Method and kit for improving lentiviral transduction efficiency and transduction stability of TIL cells

Abstract

The present invention relates to a method and a kit for improving the lentiviral transduction efficiency and transduction stability of TIL cells. The present invention relates to the field of biotechnology. Specifically, the present invention provides a method and a kit for promoting the lentiviral transduction efficiency and transduction stability of TIL cells, including adding BX-795 during the process of lentiviral infection, which can increase the transduction efficiency to more than 60%, significantly improve the transduction stability, and have no obvious effect on the viability of TIL cells. The method for improving the lentiviral transduction efficiency and transduction stability of TIL cells of the present invention can be applied to obtain genetically engineered TIL cells, and promote the development and application of TIL cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a method and a kit for improving the lentiviral transduction efficiency and transduction stability of TIL cells. Background Art

[0002] Tumor infiltrating lymphocyte (TIL) therapy refers to isolating and extracting immune cells from a patient's own tumor tissue, culturing and massively expanding them in vitro, and then re-infusing them into the patient's body to attack and destroy tumor cells. TIL cells cultured and expanded in vitro have shown great application potential and value in tumor immunotherapy due to their stronger killing function, multiple targets, strong tumor tropism and infiltration ability, and low side effects.

[0003] During the clinical application process, the immunosuppressive tumor microenvironment often severely weakens the full exertion of the therapeutic effect of the infused TIL cells in vivo. To solve this problem, gene modification or gene editing techniques are often used to modify TIL cells to further improve their resistance to immunosuppression in the tumor microenvironment and enhance their therapeutic effect on tumors. Currently, in the research of immune cell therapy products represented by CAR-T cells, common viral vectors include lentivirus and retrovirus vectors, among which lentivirus vectors are widely used due to their better safety and technical maturity. CAR-T cells are often derived from peripheral blood T lymphocytes of patients or healthy donors, and most of them are in an undifferentiated or naive state, and lentivirus vectors have a high and stable transduction efficiency for them.

[0004] However, TIL cells derived from tumor tissues, especially those from tumor tissues of advanced patients, are often in a state of terminal differentiation and exhaustion, resulting in a generally low transduction efficiency of lentivirus vectors for TIL cells, and continuously decreasing with the expansion of TIL cells, with the problem of unstable transduction efficiency. Currently, there are no cases of highly efficient (such as transduction efficiency of 50% or more) and stable gene modification and transformation of TIL cells using lentivirus vectors.

[0005] Therefore, there is an urgent need in the art to develop a method for highly efficient and stable gene modification and transformation of TIL cells using lentivirus vectors. Summary of the Invention

[0006] The object of the present invention is to provide a method for improving the lentiviral transduction efficiency and transduction stability of TIL cells.

[0007] In the first aspect of the present invention, a method for obtaining lentivirally transduced TIL cells is provided, comprising the step of adding a transduction-promoting reagent to the transduction system for lentiviral transduction of TIL cells, wherein the transduction-promoting reagent comprises BX-795.

[0008] In another preferred example, the transduction promoting reagent further comprises: other protein kinase TBK1 / IKKε inhibitors in addition to BX-795, such as Amlexanox, MRT67307, or a combination thereof.

[0009] In another preferred example, the transduction promoting reagent further comprises: LentiBoost, Polybrene, Lentiviral Transduction Enhancer, or a combination thereof.

[0010] In another preferred example, the method specifically comprises the steps of:

[0011] A. Providing TIL cells to be transduced and lentivirus;

[0012] B. Inoculating the TIL cells to be transduced into a complete expansion medium, and transducing the TIL cells with the lentivirus in the presence of a transduction promoting reagent; wherein the transduction promoting reagent comprises BX-795;

[0013] C. Collecting the transduced cells;

[0014] D. Amplifying and culturing the cells in step C in a complete expansion medium.

[0015] In another preferred example, in step B, the final concentration of BX-795 in the reaction system is 1-10 μM, preferably 1.5-6 μM, such as 1.5-3 μM, or 3-6 μM.

[0016] In another preferred example, in step B, the medium is a complete expansion medium.

[0017] In another preferred example, the components of the complete expansion medium include: a cell basal medium, a serum substitute, L-glutamine or its substitute, IL-2.

[0018] In another preferred example, the dosage of IL-2 in the complete expansion medium is 100-600 IU / ml, preferably 300 IU / mL.

[0019] In another preferred example, the cell basal medium is a medium suitable for mammalian cell culture.

[0020] In another preferred example, the cell basal medium is selected from the group consisting of: RPMI 1640 medium, AIM-V TM medium, SFM medium, X-VIVO medium, or a combination thereof.

[0021] In another preferred example, the cell basal medium includes Advanced RPMI 1640 medium and CTS TMAIM-V TM medium.

[0022] In another preferred example, the cell basal medium is a medium obtained by mixing Advanced RPMI 1640 medium and CTS TMAIM-V TM medium in a ratio of 1:1.

[0023] In another preferred example, the L-glutamine substitute is Gluta Max TM .

[0024] In another preferred example, the content of Gluta Max in the complete expansion medium is 0.5 - 2%, preferably 1%. TM

[0025] In another preferred example, the serum substitute is CTS TM Immune Cell Serum Substitute.

[0026] In another preferred example, the content of CTS Immune Cell Serum Substitute in the complete expansion medium is 1% - 5%, preferably 2.5%. TM

[0027] In another preferred example, in step A, the TIL cells to be transduced are derived from tumor tissues.

[0028] In another preferred example, the tumor tissue is preferably the tumor tissue from patients with advanced tumors.

[0029] In another preferred example, the patients with advanced tumors refer to those in stage III or IV according to the TNM staging.

[0030] In another preferred example, the TIL cells are TIL cells in a state of terminal differentiation and exhaustion.

[0031] In another preferred example, the TIL cells to be transduced are TIL cells pretreated with BX-795, and the concentration of BX-795 for pretreatment is 1 μM, and the pretreatment time is 4 h to 16 h.

[0032] In another preferred example, the TIL cells to be transduced are TIL cells that have been initially cultured.

[0033] In another preferred example, the initial culture is carried out in an initial culture medium, and the initial culture medium contains IL-2.

[0034] In another preferred example, the primary culture medium further comprises components: TIL cell basal medium, buffer, serum or serum substitute, L-glutamine or its substitute, antibiotic, and optionally a TIL cell activation reagent.

[0035] In another preferred example, the concentration of the IL-2 is 100 - 6000 IU / mL.

[0036] In another preferred example, the TIL cell basal medium includes: RPMI 1640 medium, SFM medium, X-VIVO medium.

[0037] In another preferred example, the TIL cell activation reagent includes: anti-CD3 monoclonal antibody, anti-4-1BB humanized monoclonal antibody, or a combination thereof.

[0038] In another preferred example, the final concentration of the anti-CD3 monoclonal antibody is 10 - 100 ng / mL, and the final concentration of the anti-4-1BB humanized monoclonal antibody is 1 - 50 μg / mL.

[0039] In another preferred example, the primary culture medium includes: RPMI 1640 medium, buffer Hepes with a final concentration of 2.5% V / V, serum substitute with a final concentration of 1% - 10% V / V, GlutaMax with a final concentration of 1% V / V, gentamicin with a final concentration of 0.1% V / V, and IL-2 with a concentration of 6000 IU / mL.

[0040] In another preferred example, the primary culture medium further includes: IL-2 with a concentration of 500 - 3000 IU / mL, anti-CD3 monoclonal antibody with a concentration of 10 - 100 ng / mL, and anti-4-1BB humanized monoclonal antibody with a concentration of 1 - 50 μg / mL.

[0041] In another preferred example, the lentivirus is a VSV-G lentivirus.

[0042] In another preferred example, the lentivirus is a recombinant lentivirus carrying an exogenous target gene.

[0043] In another preferred example, the exogenous target gene includes: the coding gene of an element for enhancing TIL cell function, and the element for TIL cell function includes: an exogenous cytokine with or without modification, an exogenous membrane protein with or without modification, an exogenous secreted protein with or without modification, and an exogenous antibody with or without modification.

[0044] In another preferred example, the multiplicity of infection (MOI) of the lentivirus is 0.1 - 100, preferably 1 - 50, and more preferably 5 - 20.

[0045] In another preferred example, in step B, the infection time is 12 to 48 hours, such as 24 to 48 hours, preferably 24h ± 2h.

[0046] In another preferred example, in step C, the method for collecting cells is a method known in the art, such as centrifugation, discarding the supernatant, and collecting the cell pellet.

[0047] In another preferred example, in step D, the amplification culture is carried out in a complete expansion medium.

[0048] In another preferred example, the complete expansion medium in step D is the same as the complete expansion medium in the reaction system.

[0049] In another preferred example, the amplification culture is carried out in the presence of feeder cells.

[0050] In another preferred example, the amplification culture is carried out in the presence of BX-795, and the concentration of BX-795 is 0.1 μM, and the treatment time of BX-795 is 48 to 72 hours.

[0051] In another preferred example, the feeder cells are K562 cells, preferably feeder cells treated by irradiation.

[0052] In another preferred example, the feeder cells are engineered K562 cells expressing membrane-bound IL-21 and 4-1BBL.

[0053] In another preferred example, the ratio of the TIL cells to the feeder cells is 1:2.5 to 1:50.

[0054] In another preferred example, the conditions for the amplification culture are 37°C ± 3°C, 5% CO2.

[0055] In another preferred example, the time for the amplification culture is 5 to 12 days, preferably 7 to 10 days.

[0056] In another preferred example, during the amplification culture, the medium is half-changed every 2 - 3 days.

[0057] In another preferred example, the transduction efficiency of the cells collected in step C is E0, E0 ≥ 50%, preferably E0 ≥ 55%, more preferably E0 ≥ 60%.

[0058] In another preferred example, the transduction efficiency of the cells after the amplification culture in step D is E1, E0 ≥ 50%, preferably E0 ≥ 55%, more preferably E0 ≥ 60%.

[0059] In another preferred example, after the amplification culture, the transduction efficiency does not decrease, or the decrease amplitude (E0 - E1) / E0 ≤ 20%, preferably ≤ 15%, more preferably ≤ 10%.

[0060] In another preferred example, the viability of the cells after amplification culture in step D is ≥60%, preferably ≥70%, more preferably ≥80%.

[0061] In the second aspect of the present invention, there is provided a use of BX-795 for preparing a reagent, which is added to the reaction system for lentivirus-infected TIL cells to improve the lentiviral transduction efficiency and transduction stability of TIL.

[0062] In another preferred example, in the reaction system, the concentration of BX-795 is 1-10 μM, preferably 1.5-6 μM, more preferably 3-6 μM.

[0063] In another preferred example, the reagent is added before the start of, during, and / or after the end of the reaction of lentivirus-infected TIL cells, preferably before the start of and / or during the reaction.

[0064] In another preferred example, the reagent further comprises: other protein kinase TBK1 / IKKε inhibitors in addition to BX-795, such as Amlexanox, MRT67307; and / or LentiBoost.

[0065] In another preferred example, the transduction efficiency is: the proportion of TIL cells that successfully express the exogenous target gene among the total TIL cells.

[0066] In another preferred example, the high transduction stability means that the transduction efficiency of the transduced TIL cells after amplification culture compared with the transduction efficiency before amplification culture hardly decreases or the decrease amplitude ≤20%, preferably ≤15%, more preferably ≤10%.

[0067] In the third aspect of the present invention, there is provided a kit for lentiviral transduction of TIL cells, which kit comprises: (a) a transduction-promoting reagent, which transduction-promoting reagent comprises BX-795; (b) a complete culture medium for expansion; and (c) an instruction manual.

[0068] In another preferred example, the instruction manual describes the usage method of the kit:

[0069] A. Provide the TIL cells to be transduced and lentivirus.

[0070] B. Inoculate the TIL cells to be transduced into the complete culture medium for expansion, and transduce the TIL cells with the lentivirus in the presence of the transduction-promoting reagent, wherein the transduction-promoting reagent comprises BX-795, and the final concentration of BX-795 in this transduction system is 1.5-6 μM.

[0071] C. Collect the transduced cells;

[0072] D. Amplify the cells in step C in the complete expansion medium.

[0073] In another preferred example, the complete expansion medium comprises components: cell basal medium, serum substitute, L-glutamine or its substitute, IL-2.

[0074] In another preferred example, the dosage of IL-2 in the complete expansion medium is 100 - 600 IU / ml, preferably 300 IU / mL.

[0075] In another preferred example, the cell basal medium is a medium obtained by mixing Advanced RPMI 1640 medium and CTS TMAIM-V TM media in a 1:1 ratio.

[0076] In another preferred example, the L-glutamine substitute is Gluta Max TM , with a content of 0.5 - 2%, preferably 1%.

[0077] In another preferred example, the serum substitute is CTS TM Immune Cell Serum Substitute, with a content of 1% - 5%, preferably 2.5%.

[0078] In another preferred example, the transduction-promoting reagent further comprises: other protein kinase TBK1 / IKKε inhibitors except BX-795, such as Amlexanox, MRT67307, or a combination thereof.

[0079] In another preferred example, the transduction-promoting reagent further comprises: LentiBoost, Polybrene, Lentiviral Transduction Enhancer, or a combination thereof.

[0080] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here.

[0081] The beneficial effects of the present invention include:

[0082] 1. The present invention provides a method that can greatly improve the transduction efficiency of lentivirus on TIL cells, which can increase the transduction efficiency of about 20% to about 60%, with an increase amplitude of up to 3 times.

[0083] 2. The method of adding BX-795 in the present invention can improve the transduction stability of lentivirus to TIL cells. Even after about 9 days of expansion culture, the transduction efficiency of TIL cells can still maintain a very high level and shows no significant decrease compared with that before expansion culture.

[0084] 3. After expansion culture, the cell viability of TIL cells obtained by the method of adding BX-795 in the present invention returns to a high level and is comparable to that of the control group without adding BX-795. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 Shows the detection results of transduction efficiency before and after expansion culture of TIL cells derived from donor 1 after being infected with lentivirus with or without BX-795.

[0086] Figure 2 Shows the detection results of cell viability before and after expansion culture of TIL cells derived from donor 1 after being infected with lentivirus with or without BX-795.

[0087] Figure 3 Shows the detection results of transduction efficiency before and after expansion culture of TIL cells derived from donor 2 after being infected with lentivirus with or without BX-795.

[0088] Figure 4 Shows the detection results of cell viability before and after expansion culture of TIL cells derived from donor 2 after being infected with lentivirus with or without BX-795.

[0089] Figure 5 Shows the detection results of the total number of transduction-positive cells after expansion culture of TIL cells derived from donor 1 after being infected with lentivirus with or without BX-795.

[0090] Figure 6 Shows the detection results of the total number of transduction-positive cells after expansion culture of TIL cells derived from donor 2 after being infected with lentivirus with or without BX-795.

[0091] Figure 7 Shows the flow cytometry maps of transduction efficiency before and after expansion culture of TIL cells derived from donor 1 after being infected with lentivirus with or without BX-795.

[0092] Figure 8 Shows the flow cytometry maps of transduction efficiency before and after expansion culture of TIL cells derived from donor 2 after being infected with lentivirus with or without BX-795.

[0093] Figure 9The detection results of the transduction efficiency before and after expansion of TIL cells from donor 3 after being infected with lentivirus under the conditions of different concentrations of BX-795 are shown. Detailed implementation manners

[0094] Through extensive and in-depth research, the present inventors provide a method for promoting the lentiviral transduction efficiency and transduction stability of TIL cells, including adding BX-795 during the lentiviral infection process, which can increase the transduction efficiency to more than 60%. Compared with the control group without adding BX-795, the transduction efficiency is increased by more than 3 times. In addition to significantly improving the transduction efficiency, the method of the present invention also significantly improves the transduction stability, that is, the transduced TIL cells will not cause a significant decrease in the transduction efficiency after passing through the amplification culture stage.

[0095] The present inventors also unexpectedly found that after amplification culture, the viability of the TIL cells obtained by the method of adding BX-795 in the present invention is increased instead, and can reach a level comparable to that of the control group without adding BX-795. The method of the present invention efficiently promotes the lentiviral transduction of cells and improves the transduction stability without affecting the cell viability, and is particularly suitable for the transduction of TIL cells in a terminally differentiated or exhausted state (such as TIL cells from cancer patients in the advanced stage).

[0096] Based on this, the present invention is completed.

[0097] Terms

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0099] As used herein, the term "tumor-infiltrating lymphocytes" or "TIL cells" refers to a class of immune cells present in the tumor microenvironment. TIL cells have significant advantages in the field of cancer treatment. First, they can be directly extracted from patients and re-injected into patients after in vitro expansion and activation, thus realizing individualized treatment. Second, since TIL cells are derived from the patients themselves, there is no risk of allogeneic rejection, and the safety is relatively high. In addition, TIL cell therapy has shown significant efficacy against certain refractory tumors (such as melanoma) in clinical trials.

[0100] As used herein, the term "primary culture" is also referred to as "preliminary culture" or "pre-REP", and refers to the first-stage culture of initial TIL cells in tumor tissues to obtain activated TIL cells with the potential for expansion. In the present invention, the culture medium used in the primary culture stage is referred to as "primary culture medium" or "primary complete culture medium", and the TIL cells obtained after the primary culture are referred to as "TIL primary culture cells".

[0101] In one embodiment, the components of the complete primary culture medium used in the primary culture stage are as follows: RPMI 1640 medium, Hepes with a final concentration of 2.5% V / V, serum substitute with a final concentration of 5% V / V, GlutaMax with a final concentration of 1% V / V, and gentamicin (concentration: 10 μg / ml) with a final concentration of 0.1% V / V, anti-CD3 monoclonal antibody with a final concentration of 30 ng / mL, anti-4-1BB humanized monoclonal antibody with a final concentration of 5 μg / mL, and human interleukin-2 injection (IL-2) with a final concentration of 1000 IU / mL.

[0102] In a preferred embodiment, the components of the complete primary culture medium used in the primary culture stage are as follows: RPMI 1640 medium, containing Hepes with a final concentration of 2.5% V / V, serum substitute with a final concentration of 5% V / V, GlutaMax with a final concentration of 1% V / V, and gentamicin (concentration: 10 μg / ml) with a final concentration of 0.1% V / V, and human interleukin-2 injection (IL-2) with a final concentration of 6000 IU / mL.

[0103] As used herein, the terms "expansion culture", "amplification culture", "rapid amplification culture", and "REP" are used interchangeably and refer to the amplification culture of cells after primary culture to rapidly obtain a large number of TIL cells.

[0104] In the present invention, the method in the expansion culture stage is not limited, and conventional expansion culture methods known in the art can be selected to rapidly amplify the cells after primary culture.

[0105] A preferred expansion culture method is: performing expansion culture in a complete expansion culture medium, wherein the complete expansion culture medium contains the following components: IL-2, cell basal medium, serum substitute, L-glutamine or its substitute. Among them, the dosage of IL-2 is 100 - 600 IU / ml, preferably, the dosage of IL-2 is 200 - 400 IU / ml, and more preferably 300 IU / ml.

[0106] In a preferred embodiment, the cell basal medium is a medium suitable for mammalian cell culture, such as RPMI 1640 medium, AIM-V TM medium, SFM medium, X-VIVO medium, and other media known in the art. In a preferred embodiment, the cell basal medium of the present invention is a medium obtained by mixing Advanced RPMI 1640 medium and CTS TM AIM-V TM medium at a ratio of 1:1.

[0107] In a preferred embodiment, the contents of other components in the expansion complete medium are as follows: 0.5 - 2% of L-glutamine substitute, preferably the L-glutamine substitute is Gluta MaxTM; 1% - 5% of serum substitute, such as CTSM Immune Cell Serum Substitute.

[0108] In the present invention, feeder cells can be optionally added or not added during the expansion stage. Feeder cells include commonly used feeder cells in the art, especially irradiated feeder cells. Preferred feeder cells are PBMC or K562 cells, and more preferred feeder cells are engineered feeder cells, such as feeder cells expressing mbIL21 and 4-1BBL. In a preferred embodiment of the present invention, the feeder cells used are irradiated K562 cells expressing mbIL21 and 41BBL. When feeder cells are added, the ratio of feeder cells to TIL cells is (2.5 - 100):1.

[0109] The term "transduction" refers to the transfer of nucleic acids into a host cell, for example, the transfer of an exogenous target gene into a host cell. As used herein, the term encompasses all techniques for introducing nucleic acids into cells, including but not limited to transformation with plasmid vectors, infection with viral vectors or viral particles, and introduction of naked DNA by electroporation, nucleofection, liposome transfection, or particle gun.

[0110] The term "pseudotype (vector)" refers to a vector particle carrying envelope glycoproteins from other enveloped viruses. In a non-limiting example, the lentiviral vector used for transducing TIL cells is a VSV-G pseudotyped lentiviral vector.

[0111] The term "intrinsic antiviral activity" refers to the activity of the host cell's innate immune system to inhibit viral replication and / or the expression of viral genes in the host cell. As is well known in the art, dsRNA or dsDNA checkpoints in the cytoplasm (e.g., retinoic acid-inducible gene I (RIG-I), cyclic GMP-AMP synthase) can recognize viral nucleic acids and trigger the host cell to enter an antiviral state by inducing a type I interferon response. Therefore, "a reagent capable of inhibiting intrinsic antiviral activity" refers to an inhibitor that can inhibit the development of the antiviral state in the host. In a non-limiting example, the reagent is an inhibitor against IκB kinase (IKKε) or TANK-binding kinase 1 (TBK1), such as BX795.

[0112] The term "BX-795" is a commonly used bioactive compound with the chemical name ethyl 2-amino-5-(1-ethyl-2-hydroxypropyl)-3,4-dioxazole-1-acetate. It is often used as an effective and relatively specific inhibitor of TBK1 and IKKε, acting downstream of RIG-I, MDA-5, and TLR3, and can effectively inhibit the signaling pathway of innate immune antiviral infection.

[0113] The term "transduction efficiency", also known as "transduction positive rate", refers to the proportion of TIL cells that successfully express the exogenous target gene among the total TIL cells. Transduction efficiency can be detected by conventional methods in the art such as flow cytometry analysis.

[0114] The term "transduction stability" refers to the change in transduction efficiency of infected TIL cells before and after expansion culture. For transduced TIL, the transduction efficiency usually decreases after expansion culture. When the decrease in transduction efficiency after expansion culture is very small (for example, the decrease amplitude ≤ 20%), the transduction stability can be considered very high.

[0115] Method for improving the transduction efficiency of lentivirus on TIL cells

[0116] In the present invention, a method for improving the transduction efficiency of lentivirus on TIL cells is provided, including adding BX-795 to the reaction system of lentivirus infecting TIL cells.

[0117] The method of the present invention specifically includes three stages: before infection, during infection, and after infection. Before infection: Prepare the lentivirus to be transduced and TIL cells; During infection: Conduct the reaction of lentivirus infecting TIL cells in the culture medium; After infection: Harvest the infected TIL cells and perform amplification culture on the TIL cells.

[0118] Before infection: First, it is necessary to prepare the lentiviral vector and perform preliminary culture on TIL cells. In the present invention, the lentiviral vector is a VSV-G type lentiviral vector, and the lentivirus carries the exogenous target gene. The preparation of lentivirus can adopt any method known in the art, such as the three-plasmid packaging method, but not limited thereto.

[0119] In the present invention, the source of TIL cells is not restricted, generally being TIL cells isolated from tumor tissues in tumor patients. Preferably, the TIL cells will undergo primary culture to obtain a sufficient number of isolated TIL cells for subsequent transduction. The primary culture method of the present invention is not restricted. For example, primary culture can be carried out in a primary culture medium known in the art. In a preferred embodiment, the components of the primary culture medium used in the present invention include: TIL cell basal medium, buffer, serum or serum substitute, L-glutamine or its substitute, antibiotic, IL-2, and optionally a TIL cell activation reagent.

[0120] In the present invention, the ratio of the total number of lentiviruses to the total number of TIL cells, i.e., the multiplicity of infection (MOI), is not restricted, and those skilled in the art can routinely adjust it according to needs. Preferably, the MOI is 0.1 - 100, such as 1 - 50, 5 - 20.

[0121] During infection: The reaction of lentivirus infecting TIL cells is usually carried out in the TIL cell culture medium, and BX - 795 is added to the TIL cell culture medium. In the present invention, the TIL cell culture medium is a medium conducive to the growth and expansion of TIL cells, and its components are not particularly restricted. In a preferred embodiment, the reaction of lentivirus infecting TIL cells is carried out in the TIL cell expansion culture medium.

[0122] In a preferred embodiment, the concentration of BX - 795 is 1 - 5 μM, preferably 2 - 4 μM, and more preferably 2.5 - 3.5 μM. The infection time is 12 - 48 h, preferably 24 h ± 2 h. After the infection ends, the TIL cells are collected by centrifugation, and the transduction efficiency and cell viability are detected, i.e., the transduction efficiency and cell viability before expansion culture.

[0123] Compared with the control group with the same other conditions but without adding BX - 795, the transduction efficiency of the method of the present invention is more than 3 times that of the control group, but the cell viability decreases.

[0124] After infection: The TIL cells collected after the infection ends are subjected to expansion culture to obtain a large number of TIL cells. The method of expansion culture in the present invention is not restricted and can be any method known in the art for the expansion culture of TIL cells. Usually, the expansion culture time is 7 - 12 days. The expansion culture can be carried out under the condition of having feeder cells or without feeder cells, and preferably under the condition of having feeder cells.

[0125] After the expansion culture ends, the transduction efficiency and cell viability of the TIL cells are detected again, i.e., the transduction efficiency and cell viability after expansion culture. Compared with the transduction efficiency before expansion culture, the transduction efficiency after expansion culture hardly decreases or the decrease range is very small, and the decrease range ≤ 20%, such as ≤ 15%, or ≤ 10%.

[0126] The present invention unexpectedly finds that, compared with the control group with the same other conditions but without adding BX - 795, the cell viability after adding BX - 795 and expansion culture in the present invention is equivalent to that of the control group, that is, the influence of BX - 795 on cell viability has been restored after expansion culture.

[0127] Application

[0128] In the present invention, there is also provided the use of BX-795 in the preparation of a reagent for adding to a transduction system for lentivirus transduction of TIL cells to improve the lentivirus transduction efficiency and transduction stability of TIL.

[0129] In the present invention, there is further provided a culture medium for TIL cells for lentivirus infection, wherein a transduction-promoting reagent is added to the culture medium, including BX-795, and optionally including other reagents capable of promoting transduction, such as other protein kinase TBK1 / IKKε inhibitors except BX-795, such as Amlexanox, MRT67307; and / or LentiBoost.

[0130] In the present invention, BX-795 can be used to promote the infection of lentivirus to TIL cells from various sources. In the present invention, the tumor type from which TIL cells are derived is not limited, for example, it is a solid tumor or a hematological tumor, preferably a solid tumor. In a preferred embodiment, the tumor includes but is not limited to: lung cancer, melanoma, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.

[0131] More preferably, BX-795 is particularly suitable for the transduction of TIL cells derived from advanced tumor tissues, and the term "advanced" refers to patients with a clinical stage of III or IV. TIL cells derived from advanced cancer patients are usually in a state of terminal differentiation or exhaustion, with great transduction difficulty and low efficiency. However, by using the transduction method in the present invention, the lentivirus transduction efficiency of TIL cells derived from advanced cancer patients can be significantly improved.

[0132] The method for improving the lentivirus transduction efficiency of TIL cells or the culture medium for TIL cells for lentivirus infection of the present invention can be applied to modify TIL cells, obtain genetically engineered TIL cells and apply them to TIL cell therapy.

[0133] The present invention will be further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and fractions are weight percentages and weight fractions.

[0134] Example 1: Primary culture of TIL cells

[0135] Source of TIL cells: The TIL cells used in the examples were TIL cells from the tumor tissues of tumor patients. Among them, donor 1 was a patient with squamous cell carcinoma of the lung (stage III), donor 2 was a patient with melanoma (stage IV), and donor 3 was a patient with mucosal melanoma (stage III).

[0136] Primary culture complete medium: RPMI 1640 medium, supplemented with Hepes at a final concentration of 2.5% V / V, serum substitute at a final concentration of 5% V / V, GlutaMax at a final concentration of 1% V / V, and gentamicin at a final concentration of 0.1% V / V (concentration: 10 μg / ml), anti-CD3 monoclonal antibody at a final concentration of 30 ng / mL, anti-4-1BB humanized monoclonal antibody at a final concentration of 5 μg / mL, and human interleukin-2 injection (IL-2) at a final concentration of 1000 IU / mL.

[0137] Primary culture steps: Transfer the tumor tissue to a 50 mL centrifuge tube, add tissue rinse solution to wash the tissue and soak for 1 - 5 min; transfer it to a 10 cm 2 culture dish, remove the surface blood stains and unusable tissues, and wash the tumor tissue with tissue rinse solution; transfer the tissue to a 10 cm 2 culture dish, add tissue rinse solution to wash and soak for 1 - 5 min, for a total of 1 - 5 times; after washing, cut all the sample tissues into tissue blocks about 1 - 3 mm 3 in size; take 0.02 - 0.1 g of tumor tissue and transfer it to a breathable culture bag or culture bottle, and supplement 100 mL of primary culture complete medium 1; place the breathable culture bag or culture bottle in a carbon dioxide incubator (37°C, 5% CO2) for culture for 7 - 14 days.

[0138] Example 2: Preparation of lentiviral vector

[0139] Use an endotoxin-free plasmid large-scale extraction kit (purchased from Qiagen) to prepare the expression plasmid linked with the target gene required for transfection (the pLV-HRE-mIL-15 expression plasmid of the mIL-15 gene used in this example comes from patent application CN117820493A).

[0140] Transfect HEK-293T cells with the expression plasmid pLV-HRE-mIL-15, the psPAX2 plasmid, and the pMD2.G plasmid. The transfection system is configured as follows: (1) Centrifuge tube A: 500 μL of Opti-MEM, 10 μg of pLV-HRE-mIL-15 plasmid + 5 μg of pMD2.G plasmid + 5 μg of psPAX2 plasmid and 40 μL of p3000; (2) Centrifuge tube B: 500 μL of OPti-MEM and 40 μL of Lipo3000. Slowly drip the mixture in centrifuge tube B into centrifuge tube A, gently mix with a pipette, and then let it stand at room temperature for 15 - 20 min.

[0141] Add the transfection system to HEK-293T cells. After 4 h of transfection, discard the supernatant, and add 10 mL of complete DMEM medium to each dish; place it in an incubator at 37°C and 5% CO2 for continued culture; after 48 h, collect the culture supernatant of the transfected HEK-293T cells; centrifuge at 400g for 5 min; filter with a 0.45 μm filter, and the obtained filtrate is the original solution of the recombinant lentivirus; concentrate the lentivirus using an Utra 15 centrifugal filtration device, centrifuge at 5000 rpm for 50 min; aliquot the concentrated lentivirus and store it in a refrigerator at -80°C for later use.

[0142] Example 3: Determination of lentivirus titer

[0143] Count the HEK-293T cells after washing and digestion. Dilute the concentrated lentivirus stock solution by 10-fold serial dilution (10×, 100×, 1000×, 10000×). Take 2.5×10 5 HEK-293T cells and add them to the corresponding centrifuge tubes. Each centrifuge tube contains 500 μL of medium-virus-cell mixture. After mixing, transfer it to a 24-well plate; place it in an incubator at 37°C and 5% CO2 for 24 h, then change the medium; after 48 h, collect the infected HEK-293T cells, and use a flow cytometer to detect the proportion of cells with positive transgenic expression, and calculate the virus titer. The calculation method of lentivirus titer: Virus titer = (m × 2.5×10 5 × dilution factor) / transfection volume, where m is the proportion of cells with positive transgenic expression.

[0144] Example 4: Lentivirus infection of TIL primary cultured cells

[0145] The TIL primary cultured cells obtained in Example 1 and the lentivirus prepared in Example 2 are used for the infection in this example. The specific steps are as follows:

[0146] Expansion complete medium: Prepare a 1:1 mixed culture medium of Advanced RPMI 1640 containing 1% Gluta Max, 2.5% CTS Immune Cell Serum Replacement, and 300 IU / ml recombinant human interleukin-2 for injection and CTS AIM-V Medium as the expansion complete medium.

[0147] Inoculate lentivirus (MOI = 10) and TIL primary cultured cells (1×10 6 ) into a 24-well cell culture plate, inoculating 2 wells. Add BX795 (final concentration of 3.0 μM) to only 1 of the wells, and do not add BX795 to the other well (i.e., final concentration of BX795 is 0 μM), and supplement with the expansion complete medium to a volume of 200 μL, and culture in an incubator at 37°C and 5% CO2; after 24 h of infection, collect the cells, centrifuge at 1000 rpm, discard the supernatant, and retain the cell pellet; resuspend with an appropriate amount of expansion complete medium, which is the cells before expansion.

[0148] Example 5: Expansion of TIL cells after lentivirus infection

[0149] In this example, in the presence of feeder cells, the TIL cells after lentivirus infection obtained in Example 4 were amplified and cultured with the expansion complete medium. The expansion complete medium is as described in Example 4.

[0150] The feeder cells used in the present invention are K562 cells expressing mbIL21 and 4-1BBL, irradiated, the irradiation dose is 100 Gy, and stored frozen after irradiation treatment, denoted as K562-mIL21-4-1BBL cells.

[0151] Resuscitate K562-mIL21-4-1BBL cells with the expansion complete medium in Example 4; use a G-REX 24-well plate to co-culture the TIL cells before expansion obtained in Example 4 and K562-mIL21-4-1BBL cells at a ratio of 1:25; culture in an incubator at 37°C and 5% CO2; change the medium by half every 2 - 3 days and culture for 9 days. Harvest the cells after expansion.

[0152] Example 6: Flow cytometry detection of transduction efficiency of TIL cells before and after expansion

[0153] Experimental materials:

[0154] Cells before expansion: Take a certain volume of cell suspension obtained in Example 4 and inoculate it into a cell culture plate, and continue to culture in an incubator at 37°C and 5% CO2 for 48 h. The obtained cells are used for subsequent detection of transduction efficiency and cell viability.

[0155] Expanded cells: The cells obtained in Example 5 were used for the detection of cell count, transduction efficiency, and cell viability.

[0156] Experimental procedure:

[0157] The detection procedure for transduction efficiency is as follows: Take 1×10 6 cells from each group, centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cells with 100 μL of 1× PBS solution containing 2% FBS, then add 5 μL of Fc blocking antibody for blocking, and incubate at room temperature for 10 min; Divide the cells into two parts, the control tube and the experimental tube, with a volume of 50 μL for each group of cells, that is, 5×10 5 cells; Add 17.5 μL of antibody mixture (including BV510 anti-human CD3 ((Biolegend, 317332), PB anti-human CD4 (Biolegend, 317429), APC / Cy7 anti-human CD8 (Biolegend, 344714), AF700 anti-human CD45 (Biolegend, 368514), PE / Cy7 anti-human CD56 (Biolegend, 362510), eF660 EGFR (eBioscience, 50-9509-42), take 2.5 μL of each of the above antibodies and mix evenly) into the control tube and the experimental tube, and incubate at room temperature for 8 min; After incubation; Only add 50 μL of 7-AAD mixture prepared with 1× PBS solution containing 2% FBS to the cells in the experimental tube, and incubate at room temperature for 7 min; Add 1 ml of 1× PBS solution containing 2% FBS to the control tube and the experimental tube, centrifuge at 400 g for 5 min; Discard the supernatant, add 200 μL of 1× PBS solution containing 2% FBS to resuspend the cells, and perform machine detection. The transduction efficiency obtained by detecting the cells before expansion is the initial transduction efficiency of TIL cells; The transduction efficiency obtained by detecting the expanded cells is the transduction efficiency of TIL cells after expansion. The flow cytometry maps of the transduction efficiency of each group of cells are shown in Figure 7 and Figure 8 .

[0158] The detection steps for cell viability and cell count are as follows: Samples are taken from each experimental well in a G-REX 24-well plate, 20 μL is aspirated into a 1.5 mL centrifuge tube, 20 μL of AO / PI staining solution is added, and dilution staining is performed in a 1:1 ratio. After mixing, 20 μL of the mixed solution is aspirated and added into the well of a Countstar counting plate. After standing for 2 min, it is inserted into the sample injection slot of a Countstar Rigel fully automatic cell fluorescence analyzer. The corresponding counting mode is selected according to the cell type, and the information of each well is input on the measurement information page. After information confirmation, click the start button, and the Countstar Rigel automatically measures. After the measurement is completed, the measurement results are displayed on the screen, and the results include information such as cell viability and cell density.

[0159] Statistically count the number of cells harvested after expansion culture, and calculate the total number of transduced positive cells after expansion culture through the following formula: Total number of transduced positive cells after expansion culture = Number of cells harvested after expansion culture × Transduction efficiency after expansion culture.

[0160] After separately detecting the cell transduction efficiency, cell viability before and after expansion culture, and the total number of transduced positive cells after expansion culture for the BX795 = 3.0 μM group and the BX795 = 0 μM group, the results are shown in Table 1 and Figures 1 - 6 as follows.

[0161] Table 1

[0162]

[0163] (1) Effect on transduction efficiency

[0164] For donor 1, as Figure 1 shown, when BX-795 is not added (0 μM), the initial transduction efficiency of lentivirus on TIL cells is 24.87%; when BX-795 with a concentration of 3.0 μM is added, the initial transduction efficiency can reach 75.34%. Compared with the group without BX-795 addition, the initial transduction efficiency is increased by 3 times.

[0165] For donor 2, as Figure 3 shown, when BX-795 is not added (0 μM), the initial transduction efficiency of lentivirus on TIL cells is 20.44%; when BX-795 with a concentration of 3.0 μM is added, the initial transduction efficiency can reach 66.08%. Compared with the group without BX-795 addition, the initial transduction efficiency is increased by 3.2 times.

[0166] The above results indicate that adding BX-795 can significantly improve the transduction efficiency before expansion culture (i.e., the initial transduction efficiency) of lentivirus infecting TIL cells, and the improvement amplitude can reach more than 3 times.

[0167] (2) Effect on transduction stability

[0168] For donor 1, as shown in Table 1 and Figure 1 as shown, when BX-795 was not added (0 μM), the transduction efficiency of lentivirus-infected TIL cells after 9 days of expansion culture was 14.82%. Compared with that before expansion culture (24.87%), the decrease was 40.4%, and the transduction efficiency after expansion culture decreased significantly.

[0169] When BX-795 was added at a concentration of 3.0 μM, the transduction efficiency after 9 days of expansion culture was 68.66%. Compared with that before expansion culture (75.34%), the decrease was 8.9%. Compared with the case without adding BX-795, when BX-795 was added at a concentration of 3.0 μM, the transduction efficiency after expansion culture did not decrease significantly.

[0170] For donor 2, as shown in Table 1 and Figure 3 as shown, when BX-795 was not added (0 μM), the transduction efficiency of lentivirus-infected TIL cells after 9 days of expansion culture was 9.82%. Compared with that before expansion culture (20.44%), the decrease was 52%, and the transduction efficiency after expansion culture decreased significantly.

[0171] When BX-795 was added at a concentration of 3.0 μM, the transduction efficiency after 9 days of expansion culture was 61.76%. Compared with that before expansion culture (66.08%), the decrease was 6.5%. Compared with the case without adding BX-795, when BX-795 was added at a concentration of 3.0 μM, the transduction efficiency after expansion culture did not decrease significantly.

[0172] The above results show that adding BX-795 can significantly improve the transduction stability of lentivirus-infected TIL cells, that is, the transduction efficiency does not decrease significantly after 9 days of expansion culture.

[0173] (3)Effect on cell viability

[0174] The results are as Figure 2 and Figure 4 shown. For TIL cells after lentivirus transfection and before expansion culture, the cell viability of the group with BX-795 added was lower than that of the control group without BX-795 added. That is, after adding BX-795, the cell viability decreased from 81.42% to 72.20% (donor 1) and from 60.82% to 43.29% (donor 2), indicating that adding BX-795 will cause a certain degree of decrease in the cell viability of TIL cells.

[0175] However, after 9 days of expansion culture using the method of the present invention, the cell viability of the group with BX-795 added was comparable to that of the control group without BX-795 added (donor 1: 87.50% vs 84.12%, donor 2: 76.53% vs 77.61%). This shows that after expanding the infected TIL cells, the adverse effect of BX-795 on cell viability has been restored.

[0176] The above results show that although the addition of BX-795 reduces the viability of TIL cells immediately after transduction, the cell viability will recover to a very high level after 9 days of expansion culture, indicating that the protocol of transduction with the addition of BX-795 and subsequent expansion culture in the present invention can ensure a high viability of TIL cells.

[0177] (4) Effect on the total number of transduced positive cells

[0178] For donor 1, as shown in Table 1 and Figure 5 as indicated, in the group with the addition of BX-795, the total number of positive cells harvested after transduction and expansion culture was increased by about 2.6 times compared with the group without addition.

[0179] For donor 2, as shown in Table 1 and Figure 6 as indicated, in the group with the addition of BX-795, the total number of positive cells harvested after transduction and expansion culture was increased by about 4.9 times compared with the group without addition.

[0180] The above results show that under the condition that other conditions are the same, the addition of BX-795 during transduction will significantly increase the total number of harvested positive cells, with an increase range of about 2 - 5 times.

[0181] Example 7: Transduction-promoting effect of different concentrations of BX-795

[0182] The TIL primary culture cells derived from donor 3 obtained in Example 1 and the lentivirus prepared in Example 2 were used for lentiviral infection in this example. The specific steps are as follows:

[0183] Expansion culture complete medium: Prepare a 1:1 mixed culture medium of Advanced RPMI 1640 containing 1% Gluta Max, 2.5% CTS Immune Cell Serum Replacement, 300 IU / ml recombinant human interleukin-2 for injection and CTS AIM-V Medium as the expansion culture complete medium.

[0184] The lentivirus (MOI = 10) and TIL primary culture cells (1×10 6 ) were respectively inoculated into a 24-well cell culture plate, with 4 wells inoculated. One well was without the addition of BX-795 (i.e., the final concentration of BX-795 was 0 μM), and different concentrations of BX-795 (the final concentration of BX-795 in each well was 1.5 μM, 3.0 μM, 6.0 μM respectively) were added to the other three wells, and the volume was made up to 200 μL with the expansion culture complete medium, and then cultured in an incubator at 37°C and 5% CO2; after 24 h of infection, the cells were collected, centrifuged at 1000 rpm, the supernatant was discarded, and the cell pellet was retained; an appropriate amount of expansion culture complete medium was added to resuspend, which was the cells before expansion culture.

[0185] The expansion operation of TIL cells after lentiviral infection was referred to Example 5. The determination of transduction efficiency before and after the expansion of lentivirus-infected TIL cells was referred to Example 6.

[0186] After detecting the transduction efficiency of cells before and after expansion in the groups with different concentrations of BX-795 added, the results are shown in Table 2 and Figure 7 .

[0187] Table 2

[0188]

[0189] As Figure 7 shown, when the concentration of BX-795 was 0 μM, the initial transduction efficiency of lentivirus on TIL cells was 44.70%, and the transduction efficiency after 9 days of expansion was 20.13%. Compared with that before expansion, the decline rate was 55%, and the transduction efficiency after expansion decreased significantly.

[0190] When the concentration of BX-795 added was 1.5 μM, the initial transduction efficiency could reach 62.11%. Compared with the group without BX-795 added, the increase rate of the initial transduction efficiency was 39%; the transduction efficiency after 9 days of expansion was 47.15%, and the decline rate compared with that before expansion was 24%. Compared with the group without BX-795 added, when the concentration of BX-795 added was 1.5 μM, the decline rate of the transduction efficiency after expansion decreased significantly.

[0191] When the concentration of BX-795 added was 3.0 μM, the initial transduction efficiency could reach 64.55%. Compared with the group without BX-795 added, the increase rate of the initial transduction efficiency was 44%; the transduction efficiency after 9 days of expansion was 67.97%, and the increase rate compared with that before expansion was 5%. Compared with the group without BX-795 added, when the concentration of BX-795 added was 3.0 μM, there was no significant difference in the transduction efficiency after expansion.

[0192] When the concentration of BX-795 added was 6.0 μM, the initial transduction efficiency could reach 66.13%. Compared with the group without BX-795 added, the increase rate of the initial transduction efficiency was 48%; the transduction efficiency after 9 days of expansion was 66.25%, which was the same as that before expansion. Compared with the group without BX-795 added, when the concentration of BX-795 added was 6.0 μM, the transduction efficiency after expansion remained basically unchanged.

[0193] The results of donor 3 showed that within the concentration range of BX-795 ≤ 6.0 μM, the addition of BX-795 could significantly improve the initial transduction efficiency in the primary culture of lentivirus-infected TIL cells, with an improvement amplitude of about 40% or more. At the same time, when BX-795 = 1.5, 3.0, and 6.0 μM, there was no significant difference in the improvement amplitude of the initial transduction efficiency among different concentrations. In addition, the addition of BX-795 could significantly improve the stability of the transduction efficiency of the expanded primary culture of lentivirus-infected TIL cells. Especially when the concentration of BX-795 was 3.0 μM, the maintenance effect of the transduction efficiency was the best.

[0194] Discussion

[0195] Lentiviral transduction technology has important applications in the field of cell therapy. However, the efficiency of lentiviral transduction of cells is often affected by cell types, resulting in very low transduction efficiency. For example, TIL cells derived from tumor tissues, especially those from tumor tissues of advanced patients, are often in a state of terminal differentiation and exhaustion, leading to a generally low transduction efficiency of lentiviral vectors for TIL cells, usually about 20%, and there are significant individual differences. In addition, the transduction efficiency will continuously decrease with the expansion of TIL cells, and there is a problem of unstable transduction efficiency.

[0196] Adding transduction-promoting reagents during the lentiviral transduction process can improve the transduction efficiency to a certain extent. However, for special cell populations such as TIL cells, there are few reports in the existing technology on which reagent can effectively promote transduction. In addition, the addition of transduction-promoting chemical reagents will lead to a decrease in cell viability and affect the quality of cell products. Therefore, there is an urgent need in this field to screen suitable transduction-promoting reagents and transduce TIL cells under appropriate methods, which can not only improve the transduction efficiency of lentivirus on TIL cells, but also ensure the high viability of TIL cells and the stability of transduction.

[0197] In the present invention, a method for promoting the transduction of lentivirus on TIL cells is provided, which can increase the transduction efficiency by more than 3 times, and the transfected cells can maintain a cell viability comparable to that of the control group after expansion culture. The results of the present invention comprehensively show that the method provided by the present invention can not only improve the transfection efficiency of TIL cells, but also provide transfection stability and ensure cell viability, and is particularly suitable for producing high-quality TIL cell products in TIL cell therapy.

[0198] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for improving the stability of lentiviral transduction of TIL cells, characterized in that: Includes steps: A. providing TIL cells and lentivirus to be transduced, wherein the TIL cells are TIL cells in a terminally differentiated and exhausted state; B. inoculating the TIL cells to be transduced into a complete culture medium, and transducing the TIL cells with the lentivirus in the presence of a transduction-promoting agent, the transduction time being 24-48 hours; wherein the transduction-promoting agent comprises BX-795, and the final concentration of BX-795 in the transduction system is 1.5-6 μM; C. Collect transduced cells; D. Expand and culture the cells in step C in complete expansion medium for 7 to 12 days; Among them, the transduction efficiency of the TIL cells transduced by the method after expansion culture is reduced by ≤20% compared with the transduction efficiency before expansion culture.

2. The method according to claim 1, characterized in that The transduction time for step B is 24 ± 2 hours.

3. The method according to claim 1, characterized in that The final concentration of BX-795 in the transduction system of step B is 3-6 μM.

4. The method according to claim 1, characterized in that The TIL cells to be transduced are derived from tumor tissue.

5. The method according to claim 1, characterized in that The components of the complete expansion culture medium include: cell basal culture medium, serum substitute, L-glutamine or its substitute, and IL-2.

6. The method according to claim 5, characterized in that The amount of IL-2 in the complete culture medium is 100-600 IU / ml.

7. The method according to claim 1, characterized in that The amplification culture time is 7 to 10 days.

8. The method according to claim 1, characterized in that The method has the following characteristics: (1) The transduction efficiency of the cells collected in step C is E0, E0 ≥ 50%; (2) The transduction efficiency of the cells expanded and cultured in step D is E1, E1 ≥ 50%; (3) After the expansion culture in step D, the transduction efficiency of the cells does not decrease, or the decrease is (E0-E1) / E0≤15%; and (4) The cell viability after expansion culture in step D is ≥ 60%.

9. A use of BX-795 in the preparation of a reagent for adding to a transduction system for lentiviral transduction of TIL cells to improve the lentiviral transduction stability of TIL; The improvement of the lentiviral transduction stability of TIL refers to that the transduction efficiency of the transduced TIL cells after amplification culture is reduced by ≤20% compared with the transduction efficiency before amplification culture.

10. A kit for improving the stability of lentiviral transduction of TIL cells, characterized in that: The kit comprises: (a) a transduction-promoting reagent, wherein the transduction-promoting reagent comprises BX-795; (b) a complete culture medium; and (c) an instruction manual, wherein the instruction manual describes the method for using the kit; The method comprises the steps of: A. providing TIL cells and lentivirus to be transduced, wherein the TIL cells are TIL cells in a terminally differentiated and exhausted state; B. inoculating the TIL cells to be transduced into a complete culture medium, and transducing the TIL cells with the lentivirus in the presence of a transduction-promoting agent, the transduction time being 24-48 hours; wherein the transduction-promoting agent comprises BX-795, and the final concentration of BX-795 in the transduction system is 1.5-6 μM; C. Collect transduced cells; D. Expand and culture the cells in step C in complete expansion medium for 7 to 12 days; The improvement of the lentiviral transduction stability of TIL refers to that the transduction efficiency of the transduced TIL cells after amplification culture is reduced by ≤20% compared with the transduction efficiency before amplification culture.

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