NK cell expansion culture method, method for lentiviral transduction of NK cells, and car-NK cell product

Abstract

Disclosed in the present disclosure are an NK cell expansion culture method, a method for lentiviral transduction of NK cells, and a CAR-NK cell product. The NK cell expansion culture method comprises the following steps: an expansion culture step: placing human peripheral blood mononuclear cells in a culture container filled with an activation medium and an expansion medium and carrying out static expansion culture to obtain NK cells, wherein when the human peripheral blood mononuclear cells reach a first expansion culture state during the static expansion culture, the culture container is replenished with the activation medium and the expansion medium; and when the human peripheral blood mononuclear cells reach a second expansion culture state during the static expansion culture, the culture container is replenished with the expansion medium.

Description

A NK cell expansion and culture method, a lentiviral transduction NK cell method, and a CAR-NK cell product Technical Field

[0001] The present disclosure relates to a method for expanding and culturing NK cells, a method for lentiviral transduction of NK cells, and a CAR-NK cell product. Background Art

[0002] Natural killer cells (NK cells) are a unique type of innate immune cell with broad-spectrum cytotoxicity. They can efficiently recognize and lyse malignant cells without prior sensitization or antigenic peptide recognition. Furthermore, NK cells can be genetically modified to produce chimeric antigen receptor (CAR)-NK cells, enabling efficient targeted killing and adoption in adoptive cell therapy for cancer patients. Preclinical and clinical data from various CAR-NK cell studies have shown that CAR-NK cells can effectively eliminate target cells without toxic side effects such as cytokine release syndrome or neurotoxins, demonstrating their promising application prospects. However, the biological technologies associated with genetically modified NK cells have significantly limited the progress of NK cell therapy, primarily involving in vitro expansion of NK cells, CAR structure design, and viral transduction techniques.

[0003] In this field, the main methods for genetically modifying primary NK cells include viral transduction and electroporation. Viral transduction, which primarily involves retroviruses, lentiviruses, and adenoviruses, can integrate viral genes into the host genome, allowing for permanent expression with minimal damage to cells. This method has become the primary method for preparing CAR-NK cells in clinical research.

[0004] However, in related technologies, either the proportion of NK cells expanded and cultured in vitro is low, affecting viral transduction efficiency; or when using viral transduction methods to transduce primary NK cells, a high viral titer is often required, limiting the application of viral transduction methods. In addition, the transduction efficiency of viral transduction methods is often relatively low, and the proportion of CAR-NK cells is also low, ultimately resulting in less than ideal transduction results of viral transduction methods.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute prior art known to ordinary technicians in the field.

[0006] Summary of the Invention

[0007] The technical problem of poor transduction effect of viral transduction method is solved by utilizing one or more embodiments of the present disclosure.

[0008] An embodiment of the present disclosure provides a method for expanding and culturing NK cells, comprising the following steps: an expansion and culturing step, placing human peripheral blood mononuclear cells in a culture container containing an activation culture medium and an expansion culture medium, and statically expanding and culturing them to obtain NK cells; wherein, when the human peripheral blood mononuclear cells are statically expanded and cultured to a first expansion and culture state, the activation culture medium and the expansion culture medium are supplemented into the culture container; when the human peripheral blood mononuclear cells are statically expanded and cultured to a second expansion and culture state, the expansion culture medium is supplemented into the culture container.

[0009] In some embodiments, the first expansion culture state is expansion culture to 72 hours to 96 hours; the second expansion culture state is expansion culture to more than 96 hours; and the first expansion culture cycle is 44 hours to 52 hours.

[0010] In some embodiments, when the human peripheral blood mononuclear cells are frozen human peripheral blood mononuclear cells, the following steps are further included before the expansion culture step: a recovery culture step, in which the frozen human peripheral blood mononuclear cells are resuscitated to obtain resuscitated human peripheral blood mononuclear cells, and the resuscitated human peripheral blood mononuclear cells are placed in a culture container containing the expansion culture medium and statically recovered and cultured until the recovery culture state is reached, and then the expansion culture step is performed.

[0011] In some embodiments, the resuscitation culture state is static resuscitation culture to the 8th to 16th hour.

[0012] In some embodiments, the activation medium comprises Retronectin, anti-human CD16 monoclonal antibody, anti-human HER-2 monoclonal antibody, OK432, recombinant human FLT3 Ligand protein, recombinant human IL3 protein, recombinant human IL7 protein, recombinant human IL18 protein, recombinant human IL21 protein, recombinant human IL1b protein, anti-human CD52 monoclonal antibody, anti-human CD137 monoclonal antibody, recombinant human SCF protein, recombinant human IL12 protein, recombinant human OX40 Ligand protein, recombinant human 4-1BB protein, recombinant human IL-15 protein and human interleukin-2 for injection; the expansion medium comprises recombinant human IL-15 protein and human interleukin-2 for injection.

[0013] The present disclosure provides a method for lentiviral transduction of NK cells, comprising the following steps: a coating step, using a recombinant human fibronectin fragment working solution as a coating solution to coat a culture container to obtain a coated culture container; a lentiviral mixture preparation step, mixing a lentiviral stock solution, a polybrene working solution, and a BX795 working solution to obtain a lentiviral mixture; a lentiviral inoculation step, adding the lentiviral mixture to the coated culture container, and centrifuging and inoculating to obtain an inoculated culture container; a viral transduction culture step, collecting NK cells that have been amplified and cultured to a third amplification culture state and adding the amplification culture medium to the inoculated culture container for viral transduction culture; and a transduction amplification culture step, when the viral transduction culture reaches a first transduction state, removing the culture medium originally containing the lentiviral mixture, adding the amplification culture medium, and then transducing and amplifying the culture to a second transduction state to obtain CAR-NK cells.

[0014] Wherein, the NK cells are obtained by amplification and culture using the above-mentioned NK cell amplification and culture method.

[0015] In some embodiments, the coating concentration of the recombinant human fibronectin fragment working solution is 0.1 μg / cm 2 ~50.0μg / cm 2 ; The working concentration of the polybrene working solution is 0.5μg / mL~20.0μg / mL; the working concentration of the BX795 working solution is 0.5μmol / mL~20.0μmol / mL; the virus infection multiplicity of the lentivirus stock solution is 1~50.

[0016] In some embodiments, in the lentiviral mixture, the volume ratio of the lentiviral stock solution, the polybrene working solution, and the BX795 working solution is (0.12-0.18): (400-600): (0.4-0.6).

[0017] In some embodiments, the first transduction state is virus transduction culture until 12h to 16h; the second transduction state is transduction amplification culture until 24h to 120h.

[0018] In some embodiments, the third expansion culture state is expansion culture from 12.0 hours to 336.0 hours.

[0019] In some embodiments, the third expansion culture state is expansion culture from 24.0 hours to 192.0 hours.

[0020] In some embodiments, the third expansion culture state is expansion culture to 48.0 hours to 144.0 hours.

[0021] In some embodiments, the second transduction state is transduction amplification culture to 48h to 96h.

[0022] In some embodiments, the second transduction state is transduction amplification culture to 60 h to 84 h.

[0023] In some embodiments, the lentiviral stock solution contains a CAR-V1052-reporter gene lentiviral vector and / or a CAR-V2195-reporter gene lentiviral vector.

[0024] The present disclosure provides a CAR-NK cell product, which is prepared by the above-mentioned lentiviral transduction method for NK cells.

[0025] The above-mentioned NK cell expansion and culture method, by adding activation medium and expansion medium during the expansion and culture process, that is, adopting a pure factor expansion and culture method, can improve the expansion efficiency of human peripheral blood mononuclear cells, so that NK cells can reach the rapid growth stage as soon as possible. Then, after lentiviral transduction, the lentivirus can be rapidly amplified as the NK cells expand, and the requirement for the amount of lentivirus used during viral transduction can be reduced. A large number of CAR-NK cells can be transduced using less lentivirus.

[0026] The above-mentioned NK cell expansion and culture method, by controlling the type of culture medium used in different expansion and culture states, can cause NK cells to be in a rapid growth period from the 2nd day to the 6th day. Then, after lentiviral transduction, the lentivirus can be rapidly amplified as the NK cells expand. During viral transduction, the requirement for the amount of lentivirus can be reduced, and the time of NK cell expansion and culture can be shortened, thereby improving the efficiency of NK cell expansion and culture.

[0027] The above-mentioned lentiviral transduction method for NK cells, by optimizing the expansion culture state of NK cells, the transduction state of lentiviral transduction culture, and the type and dosage of transduction reagents, can not only improve the activity and expansion rate of NK cells, but also improve the transduction efficiency and gene integration efficiency of NK cells. At the same time, it can also improve the cell activity of CAR-NK cells, so that they can continue to expand after viral transduction culture, thereby reducing the demand for lentiviral dosage and lowering the preparation cost of CAR-NK cells. This lentiviral transduction method is simple and rapid, and has good application prospects.

[0028] The above-mentioned CAR-NK cell product is prepared using the method of lentiviral transduction of NK cells disclosed in the present invention, which can make the CAR-NK cells have higher activity, be more conducive to in vitro expansion, and have the advantages of high activity and low preparation cost, thus having better application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, a brief introduction will be given below to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0030] FIG1 shows fluorescent photographs of CAR-V1052-EGFP cells transduced and amplified and cultured at different time points in Example 1 of the present disclosure;

[0031] FIG2 shows fluorescent photographs of CAR-V2195-EGFP cells transduced and amplified and cultured at different time points in Example 1 of the present disclosure;

[0032] FIG3 shows the expression of GFP in NK cells detected by flow cytometry in Example 1 of the present disclosure;

[0033] FIG4 shows fluorescent photographs of CAR-V1052-EGFP cells transduced and amplified and cultured at different time points in Example 2 of the present disclosure;

[0034] FIG5 shows fluorescent photographs of CAR-V2195-EGFP cells transduced and amplified and cultured at different time points in Example 2 of the present disclosure;

[0035] FIG6 shows the expression of GFP in NK cells detected by flow cytometry in Example 2 of the present disclosure;

[0036] FIG7 shows fluorescent photographs of CAR-V1052-EGFP cells transduced and amplified and cultured at different time points in Example 3 of the present disclosure;

[0037] FIG8 shows fluorescent photographs of CAR-V2195-EGFP cells transduced and amplified and cultured at different time points in Example 3 of the present disclosure;

[0038] FIG9 shows a diagram of flow cytometry detection of NK cells in Example 3 of the present disclosure;

[0039] FIG10 shows the expression of GFP in NK cells detected by flow cytometry in Example 3 of the present disclosure;

[0040] FIG11 shows a diagram of NK cells before lentiviral transduction in Example 4 of the present disclosure;

[0041] FIG12 shows micrographs of PB-NK cells in FIG11 at different magnifications;

[0042] FIG13 shows a fluorescent photograph of NK cells at 24 hours after transduction and expansion culture in Example 4 of the present disclosure;

[0043] FIG14 shows a flow cytometry analysis of NK cells at 24 hours after transduction and expansion in Example 4 of the present disclosure;

[0044] FIG15 shows the expression of GFP in NK cells detected by flow cytometry at 24 hours after transduction and amplification culture in Example 4 of the present disclosure;

[0045] FIG16 shows a fluorescent photograph of NK cells at 48 hours after transduction and expansion culture in Example 4 of the present disclosure;

[0046] FIG17 shows a flow cytometry analysis of NK cells at 48 hours after transduction and expansion in Example 4 of the present disclosure;

[0047] FIG18 shows the expression of GFP in NK cells detected by flow cytometry at 48 hours after transduction and amplification culture in Example 4 of the present disclosure;

[0048] FIG19 shows a fluorescent photograph of NK cells at 72 hours after transduction and expansion culture in Example 4 of the present disclosure;

[0049] FIG20 shows the flow cytometry detection of NK cells at 72 hours after transduction and expansion culture in Example 4 of the present disclosure;

[0050] FIG21 shows the expression of GFP in NK cells detected by flow cytometry at 72 hours after transduction and amplification culture in Example 4 of the present disclosure;

[0051] FIG22 shows a fluorescent photograph of NK cells at 24 hours after transduction and expansion culture in Example 5 of the present disclosure;

[0052] FIG23 shows the flow cytometry detection of NK cells at 24 hours after transduction and expansion culture in Example 5 of the present disclosure;

[0053] FIG24 shows the expression of GFP in NK cells detected by flow cytometry at 24 hours after transduction and amplification culture in Example 5 of the present disclosure;

[0054] FIG25 shows a fluorescent photograph of NK cells at 48 hours after transduction and expansion culture in Example 5 of the present disclosure;

[0055] FIG26 shows the flow cytometry detection of NK cells at 48 hours after transduction and expansion culture in Example 5 of the present disclosure;

[0056] FIG27 shows the expression of GFP in NK cells detected by flow cytometry at 48 hours after transduction and amplification culture in Example 5 of the present disclosure;

[0057] FIG28 shows a fluorescent photograph of NK cells at 72 hours after transduction and expansion culture in Example 5 of the present disclosure;

[0058] FIG29 shows the flow cytometry detection of NK cells at 72 hours after transduction and expansion culture in Example 5 of the present disclosure;

[0059] FIG30 shows the expression of GFP in NK cells detected by flow cytometry at 72 hours after transduction and amplification culture in Example 5 of the present disclosure;

[0060] FIG31 shows a fluorescent photograph of NK cells at 24 hours after transduction and expansion culture in Example 6 of the present disclosure;

[0061] FIG32 shows the flow cytometry detection of NK cells at 24 hours after transduction and expansion culture in Example 6 of the present disclosure;

[0062] FIG33 shows the expression of GFP in NK cells detected by flow cytometry at 24 hours after transduction and expansion culture in Example 6 of the present disclosure;

[0063] FIG34 shows a fluorescent photograph of NK cells at 48 hours after transduction and expansion culture in Example 6 of the present disclosure;

[0064] FIG35 shows the flow cytometry detection of NK cells at 48 hours after transduction and expansion culture in Example 6 of the present disclosure;

[0065] FIG36 shows the expression of GFP in NK cells detected by flow cytometry at 48 hours after transduction and expansion culture in Example 6 of the present disclosure;

[0066] FIG37 shows a fluorescent photograph of NK cells at 72 hours after transduction and expansion culture in Example 6 of the present disclosure;

[0067] FIG38 shows the flow cytometry detection of NK cells at 72 hours after transduction and expansion culture in Example 6 of the present disclosure;

[0068] Figure 39 shows the expression of GFP in NK cells detected by flow cytometry at 72 hours after transduction and amplification culture in Example 6 of the present disclosure. DETAILED DESCRIPTION

[0069] The following will be combined with the accompanying drawings in the embodiments of the present disclosure to clearly and completely describe the technical solutions in the embodiments of the present disclosure. Obviously, the embodiments described are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present disclosure.

[0070] In addition, the present disclosure may repeat reference numbers and / or reference letters in different examples. This repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or arrangements discussed. In addition, the present disclosure provides examples of various specific processes and materials, but those skilled in the art will recognize the application of other processes and / or the use of other materials.

[0071] The present disclosure is described below with reference to specific embodiments and in conjunction with the accompanying drawings:

[0072] Some embodiments of the present disclosure provide a method for expanding and culturing NK cells, comprising the following steps:

[0073] In the expansion culture step, human peripheral blood mononuclear cells are placed in a culture container containing activation culture medium and expansion culture medium, and statically expanded and cultured to obtain NK cells; wherein, when the human peripheral blood mononuclear cells are statically expanded and cultured to a first expansion culture state, the activation culture medium and the expansion culture medium are supplemented into the culture container; when the human peripheral blood mononuclear cells are statically expanded and cultured to a second expansion culture state, the expansion culture medium is supplemented into the culture container during each first expansion culture cycle.

[0074] The NK cell expansion and culture method of the disclosed embodiment can improve the expansion efficiency of human peripheral blood mononuclear cells by adding activation medium and expansion medium during the expansion and culture process, that is, adopting a pure factor expansion and culture method, so that NK cells can reach the rapid growth stage as soon as possible. Furthermore, after lentiviral transduction, the lentivirus can be rapidly amplified as the NK cells expand, and the requirement for the amount of lentivirus used during viral transduction can be reduced, and a large number of CAR-NK cells can be transduced using less lentivirus.

[0075] In some embodiments of the present disclosure, human peripheral blood mononuclear cells (PBMCs) are cells with a single nucleus in human peripheral blood, including lymphocytes and monocytes. In the embodiments of the present disclosure, the expansion and culture of human peripheral blood mononuclear cells are mainly used to obtain natural killer cells (NK cells) from lymphocytes.

[0076] In some embodiments of the present disclosure, the conditions for static expansion culture of human peripheral blood mononuclear cells are to place them in an incubator at 37°C and 5% CO2 for static expansion culture. During the static expansion culture process, no further manipulation is required; only the corresponding culture medium needs to be replenished when the static expansion culture reaches the first expansion culture state and the second expansion culture state.

[0077] In some embodiments of the present disclosure, the culture container used in the NK cell expansion culture method can be selected from any one of a cell culture flask, a triangular cell culture shake flask, a culture dish, and a cell culture plate. When used, an appropriate culture container can be selected according to the capacity and purpose required for the expansion culture.

[0078] In some embodiments of the present disclosure, cell culture plates may be used for expansion culture.

[0079] As an optional embodiment, in the NK cell expansion and culture method of the disclosed embodiment, the first expansion and culture state is the expansion and culture to the 72nd to 96th hour; the second expansion and culture state is the expansion and culture to more than the 96th hour; and the first expansion and culture cycle is 44h to 52h.

[0080] The NK cell expansion and culture method of the disclosed embodiment can cause NK cells to be in a rapid growth period from the 2nd day to the 6th day by controlling the type of culture medium used in different expansion and culture states. Then, after lentiviral transduction, the lentivirus can be rapidly expanded along with the expansion of NK cells. During viral transduction, the requirement for the amount of lentivirus can be reduced, and the time of NK cell expansion and culture can be shortened, thereby improving the efficiency of NK cell expansion and culture.

[0081] It should be noted that the first expansion culture state is the expansion culture from 72h to 96h, which is calculated from the time when the human peripheral blood mononuclear cells are placed in the culture container containing activation medium and expansion medium; the second expansion culture state is the expansion culture from 96h onwards, which is also calculated from the time when the human peripheral blood mononuclear cells are placed in the culture container containing activation medium and expansion medium; the first expansion culture cycle is 44h to 52h, which is calculated from the time when the expansion medium is replenished in the culture container.

[0082] In some embodiments of the present disclosure, the first expansion culture state is the expansion culture to any period between 72h and 96h, for example, the first expansion culture state is the expansion culture to integer periods of 72h, 75h, 78h, 81h, 84h, 87h, 90h, 93h and 96h; it can also be the expansion culture to a non-integer period, for example, the first expansion culture state is the expansion culture to non-integer periods such as 72.5h, 78.4h, 81.6h, 84.7h, 87.9h, 90.1h, 93.3h and 95.8h.

[0083] In some embodiments of the present disclosure, the second expansion culture state is the expansion culture to any integer period above 96 hours, for example, the second expansion culture state is the expansion culture to integer periods such as 96 hours, 97 hours, 98 hours, 99 hours, 100 hours, 103 hours, 106 hours, 110 hours, 115 hours and 120 hours; it can also be the expansion culture to any non-integer period above 96 hours, for example, the second expansion culture state is the expansion culture to integer periods such as 96.1 hours, 97.8 hours, 98.3 hours, 99.6 hours, 100.4 hours, 103.5 hours, 106.2 hours, 110.8 hours, 11.65 hours and 120.7 hours.

[0084] In some embodiments of the present disclosure, the first amplification culture cycle is any period between 44h and 52h, for example, the first amplification culture cycle is an integer period such as 44h, 45h, 46h, 47h, 48h, 49h, 50h, 51h and 52h; it can also be a non-integer period, for example, the first amplification culture cycle is an integer period such as 44.3h, 45.8h, 46.2h, 47.9h, 48.7h, 49.6h, 50.4h, 51.3h and 51.9h.

[0085] In the NK cell expansion and culture method of the embodiment of the present disclosure, human peripheral blood mononuclear cells can be fresh human peripheral blood mononuclear cells or frozen human peripheral blood mononuclear cells.

[0086] As some optional embodiments, when the human peripheral blood mononuclear cells are frozen human peripheral blood mononuclear cells, the following steps are further included before the expansion and culture step:

[0087] The resuscitation culture step is to resuscitate the frozen human peripheral blood mononuclear cells to obtain resuscitated human peripheral blood mononuclear cells, and the resuscitated human peripheral blood mononuclear cells are placed in a culture container filled with expansion culture medium and statically resuscitated and cultured until the resuscitation culture state is reached, and then the expansion culture step is performed.

[0088] In some embodiments of the present disclosure, the conditions for the static recovery culture in the recovery culture step are static culture in a 37° C., 5% CO 2 incubator.

[0089] In some embodiments of the present disclosure, the resuscitation culture state is static resuscitation culture to the 8th to 16th hour.

[0090] In some embodiments, before expansion culture, the revived human peripheral blood mononuclear cells can be statically cultured in an expansion medium for 8 to 16 hours to fully revive the NK cells in the human peripheral blood mononuclear cells, thereby increasing the activity of the NK cells and improving the survival rate of the frozen human peripheral blood mononuclear cells, thereby improving the efficiency of the expansion culture.

[0091] In some embodiments of the present disclosure, the recovery culture state can be static recovery culture to any period between 8h and 16h, for example, the recovery culture state is static recovery culture to any integer period of 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h and 16h; it can also be a non-integer period, for example, the recovery culture state is static recovery culture to any non-integer period of 8.2h, 9.7h, 10.6h, 11.4h, 12.5h, 13.1h, 14.8h, 15.9h and 16h. In some embodiments, the NK cell expansion culture method and the working hours of the staff are adaptively adjusted. For example, after the recovery treatment, the recovery culture can be statically cultured overnight so that the expansion culture step can be performed the next day.

[0092] In some embodiments of the present disclosure, the resuscitation treatment can be to place the frozen human peripheral blood mononuclear cells in a 37°C water bath, and complete the thawing of the frozen human peripheral blood mononuclear cells within 1 to 2 minutes. The thawed cell suspension can then be inoculated into a culture container containing culture medium for static resuscitation culture.

[0093] In some embodiments of the present disclosure, optionally, after thawing and melting of human peripheral blood mononuclear cells, the thawed cell suspension can be placed in a centrifuge tube, centrifuged at 1200 rpm for 3 minutes, and the supernatant can be discarded. The cells can be reselected with fresh culture medium and then inoculated into a culture container containing culture medium for static recovery culture. By centrifuging and discarding the supernatant, the freezing solution in the cell suspension can be removed, thereby preventing the freezing solution from producing cytotoxicity to the human peripheral blood mononuclear cells and affecting the subsequent expansion and culture of the human peripheral blood mononuclear cells.

[0094] As an optional embodiment, the activation medium includes Retronectin, anti-human CD16 monoclonal antibody, anti-human HER-2 monoclonal antibody, OK432, recombinant human FLT3 Ligand protein, recombinant human IL3 protein, recombinant human IL7 protein, recombinant human IL18 protein, recombinant human IL21 protein, recombinant human IL1β protein, anti-human CD52 monoclonal antibody, anti-human CD137 monoclonal antibody, recombinant human SCF protein, recombinant human IL12 protein, recombinant human OX40 Ligand protein, recombinant human 4-1BB protein, recombinant human IL-1α protein, recombinant human IL-15 protein and human interleukin-2 for injection; the expansion medium includes recombinant human IL-15 protein and human interleukin-2 for injection.

[0095] In some embodiments of the present disclosure, an optional NK cell expansion method is as follows:

[0096] Place the frozen human peripheral blood mononuclear cells in a 37°C water bath. After complete thawing within 1 to 2 minutes, inoculate them into a culture container containing a culture medium with 10% autologous plasma and culture them in a 37°C, 5% CO2 incubator for recovery overnight.

[0097] After overnight static thawing culture, human peripheral blood mononuclear cells are seeded into cell culture plates containing activation medium and expansion medium. Incubate in a 37°C, 5% CO2 incubator for static expansion until the first expansion state, for example, until the 72nd hour. Do not perform any manipulation during the static culture to the 72nd hour. If lentiviral transduction is required, remove one or more groups of samples for lentiviral transduction at an appropriate time, while the other groups remain untreated.

[0098] For human peripheral blood mononuclear cells expanded to the first expansion culture state, for example, human peripheral blood mononuclear cells expanded to the 72th hour of static expansion culture, equivalent to the 4th day of static expansion culture, add appropriate amounts of activation medium and expansion medium to each well of the culture plate and continue static expansion culture in a 37°C, 5% CO2 incubator. Optionally, the medium can be added at a volume ratio of 1:1 between the original medium and the fresh medium.

[0099] For human peripheral blood mononuclear cells that have been expanded and cultured to the second expansion culture state, such as human peripheral blood mononuclear cells that have been statically expanded and cultured for more than 96 hours, which is equivalent to the expansion culture to the 5th day and after the 5th day, an appropriate amount of expansion culture medium is added to each well of the culture plate every first expansion culture cycle, for example, an appropriate amount of expansion culture medium is added to each well of the culture plate every 48 hours, and static expansion culture is continued in a 37°C, 5% CO2 incubator. Optionally, the culture medium is added at a ratio of 1:1 in volume of the original culture medium to the fresh culture medium.

[0100] During the NK cell expansion process, human peripheral blood mononuclear cells from the corresponding group can be taken at an appropriate time to perform relevant lentiviral transduction operations according to the time conditions for lentiviral transduction of NK cells.

[0101] The present disclosure provides a method for lentiviral transduction of NK cells, comprising the following steps:

[0102] a coating step, using a recombinant human fibronectin fragment working solution as a coating solution to coat the culture container to obtain a coated culture container;

[0103] The lentivirus mixture preparation step comprises mixing the lentivirus stock solution, the polybrene working solution, and the BX795 working solution to prepare the lentivirus mixture;

[0104] The lentivirus inoculation step comprises adding the lentivirus mixture to the coated culture container, and performing a centrifugal inoculation process to obtain an inoculated culture container;

[0105] a viral transduction culture step, collecting the NK cells that have been expanded and cultured to the third expansion culture state, adding them to an inoculated culture container, and adding expansion medium to the inoculated culture container for viral transduction culture; and

[0106] In the transduction and amplification culture step, when the virus transduction culture reaches the first transduction state, the original culture medium containing the lentiviral mixture is removed, and the amplification medium is added and the cells are transduced and amplified to the second transduction state to obtain CAR-NK cells;

[0107] Wherein, NK cells are obtained by amplification and culture using the above-mentioned NK cell amplification and culture method.

[0108] The disclosed embodiments of the lentiviral transduction method for NK cells, by optimizing the expansion culture state of NK cells, the transduction state of lentiviral transduction culture, and the type and dosage of transduction reagents, can not only improve the activity and expansion rate of NK cells, but also improve the transduction efficiency and gene integration efficiency of NK cells. It can also improve the cell activity of CAR-NK cells, allowing them to continue to expand after viral transduction culture, thereby reducing the demand for lentiviral dosage and lowering the preparation cost of CAR-NK cells. This lentiviral transduction method is simple and rapid, and has good application prospects.

[0109] In the embodiments disclosed herein, it was found through research that the application of recombinant human fibronectin fragments (Retronectin) in the process of lentiviral transduction can increase the lentiviral titer and cell number per unit space of the cell culture plate, so that NK cells and lentiviral vectors coexist at high concentrations, significantly improving the efficiency of lentiviral transduction into NK cells, while reducing the usage and cytotoxicity of other reagents. In addition, the activity of single NK cells can be significantly improved, which is beneficial to the in vitro expansion of CAR-NK cells. Hexadimethrine bromide (Polybrene) is a polycationic polymer that can be widely used in retroviral-mediated gene transduction. Its mechanism of action may be to promote viral adsorption by neutralizing the electrostatic repulsion between cell surface sialic acid and viral particles. BX795, also known as N-[3-[[5-iodo-4-[[3-[(2-thienylcarbonyl)amino]propyl]amino]-2-pyrimidinyl]amino]phenyl]-1-pyrrolidinecarboxamide (CAS number 702675-74-9), may stimulate the action of lentiviral VSVG envelope protein to bind to ligands TRAM and TRIF via Toll-like receptor 4 on the surface of NK cells, activating downstream signaling molecules TBK1 and IKKε, which in turn activates the transcription factor IRF3, inducing NK cells to produce IFN-β and various proinflammatory cytokines and chemokines, thereby limiting viral replication and spread and achieving an antiviral effect. BX795 selectively inhibits TBK1 and IKKε molecules, blocking TBK1- and IKKε-mediated IRF3 activation and IFN-β production, thereby blocking signal transmission and limiting the antiviral response of NK cells, thereby improving the efficiency of viral transduction of NK cells.

[0110] In some embodiments of the present disclosure, when recombinant human fibronectin fragment (Retronectin) and BX795 are used in combination, the problem of low transduction efficiency of lentiviral transduction of NK cells can be significantly solved, and the efficiency of lentiviral transduction of NK cells and gene integration efficiency can be improved to a certain extent, thereby reducing the amount of lentivirus used and reducing the preparation cost of CAR-NK cells; at the same time, the method of lentiviral transduction of NK cells is simple and convenient to operate, and can also improve the activity of CAR-NK cells, and has good application prospects.

[0111] The method for lentiviral transduction of NK cells according to the embodiments of the present disclosure can significantly improve the efficiency of lentiviral transduction of NK cells compared to the conventional lentiviral transduction efficiency of less than 5%, and can even increase the lentiviral transduction efficiency to 50% to 60%.

[0112] In some embodiments of the present disclosure, the culture container used in the method of lentiviral transduction of NK cells can be selected from any one of a cell culture flask, a triangular cell culture shake flask, a culture dish, and a cell culture plate. When used, an appropriate culture container can be selected according to the capacity and purpose required for lentiviral transduction of NK cells.

[0113] In some embodiments of the present disclosure, cell culture plates may be used for expansion culture.

[0114] As an optional embodiment, the coating concentration of the recombinant human fibronectin fragment working solution is 0.1 μg / cm 2 ~50.0μg / cm 2 .

[0115] In some embodiments of the present disclosure, the recombinant human fibronectin fragment working solution can use PBS as the solute or be diluted with PBS; optionally, the recombinant human fibronectin fragment working solution is prepared and used immediately.

[0116] In some embodiments of the present disclosure, the coating concentration of the recombinant human fibronectin fragment working solution can be 2.0 μg / cm 2 ~20.0μg / cm 2 In some embodiments of the present disclosure, the coating concentration of the recombinant human fibronectin fragment working solution can be 5.0 μg / cm 2 ~15.0 μg / cm 2 For example, the coating concentration of the recombinant human fibronectin fragment working solution can be 0.1 μg / cm 2 , 0.3μg / cm 2 , 0.5μg / cm 2 , 0.7μg / cm 2 , 0.9μg / cm 2 , 1.0μg / cm 2 , 2.0μg / cm 2 , 3.0μg / cm 2 , 4.0μg / cm 2 , 5.0μg / cm 2 , 6.0μg / cm 2 , 7.0μg / cm 2 , 8.0μg / cm 2 , 9.0μg / cm 2 , 10.0μg / cm 2 , 15.0μg / cm 2 , 20.0μg / cm 2 , 25.0μg / cm 2 , 30.0μg / cm 2 、35.0μg / cm2 , 40.0μg / cm 2 , 45.0μg / cm 2 and 50.0 μg / cm 2 .

[0117] As an optional embodiment, the working concentration of the polybrene working solution is 0.5 μg / mL to 20.0 μg / mL.

[0118] In some embodiments of the present disclosure, the polybrene working solution can use culture medium as a solute or be diluted with culture medium; alternatively, the polybrene working solution is prepared and used immediately.

[0119] In some embodiments of the present disclosure, the working concentration of the polybrene working solution can be 2.0 μg / mL to 18.0 μg / mL; in some embodiments of the present disclosure, the working concentration of the polybrene working solution can be 5.0 μg / mL to 15.0 μg / mL. For example, the working concentration of the polybrene working solution can be 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL, 2.0 μg / mL, 2.5 μg / mL, 3.0 μg / mL, 3.5 μg / mL, 4.0 μg / mL, 4.5 μg / mL, 5.0 μg / mL, 5.5 μg / mL, 6.0 μg / mL, 6.5μg / mL, 7.0μg / mL, 7.5μg / mL, 8.0μg / mL, 8.5μg / mL, 9.0μg / mL, 9.5μg / mL, 1 0.0μg / mL, 10.5μg / mL, 11.0μg / mL, 11.5μg / mL, 12.0μg / mL, 12.5μg / mL, 13.0μg / mL, 1 3.5μg / mL, 14.0μg / mL, 14.5μg / mL, 15.0μg / mL, 15.5μg / mL, 16.0μg / mL, 16.5μg / mL, 1 7.0μg / mL, 170.5μg / mL, 18.0μg / mL, 18.5μg / mL, 19.0μg / mL, 19.5μg / mL and 20.0μg / mL.

[0120] As an optional embodiment, the working concentration of the BX795 working solution is 0.5 μmol / mL to 20.0 μmol / mL.

[0121] In some embodiments of the present disclosure, the BX795 working solution may use culture medium as a solute or be diluted with culture medium; alternatively, the BX795 working solution is prepared and used immediately.

[0122] In some embodiments of the present disclosure, the working concentration of the BX795 working solution can be 2.0 μmol / mL to 18.0 μmol / mL; in some embodiments of the present disclosure, the working concentration of the BX795 working solution can be 5.0 μmol / mL to 15.0 μmol / mL. For example, the working concentration of the BX795 working solution can be 0.5 μmol / mL, 1.0 μmol / mL, 1.5 μmol / mL, 2.0 μmol / mL, 2.5 μmol / mL, 3.0 μmol / mL, 3.5 μmol / mL, 4.0 μmol / mL, 4.5 μmol / mL, 5.0 μmol / mL, 5.5 μmol / mL, 6.0 μmol / mL, 6.5 μmol / mL, 7.0 μmol / mL, 7.5 μmol / mL, 8.0 μmol / mL, 8.5 μmol / mL, 9.0 μmol / mL, 9.5 μmol / mL, 10.0 μmol / mL, 11.0 μmol / mL, 12.0 μmol / mL, 13.0 μmol / mL, 14.0 μmol / mL, 15.0 μmol / mL, 16.0 μmol / mL, 17.0 μmol / mL, 18.0 μmol / mL, 19.0 μmol / mL, 20.0 μmol / mL, 21.0 μmol / mL, 22.0 μmol / mL, 23.0 μmol / mL, 24.0 μmol / mL, 25.0 μmol / mL, 26.0 μmol / mL, 27.0 μmol / mL, 28.0 μmol / mL, 29.0 μmol / mL, 30.0 μmol / mL, 31.0 μmol / mL, 32.0 μmol / mL, 33.0 μmol / mL, 0.5μmol / mL, 11.0μmol / mL, 11.5μmol / mL, 12.0μmol / mL, 12.5μmol / mL, 13.0μmol / mL, 13.5μmol / mL, 14.0μmol / mL, 14.5μmol / mL, 15.0μmol / mL, 1 5.5 μmol / mL, 16.0 μmol / mL, 16.5 μmol / mL, 17.0 μmol / mL, 170.5 μmol / mL, 18.0 μmol / mL, 18.5 μmol / mL, 19.0 μmol / mL, 19.5 μmol / mL, and 20.0 μmol / mL.

[0123] As an optional embodiment, the multiplicity of infection of the lentiviral stock solution is 1-50.

[0124] In some embodiments of the present disclosure, the multiplicity of infection of the lentiviral stock solution may be 10 to 40. In some embodiments of the present disclosure, the multiplicity of infection of the lentiviral stock solution may be 20 to 30. For example, the multiplicity of infection of the lentiviral stock solution may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50.

[0125] As an optional embodiment, in the lentiviral mixture, the volume ratio of the lentiviral stock solution, the polybrene working solution and the BX795 working solution is (0.12-0.18): (400-600): (0.4-0.6).

[0126] In some embodiments of the present disclosure, the volume ratio of the lentiviral stock solution, polybrene working solution, and BX795 working solution in the lentiviral mixture is (0.14-0.16): (450-550): (0.45-0.65); in some embodiments of the present disclosure, the volume ratio of the lentiviral stock solution, polybrene working solution, and BX795 working solution in the lentiviral mixture is 0.15:500:0.5. For example, when using a cell culture plate to perform a method for lentiviral transduction of NK cells, the amount of the lentiviral stock solution used is 0.15 μL / well, the amount of the polybrene working solution used is 500 μL / well, and the amount of the BX795 working solution used is 0.5 μL / well.

[0127] As an optional embodiment, the first transduction state is virus transduction culture until the 12th to 16th hour; the second transduction state is transduction amplification culture until the 24th to 120th hour.

[0128] In some embodiments of the present disclosure, before the viral transduction culture reaches the first transduction state, the lentiviral transduction NK cell process mainly occurs, that is, the process of integrating the genes carried by the lentivirus into the genome of the NK cells; before the viral transduction culture reaches the second transduction state, the CAR-NK cell expansion process mainly occurs, that is, the process of integrating the genes carried by the lentivirus and the in vitro expansion of NK cells.

[0129] It should be noted that the first transduction state refers to the viral transduction culture period from 12h to 16h, which is calculated from the time when NK cells are added to the inoculated culture vessel and the expansion medium is added; the second transduction state refers to the viral transduction culture period from 24h to 120h, which is also calculated from the time when NK cells are added to the inoculated culture vessel and the expansion medium is added.

[0130] In some embodiments of the present disclosure, the first transduction state is the virus transduction culture to any integer period between 12h and 16h, for example, the first transduction state is the virus transduction culture to integer periods such as 12h, 13h, 14h, 15h and 16h; it can also be the virus transduction culture to any non-integer period between 12h and 16h, for example, the first transduction state is the virus transduction culture to non-integer period such as 12.3h, 13.7h, 14.5h and 15.9h.

[0131] In some embodiments of the present disclosure, the second transduction state is any integer period from 24h to 120h of viral transduction culture. For example, the second transduction state is any integer period from 24h to 120h of viral transduction culture. For example, the second transduction state is any integer period from 24h to 120h of viral transduction culture. The virus can be transduced and cultured to an integer time period such as 0 h, or to a non-integer time period such as 25.3 h, 29.7 h, 37.8 h, 40.2 h, 49.6 h, 55.1 h, 62.4 h, 66.7 h, 70.2 h, 77.9 h, 82.5 h, 93.6 h, 98.1 h, 101.2 h, 107.8 h, 115.7 h and 118.9 h.

[0132] As an optional embodiment, the third expansion culture state is expansion culture to 12.0 hours to 336.0 hours.

[0133] In one embodiment of the present disclosure, the NK cells expanded and cultured to the third expansion and culture state are NK cells collected from the 12th to the 336th hour after the human peripheral blood mononuclear cells are placed in a culture container filled with activation medium and expansion medium. The NK cells collected at this time have high activity and expansion capacity, and can be transduced with lentivirus to generate CAR-NK cells, and can be expanded in vitro after transduction. That is to say, the generated CAR-NK cells can be well expanded in vitro, so that a large number of CAR-NK cells can be collected to increase the yield of CAR-NK cells.

[0134] It should be noted that the third expansion culture state is the expansion culture from 12.0 hours to 336.0 hours, which is calculated from the time when the human peripheral blood mononuclear cells are placed in the culture container containing the activation medium and the expansion medium.

[0135] In some embodiments of the present disclosure, the third expansion culture state is the expansion culture to any integer period between 12.0 hours and 336.0 hours, or it can be the expansion culture to a non-integer period between 12.0 hours and 336.0 hours, which are not listed here one by one.

[0136] As an optional embodiment, the third expansion culture state is expansion culture to 24.0 hours to 192.0 hours.

[0137] In some embodiments of the present disclosure, the third expansion culture state is the expansion culture to any integer period between 24.0 hours and 192.0 hours, or it can be the expansion culture to a non-integer period between 24.0 hours and 192.0 hours, which are not listed here one by one.

[0138] As an optional embodiment, the third expansion culture state is expansion culture to 48.0 hours to 144.0 hours.

[0139] In some embodiments of the present disclosure, the third expansion culture state is any integer period of expansion culture from 48.0 hours to 144.0 hours, or a non-integer period of expansion culture from 48.0 hours to 144.0 hours, which are not listed here one by one.

[0140] As an optional embodiment, the second transduction state is transduction amplification culture to 48 hours to 96 hours.

[0141] In some embodiments of the present disclosure, the second transduction state is any integer period from 48h to 96h of viral transduction culture, or any non-integer period from 48h to 96h of viral transduction culture, which are not listed here one by one.

[0142] As an optional embodiment, the second transduction state is transduction amplification culture to 60 hours to 84 hours.

[0143] In some embodiments of the present disclosure, the second transduction state is any integer period from 60h to 84h of viral transduction culture, or any non-integer period from 60h to 84h of viral transduction culture, which are not listed here one by one.

[0144] As an optional embodiment, the lentiviral stock solution contains a CAR-V1052-reporter gene lentiviral vector and / or a CAR-V2195-reporter gene lentiviral vector.

[0145] In some embodiments of the present disclosure, the reporter gene of the CAR-V1052-reporter gene lentiviral vector and / or the CAR-V2195-reporter gene lentiviral vector may be a fluorescent reporter gene.

[0146] In some embodiments of the present disclosure, the fluorescent reporter gene is selected from any one of GFP, EGFP, RFP, mCherry, mStrawberry, Luciferase, mApple, mRuby and EosFP.

[0147] For example, in some embodiments of the present disclosure, the CAR-V1052-reporter gene lentiviral vector can be a CAR-V1052-EGFP lentiviral vector, and the CAR-V2195-reporter gene lentiviral vector can be a CAR-V2195-EGFP lentiviral vector. In other words, both the CAR-V1052-EGFP lentiviral vector and the CAR-V2195-EGFP lentiviral vector carry a GFP fluorescent indicator protein.

[0148] In some embodiments of the present disclosure, during the packaging and preparation of the CAR-V1052-EGFP lentiviral vector and / or the CAR-V2195-EGFP lentiviral vector, the envelope plasmids used include pSPAX and / or pMD2.G. Optionally, the nucleic acid sequence of pSPAX is the sequence shown in SEQ ID NO: 1, and / or the nucleic acid sequence of pMD2.G is the sequence shown in SEQ ID NO: 2.

[0149] In some embodiments of the present disclosure, the nucleic acid sequence of pSPAX is a modified sequence of the sequence shown in SEQ ID NO: 1, and / or the nucleic acid sequence of pMD2.G is a modified sequence of the sequence shown in SEQ ID NO: 2.

[0150] In some embodiments of the present disclosure, the sequence of the target plasmid in the CAR-V1052-EGFP lentiviral vector is the sequence shown in SEQ ID NO: 3; the sequence of the target plasmid in the CAR-V2195-EGFP lentiviral vector is the sequence shown in SEQ ID NO: 4.

[0151] Based on the same inventive concept, the disclosed embodiments further provide a CAR-NK cell product, which is prepared by the above-mentioned method of lentiviral transduction of NK cells.

[0152] Since the CAR-NK cells provided by the present disclosure include CAR-NK cells prepared by the method of lentiviral transduction of NK cells in the above-mentioned technical solution, the CAR-NK cell products provided by the present disclosure have all the beneficial effects of the above-mentioned method of lentiviral transduction of NK cells, which will not be described in detail here.

[0153] The CAR-NK cell product of the embodiment of the present disclosure is prepared by the method of lentiviral transduction of NK cells disclosed herein, which can make the CAR-NK cells have higher activity, be more conducive to in vitro expansion, and have the advantages of high activity and low preparation cost, thus having better application prospects.

[0154] In an embodiment of the present disclosure, optionally, a method for preparing lentivirus packaging is as follows:

[0155] 24 h before plasmid transfection, 293T cells in logarithmic growth phase were digested with TrypLETM Select (1x) without Phenol Red and resuspended in 10 mL of DMEM medium containing 10% serum at 6 × 10 6 The cells were seeded in 10 cm cell culture dishes and cultured in a 37°C, 5% CO2 incubator.

[0156] 2 h before plasmid transfection, the above DMEM medium was replaced with DMEM medium containing 2% serum.

[0157] Add 24 μg of the prepared lentiviral packaging DNA solution to the first sterile centrifuge tube, wherein the lentiviral packaging DNA solution includes pMD2.G plasmid, pSPAX2 plasmid and target plasmid. Optionally, the mass ratio of pMD2.G plasmid: pSPAX2 plasmid: target plasmid is 1:3:4. Dilute with 500 μL Opti-MEM medium and mix well. Incubate at room temperature for 5 minutes.

[0158] Add 90 μL of Lipofectamine 2000 reagent to the second sterile centrifuge tube, dilute with 500 μL of Opti-MEM medium, mix well, and incubate at room temperature for 5 minutes.

[0159] Mix the diluted lentiviral packaging DNA solution with the diluted Lipofectamine 2000 solution by gently pipetting 20 times without shaking. Incubate at room temperature for 20 minutes to allow the DNA and Lipofectamine 2000 dilution solution to form a transfection complex.

[0160] Transfer the transfection complex to a 293T cell culture dish, mix thoroughly, and incubate in a 37°C, 5% CO2 incubator. After 6-7 hours of incubation, discard the culture medium containing the transfection complex. Add 5 mL of PBS to each cell culture dish, gently shake the dish to wash away any remaining transfection mixture, and then discard. Add 10 mL of DMEM medium supplemented with 5% serum to each cell culture dish and incubate in a 37°C, 5% CO2 incubator.

[0161] After 24 hours of cell culture, collect the cell supernatant and replace with 10 mL of fresh DMEM medium containing 5% serum. Centrifuge the collected cell supernatant at 300g for 5 minutes to remove cell debris. Filter the supernatant through a 0.45μm PVDF filter into a 50mL centrifuge tube and store at 4°C for short-term storage.

[0162] After 48 hours of cell culture, the cell supernatant was collected and centrifuged at 300g for 5 minutes to remove cell debris. The supernatant was then filtered through a 0.45μm PVDF filter into a 50mL centrifuge tube. The cell supernatants collected at 24 and 48 hours were combined and subsequently concentrated and purified to obtain lentiviral particles.

[0163] In an embodiment of the present disclosure, optionally, a method for concentrating and purifying lentivirus is as follows:

[0164] To prepare 5X PEG-3000 NaCl stock solution, add 8.766g sodium chloride and 50g PEG-3000 to 200mL pure water, mix and dissolve, then sterilize at 121℃ for 30min, let it stand to room temperature, and then store at 4℃.

[0165] The cell supernatant collected by the above lentiviral packaging preparation method was filtered, and 2.5 mL of 5X PEG-3000 NaCl stock solution was slowly added dropwise to every 10 mL of filtered cell supernatant. The mixture was incubated at 4°C. Mixing was performed every 20 to 30 minutes for 3 to 5 times, and the mixture was incubated at 4°C overnight.

[0166] Centrifuge at 4°C, 4000g for 20 minutes to allow the lentiviral particles to accumulate at the bottom of the tube, resulting in a distinct white precipitate. Discard the supernatant and centrifuge again at 4°C, 4000g for 5 minutes, or allow the tube to stand for 1-2 minutes to remove any remaining liquid.

[0167] After removing the residual liquid, add an appropriate amount of culture medium / HBSS / PBS / sterile double-distilled water to the centrifuge tube to dissolve the lentiviral particle precipitation. Collect the precipitated lentiviral particles and dispense them into cryopreservation tubes to prepare the lentiviral stock solution. For example, dispense them into 10 μL / tube, store them at -80°C, and use them as needed.

[0168] In an embodiment of the present disclosure, optionally, a method for determining the titer of a lentivirus is as follows:

[0169] HEK293T cells that are growing well were digested, counted, and diluted to 1×10 5 / mL, according to the dosage of 100 μL / well (1×10 4 10 cells / well) were added to a 96-well culture plate. Alternatively, each virus requires 6 wells and cultured in DMEM medium containing 10% FBS in a 37°C, 5% CO2 incubator overnight. The next day, the cell supernatant was discarded to prepare HEK293T cells.

[0170] The lentiviral stock solution prepared by the above lentiviral concentration and purification method was serially diluted 10-fold using the limiting dilution method in FBS-free DMEM medium. A 5 μg / mL polybrene working solution was added to prepare six serial dilutions of the virus solution. The virus solution was incubated at 37°C in a 5% CO2 incubator. The limiting dilution method was as follows: Prepare six 1.5mL EP tubes for each virus, add 90 μL of complete medium to each tube, add 10 μL of the lentiviral stock solution to each tube, mix thoroughly, then pipette 10 μL into the second tube and mix thoroughly. Repeat this process.

[0171] Incubate the diluted virus solution and HEK293T cells in a 37°C, 5% CO2 incubator. After 6-8 hours of incubation, discard the virus-containing culture medium and add 100 μL of DMEM medium containing 10% FBS to each well to facilitate cell growth. Incubate overnight in a 37°C, 5% CO2 incubator. Observe GFP expression under a fluorescence microscope after 48-72 hours of incubation.

[0172] Observe the results under a fluorescence microscope, count the cells in wells with an appropriate fluorescence ratio (between 10% and 30%), and perform flow cytometry to detect the GFP ratio, and calculate the viral titer. The calculation formula is as follows:

[0173] Virus titer (TU / ml) = number of cells × percentage of GFP fluorescence × 10 3 / Volume of virus stock solution (μL).

[0174] In an embodiment of the present disclosure, optionally, a NK cell flow cytometry detection method is as follows:

[0175] Take 1×10 6 Transfer the NK cells to a 1 mL centrifuge tube and centrifuge at 3000 rpm for 3-5 minutes. Discard the supernatant. Add 1 mL of FACS solution, mix thoroughly by pipetting, wash the cells, and centrifuge at 3000 rpm for 3-5 minutes. Discard the supernatant. FACS solution (PBS containing 1% FBS) is prepared by adding 1 mL of fetal bovine serum (FBS) to 49 mL of PBS and mixing thoroughly.

[0176] Add 100 μL of FACS solution to block the Fc receptors on the surface of NK cells and place at 4°C for 15 minutes.

[0177] Add 1 μL FITC-CD3 and 1 μL AF647-CD56 flow cytometry antibodies, mix well by pipetting, and place at 4°C for 30 min.

[0178] Add 1 mL of FACS solution, mix thoroughly by pipetting, wash the cells, centrifuge at 3000 rpm for 3 to 5 minutes, and discard the cell supernatant.

[0179] Add 200 μL FACS solution and detect NK cells (CD3 - CD56 + ) ratio.

[0180] FlowJO flow cytometry software was used for data analysis.

[0181] In an embodiment of the present disclosure, optionally, a CAR-NK cell flow cytometry detection method is as follows:

[0182] Collect CAR-NK cells into a 1 mL centrifuge tube, centrifuge at 3000 rpm for 3-5 minutes, and discard the cell supernatant.

[0183] Add 1 mL of FACS solution, mix thoroughly by pipetting, wash the cells, centrifuge at 3000 rpm for 3 to 5 minutes, and discard the cell supernatant.

[0184] Add 100 μL of FACS solution to block the Fc receptors on the surface of NK cells and place at 4°C for 15 minutes.

[0185] Add 1 μL of APC-Cy7-CD3 and 1 μL of AF647-CD56 flow cytometry antibody, mix well by pipetting, and place at 4°C for 30 min.

[0186] Add 1 mL of FACS solution, mix thoroughly by pipetting, wash the cells, centrifuge at 3000 rpm for 3 to 5 minutes, and discard the cell supernatant.

[0187] Add 200 μL FACS solution and detect NK cells (CD3 - CD56 + ) and the proportion of GFP+ cells.

[0188] FlowJO flow cytometry software was used for data analysis.

[0189] In some embodiments of the present disclosure, the lentiviral vectors used can be the CAR-V1052-EGFP lentiviral vector and the CAR-V2195-EGFP lentiviral vector prepared by our company. Both lentiviral vectors carry GFP fluorescent indicator protein, and the viral titer can be 1×10 8 TU / ml.

[0190] In some embodiments of the present disclosure, the recombinant human fibronectin fragment (Takara, GT-T551H3) stock solution is diluted with PBS to prepare a recombinant human fibronectin fragment working solution, which is prepared and used immediately.

[0191] In some embodiments of the present disclosure, a polybrene stock solution is diluted with culture medium to prepare a polybrene working solution, which is prepared and used immediately.

[0192] In some embodiments of the present disclosure, BX795 (Cayman Chemical Company, 14932) is diluted with culture medium to prepare a BX795 working solution, which is used immediately after preparation.

[0193]

[0194] Example 1

[0195] (1) Add a certain amount of recombinant human fibronectin fragment stock solution into PBS, pipette and mix, and prepare it to 5.0ug / cm 2 The recombinant human fibronectin fragment working solution at the coating concentration was used as the coating solution, and the coating solution was transferred to a 24-well cell culture plate at a volume of 500 μL / well. After coating at 4° C. for 12.0 h to 24.0 h, the coating solution was discarded to prepare a coated culture plate.

[0196] (2) Add 0.15 μL / well of lentiviral stock solution with an MOI of 50, 500 μL / well of polybrene working solution with a working concentration of 8 μg / mL, and 0.5 μL / well of BX795 working solution with a working concentration of 0.5 μmol / mL to the amplification medium, and pipette and mix 20 times to prepare a lentiviral mixture. The lentiviral stock solution contains CAR-V1052-EGFP lentiviral vector and CAR-V2195-EGFP lentiviral vector.

[0197] (3) Add the lentiviral mixture to the coated culture plate and centrifuge at 32°C, 2000g for 2.0 h.

[0198] (4) Collect the PB-NK cells that were cultured to 48.0 hours after the aforementioned NK cell expansion method, and culture the PB-NK cells at 3×10 5 / well was added to a 24-well cell culture plate containing the lentivirus mixture, and amplification medium was added at a rate of 1 mL / well for lentiviral transduction culture.

[0199] (5) Cultivate in a 37°C, 5% CO2 incubator until 12.0 h to 16.0 h, remove the culture medium containing the lentivirus mixture, and replace it with fresh amplification medium at a volume of 1 mL / well.

[0200] (6) The cells were cultured in a 37°C, 5% CO2 incubator until 24.0 h, 48.0 h, 72.0 h, and 96.0 h, and fluorescence microscopy was used to observe the expression of GFP in the PB-NK cells that were successfully transduced with the lentivirus at the above time points.

[0201] As shown in Figure 1, fluorescence images of CAR-V1052-EGFP cells at different time points are shown. As shown in Figure 2, fluorescence images of CAR-V2195-EGFP cells at different time points are shown. As can be seen from Figures 1 and 2, the stronger the fluorescence reaction, the more successful the lentiviral transduction.

[0202] As shown in Figure 3, the expression of GFP in CAR-NK cells detected by flow cytometry at 96.0 hours after lentiviral transduction culture is shown. As can be seen from Figure 3, NK cells account for 1.64%, CAR-V1052-EGFP NK cells account for 56.4%, and CAR-V2195-EGFP NK cells account for 19.8%.

[0203] Example 2

[0204] (1) Add a certain amount of recombinant human fibronectin fragment stock solution into PBS, pipette and mix, and prepare it to 5.0ug / cm 2 A working solution of recombinant human fibronectin fragment at a working concentration was used as a coating solution. The coating solution was transferred to a 24-well cell culture plate at a volume of 500 μL / well. After coating at 4° C. for 12.0 h to 24.0 h, the coating solution was discarded to prepare a coated culture plate.

[0205] (2) Add 0.15 μL / well of lentiviral stock solution with an MOI of 50, 500 μL / well of polybrene working solution with a working concentration of 8 μg / mL, and 0.5 μL / well of BX795 working solution with a working concentration of 0.5 μmol / mL to the amplification medium, and pipette and mix 20 times to prepare a lentiviral mixture. The lentiviral stock solution contains CAR-V1052-EGFP lentiviral vector and CAR-V2195-EGFP lentiviral vector.

[0206] (3) Add the lentiviral mixture to the coated culture plate and centrifuge at 32°C, 2000g for 2.0 h.

[0207] (4) The PB-NK cells that were cultured to 96.0 h after the aforementioned NK cell expansion method were collected and cultured at 3×10 5 / well was added to a 24-well cell culture plate containing the lentivirus mixture, and amplification medium was added at a rate of 1 mL / well for lentiviral transduction culture.

[0208] (5) Cultivate in a 37°C, 5% CO2 incubator until 12.0 h to 16.0 h, remove the culture medium containing the lentivirus mixture, and replace it with fresh amplification medium at a volume of 1 mL / well.

[0209] (6) The cells were cultured in a 37°C, 5% CO2 incubator until 24.0 h, 48.0 h, 72.0 h, and 96.0 h, and fluorescence microscopy was used to observe the expression of GFP in the PB-NK cells that were successfully transduced with the lentivirus at the above time points.

[0210] As shown in Figure 4, fluorescence images of CAR-V1052-EGFP cells at different time points are shown. As shown in Figure 5, fluorescence images of CAR-V2195-EGFP cells at different time points are shown. As can be seen from Figures 4 and 5, a stronger fluorescence reaction indicates a more successful lentiviral transduction.

[0211] As shown in FIG6 , the expression of GFP in CAR-NK cells was detected by flow cytometry at 96.0 h after lentiviral transduction. As can be seen from FIG6 , CAR-V1052-EGFP NK cells accounted for 65.3% and CAR-V2195-EGFP NK cells accounted for 52.9%.

[0212] Example 3

[0213] (1) Add a certain amount of recombinant human fibronectin fragment stock solution into PBS, pipette and mix, and prepare it to 5.0ug / cm 2 A working solution of recombinant human fibronectin fragment at a working concentration was used as a coating solution. The coating solution was transferred to a 24-well cell culture plate at a volume of 500 μL / well. After coating at 4° C. for 12.0 h to 24.0 h, the coating solution was discarded to prepare a coated culture plate.

[0214] (2) Add 0.15 μL / well of lentiviral stock solution with an MOI of 50, 500 μL / well of polybrene working solution with a working concentration of 8 μg / mL, and 0.5 μL / well of BX795 working solution with a working concentration of 0.5 μmol / mL to the amplification medium, and pipette and mix 20 times to prepare a lentiviral mixture. The lentiviral stock solution contains CAR-V1052-EGFP lentiviral vector and CAR-V2195-EGFP lentiviral vector.

[0215] (3) Add the lentiviral mixture to the coated culture plate and centrifuge at 32°C, 2000g for 2.0 h.

[0216] (4) The PB-NK cells that were cultured to 144.0 hours after the aforementioned NK cell expansion method were collected and cultured at 3×10 5 / well was added to a 24-well cell culture plate containing the lentivirus mixture, and amplification medium was added at a rate of 1 mL / well for lentiviral transduction culture.

[0217] (5) Cultivate in a 37°C, 5% CO2 incubator until 12.0 h to 16.0 h, remove the culture medium containing the lentivirus mixture, and replace it with fresh amplification medium at a volume of 1 mL / well.

[0218] (6) The cells were cultured in a 37°C, 5% CO2 incubator until 24.0 h, 48.0 h, 72.0 h, and 96.0 h, and fluorescence microscopy was used to observe the expression of GFP in the PB-NK cells that were successfully transduced with the lentivirus at the above time points.

[0219] As shown in Figure 7, fluorescence images of CAR-V1052-EGFP cells at different time points are shown. As shown in Figure 8, fluorescence images of CAR-V2195-EGFP cells at different time points are shown. As can be seen from Figures 7 and 8, the strong fluorescence reaction indicates that the lentiviral transduction was successful.

[0220] As shown in Figure 9, the expression of NK cells was detected by flow cytometry at 96.0 hours after lentiviral transduction. As shown in Figure 10, the expression of GFP in CAR-NK cells was detected by flow cytometry at 96.0 hours after lentiviral transduction. As can be seen from Figure 10, NK cells accounted for 0%, CAR-V1052-EGFP NK cells accounted for 14.3%, and CAR-V2195-EGFP NK cells accounted for 4.9%.

[0221] Comparative Example 1

[0222] (1) Add a certain amount of recombinant human fibronectin fragment stock solution into PBS, pipette and mix, and prepare it to 5.0ug / cm 2 A working solution of recombinant human fibronectin fragment at a working concentration was used as a coating solution. The coating solution was transferred to a 24-well cell culture plate at a volume of 500 μL / well. After coating at 4° C. for 12.0 h to 24.0 h, the coating solution was discarded to prepare a coated culture plate.

[0223] (2) Add 0.15 μL / well of lentiviral stock solution with an MOI of 10 and 500 μL / well of polybrene working solution with a working concentration of 8 μg / mL to the amplification culture medium, pipette and mix 20 times to prepare a lentiviral mixture. The lentiviral stock solution contains CAR-V1052-EGFP lentiviral vector and CAR-V2195-EGFP lentiviral vector. (3) Add the lentiviral mixture to the coated culture plate and centrifuge at 32°C, 2000g for 2.0 hours.

[0224] (4) PB-NK cells that were cultured and expanded by the aforementioned NK cell expansion method until 336.0 hours were collected. The flow cytometry results of PB-NK cells at this time are shown in FIG11 , which show that the proportion of NK cells in PB-NK cells reached more than 95% before lentiviral transduction; microscopic photos at different magnifications are shown in FIG12 , which show that the cells are in good condition. PB-NK cells were cultured at 3×10 5 / well was added to a 24-well cell culture plate containing the lentivirus mixture, and amplification medium was added at a rate of 1 mL / well for lentiviral transduction culture.

[0225] (5) Cultivate in a 37°C, 5% CO2 incubator until 12.0 h to 16.0 h, remove the culture medium containing the lentivirus mixture, and replace it with fresh amplification medium at a volume of 1 mL / well.

[0226] (6) The cells were cultured in a 37°C, 5% CO2 incubator until 24.0 h, 48.0 h, 72.0 h, and 96.0 h, and fluorescence microscopy was used to observe the expression of GFP in the PB-NK cells that were successfully transduced with the lentivirus at the above time points.

[0227] Figure 13 shows a fluorescence image of PB-NK cells 24 hours after transduction and culture; Figure 16 shows a fluorescence image of PB-NK cells 48 hours after transduction and culture; and Figure 19 shows a fluorescence image of PB-NK cells 72 hours after transduction and culture. As can be seen from the figures, the strong fluorescence reaction indicates successful lentiviral transduction.

[0228] As shown in FIG14 , it shows the expression of NK cells detected by flow cytometry at 24.0 h after lentiviral transduction and culture.

[0229] As shown in Figure 15, the expression of GFP in CAR-NK cells was detected by flow cytometry at 24.0 hours after lentiviral transduction culture. As can be seen from Figure 15, NK cells accounted for 1.78%, CAR-V1052-EGFP NK cells accounted for 13.2%, and CAR-V2195-EGFP NK cells accounted for 12.3%.

[0230] As shown in FIG17 , it shows the expression of NK cells detected by flow cytometry at 48.0 h after lentiviral transduction and culture.

[0231] As shown in Figure 18, the expression of GFP in CAR-NK cells was detected by flow cytometry at 48.0 hours after lentiviral transduction culture. As can be seen from Figure 18, NK cells accounted for 1.69%, CAR-V1052-EGFP NK cells accounted for 12.9%, and CAR-V2195-EGFP NK cells accounted for 11.0%.

[0232] As shown in FIG20 , the expression of NK cells detected by flow cytometry at 72.0 h after lentiviral transduction culture is shown.

[0233] As shown in Figure 21, the expression of GFP in CAR-NK cells was detected by flow cytometry at 72.0 hours after lentiviral transduction culture. As can be seen from Figure 21, NK cells accounted for 1.01%, CAR-V1052-EGFP NK cells accounted for 11.1%, and CAR-V2195-EGFP NK cells accounted for 10.7%.

[0234] Comparative Example 2

[0235] (1) Add a certain amount of recombinant human fibronectin fragment stock solution into PBS, pipette and mix, and prepare it to 5.0ug / cm 2 A working solution of recombinant human fibronectin fragment at a working concentration was used as a coating solution. The coating solution was transferred to a 24-well cell culture plate at a volume of 500 μL / well. After coating at 4° C. for 12.0 h to 24.0 h, the coating solution was discarded to prepare a coated culture plate.

[0236] (2) Add 0.15 μL / well of a lentiviral stock solution with an MOI of 50 and 500 μL / well of a polybrene working solution with a working concentration of 8 μg / mL to the amplification medium, pipet and mix 20 times to prepare a lentiviral mixture. The lentiviral stock solution contains the CAR-V1052-EGFP lentiviral vector and the CAR-V2195-EGFP lentiviral vector.

[0237] (3) Add the lentiviral mixture to the coated culture plate and centrifuge at 32°C, 2000g for 2.0 h.

[0238] (4) PB-NK cells that were cultured and expanded by the aforementioned NK cell expansion method until 336.0 hours were collected. The flow cytometry results of PB-NK cells at this time are shown in FIG11 , which show that the proportion of NK cells in PB-NK cells reached more than 95% before lentiviral transduction; microscopic photos at different magnifications are shown in FIG12 , which show that the cells are in good condition. PB-NK cells were cultured at 3×10 5 / well was added to a 24-well cell culture plate containing the lentivirus mixture, and amplification medium was added at a rate of 1 mL / well for lentiviral transduction culture.

[0239] (5) Cultivate in a 37°C, 5% CO2 incubator until 12.0 h to 16.0 h, remove the culture medium containing the lentivirus mixture, and replace it with fresh amplification medium at a volume of 1 mL / well.

[0240] (6) The cells were cultured in a 37°C, 5% CO2 incubator until 24.0 h, 48.0 h, and 72.0 h, and fluorescence microscopy was used to observe the expression of GFP in the PB-NK cells that were successfully transduced with the lentivirus at the above time points.

[0241] Figure 22 shows a fluorescence image of PB-NK cells 24 hours after transduction and culture; Figure 25 shows a fluorescence image of PB-NK cells 48 hours after transduction and culture; and Figure 28 shows a fluorescence image of PB-NK cells 72 hours after transduction and culture. As can be seen from the figures, the strong fluorescence reaction indicates successful lentiviral transduction.

[0242] As shown in FIG23 , it shows the expression of NK cells detected by flow cytometry at 24.0 hours after lentiviral transduction and culture.

[0243] As shown in Figure 24, the expression of GFP in CAR-NK cells was detected by flow cytometry at 24.0 hours after lentiviral transduction culture. As can be seen from Figure 24, NK cells accounted for 1.78%, CAR-V1052-EGFP NK cells accounted for 40.4%, and CAR-V2195-EGFP NK cells accounted for 21.6%.

[0244] As shown in FIG26 , it shows the expression of NK cells detected by flow cytometry at 48.0 h after lentiviral transduction and culture.

[0245] As shown in Figure 27, the expression of GFP in CAR-NK cells was detected by flow cytometry at 48.0 hours after lentiviral transduction culture. As can be seen from Figure 27, NK cells accounted for 1.69%, CAR-V1052-EGFP NK cells accounted for 34.6%, and CAR-V2195-EGFP NK cells accounted for 17.3%.

[0246] As shown in FIG29 , the expression of NK cells detected by flow cytometry at 72.0 hours after lentiviral transduction culture is shown.

[0247] As shown in Figure 30, the expression of GFP in CAR-NK cells was detected by flow cytometry at 72.0 hours after lentiviral transduction culture. As can be seen from Figure 30, NK cells accounted for 1.01%, CAR-V1052-EGFP NK cells accounted for 26.4%, and CAR-V2195-EGFP NK cells accounted for 23.8%.

[0248] Comparative Example 3

[0249] (1) Add a certain amount of recombinant human fibronectin fragment stock solution into PBS, pipette and mix, and prepare it to 5.0ug / cm 2A working solution of recombinant human fibronectin fragment at a working concentration was used as a coating solution. The coating solution was transferred to a 24-well cell culture plate at a volume of 500 μL / well. After coating at 4° C. for 12.0 h to 24.0 h, the coating solution was discarded to prepare a coated culture plate.

[0250] (2) Add 0.15 μL / well of lentiviral stock solution with an MOI of 50, 500 μL / well of polybrene working solution with a working concentration of 8 μg / mL, and 0.5 μL / well of BX795 working solution with a working concentration of 0.5 μmol / mL to the amplification medium, and pipette and mix 20 times to prepare a lentiviral mixture. The lentiviral stock solution contains CAR-V1052-EGFP lentiviral vector and CAR-V2195-EGFP lentiviral vector.

[0251] (3) Add the lentiviral mixture to the coated culture plate and centrifuge at 32°C, 2000g for 2.0 h.

[0252] (4) PB-NK cells that were cultured and expanded by the aforementioned NK cell expansion method until 336.0 hours were collected. The flow cytometry results of PB-NK cells at this time are shown in FIG11 , which show that the proportion of NK cells in PB-NK cells reached more than 95% before lentiviral transduction; microscopic photos at different magnifications are shown in FIG12 , which show that the cells are in good condition. PB-NK cells were cultured at 3×10 5 / well was added to a 24-well cell culture plate containing the lentivirus mixture, and amplification medium was added at a rate of 1 mL / well for lentiviral transduction culture.

[0253] (5) Cultivate in a 37°C, 5% CO2 incubator until 12.0 h to 16.0 h, remove the culture medium containing the lentivirus mixture, and replace it with fresh amplification medium at a volume of 1 mL / well.

[0254] (6) The cells were cultured in a 37°C, 5% CO2 incubator until 24.0 h, 48.0 h, and 72.0 h, and fluorescence microscopy was used to observe the expression of GFP in the PB-NK cells that were successfully transduced with the lentivirus at the above time points.

[0255] Figure 31 shows a fluorescence photograph of PB-NK cells 24 hours after transduction and culture; Figure 34 shows a fluorescence photograph of PB-NK cells 48 hours after transduction and culture; and Figure 37 shows a fluorescence photograph of PB-NK cells 72 hours after transduction and culture. As can be seen from the figures, the strong fluorescence reaction indicates successful lentiviral transduction.

[0256] As shown in FIG32 , it shows the expression of NK cells detected by flow cytometry at 24.0 hours after lentiviral transduction and culture.

[0257] As shown in Figure 33, the expression of GFP in CAR-NK cells was detected by flow cytometry at 24.0 hours after lentiviral transduction culture. As can be seen from Figure 22, NK cells accounted for 1.78%, CAR-V1052-EGFP NK cells accounted for 26.7%, and CAR-V2195-EGFP NK cells accounted for 21.6%.

[0258] As shown in FIG35 , it shows the expression of NK cells detected by flow cytometry at 48.0 h after lentiviral transduction and culture.

[0259] As shown in Figure 36, the expression of GFP in CAR-NK cells was detected by flow cytometry at 48.0 hours after lentiviral transduction culture. As can be seen from Figure 36, NK cells accounted for 1.69%, CAR-V1052-EGFP NK cells accounted for 20.0%, and CAR-V2195-EGFP NK cells accounted for 17.3%.

[0260] As shown in FIG38 , it shows the expression of NK cells detected by flow cytometry at 72.0 hours after lentiviral transduction and culture.

[0261] As shown in Figure 39, the expression of GFP in CAR-NK cells was detected by flow cytometry at 72.0 hours after lentiviral transduction culture. As can be seen from Figure 39, NK cells accounted for 1.01%, CAR-V1052-EGFP NK cells accounted for 17.1%, and CAR-V2195-EGFP NK cells accounted for 23.8%.

[0262] The differences and transduction effects between the examples of the present disclosure and the comparative examples are shown in Table 1.

[0263] Table 1 Differences and transduction effects of Examples 1 to 6

[0264] It can be seen from the above embodiments of the present disclosure that in Examples 1 to 3, human peripheral blood mononuclear cells obtained by culturing the NK cell expansion culture method from the 2nd day to the 6th day, especially human peripheral blood mononuclear cells obtained by expansion culture from the 48th hour to the 96th hour, are in a rapid growth period at this time. After lentiviral transduction, the lentivirus can be rapidly amplified along with the amplification of NK cells, which can reduce the requirement for the amount of lentivirus used during viral transduction. A large number of CAR-NK cells can be prepared by using less lentivirus, thereby improving the viral transduction rate.

[0265] It can be seen from Comparative Examples 1 to 3 that either BX795 was not added during viral transduction, resulting in a low viral transduction rate, or the human peripheral blood mononuclear cells obtained after culturing for 10 days using the NK cell expansion culture method, at which time the NK cell expansion rate tends to be slow and non-expanding. After successful lentiviral transduction, they become CAR-NK cells and cannot be expanded in large quantities, resulting in poor transduction effect.

[0266] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

[0267] In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in this field can implement them. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this disclosure.

[0268] Although the embodiments of the present disclosure have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations may be made to the embodiments without departing from the principles and spirit of the present disclosure, the scope of which is defined by the claims and their equivalents.

[0269]

Claims

1. A method for expanding and culturing NK cells, comprising the following steps: An expansion and culture step of placing human peripheral blood mononuclear cells in a culture container containing an activation medium and an expansion medium, and statically expanding and culturing to obtain NK cells; wherein, When the human peripheral blood mononuclear cells are statically expanded and cultured to the first expansion and culture state, the activation medium and the expansion medium are supplemented into the culture container; When the human peripheral blood mononuclear cells are statically expanded and cultured to the second expansion and culture state, the expansion medium is supplemented into the culture container.

2. The method for expanding and culturing NK cells according to claim 1, wherein, The first expansion and culture state is the expansion and culture to the 72nd hour to the 96th hour; the second expansion and culture state is the expansion and culture for more than the 96th hour; the first expansion and culture cycle is 44 hours to 52 hours.

3. The method for expanding and culturing NK cells according to claim 1, wherein, When the human peripheral blood mononuclear cells are cryopreserved human peripheral blood mononuclear cells, before the expansion and culture step, the following steps are further included: A resuscitation and culture step of resuscitating the cryopreserved human peripheral blood mononuclear cells to obtain resuscitated human peripheral blood mononuclear cells, and placing the resuscitated human peripheral blood mononuclear cells in a culture container containing the expansion medium and statically resuscitating and culturing until the resuscitation and culture state is reached, and then performing the expansion and culture step.

4. The method for expanding and culturing NK cells according to claim 3, wherein, The resuscitation and culture state is statically resuscitating and culturing to the 8th hour to the 16th hour.

5. The method for expanding and culturing NK cells according to any one of claims 1 to 4, wherein, The activation medium includes Retronectin, anti-human CD16 monoclonal antibody, anti-human HER-2 monoclonal antibody, OK432, recombinant human FLT3 Ligand protein, recombinant human IL3 protein, recombinant human IL7 protein, recombinant human IL18 protein, recombinant human IL21 protein, recombinant human IL1b protein, anti-human CD52 monoclonal antibody, anti-human CD137 monoclonal antibody, recombinant human SCF protein, recombinant human IL12 protein, recombinant human OX40 Ligand protein, recombinant human 4-1BB protein, recombinant human IL-15 protein, and human interleukin-2 for injection; the expansion medium includes recombinant human IL-15 protein and human interleukin-2 for injection.

6. A method for lentiviral transduction of NK cells, comprising the following steps: A coating step of coating a culture container with a recombinant human fibronectin fragment working solution as a coating solution to obtain a coated culture container; A lentiviral mixture preparation step of mixing a lentiviral stock solution, a polybrene working solution, and a BX795 working solution to obtain a lentiviral mixture; A lentiviral inoculation step of adding the lentiviral mixture to the coated culture container and performing centrifugal inoculation treatment to obtain an inoculated culture container; Viral transduction culture step: Collect NK cells amplified and cultured to the third amplified culture state and add them to the inoculated culture container, and add the amplification medium to the inoculated culture container for viral transduction culture; and, Transduction amplification culture step: When the viral transduction culture reaches the first transduction state, remove the original medium containing the lentivirus mixture, add the amplification medium, and perform transduction amplification culture until the second transduction state to obtain CAR-NK cells; Among them, the NK cells are obtained by amplification culture using the NK cell amplification culture method described in any one of claims 1 to 5.

7. The lentivirus transduction NK cell method according to claim 6, wherein, The coating concentration of the recombinant human fibronectin fragment working solution is 0.1 μg / cm 2 ~50.0 μg / cm 2 ; the working concentration of the polybrene working solution is 0.5 μg / mL to 20.0 μg / mL; the working concentration of the BX795 working solution is 0.5 μmol / mL to 20.0 μmol / mL; the multiplicity of infection of the lentivirus stock solution is 1 to 50.

8. The lentivirus transduction NK cell method according to claim 7, wherein, in the lentivirus mixture, the volume ratio of the lentivirus stock solution, the polybrene working solution, and the BX795 working solution is (0.12 - 0.18):(400 - 600):(0.4 - 0.6).

9. The lentivirus transduction NK cell method according to claim 6, wherein, the first transduction state is when the viral transduction culture reaches 12 h to 16 h; the second transduction state is when the transduction amplification culture reaches 24 h to 120 h.

10. The lentivirus transduction NK cell method according to claim 6, wherein, the third amplified culture state is when the amplification culture reaches 12.0 h to 336.0 h.

11. The lentivirus transduction NK cell method according to claim 10, wherein, the third amplified culture state is when the amplification culture reaches 24.0 h to 192.0 h.

12. The lentivirus transduction NK cell method according to claim 11, wherein, the third amplified culture state is when the amplification culture reaches 48.0 h to 144.0 h.

13. The lentivirus transduction NK cell method according to claim 10, wherein, the second transduction state is when the transduction amplification culture reaches 48 h to 96 h.

14. The lentivirus transduction NK cell method according to claim 13, wherein, the second transduction state is when the transduction amplification culture reaches 60 h to 84 h.

15. The lentivirus transduction NK cell method according to any one of claims 6 to 14, wherein, the lentivirus stock solution contains a CAR-V1052-reporter gene lentiviral vector and / or a CAR-V2195-reporter gene lentiviral vector.

16. A CAR-NK cell product, wherein, the CAR-NK cell product is prepared by the lentivirus transduction NK cell method described in any one of claims 6 to 15.

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