells growing adherently, and Figure B is AZ293TS cells growing in suspension after domestication. The scale bar is 100 μm.

[0048] Figure 2 It is a fluorescence photograph of lentivirus expressing GFP (VSVG envelope protein) prepared by an adherent or suspension system for Example 2 infecting HT1080 cells under the condition of adding a co-infection reagent (Polybrene). The scale bar is 100 μm.

[0049] Figure 3A It is a fluorescence photograph of lentivirus expressing GFP (BaEV envelope protein) prepared by an adherent or suspension system for Example 3 infecting NK-92MI cells under the condition of not adding or adding a co-infection reagent (Vectofusin-1). The illustrated results show that the suspension system is superior to the adherent system.

[0050] Figure 3B It is a flow cytometry analysis result diagram of lentivirus expressing GFP (BaEV envelope protein) prepared by an adherent or suspension system for Example 3 infecting NK-92MI cells under the condition of not adding or adding a co-infection reagent (Vectofusin-1). Under the condition of adding the co-infection reagent Vectofusin-1, the infection efficiency of the lentivirus (BaEV envelope protein) prepared by the suspension system for NK-92MI cells can reach 96%, and under the condition of not adding the co-infection reagent Vectofusin-1, the infection efficiency for NK-92MI cells can also reach 82%.

[0051] Figure 4 It is a result diagram of lentivirus expressing GFP (VSVG or BaEV envelope protein) prepared by a suspension system for Example 4 infecting NK-92MI cells under the condition of not adding or adding different co-infection reagents (Polybrene or RetroNectin). Figure A is a fluorescence photograph, and Figure B is a flow cytometry analysis result diagram.

[0052] Figure 5The figure showing the results of infecting NK-92MI cells with the lentivirus expressing CD19 CAR-P2A-GFP (BaEV envelope protein) prepared by the suspension system in Example 5 in the presence of the co-staining reagent Polybrene. Panel A is the fluorescence photograph, and panel B is the flow cytometry analysis result.

[0053] Figure 6 The figure showing the results of infecting NK-92MI cells with the lentivirus expressing GFP (RD114 envelope protein) prepared by the suspension system in Example 6 without or in the presence of different co-staining reagents (Polybrene or RetroNectin). Panel A is the fluorescence photograph, and panel B is the flow cytometry analysis result. Detailed implementation manners

[0054] To further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0055] For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0056] Term explanations

[0057] Lentivirus: Belongs to the Retroviridae family, with a particle size of 80 - 120 nm, having an envelope and containing two identical single-stranded positive-strand RNAs inside. Before infected individuals show typical clinical symptoms, most experience a latent period of up to several years, and then develop the disease slowly. Therefore, these pathogens are called lentiviruses. Human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIA), and feline immunodeficiency virus (FIV) all belong to lentiviruses.

[0058] Lentiviral Vector (LV): A gene transfer vector developed based on HIV-1 (human immunodeficiency virus type I). Different from general retroviral vectors, it has the ability to infect both dividing and non-dividing cells. LV can stably integrate the exogenous genes it carries into the host cell genome, enabling the exogenous genes to be persistently expressed in the host cells. LV can effectively infect various types of cells such as neuronal cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells. LV is widely used in biological and clinical treatment research, such as: construction of stable cell lines, screening of gRNA libraries, preparation of transgenic animals, cell and gene therapy research, etc.

[0059] Tumor: It refers to a new growth formed by the hyperplasia of local tissue cells under the action of various tumorigenic factors. According to the cell characteristics of the new growth and the degree of harm to the body, tumors are divided into two major categories: benign tumors and malignant tumors. Malignant tumors can be further divided into carcinomas and sarcomas.

[0060] Immune cells: Cells that participate in or are related to immune responses. They include lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Lymphocytes are widely distributed in the body, mainly including T cells, B cells, and NK cells.

[0061] NK cell: The full name is Natural Killer Cell, which has dual functions of cytotoxicity and immune regulation. Like T cells, NK cells have numerous receptors on their surfaces, which are divided into two categories: activating receptors and inhibitory receptors. When an NK cell contacts a healthy cell, the MHC molecules of the healthy cell will activate the inhibitory receptors of the NK cell, preventing the activation of the NK cell from accidentally injuring healthy cells. However, when an NK cell contacts a tumor cell with downregulated MHC molecules, the lack of inhibitory signals can cause the NK cell to kill the tumor. At the same time, when the activation signals of certain tumor cells are upregulated, it will also cause the activation of NK cells and the killing of tumor cells. All in all, NK cells are an indispensable part of the anti-cancer defense line in the human body and directly participate in the process of killing tumor cells in the human body.

[0062] CAR-T: Chimeric Antigen Receptor (CAR) is a receptor protein that endows immune cells with new abilities to target specific antigen proteins. Chimeric Antigen Receptor T Cell (CAR-T) therapy refers to the transfer of genetic material with specific antigen recognition domains and T cell activation signals into T cells through genetic engineering modification technology, enabling T cells to directly bind to specific antigens on the surface of tumor cells and be activated. By releasing perforin, granzyme B, etc., it directly kills tumor cells. At the same time, it also recruits endogenous immune cells in the human body to kill tumor cells by releasing cytokines, thereby achieving the purpose of treating tumors. Moreover, it can also form immune memory T cells, thereby obtaining a specific long-term anti-tumor mechanism. CAR-T cell immunotherapy has achieved great success in the treatment of hematological tumors such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and lymphoma.

[0063] CAR-NK: It refers to genetically engineered modification technology that enables NK cells to express CAR, which can break through the limitations of inhibitory receptors and activate NK cells, thereby enhancing the specific killing of target cells by NK cells. Although CAR-T cell immunotherapy has developed rapidly, there are still some deficiencies in clinical applications. For example, it shows very low efficacy in the treatment of solid tumors, and there are also problems such as graft-versus-host disease (GVHD) and cytokine release syndrome. The latest research shows that NK cells expressing CAR may overcome the above defects of CAR-T cells and show significant anti-tumor effects. CAR-NK has broad prospects in tumor immunotherapy.

[0064] In the specific embodiments of the present invention, 293T cells (Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; HEK293T), DMEM medium (KEL; DMEM High Glucose), FBS medium (Viva Cell; Fetal Bovine Serum), and OPM medium (OPM; OPM-293CD05 Medium) are used.

[0065] In the specific embodiments of the present invention, taking GFP protein or CD19CAR-P2A-GFP protein as the target protein as an example, the infection effect is verified.

[0066] Example 1

[0067] In this example, suspension-adapted 293T cells are domesticated.

[0068] (1) Resuscitate 293T cells in a T25 culture flask containing 90% DMEM and 10% FBS medium (for simplicity of writing, it can be written as 90% DMEM + 10% FBS medium, and % refers to volume percentage. For example, to prepare 100 mL of complete medium, 90 mL of DMEM and 10 mL of FBS are mixed and reserved for later use, and so on for the following). Place it in an incubator (37°C, 5% CO2) and culture until it reaches 80% - 90% confluence.

[0069] (2) Discard the medium, add 0.05% trypsin for digestion, add medium to terminate digestion, transfer the digested cells to a centrifuge tube, centrifuge at 1000 rpm for 5 min, and collect the cell pellet.

[0070] (3) Resuspend the cell pellet with 80% DMEM + 10% OPM + 10% FBS medium and transfer it to a T25 flask. Place it in an incubator (37°C, 5% CO2) and culture until it reaches 80% - 90% confluence. Collect the cell pellet using the method in (2).

[0071] (4) Resuspend the cell pellet with 50% DMEM + 40% OPM + 10% FBS medium and transfer it into a T25 flask. Incubate it in an incubator (37°C, 5% CO2) until it reaches 80% - 90% confluence. Collect the cell pellet using the method in (2).

[0072] (5) Resuspend the cell pellet with 10% DMEM + 80% OPM + 10% FBS medium and transfer it into a T25 flask. Incubate it in an incubator (37°C, 5% CO2) until it reaches 80% - 90% confluence. Collect the cell pellet using the method in (2).

[0073] (6) Resuspend the cell pellet with 90% OPM + 10% FBS medium and transfer it into a T25 flask. Incubate it in an incubator (37°C, 5% CO2) until it reaches 80% - 90% confluence. At this time, the cells are still adherent. Collect the cell pellet using the method in (2).

[0074] (7) Resuspend the cell pellet with OPM + 1% FBS medium and transfer it into a T25 flask. Incubate it in an incubator (37°C, 5% CO2) until it reaches 80% - 90% confluence. At this time, the cells are still adherent. Collect the cell pellet using the method in (2).

[0075] (8) Resuspend the cell pellet completely with OPM medium and transfer it into a T25 flask. Incubate it in an incubator (37°C, 5% CO2) for 60 h. At this time, the cells form a sheet and float.

[0076] (9) After gently pipetting the above cells to make them uniform, transfer them into a 125 mL shake flask at 5.0E+05 cells / mL. Incubate it on a shaker (37°C, 120 rpm, 8% CO2). The cells are completely transformed into a suspension state, and then expanded in culture. The cell viability is restored to more than 95%, and then cryopreserved. Name the cells AZ293TS cells. The microscopic examination results are as Figure 1 shown.

[0077] Example 2

[0078] In this example, lentivirus preparation and titer detection were carried out.

[0079] (1) Construct lentiviral packaging plasmids encoding BaEV or RD114 envelope proteins

[0080] The human - derived sequences were optimized and the genes BaEV or RD114 were synthesized conventionally (synthesized by Genewiz). The genes were cloned into the vector pMD2.G using recombinant methods. After digestion with enzymes and sequencing identification, lentiviral packaging plasmids pMD2.BaEV or pMD2.RD114 were constructed. The constructed plasmids were extracted and endotoxin - removed for lentiviral packaging.

[0081] (2) Construction of lentiviral transfer plasmids encoding GFP or CD19 CAR-P2A-GFP proteins

[0082] The lentiviral transfer plasmid encoding GFP protein uses the pGWLV10-new vector constructed by our company. The human-derived sequence is optimized and the gene CD19 CAR-P2A is synthesized routinely, with restriction enzyme sites 5' NheI and 3' EcoRI added. It is cloned into the vector pGWLV10-new through the restriction enzyme sites 5' NheI and 3' EcoRI, and after digestion and sequencing identification, the lentiviral transfer plasmid CD19 CAR-P2A-GFP in pGWLV10-new is constructed. The constructed plasmid is extracted and endotoxin is removed for lentiviral packaging.

[0083] (3) Lentivirus preparation

[0084] a) Adherent system

[0085] Prepare adherent cells 293T, and package the virus when the cell confluence reaches 80-90%. Mix the VSVG or BaEV packaging plasmid (5 μg), the transfer plasmid expressing GFP (8 μg), and the psPAX2 packaging plasmid (20 μg) and add them to a centrifuge tube containing Opti-MEM medium. Add the transfection reagent PEI to another centrifuge tube containing Opti-MEM medium. Mix the two tubes of solutions and let them stand in the dark at room temperature for 15 min, then add them to 25 mL of DMEM + 4% FBS medium, mix well and slowly add them to a T175 culture flask containing adherent cells 293T, and culture them in an incubator (37 °C, 5% CO2). Collect the virus supernatant after 48-72 h, after concentration and ultracentrifugation (25000 rpm, 8 °C, 2.5 h), resuspend the virus pellet with virus preservation solution, and filter and sterilize it through a 0.22 μm filter membrane to obtain the lentivirus expressing GFP (VSVG or BaEV envelope protein).

[0086] b) Suspension system

[0087] Prepare suspension cells AZ293TS. When the cell density reaches 3.0E+06 cells / mL, package the virus. Mix the VSVG, BaEV or RD114 packaging plasmid (5 μg), the transfer plasmid expressing GFP or CD19CAR-P2A-GFP (8.5 μg), and the psPAX2 packaging plasmid (20 μg), and add them to a centrifuge tube containing Opti-MEM medium. Add the transfection reagent PEI to another centrifuge tube containing Opti-MEM medium. Mix the two tubes of solutions, let them stand in the dark at room temperature for 15 min, then slowly add them to a shaking flask containing suspension cells AZ293TS, gently shake to mix evenly, and place them in a shaker (37 °C, 120 rpm, 8% CO2) for culture. After 48 - 72 h, collect the virus supernatant. After concentration and ultracentrifugation (25000 rpm, 8 °C, 2.5 h), resuspend the virus pellet with virus preservation solution, and filter and sterilize it through a 0.22 μm filter membrane to obtain lentivirus expressing GFP or CD19CAR-P2A-GFP (VSVG, BaEV or RD114 envelope protein).

[0088] (4) Titer detection

[0089] Detect the infectious activity titer by qPCR method: Use HT1080 cells as the tool cells. The basic process includes: cell plating, virus infection, cell harvesting, genomic extraction, and qPCR detection, etc. Specifically: One day before infection, add 8.0E+04 HT1080 cells to each well of a 24-well plate. The number of cells doubles on the day of infection (24 h after plating), which is 1.6E+05 cells per well. Add an appropriate volume of lentivirus to each well, add Polybrene (final concentration 8 μg / mL), and place it in an incubator (37 °C, 5% CO2) for culture. 72 h after virus infection, harvest the cells and extract the genome, and use the qPCR method to detect the virus titer (TU / mL).

[0090] Detect the expression activity titer by flow cytometry: Dilute NK-92MI cells with medium containing Polybrene (final concentration 8 μg / mL). Add 1.6E+05 NK-92MI cells to each well of a 24-well plate, and add different dilution volumes of lentivirus respectively, and place it in an incubator (37 °C, 5% CO2) for culture. 72 h after virus infection, use flow cytometry to detect the proportion of GFP-positive cells in each gradient, select the gradient with a GFP-positive cell proportion of 10 - 50%, and calculate the virus titer (TU / mL).

[0091] The titer data is shown in Table 1.

[0092] Table 1

[0093] sample Titer (TU / mL) Detection method Scope of use Adherent / VSVG / GFP 3.37E+08 qPCR method (HT1080) Example 2 Suspension / VSVG / GFP 1.21E+09 qPCR method (HT1080) Examples 2 and 4 Adherent / BaEV / GFP 2.64E+08 qPCR method (HT1080) Example 3 Suspension / BaEV / GFP 7.55E+08 qPCR method (HT1080) Examples 3 and 4 Suspension / BaEV / CD19CAR-P2A-GFP 3.17E+08 qPCR method (HT1080) Example 5 Suspension / RD114 / GFP 8.45E+08 qPCR method (HT1080) Example 6 Suspension / BaEV / GFP 5.67E+07 Flow cytometry (NK-92MI) Examples 3 and 4

[0094] Table 1 results show that the lentivirus titer in the suspension system packaging is better than that in the adherent system. Take 0.5 μL of the lentivirus expressing GFP (VSVG envelope protein) prepared by the above adherent system or suspension system respectively to infect 1.6E+05 HT1080 cells (adding the co-staining reagent Polybrene), and culture them in the incubator (37 °C, 5% CO2) using OPM-293CD05 medium. After 3 days, take pictures to observe the GFP fluorescence expression. Set the HT1080 cells not infected with lentivirus (adding the co-staining reagent Polybrene) as the control. The results show that the lentivirus expressing GFP (VSVG envelope protein) prepared by the adherent system or suspension system can effectively infect HT1080 cells and highly express GFP protein, and the infection effect of the suspension system is stronger ( Figure 2 ).

[0095] In addition, it should be noted that in the subsequent examples, the NK-92MI cells were infected at an MOI = 100 according to the qPCR infectious activity titer (suspension / BaEV / GFP). According to the expression activity titer detected by flow cytometry in the above table, it can be calculated that the actual MOI is 7.5, which is basically consistent with the literature (in the literature, flow cytometry was used for detection and the infection was at an MOI = 10). The relatively high virus MOI in the subsequent examples is related to the titer detection method. To avoid misunderstanding, it is hereby declared.

[0096] Example 3

[0097] Take the lentivirus expressing GFP (BaEV envelope protein) prepared by the adherent system or suspension system in Example 2 and infect 1.6E+05 NK-92MI cells at an MOI = 100 (with or without adding the co-staining reagent Vectofusin-1), and culture them in the incubator (37 °C, 5% CO2). After 3 days, use flow cytometry to detect the infection efficiency (proportion of GFP-positive cells). Set the NK-92MI cells not infected with lentivirus (with or without adding the co-staining reagent Vectofusin-1) as the control.

[0098] The results showed that the efficiency of lentivirus (BaEV envelope protein) prepared using the suspension system in infecting NK-92MI cells was significantly higher than that of the adherent system. It is worth noting that according to the method described in the literature (Bari R, Granzin M, Tsang KS, Roy A, Krueger W, Orentas R, et al. A Distinct Subset of Highly Proliferative and Lentiviral Vector (LV)-Transducible NK Cells Define a Readily Engineered Subset for Adoptive Cellular Therapy. Front Immunol (2019) 10:102975. doi:10.3389 / fimmu.2019.02001), when the lentivirus (BaEV envelope protein) prepared using the adherent system was used to infect NK-92MI cells, even in the presence of the co-infection reagent Vectofusin-1, the infection efficiency was only 2-3%. However, for the lentivirus (BaEV envelope protein) prepared using the suspension system of the present invention, in the presence of the co-infection reagent Vectofusin-1, the infection efficiency for NK-92MI cells could reach 96%; in the absence of the co-infection reagent Vectofusin-1, the infection efficiency for NK-92MI cells could also reach 82% ( Figure 3A and Figure 3B ), indicating that the strategy of packaging lentivirus using suspension cells in the present invention can significantly improve the efficiency of lentivirus in infecting NK cells.

[0099] Example 4

[0100] The GFP-expressing lentivirus (VSVG or BaEV envelope protein) prepared using the suspension system in Example 2 was used to infect 1.6E+05 NK-92MI cells at an MOI of 100 (without or with the co-infection reagents Polybrene or RetroNectin. If the co-infection reagent RetroNectin is used, the well plate needs to be pre-coated with RetroNectin in advance), and then cultured in an incubator (37 °C, 5% CO2). 3-5 days later, flow cytometry was used to detect the infection efficiency (the proportion of GFP-positive cells), and NK-92MI cells not infected with lentivirus (without or with the co-infection reagents Polybrene or RetroNectin) were set as controls.

[0101] The results showed that the efficiency of lentivirus with BaEV envelope protein prepared by the suspension system in infecting NK-92MI cells was significantly higher than that of lentivirus with VSVG envelope protein. Without adding a co-infection reagent, the infection efficiency of lentivirus with BaEV envelope protein prepared by the suspension system in infecting NK-92MI cells could reach 80%. Adding co-infection reagents such as Polybrene or RetroNectin could significantly improve the infection efficiency of lentivirus with BaEV envelope protein prepared by the suspension system in infecting NK-92MI cells. When adding the co-infection reagent Polybrene, the infection efficiency of lentivirus with BaEV envelope protein prepared by the suspension system in infecting NK-92MI cells could reach 98%. When adding the co-infection reagent RetroNectin, the infection efficiency of lentivirus with BaEV envelope protein prepared by the suspension system in infecting NK-92MI cells could reach 94%( Figure 4 ).

[0102] Since the use cost of the co-infection reagent Polybrene was significantly lower than that of Vectofusin-1 and RetroNectin, and the culture plate needed to be coated with RetroNectin before using the co-infection reagent RetroNectin, with cumbersome operations, using Polybrene as the co-infection reagent had cost advantages and was easy to operate.

[0103] Example 5

[0104] The lentivirus expressing CD19CAR-P2A-GFP (BaEV envelope protein) prepared by the suspension system in Example 2 was used to infect 1.6E+05 NK-92MI cells (adding the co-infection reagent Polybrene) at an MOI of 100 and cultured in an incubator (37 °C, 5% CO2). After 3 days, flow cytometry was used to detect the infection efficiency (the proportion of GFP-positive cells), and NK-92MI cells not infected with lentivirus were set as the control.

[0105] The results showed that the lentivirus expressing CD19CAR-P2A-GFP (BaEV envelope protein) prepared by the suspension system, when adding the co-infection reagent Polybrene, the infection efficiency in infecting NK-92MI cells could reach 97%( Figure 5 ).

[0106] Example 6

[0107] The lentivirus expressing GFP (RD114 envelope protein) prepared by the suspension system in Example 2 was used to infect 1.6E+05 NK-92MI cells at an MOI of 100 (without adding or adding the co-staining reagent Polybrene or RetroNectin. If the co-staining reagent RetroNectin is used, the well plate needs to be pre-coated with RetroNectin), and then cultured in an incubator (37 °C, 5% CO2). 3 to 5 days later, flow cytometry was used to detect the infection efficiency (the proportion of GFP-positive cells), and NK-92MI cells not infected with lentivirus were set as the control.

[0108] The results showed that in the case of not adding the co-staining reagent, the infection efficiency of the RD114 envelope protein lentivirus prepared by the suspension system on NK-92MI cells could reach 57%. In the case of adding the co-staining reagent Polybrene, the infection efficiency of the RD114 envelope protein lentivirus prepared by the suspension system on NK-92MI cells could reach 95%. In the case of adding the co-staining reagent RetroNectin, the infection efficiency of the RD114 envelope protein lentivirus prepared by the suspension system on NK-92MI cells could reach 98% ( Figure 6 ).

[0109] In summary, the present invention discovers that packaging and preparing lentivirus using suspension-cultured 293T cells can significantly improve the infection efficiency of lentivirus on NK cells, and compared with the lentivirus prepared using adherent cells, it has a higher virus titer and does not require adding serum to the culture medium, which can reduce the usage amount of lentivirus in subsequent infection experiments, thereby saving costs and improving safety. In addition, the operation is simple, time-saving, and conducive to large-scale production.

[0110] The applicant declares that the present invention uses the above examples 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 to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A reagent combination for improving the efficiency of lentivirus infection of NK cells, characterized in that The reagent combination includes suspension-cultured 293T cells, a lentiviral transfer plasmid, a lentiviral packaging plasmid, and a lentiviral packaging helper plasmid.

2. The reagent combination for improving the efficiency of lentivirus infection of NK cells according to claim 1, wherein, The lentiviral packaging plasmid includes the pMD2.G plasmid containing the VSVG sequence; Preferably, the lentiviral packaging helper plasmid includes the psPAX2 plasmid; Preferably, the lentiviral transfer plasmid further contains a CAR structural sequence and / or GFP protein; Preferably, the lentiviral packaging plasmid further contains the coding gene of BaEV envelope protein and / or RD114 envelope protein.

3. The reagent combination for improving the efficiency of lentivirus infecting NK cells according to claim 1 or 2, characterized in that The reagent combination further includes a co-infection agent; Preferably, the co-infection agent includes any one or a combination of at least two of RetroNectin, Polybrene, or Vectofusin-1.

4. Use of the reagent combination according to any one of claims 1-3 for improving the efficiency of lentiviral infection of NK cells in the lentiviral infection of NK cells.

5. A method for improving the efficiency of lentivirus infection of NK cells, characterized in that, The method for improving the efficiency of lentiviral infection of NK cells includes preparing lentivirus using the reagent combination according to any one of claims 1-3 for improving the efficiency of lentiviral infection of NK cells and infecting NK cells.

6. The method for improving the efficiency of lentivirus infection of NK cells according to claim 5, characterized in that, The method for improving the efficiency of lentiviral infection of NK cells includes: Inserting a target gene into the lentiviral transfer plasmid, co-transfecting suspension-cultured 293T cells with the lentiviral packaging plasmid and the lentiviral packaging helper plasmid, performing lentiviral packaging, and collecting the lentivirus; Infecting NK cells with the lentivirus.

7. The method for improving the efficiency of lentivirus infection of NK cells according to claim 6, wherein The construction method of the lentiviral packaging plasmid includes replacing the VSVG sequence in pMD2.G with the coding sequence of BaEV envelope protein and / or RD114 envelope protein.

8. The method for improving the efficiency of lentivirus infection of NK cells according to claim 6 or 7, characterized in that, The target gene includes the coding gene of the CAR structure.

9. The method for improving the efficiency of lentivirus infection of NK cells according to any one of claims 6-8, characterized in that, The step of infecting NK cells further includes adding a co-infection agent.

10. The method for improving the lentivirus infection efficiency of NK cells according to any one of claims 5-9, characterized in that The method includes the following steps: (1) Inserting a target gene into the lentiviral transfer plasmid, co-transfecting suspension-cultured 293T cells with the lentiviral packaging plasmid containing the coding gene of BaEV envelope protein and / or RD114 envelope protein and the lentiviral packaging helper plasmid, performing lentiviral packaging, and collecting the lentivirus; (2) Mixing the lentivirus with the co-infection agent and NK cells for infection.

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